Methods and devices for multiphoton imaging and laser-tissue interaction

Abstract

Methods, systems, and adapters are provided for multiphoton imaging of non-transparent tissues, e.g., ocular and periocular tissues, and laser-tissue interaction with non-transparent tissues, e.g., ocular and periocular tissues. Aspects of the invention include a method of imaging a structure through non-transparent tissue, e.g., ocular or periocular tissue, comprising: deploying an excitation source to transmit light energy to the structure through the non-transparent tissue, e.g., ocular or periocular tissue; detecting light emitted from the structure through the non-transparent tissue, e.g., ocular or periocular tissue, via multiphoton excitation; and imaging the structure based on the detected light. Aspects of the invention also include a method of treating tissue, e.g., ocular or periocular tissue, or dermatological tissue, by manipulating the imaged structure, e.g., providing non-incisional therapy, light-tissue interaction, multiphoton-mediated damage, e.g., thermal damage, photodisruption, photocrosslinking, vascular coagulation, or tissue ablation. Aspects of the invention also include a method of imaging dermatological tissue or a method of providing a dermatological treatment. Aspects of the invention also include a method of providing a cosmetic treatment. Aspects of the invention further include methods for guiding the delivery of gene or cell therapy in ocular or periocular tissues. Also provided are systems and adapters for carrying out the methods described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 431,428, filed December 9, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Introduction Techniques for imaging and manipulating tissues, e.g., ocular and periocular tissues, provide a means to visualize structures, e.g., ocular structures, study photoreceptors and their function, and study and alter biology and disease dynamics, e.g., ocular biology and disease dynamics. However, existing methods for imaging and manipulating tissues, e.g., ocular and periocular tissues, have limited resolution and are unable to image deeper or peripheral structures, e.g., the choroid, peripheral retina, or ciliary body. This is due to various limitations, e.g., the inherent constraints of transpupillary imaging and the high optical absorption and scattering of the uvea and periocular tissues. Certain optical tools for intraocular imaging and therapy rely exclusively on transpupillary approaches or require open surgical procedures in an operating room.

[0003] Additionally, existing dermatological or cosmetic treatments, particularly laser-based dermatological or cosmetic treatments, are limited by existing optical techniques, which may be limited in terms of the depth to which tissue can be manipulated, the precision available for manipulating tissue, or insofar as they are only capable of delivering heat-based treatments for manipulating tissue and not other types of treatments therefor. Summary of the Invention

[0004]

[0003] Thus, there is a need for improved and useful methods and systems for imaging and manipulating tissue, e.g., ocular and periocular tissue, including improved techniques for delivering dermatological and cosmetic treatments or therapies. In particular, there is a need for imaging and manipulating tissue, e.g., ocular and periocular tissue, deeper and through non-transparent tissue than is available through existing techniques. There is also a need for manipulating tissue, including deeper within tissue, using techniques other than heat-based treatments, e.g., providing or applying non-incisional treatments, light-tissue interactions, laser-tissue interactions, laser tissue perturbation, photodisruption, vascular coagulation, ablation, etc. There is also a need for delivering heat-based treatments with greater accuracy and precision than the application of existing heat-based treatments, e.g., multiphoton-mediated thermal damage. The present invention provides such new and useful methods and systems that address the limitations noted above. To achieve this, the present invention utilizes next-generation techniques, such as multiphoton microscopy, including three-photon excited fluorescence (3PEF) microscopy, second harmonic generation (2HG), and third harmonic generation (3HG), to image structures and utilize multiphoton tissue interactions to manipulate structures, in each case through non-transparent tissues, such as ocular or periocular tissues, thereby accessing deeper and peripheral structures not previously accessible using existing techniques, or with greater precision or finer granularity than existing techniques.

[0005] Embodiments of the present invention are capable of visualizing any convenient structure, e.g., ocular and periocular structures, including anterior and superficial structures as well as deep and peripheral structures, e.g., ocular structures such as the chorioretinal vasculature, retinal pigment epithelium, and ciliary body, in each case with excellent cellular detail. To the inventors' knowledge, the present invention is the first to successfully use such optical techniques to visualize peripheral ocular structures, e.g., the ciliary body, in a living eye, providing such high-resolution images of deeper ocular structures. Unlike existing techniques, embodiments of the present invention do not need to rely on transpupillary techniques. Embodiments of the present invention provide imaging tools for high-resolution visualization of ocular structures, including intraocular structures including the ciliary body, peripheral retina, and choroid, previously inaccessible by optical techniques, and can do so with cellular resolution and functional imaging capabilities. Additionally, embodiments of the present invention provide tools for studying cellular and molecular biology in the living eye and periocular cavity. Embodiments of the present invention provide techniques for aiding in the diagnosis of disease, e.g., cancerous tissue. Embodiments of the present invention provide techniques for the treatment of certain conditions, such as breaking up clotted fluid that blocks tear ducts and causes the symptoms of dry eye.Embodiments of the present invention provide techniques for dermatological applications, such as performing biopsy procedures or other surgical procedures or treating scars.Embodiments of the present invention provide techniques for cosmetic applications, such as manipulating the shape or volume of fat deposits, such as those around the eye.

[0006] Background information, including background information on aspects of two-photon microscopy, is provided in Helmchen, F. and W. Denk, Deep tissue two-photon microscopy. Nat Methods, 2005. 2(12): pp. 932-40, and Zipfel, W. R. Williams, and W. Webb, Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol, 2003. 21(11): pp. 1369-77, the disclosures of which are incorporated herein in their entirety. In general, the depth of multiphoton imaging is inherently limited to approximately five times the attenuation distance of the excitation wavelength within tissue, and imaging through highly scattering and absorbing tissues (e.g., bone or sclera) is a difficult challenge. Therefore, 3PEF imaging and other advanced techniques enable imaging and manipulation of much deeper tissues. Background information, including background information on aspects of three-photon microscopy, can be found in Wang T, et al. Three-photon imaging of mouse brain structure and function through the intact skull. Nat Methods. 2018 Oct;15(10):789-792. doi:10.1038 / s41592-018-0115-y. Epub 2018 Sep 10. PMID:30202059; PMCID:PMC6188644, and Cheng YT, Lett KM, Schaffer CB. Surgical preparations, labeling strategies, and optical techniques for cell-resolved, in vivo imaging in the mouse spinal cord. Exp Neurol. 2019 Aug;318:192-204. doi:10.1016 / j.expneurol.2019.05.010. Epub 2019 May 13. PMID: 31095935; PMCID: PMC6588420, the disclosures of which are incorporated herein in their entireties.Background information, including background information on imaging results obtained using second harmonic generation, is provided in Campagnola PJ, Loew LM. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol. 2003 Nov;21(11):1356-60. doi:10.1038 / nbt894. PMID:14595363.

[0007] Additionally, embodiments of the present invention provide non-incisional therapy tools for use in a variety of applications previously not possible or limited to incisional therapy, such as (i) precisely targeting cells and / or tissue ablation of the ciliary body to controllably and safely reduce aqueous humor production in the eye to treat glaucoma, (ii) performing transscleral laser trabeculotomy and / or trabeculoplasty and / or sclerotomy to increase aqueous humor outflow via traditional drainage pathways to treat glaucoma, (iii) identifying and providing photocoagulation therapy to peripheral retinal breaks or irregularities to treat and prevent retinal detachment, and (v) identifying and providing photocoagulation and thermal therapy to ciliary body tumors and peripheral choroidal tumors. (vii) photocrosslinking scleral tissue to prevent myopia, (viii) identifying and implementing targeted alterations in extraocular muscle function, (ix) and visualization of orbital fat and targeted thermal and photocoagulation therapy, (x) dermatological applications, such as imaging skin tissue, distinguishing cancerous versus non-cancerous tissue, diagnosing melanoma, cauterizing or otherwise destroying cancerous tissue, e.g., melanoma, performing surgical skin biopsies, or other dermatological procedures and applications, or (xi) cosmetic applications, such as affecting the shape or volume of fat deposits within tissue, affecting the appearance of skin, e.g., skin tightening, or affecting skin color, e.g., removing discoloration or scars or tattoos, or other cosmetic applications. Embodiments of the present invention provide tools for use in such drug, cell, or gene delivery by enabling high-resolution imaging of previously inaccessible structures to enable precise localization (e.g., within ocular or periocular tissues) for delivery of drugs, cells, genes, etc.

[0008] As described, optical imaging of the eye is important for ophthalmic care and is also a powerful technique for assessing systemic health and disease. For example, capturing images showing subtle changes in the neurovascular retina can be analyzed to provide accurate insight into both ocular and systemic conditions ranging from cardiovascular health to metabolic disorders. However, conventional methods for imaging through the pupil provide only a limited view of the retina, with many important intraocular structures obscured by opaque, highly pigmented tissue. For example, conventional optical imaging provides only a limited view of the retina, with the majority of intraocular tissues, such as the ciliary body, choroid, peripheral retina, and peripheral retinal pigment epithelium (RPE), either out of reach or inadequately visualized. These hidden regions play important roles in the development and progression of ocular diseases. For example, the ciliary body produces aqueous humor for the eye and critically regulates glaucoma pathophysiology. The choroid is a highly vascularized tissue that supports photoreceptors, the RPE, and provides immune surveillance in the eye. Changes in the choroid can reflect systemic conditions, including autoimmune diseases, metastatic cancer, systemic infections, blood disorders, hypertension, and diabetes. Embodiments of the present invention utilizing multiphoton microscopy are capable of deep imaging with subcellular resolution, including of such regions. Embodiments of the present invention further utilizing adaptive optics and laser techniques enable higher-order nonlinear processes, such as three-photon excited fluorescence and third harmonic generation, further into such tissues, including regions of highly scattering tissue, or even directly through, for example, the mouse skull or the opaque scleral wall of the eye.

[0009] Embodiments including laser sources configured for higher-order nonlinear processes, such as three-photon excited fluorescence (3PEF) and third harmonic generation (THG), enable much deeper imaging. Their longer excitation wavelengths have dramatically less scattering and phototoxicity, resulting in greater penetration and imaging through the intact skull. Embodiments utilizing 3PEF provide superior vascular imaging when combined with intravenous injection of fluorescent dyes, while embodiments utilizing THG provide label-free contrast of blood vessel-containing tissues. In ocular tissues, embodiments utilizing THG can be configured to visualize the inner nuclear layer, outer nuclear layer, ganglion cell layer, and retinal pigment epithelium. Rim, TH, et al., Prediction of systemic biomarkers from retinal photographs: development and validation of deep-learning algorithms. Lancet Digit Health, 2020.2(10):p.e526-e536, Wagner, SK, et al., Insights into Systemic Disease through Retinal Imaging-Based Oculomics. Transl Vis Sci Technol,2020.9(2):p.6,Poplin,R.,et al.,Prediction of cardiovascular risk factors from retinal fundus photographs via deep learning.Nat Biomed Eng,2018.2(3):p.158-164,Anand,N.,et al.,A Review of Cyclodestructive Procedures for the Treatment of Glaucoma.Semin Ophthalmol,2020.35(5-6):p.261-275, Kiel, JWand HAReitsamer,Relationship between ciliary blood flow and aqueous production:does it play a role in glaucoma therapy?J Glaucoma,2006.15(2):p.172-81、Nickla,D.L.andJ.Wallman,The multifunctional choroid.Prog Retin Eye Res,2010.29(2):p.144-68、Kongwattananon,W.,T.Pothikamjorn,and T.Somkijrungroj,Posterior segment manifestations of ocular metastasis.Curr Opin Ophthalmol,2023、Nowinska,A.K.,et al.,Ocular Manifestations of Systemic Diseases.J Ophthalmol,2018.2018:p.7851691、Helmchen,F.and W.Denk,Deep tissue two-photon microscopy.Nat Methods,2005.2(12):p.932-40、Zipfel,W.R.,R.M.Williams,and W.W.Webb,Nonlinear magic:multiphoton microscopy in the biosciences.Nat Biotechnol,2003.21(11):p.1369-77、Wang,T.,et al.,Three-photon imaging of mouse brain structure and function through the intact skull.Nat Methods,2018.15(10):p.789-792、Ouzounov,D.G.,et al.,In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain.Nat Methods,2017.14(4):p.388-390、Yildirim,M.,et al.,Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy.Nat Commun,2019.10(1):p.177、Masihzadeh,O.,et al.,Third harmonic generation microscopy of a mouse retina.Mol Vis,2015.21:p.538-47、Wu,J.,et al.,Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.Nat Methods,2020.17(3):p.287-290、Kim,T.N.,et al.,Line-scanning particle image velocimetry:an optical approach for quantifying a wide range of blood flow speeds in live animals.PLoS One,2012.7(6):p.e38590、Briers,J.D.,Laser Doppler,speckle and related techniques for blood perfusion mapping and imaging.Physiol Meas,2001.22(4):p.R35-66、Hu,S.and L.V.Wang,Photoacoustic imaging and characterization of the microvasculature.J Biomed Opt,2010.15(1):p.011101、Meng,G.,et al.,Ultrafast two-photon fluorescence imaging of cerebral blood circulation in the mouse brain in vivo.Proc Natl Acad Sci U S A,2022.119(23):p.e2117346119、Ji,N.,Adaptive optical fluorescence microscopy.Nat Methods,2017.14(4):p.374-380, Zhang,Q.,et al.,Adaptive optics for optical microscopy[Invited].Biomed Opt Express,2023.14(4):p.1732-1756,Wang,K.,et al.,Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue.Nat Commun,2015.6:p.7276, Rodriguez,C.,et al.,An adaptive optics module for deep tissue multiphoton imaging in vivo.Nat Methods,2021.18(10):p.1259-1264,Wang,C.,et al.,Multiplexed aberration measurement for deep tissue imaging in vivo.Nat Methods,2014.11(10):p.1037-40, Jung,S.,et al.,Analysis of See, for example, fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol, 2000. 20(11): pp. 4106-14; Kang I, ZQ, Yu SX, and Ji N, Coordinate-based neural representations for computational adaptive optics in widefield microscopy. bioRxiv, 2023, which are incorporated herein by reference.

[0010] Methods, systems, and adapters are provided for multiphoton imaging of non-transparent tissues, e.g., ocular and periocular tissues, and laser-tissue interaction with non-transparent tissues, e.g., ocular and periocular tissues. Aspects of the invention include a method of imaging a structure through non-transparent tissue, e.g., ocular or periocular tissue, comprising: deploying an excitation source to transmit light energy to the structure through the non-transparent tissue, e.g., ocular or periocular tissue; detecting light emitted from the structure through the non-transparent tissue, e.g., ocular or periocular tissue, via multiphoton excitation; and imaging the structure based on the detected light. Aspects of the invention also include a method of treating tissue, e.g., ocular or periocular tissue, or dermatological tissue, by manipulating the imaged structure, e.g., providing non-incisional therapy, light-tissue interaction, multiphoton-mediated damage, e.g., thermal damage, photodisruption, photocrosslinking, vascular coagulation, or tissue ablation. Aspects of the invention also include a method of imaging dermatological tissue or a method of providing a dermatological treatment. Aspects of the invention also include a method of providing a cosmetic treatment. Aspects of the invention further include methods for guiding the delivery of gene or cell therapy in ocular or periocular tissues. Also provided are systems and adapters for carrying out the methods described herein. [Brief explanation of the drawings]

[0011] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which:

[0012] [Figure 1A] 1 shows a flow chart of a method for imaging a structure through a non-transparent tissue, e.g., an ocular or periocular tissue, and a method for utilizing the imaging results to perform flow analysis, in each case according to an embodiment of the present invention. [Figure 1B] 1 shows a flow chart of a method for imaging a structure through a non-transparent tissue, e.g., an ocular or periocular tissue, and a method for utilizing the imaging results to perform flow analysis, in each case according to an embodiment of the present invention. [Figure 1C] 1 shows a flow chart of a method for imaging a structure through a non-transparent tissue, e.g., an ocular or periocular tissue, and a method for utilizing the imaging results to perform flow analysis, in each case according to an embodiment of the present invention. [Figure 1D] 1 shows a flow chart of a method for imaging a structure through a non-transparent tissue, e.g., an ocular or periocular tissue, and a method for utilizing the imaging results to perform flow analysis, in each case according to an embodiment of the present invention. [Figure 1E] 1 shows a flow chart of a method for imaging a structure through a non-transparent tissue, e.g., an ocular or periocular tissue, and a method for utilizing the imaging results to perform flow analysis, in each case according to an embodiment of the present invention. [Figure 2A] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2B] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2C] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2D] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2E] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2F]1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2G] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2H] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2I] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2J] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2K] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2L] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2M] 1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 2N]1 illustrates embodiments of an adapter for coupling an optical system to non-transparent ocular tissue, as well as aspects relating to immersion media and gels used in connection with the adapter embodiments, in each case according to the present invention. [Figure 3] 1 shows a schematic diagram of an exemplary system for imaging structures through non-transparent ocular or periocular tissue, in accordance with an embodiment of the present invention. [Figure 4A] 1 illustrates an exemplary system for imaging structures through non-transparent ocular or periocular tissue, according to an embodiment of the present invention. [Figure 4B] 1 illustrates an exemplary system for imaging structures through non-transparent ocular or periocular tissue, according to an embodiment of the present invention. [Figure 5A] 1 shows the imaging results of 3PEF transscleral imaging to visualize the chorioretinal vasculature and chorioretinal anastomosis. [Figure 5B] 1 shows the imaging results of 3PEF transscleral imaging to visualize the chorioretinal vasculature and chorioretinal anastomosis. [Figure 5C] 1 shows the imaging results of 3PEF transscleral imaging to visualize the chorioretinal vasculature and chorioretinal anastomosis. [Figure 5D] 1 shows the imaging results of 3PEF transscleral imaging to visualize the chorioretinal vasculature and chorioretinal anastomosis. [Figure 5E] 1 shows the imaging results of 3PEF transscleral imaging to visualize the chorioretinal vasculature and chorioretinal anastomosis. [Figure 5F] 1 shows the imaging results of 3PEF transscleral imaging to visualize the chorioretinal vasculature and chorioretinal anastomosis. [Figure 6] Imaging results of third harmonic generation (THG) transscleral imaging to visualize the retinal pigment epithelium are shown. [Figure 7A] The results of THG transscleral imaging of the ciliary body are shown. [Figure 7B] The results of THG transscleral imaging of the ciliary body are shown. [Figure 8A]1 shows the imaging results of in vivo cell imaging in a living eye. [Figure 8B] 1 shows the imaging results of in vivo cell imaging in a living eye. [Figure 8C] 1 shows the imaging results of in vivo cell imaging in a living eye. [Figure 8D] 1 shows the imaging results of in vivo cell imaging in a living eye. [Figure 8E] 1 shows the imaging results of in vivo cell imaging in a living eye. [Figure 9] 1 shows an example of imaging of chorioretinal vascular dysplasia. [Figure 10A] 1 shows the results of imaging choroidal blood flow and reconstruction. [Figure 10B] 1 shows the results of imaging choroidal blood flow and reconstruction. [Figure 10C] 1 shows the results of imaging choroidal blood flow and reconstruction. [Figure 10D] 1 shows the results of imaging choroidal blood flow and reconstruction. [Figure 10E] 1 shows the results of imaging choroidal blood flow and reconstruction. [Figure 10F] 1 shows the results of imaging choroidal blood flow and reconstruction. [Figure 11] 1 illustrates an exemplary quantitative analysis technique for blood flow analysis based on collected imaging data, according to an embodiment of the present invention. [Figure 12A] Hemodynamic analysis with line-scanning particle image velocimetry is shown. [Figure 12B] Hemodynamic analysis with line-scanning particle image velocimetry is shown. [Figure 12C] Hemodynamic analysis with line-scanning particle image velocimetry is shown. [Figure 12D] Hemodynamic analysis with line-scanning particle image velocimetry is shown. [Figure 13A] 1 illustrates aspects of ophthalmic imaging and therapeutic applications of embodiments of the present invention. [Figure 13B]1 illustrates aspects of ophthalmic imaging and therapeutic applications of embodiments of the present invention. [Figure 13C] 1 illustrates aspects of ophthalmic imaging and therapeutic applications of embodiments of the present invention. [Figure 14A] A review of alternative ophthalmic imaging and therapy applications of embodiments of the present invention is presented. [Figure 14B] A review of alternative ophthalmic imaging and therapy applications of embodiments of the present invention is presented. [Figure 14C] A review of alternative ophthalmic imaging and therapy applications of embodiments of the present invention is presented. [Figure 15A] 1 presents a schematic diagram of another ophthalmic imaging and therapy application of an embodiment of the present invention. [Figure 15B] 1 presents a schematic diagram of another ophthalmic imaging and therapy application of an embodiment of the present invention. [Figure 16] 1 shows an overview of the arrangement of elements of an embodiment of the present invention for use in imaging ocular tissue in a clinical setting. [Figure 17] 1 shows an overview of potential configurations for use with embodiments of the present invention relating to imaging of ocular tissues. [Figure 18] 1 shows the behavior of three-photon excited fluorescence (3PEF) compared to two-photon excited fluorescence (2PEF). [Figure 19A] An overview of another ophthalmic imaging and therapy application of an embodiment of the present invention is presented. [Figure 19B] An overview of another ophthalmic imaging and therapy application of an embodiment of the present invention is presented. DETAILED DESCRIPTION OF THE INVENTION

[0013] Methods, systems, and adapters are provided for multiphoton imaging of non-transparent tissues, e.g., ocular and periocular tissues, and laser-tissue interaction with non-transparent tissues, e.g., ocular and periocular tissues. Aspects of the invention include a method of imaging a structure through non-transparent tissue, e.g., ocular or periocular tissue, comprising: deploying an excitation source to transmit light energy to the structure through the non-transparent tissue, e.g., ocular or periocular tissue; detecting light emitted from the structure through the non-transparent tissue, e.g., ocular or periocular tissue, via multiphoton excitation; and imaging the structure based on the detected light. Aspects of the invention also include a method of treating tissue, e.g., ocular or periocular tissue, or dermatological tissue, by manipulating the imaged structure, e.g., providing non-incisional therapy, light-tissue interaction, multiphoton-mediated damage, e.g., thermal damage, photodisruption, photocrosslinking, vascular coagulation, or tissue ablation. Aspects of the invention also include a method of imaging dermatological tissue or a method of providing a dermatological treatment. Aspects of the invention also include a method of providing a cosmetic treatment. Aspects of the present invention further include methods of providing gene therapy in ocular or periocular tissues, i.e., methods of guiding the delivery of gene therapy. Also provided are systems and adapters for carrying out the methods described herein.

[0014] Before the present invention is described in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0015] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specific excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0016]

[0023] In this specification, certain ranges are presented with the term "about" before the numerical values. In this specification, the term "about" is used to provide literal support for the exact number it precedes, as well as a number that is near or approximately the number it precedes. In determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context provided, provides substantial equivalence to the specifically recited number.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0018] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and incorporation by reference herein discloses and describes the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0019] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate to the use of such exclusive terminology as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0020] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0021] Systems and methods may be described for grammatical eloquence and functional description, and the claims are in no way construed as necessarily limited by "means" or "step" limitations unless expressly recited under 35 U.S.C. 112; the claims are to be given the full scope of legal equivalents and meaning of the definitions provided by the claims, and in any case where a claim is expressly recited under 35 U.S.C. 112, the claim is to be expressly understood to be given the full legal equivalents under 35 U.S.C. 112.

[0022] Method for imaging and manipulating structures through non-transparent tissues - Patent Application 20070122997 Aspects of the present disclosure include methods for imaging and manipulating a structure through a non-transparent tissue, e.g., an ocular or periocular tissue. In particular, the present disclosure includes a method of imaging a structure through a non-transparent tissue, e.g., an ocular or periocular tissue, comprising: deploying an excitation source to deliver light energy to the structure through the non-transparent tissue, e.g., the ocular or periocular tissue; detecting light emitted from the structure through the non-transparent tissue, e.g., the ocular or periocular tissue, via multiphoton excitation; and imaging the structure based on the detected light.

[0023] 1A shows a flow chart 100 for imaging a structure through non-transparent tissue, e.g., ocular or periocular tissue, in accordance with one embodiment of the present invention. The embodiment of the present invention shown in FIG. 1A relates to imaging any convenient structure accessible through non-transparent tissue, e.g., ocular or periocular tissue, in accordance with the present techniques, as such may vary. Flow chart 100 is an exemplary embodiment of the present invention provided for illustrative purposes, and the applied structures and optical techniques may vary as desired in embodiments of the present invention.

[0024] Flowchart 100 begins at step 102. From starting step 102, the process then proceeds to step 104.

[0025] In step 104, the excitation source is deployed. Deploying the excitation source means that the excitation source is operably interfaced with non-transparent tissue, e.g., ocular or periocular tissue, such that the structure is visualized through the non-transparent tissue. That is, the excitation source may be positioned relative to the non-transparent tissue, e.g., ocular or periocular tissue, in any convenient manner, such that light emitted from the excitation source is transmitted through the non-transparent tissue, e.g., ocular or periocular tissue. In embodiments, the excitation source may be positioned on or proximal to the surface of the non-transparent tissue, e.g., ocular or periocular tissue. The operable interface between the excitation source and the non-transparent tissue, e.g., ocular or periocular tissue, may be located, for example, on the surface of the eye, e.g., on the scleral tissue or partially on the scleral tissue or partially on the cornea, or on the surface of the periocular tissue, e.g., on one or more fat pads under the eye, or in any other convenient location that allows the excitation source to transmit light energy to the structure through the non-transparent tissue, e.g., ocular or periocular tissue. Such locations may be determined, for example, by the clinician, taking into account anatomical or physiological constraints that allow access to the structures intended to be imaged by the present invention.

[0026] Non-transparent tissue: With respect to non-transparent tissues, e.g., ocular or periocular tissues, in embodiments, the non-transparent tissues, e.g., ocular or periocular tissues, can include any tissue through which optical energy can be transmitted to a structure intended to be imaged by the present invention, and such can vary. The non-transparent ocular or periocular tissues can include exclusively ocular tissues, exclusively periocular tissues, other tissues, e.g., dermatological tissues, or combinations thereof. In embodiments, the non-transparent ocular tissues include one or more of the following: sclera tissue, retinal pigment epithelium (RPE), uvea, conjunctiva, Tenon's capsule, ocular muscles, or ciliary body, or tissues or structures proximal thereto. In other embodiments, the non-transparent periocular tissues include one or more of the palpebral conjunctiva, orbital septum, capsulopalvebral fascia, tarsal plate, tarsal gland, periocular adipose tissue, or dermis, or tissues or structures proximal thereto. In embodiments, the non-transparent tissue comprises dermatological tissue, such as the epidermis, dermis, or subcutaneous tissue.

[0027] In embodiments, the non-transparent tissue, e.g., ocular or periocular tissue, comprises light-scattering tissue. Light scattering means that light impinging on the non-transparent tissue results in light being emitted in a direction other than the direction of incidence. In other embodiments, the non-transparent tissue, e.g., ocular or periocular tissue, comprises light-absorbing tissue. In yet other embodiments, the non-transparent tissue, e.g., ocular or periocular tissue, comprises pigmented uveal tissue. In other embodiments, non-transparent tissue refers to tissue that is non-transparent to typical wavelengths in the visible spectrum.

[0028] Structures imaged: Regarding the structure to be imaged, i.e., a structure intended to be imaged through a non-transparent tissue, e.g., an ocular or periocular tissue, in embodiments, the structure to be imaged includes any convenient structure accessible through a non-transparent tissue, e.g., an ocular or periocular tissue, and such may vary. In embodiments, the structure to be imaged includes one or more of the following: scleral tissue, corneal tissue, ocular vasculature, suprachoroidal space, choroid, chorioretinal vasculature, choriocapillaris, retinal pigment epithelium (RPE), photoreceptors, uvea, conjunctiva, Tenon's capsule, ocular muscles, ciliary body, or peripheral retina, or tissues or structures proximal thereto. In other embodiments, the structure to be imaged includes one or more of the palpebral conjunctiva, orbital septum, capsulopalveolar fascia, tarsal plate, tarsal gland, periocular adipose tissue, or dermis, or tissues or structures proximal thereto. In yet other embodiments, the imaged structure includes a choroidal capillary, venule, vein, arteriole, or artery, or tissue or structure proximal thereto, e.g., other vasculature. In other embodiments, the imaged structure includes a fat deposit, a nerve structure, or a pain receptor, or a gland, e.g., a tear duct.

[0029] The imaged structure may be a dynamic structure, i.e., the imaged structure may include an image or series of images showing movement or change in the structure. In embodiments, the imaged structure includes circulating cells. In embodiments, the imaged structure includes fluid flow within tissue.

[0030] In embodiments, the structure being imaged is anterior to the retinal pigment epithelium (RPE). In other embodiments, the structure being imaged is posterior to the retinal pigment epithelium (RPE).

[0031] In some cases, the imaged structures include light-scattering or light-absorbing structures, or a combination thereof.

[0032] In certain embodiments, the method further includes introducing a fluorescent contrast agent or tag into the structure to be imaged or applying other labeling techniques to the structure to be imaged. In some cases, the method further includes labeling the plasma present in the structure to be imaged with a fluorescent dye. Any convenient fluorescent dye, such as a particle, e.g., a fluorophore, that emits stimulated light in response to receiving light transmitted from an excitation source, can be applied. Any convenient commercially available fluorophore can be applied, and such can vary. Fluorophores of interest include, for example, fluorescein, Texas Red, Alexa 680, quantum dots, etc. Fluorophores can be tagged to dextran, antibodies, cells, drugs, molecular agents, etc. These fluorescent tagging agents can be introduced into the plasma via intravenous injection. In embodiments, longer excitation wavelengths, i.e., longer excitation wavelengths delivered by the excitation source, can induce higher-order nonlinear processes to excite conventional fluorophores (e.g., an excitation wavelength of 1.3 μm can three-photon pump green fluorescent molecules, e.g., GFP and FITC, while 1.7 μm light can efficiently pump red fluorescent molecules, e.g., RFP and Texas Red).

[0033] Deploying the excitation source as in step 104 further means that the excitation source is activated, i.e., turned on, causing the excitation source to emit energy, e.g., radiant energy, e.g., optical energy. In embodiments, the excitation source is deployed to transmit optical energy through non-transparent tissue, e.g., ocular or periocular tissue, to a structure intended to be imaged.

[0034] Excitation source: Regarding the excitation source and the light energy, i.e., radiant energy, transmitted therefrom, the excitation source may comprise one or more light energy sources. Any convenient one or more excitation sources capable of transmitting sufficient light energy through non-transparent tissue, such as ocular or periocular tissue, may be applied, and such may vary. Sufficient light energy refers to light energy that, first, can be transmitted through non-transparent tissue, such as ocular or periocular tissue, and second, can cause light to be emitted from the structure via multiphoton excitation. Furthermore, the light energy transmitted to the structure through the non-transparent tissue, such as ocular or periocular tissue, must be sufficient to cause appropriate light to be emitted from the structure via multiphoton excitation, and the emitted light itself must be detectable through the non-transparent tissue, such as ocular or periocular tissue. Examples of multi-photon excitations of interest include, for example, multi-photon excitations that involve excitation of a molecule resulting in the release / induction of light energy via two or more photons, such as two-photon excited fluorescence (2PEF), second harmonic generation (SHG), three-photon excited fluorescence (3PEF), third harmonic generation (THG), four-photon excited fluorescence, fourth harmonic generation, or other higher order multi-photon processes.

[0035] In embodiments, the excitation source comprises one or more laser systems. Any convenient laser capable of delivering sufficient optical energy and intensity at an appropriate wavelength through non-transparent tissue, such as ocular or periocular tissue, may be applied, and such may vary. In some embodiments, the laser system comprises a solid-state laser. In other embodiments, the laser system comprises a fiber laser. In yet other embodiments, the laser system comprises an ytterbium-doped fiber laser. The laser system may be configured to generate optical energy having any convenient wavelength and / or pulse duration and / or power output, and such may vary. In embodiments, the laser system is configured to generate optical energy having a wavelength in the range of 350 nm to 5.0 μm, a pulse duration of 1 to 1,000 femtoseconds, a maximum power output of greater than 100 watts, and a pulse repetition rate in the range of 1 Hz to 1,000 MHz. In certain contexts, the useful wavelength range of optical energy generated by the laser system is approximately 600 to 1,700 nm. In embodiments, the per pulse energy may vary depending on the laser pulse rate. Higher average power (i.e., wattage) is a more broadly applicable metric for a given laser. Lasers capable of or emitting higher powers are desirable because such emitted power can be attenuated to any desired level. Higher powers may also be desirable to enable faster pulse rates, i.e., to divide the wattage of emitted optical energy per pulse energy. Lasers capable of faster pulse rates (with sufficient energy) enable faster imaging and / or laser therapy. In other embodiments, the laser system includes dispersion compensation. Dispersion compensation refers to the application of any convenient technique, e.g., optical elements, to control, cancel, or compensate for chromatic dispersion of the optical energy emitted by the laser system and / or generated by the optical system and tissue downstream of the laser system. Dispersion compensation can be achieved, for example, by manually applying optical components internal or external to the laser.Further details regarding laser systems of interest are provided in RP Photonics Encyclopedia: Femtosecond Lasers, https: / / www.rp-photonics.com / femtosecond_lasers.html (last accessed October 25, 2022), the disclosure of which is incorporated herein in its entirety. Other known or yet to be discovered or implemented pump sources or lasers or laser systems or optical systems may be applied as desired.

[0036] In embodiments, the excitation source comprises a first laser. Any convenient laser capable of transmitting sufficient optical energy at an appropriate wavelength through non-transparent tissue, such as ocular or periocular tissue, may be applied, and such may vary. In some embodiments, the laser system comprises a solid-state laser. In other embodiments, the laser system comprises a fiber laser. In yet other embodiments, the laser system comprises an ytterbium-doped fiber laser. The laser system may be configured to generate optical energy having any convenient wavelength and / or pulse duration and / or power output, and such may vary. In embodiments, the laser system is configured to generate optical energy having a wavelength in the range of 350 nm to 5.0 μm, a pulse duration of 1 to 1,000 femtoseconds, a maximum power output of greater than 100 watts, and a pulse repetition rate in the range of 1 Hz to 1,000 MHz. In yet other embodiments, the laser system includes dispersion compensation. Dispersion compensation refers to the application of any convenient technique, e.g., optical elements, to control, cancel, or compensate for chromatic dispersion of the optical energy emitted by the laser system and / or generated by the optical system and structures downstream of the laser system. Dispersion compensation can be achieved, for example, by manually applying optical components internal or external to the laser.

[0037] In embodiments, the excitation source includes a second laser. Any convenient laser capable of transmitting sufficient optical energy at an appropriate wavelength through non-transparent tissue, such as ocular or periocular tissue, may be used, and such may vary. Such a second laser may emit optical energy together with, separately from, or otherwise complementary to the optical energy emitted from a first laser, such as the first laser described above. In some cases, the second laser is a titanium-doped sapphire laser. In other cases, the second laser is a fixed wavelength laser. For example, the second laser is a fixed 1,045 nm laser. In embodiments, the second laser is configured to generate optical energy having a specified power output. For example, the second laser may be configured to generate optical energy having a maximum power output of greater than 100 W. In other embodiments, the second laser includes a specified usable power band. For example, the second laser may include a usable power band from 350 nm to 5.0 μm. In certain cases, the second laser may be configured for imaging and / or laser therapy, while in other cases, the second laser may be an OPA (optical parametric amplifier) ​​configured to extend the wavelength range of the first laser.

[0038] In some embodiments, the excitation source includes a third component. Any convenient third component capable of transmitting sufficient optical energy through non-transparent tissue, such as ocular or periocular tissue, may be used, and such third component may vary. Such a third component may emit optical energy together with, separately from, or otherwise complementary to the optical energy emitted from the first and second lasers, e.g., one or both of the first and second lasers described above. In some cases, the third component includes a fixed wavelength excitation source. For example, the third component may include a 1.7 μm excitation source. In other cases, the third component includes the Raman shift of 1.5 μm light in a large-mode-area photonic crystal rod. In still other cases, the third component emits an excitation wavelength at a fixed wavelength or within that range. For example, the third component may emit an excitation wavelength between 350 nm and 5 μm. Other known or yet to be discovered or implemented excitation sources or lasers or laser systems or optical systems may be applied for the excitation sources of embodiments of the present invention, e.g., the first, second, or third lasers described above, as desired.

[0039] Contemplated excitation sources include two-part laser systems that allow imaging of tissue through non-transparent tissue, e.g., ocular or periocular tissue (and manipulating tissue, e.g., providing laser treatment of tissue, as described in detail herein). Such embodiments may include an optical parametric amplifier (OPA) used to extend the tuning range of an ytterbium-amplified laser. Optical parametric amplifier (OPA) refers to a laser light source, e.g., a commercially available laser technology, including common commercial light sources, including those used for photodisruption or micromachining, that emits light of tunable wavelengths via any convenient optical parametric amplification process. In some cases, the tuning range of an excitation source comprising such a two-part laser system includes 350 nm to 5 μm. However, the tuning range may vary depending on the specific characteristics of the underlying technology and is expected to change as the underlying technology changes and evolves.

[0040] In some embodiments, the excitation source, e.g., the two-part laser system (i.e., the first and second lasers) described above, may include a wavelength tuning range of 350 nm to 5 μm. With respect to such tuning ranges corresponding to certain embodiments, a useful window or bandwidth for imaging through non-transparent ocular or periocular tissues includes approximately 1,300 to 1,700 nm. Other frequency windows or bandwidths within such tuning ranges may be applicable in connection with manipulating tissue, e.g., providing laser treatment of tissue, particularly in connection with thermal energy deposition, as described in more detail below.

[0041] In embodiments, the pump source may comprise one or more next-generation laser sources. In some cases, the pump source comprises a conventional titanium-doped sapphire laser with a usable power bandwidth ranging from 690 to 970 nm, such as a SpectraPhysics MaiTai HP DeepSee or similar product. In other cases, the pump source comprises an extended-infrared ytterbium-doped fiber laser with a usable power bandwidth ranging from 690 to 1300 nm, such as a SpectraPhysics Insight X3 or similar product. In yet other cases, the pump source comprises an optical parametrically amplified ytterbium laser with a usable power bandwidth ranging from 600 to 2500 nm, such as a Coherent Monaco-Opera F or similar product.

[0042] In some embodiments, deploying the excitation source includes directing the focus of the excitation source over a predetermined region, i.e., a region of interest. For example, the excitation source may move over a designated region to deliver light energy to structures corresponding to the designated region through which the excitation source moves, thereby allowing structures to be imaged over such corresponding designated region. Similarly, in other embodiments, deploying the excitation source includes articulating the excitation source around non-transparent tissue, e.g., ocular or periocular tissue. That is, the excitation source may articulate around non-transparent tissue, e.g., ocular or periocular tissue, to deliver light energy to structures corresponding to the region through which the excitation source articulates, thereby allowing structures to be imaged over such corresponding region. Articulating the excitation source means moving the excitation source in any desired manner, e.g., any combination of translation and / or rotation in three dimensions of the excitation source.

[0043] In embodiments, the excitation source of interest comprises one or more pulsed lasers. By pulsed laser, we mean any convenient laser that is not a continuous laser. For example, a pulsed laser can be configured such that optical power is delivered in pulses of any duration at any repetition rate or specified duty cycle. In some embodiments, the pulsed laser can generate optical energy having a pulse duration lasting 1 to 1,000 femtoseconds. In other embodiments, the pulsed laser can generate optical energy having a pulse duration lasting less than 300 femtoseconds, e.g., 30 to 300 femtoseconds. In some embodiments, pulse durations lasting 40 to 150 femtoseconds can be applied in connection with imaging structures through non-transparent tissues, e.g., ocular or periocular tissues. In embodiments, the pulse repetition rate of the pulsed laser is approximately 1 MHz to provide sufficient energy to image structures through non-transparent tissues, e.g., ocular or periocular tissues. However, any convenient pulse repetition rate may be applied, and such may vary, for example, based on the underlying technique or based on the underlying non-translucent tissue, e.g., ocular or periocular tissue, i.e., to provide sufficient pulse energy therefor. In other embodiments, the pulse repetition rate of the pulsed laser may range from 1 Hz to 1,000 MHz, e.g., 1 Hz to 65 MHz. Certain embodiments may deliver optical energy with a maximum per pulse energy of approximately 700 μJ, e.g., a pulsed laser having a pulse energy of approximately 600 μJ, corresponding to a low pulse repetition rate of less than 5 kHz, e.g., 2 kHz. Other embodiments may deliver optical energy with an average power output of greater than 100 watts.

[0044] In embodiments, the excitation source includes additional optical components. Such additional optical components may transmit optical energy to a structure through non-transparent tissue, such as ocular or periocular tissue, to facilitate imaging of such a structure. Exemplary optical components include, but are not limited to, a laser scanner for moving the laser focal point in any desired scanning pattern or raster across the image field. Other exemplary optical components include, but are not limited to, a piezoelectric component for rapidly controlling the focal point position along the optical axis, a pulse compressor for shortening the pulse width, a power attenuator, or adaptive optics for wavefront shaping, as described in detail herein. Exemplary optical components include those used in connection with LASIK and femto-mediated cataract surgery to rapidly direct the laser to different locations; such optical components are known in the art. In embodiments, such optical components may be implemented with the laser of the excitation source of the embodiments, but may not be essential to the laser technology itself.

[0045] Upon completion of the deployment of the excitation source in step 104, the process then proceeds to step .

[0046] Emitted Light: In step 106, light emitted from the structure is detected. That is, the effect of the light energy transmitted through non-transparent tissue, e.g., ocular or periocular tissue, to the structure in step 104 is that light is emitted from the structure via multi-photon excitation. In embodiments, the light emitted from the structure via multi-photon excitation is itself transmitted through non-transparent tissue, e.g., ocular or periocular tissue, and then detected, as described above.

[0047] In embodiments, light emitted from a structure via multi-photon excitation includes stimulated light. Stimulated light, in embodiments, means that light transmitted from an excitation source interacts with particles at the molecular or subatomic level, liberating energy and thereby generating photons. In some embodiments, the stimulated light is excited via a high-order nonlinear process. In some cases, the high-order nonlinear process includes one or more of two-photon excited fluorescence (2PEF), second harmonic generation (SHG), three-photon excited fluorescence (3PEF), third harmonic generation (THG), or excitation of molecules to emit / stimulate light generation via more than three photons (e.g., four-photon excitation (4PEF) or fourth harmonic generation (4HG)).

[0048] In certain embodiments, the light emitted via multiphoton excitation includes light emitted from endogenous fluorophores present in the structure being imaged. In other embodiments, the light emitted via multiphoton excitation includes light emitted from exogenous fluorophores present in the structure being imaged. In such embodiments, any convenient endogenous or exogenous fluorophore may be utilized or introduced into the structure, as the case may be. In some cases, the fluorophore emits one or more of ultraviolet, blue, green, red, or far-red light.

[0049] With respect to multi-photon excitation, in some embodiments, the light emitted from the structure comprises light emitted via two-photon excitation (2PEF) and further comprises a second harmonic generation (SHG) signal. In other embodiments, the light emitted from the structure comprises light emitted via three-photon excitation (3PEF) and further comprises a third harmonic generation (THG) signal.

[0050] In some embodiments, the longer laser wavelength of the excitation source allows for penetration of the excitation light through non-transparent tissues, such as the sclera, etc. In one embodiment, depending on the fluorophore applied, the same excitation wavelength can be used to excite the fluorophore with two photons, three photons, or more than three photons.

[0051] In some embodiments, the same lens, e.g., an objective lens, can be used to (i) transmit light energy from the excitation source to the structure through non-transparent tissue, e.g., ocular or periocular tissue, and (ii) transmit light emitted from the structure through non-transparent tissue, e.g., ocular or periocular tissue, via multiphoton excitation, i.e., to one or more sensors or detectors. In other embodiments, the same objective lens does not need to be used for both such optical paths. In some cases, the signal is collected (for detection via a detector) at a location different from where the light emitted by the excitation source is transmitted into the non-transparent tissue, e.g., ocular or periocular tissue. In some cases, the signal is collected (for detection via a detector) at any convenient location, and such locations can vary. For example, the signal can be collected by placing a sensor on the cornea. In embodiments, such sensors collect signal light emitted deep within tissue, e.g., ocular or periocular tissue, by means other than signal light collected by an objective lens used to transmit light emitted by an excitation source to non-transparent tissue, e.g., ocular or periocular tissue. Light emitted deep within tissue, e.g., ocular or periocular tissue, refers to signal light generated by multiphoton excitation emitted at a laser focal point. In some cases, signal light may be collected from multiple detectors at any convenient location. For example, some embodiments include both a sensor on the cornea and signal light collected through the same objective lens used to transmit light from the excitation source into the tissue. Generally, in embodiments, an image of a structure is formed by moving the focal point of light emitted by an excitation source through a tissue, e.g., ocular or periocular tissue, over a three-dimensional volume. The signal generated at the focal point of the excitation source may be collected by a sensor. Such a sensor may be positioned anywhere; generally, the more light collected, the better the resulting image quality. When the focal point of light emitted from the excitation source is moved (e.g., by a scanning mirror or a translation device or other convenient mechanism) across a three-dimensional volume, the detected optical signal correlates with the position of the focal point of the light emitted by the excitation source.Such a process can be used to digitally assemble an image voxel by voxel, and unlike conventional cameras, for example, such image formation does not depend on a specific mode or location of signal light collection.

[0052] In some cases, adaptive optics techniques are applied to the light energy emitted from the excitation source and / or the light guided at the structure being imaged. Any convenient adaptive optics technique may be applied. Adaptive optics techniques of interest are described in detail herein.

[0053] Upon completion of detecting light emitted from the structure in step 106, the process then proceeds to step 108.

[0054] Imaging / Detection: In step 108, the structure is imaged based on the light detected in step 106. That is, the light emitted and detected from the structure is used to generate an image of the structure (i.e., accumulated, combined, and correlated over a specified area, volume, or time period). In embodiments, the imaging and / or detecting steps may be computer-implemented, that is, a computer processing device may be programmed or otherwise used to combine, sequence, or otherwise arrange the light detected from the structure in step 106 to generate one or more images of the structure. Any convenient technique or algorithm may be used to resolve the light emitted and detected from the structure in step 106 into an image of the structure in step 108.

[0055] That is, in embodiments, an image can be formed by moving the focus of light (e.g., a laser focus) transmitted from an excitation source through a tissue, e.g., an ocular or periocular tissue, in three-dimensional space, i.e., throughout a volume containing the structure to be imaged. The signal generated at the focus of the light transmitted by the excitation source is collected by a sensor, i.e., a detector, as described herein, which can be positioned in any convenient location capable of receiving the stimulated light emitted by the structure to be imaged. Such a detector can include, for example, a contact lens positioned directly on the cornea, a filter, a photomultiplier tube (PMT), and the like. In embodiments, a conventional detection device can be applied, in which light is collected through the same objective lens used to deliver the excitation light. When the focus of the excitation source is moved in three-dimensional space, e.g., by translation or articulation of the optical system relative to the tissue, e.g., an ocular or periocular tissue, and / or a scanning mirror, the detected signal can be correlated to the position of the focus of the excitation source, which is used to digitally assemble the image pixel by pixel. For visualization purposes, in embodiments, imaging a structure by such pixel-by-pixel assembly is analogous to applying a very high resolution dot matrix printer in three dimensions.

[0056] In embodiments, the imaging includes subcellular resolution.

[0057] Spatial Imaging: Certain embodiments of the present invention include imaging a structure over a specified volume. Any convenient volume may be selected, and such may vary depending, for example, on the volume of the structure or phenomenon being imaged. In some cases, imaging a structure over a specified volume includes accumulating imaging data at a specified spatial resolution. Any convenient specified spatial resolution may be applied, and such may vary depending, for example, on the feature size of the structure that is desired to be imaged.

[0058] In some cases, deploying the excitation source to deliver optical energy to the structure includes spatially guiding the excitation source to deliver optical energy through non-transparent tissue, such as ocular or periocular tissue. In some cases, spatially guiding the excitation source to deliver optical energy to the structure includes guiding the excitation source using a multimodal laser scanner. That is, a laser scanner can be used to direct the focus of the excitation laser to different locations. In embodiments, since the relative position of the laser focus is known, an image is computed by moving the laser focus across a desired field of view, thereby essentially generating the image pixel by pixel by moving the focus back and forth (i.e., "laser scanning"). Any convenient multimodal laser scanner or technique for laser scanning can be used, and such can vary. In some cases, the multimodal laser scanner is switchable between resonant imaging and patterned point scanning.

[0059] Embodiments of the present invention may apply laser scanning techniques, such as patterned laser scanning, together with computational methods for combining the results of the laser scanning. Some embodiments further include using line-scanning particle image velocimetry (LS-PIV). In such embodiments, using line-scanning particle image velocimetry (LS-PIV) may include measuring fluid flow through a particular tissue or structure, such as measuring blood flow. In such cases, measuring blood flow may include measuring choroidal blood flow. Generally, patterned laser scanning techniques enable organized and / or systematic laser scanning of an excitation laser. Such techniques enable measurement of biological phenomena, such as the velocity of blood cells within blood vessels. A next step in applying such patterned scanning techniques may include applying computational methods to a unique image dataset obtained via patterned scanning. Embodiments using such laser scanning and computational analysis techniques may be referred to as applying particle image velocimetry.

[0060] In connection with imaging structures over a specified volume, one embodiment of the present invention is a method of imaging neural structures, i.e., embodiments of the present invention may relate to visualizing neural structures, in some cases in relation to pain management, i.e., to prescribing treatments or procedures that would benefit from visualizing neural structures or pain receptors.

[0061] Embodiments of the systems, devices, and methods disclosed herein are configured to acquire and process, i.e., analyze, images such that clinically relevant imaging latencies can be obtained. That is, spatial imaging can be acquired with clinically relevant processing times using embodiments of the claimed invention. In some cases, real-time or near real-time spatial imaging can be acquired using embodiments of the invention.

[0062] Time Imaging: Certain embodiments of the present invention include imaging a structure over a specified period of time. Any convenient period of time may be selected, and such may vary depending, for example, on the time scale of the phenomenon that is desired to be imaged. In some cases, imaging a structure over a specified period of time includes accumulating imaging data at a specified temporal resolution or a specified pulse repetition rate. In embodiments, the pulse repetition rate is related to the rate of imaging or laser treatment. Any convenient temporal resolution or pulse repetition rate may be applied, and such may vary depending, for example, on the temporal resolution of the phenomenon being imaged. In certain cases, the pulse repetition rate is between 1 Hz and 1,000 MHz, e.g., between 1 kHz and 1,000 kHz.

[0063] In connection with imaging structures over a specified period of time, an embodiment of the present invention is a method for imaging cell dynamics. In some cases, an embodiment of the present invention is a method for imaging neural activity, e.g., imaging neural activity with calcium indicators. In still other cases, an embodiment of the present invention is a method for imaging hemodynamics. In still other cases, an embodiment of the present invention is a method for imaging blood flow in the microvasculature.

[0064] Embodiments of the systems, devices, and methods disclosed herein are configured to obtain and process, i.e., analyze, images such that clinically relevant imaging latencies can be obtained. That is, temporal imaging can be obtained with clinically relevant processing times using embodiments of the claimed invention. In some cases, real-time or near real-time temporal imaging can be obtained using embodiments of the invention.

[0065] CBC - Complete blood components analyze morphological features, cell size, histological staining, etc.: Embodiments of the systems, devices, and methods disclosed herein, including those related to blood flow or flow analysis, are capable of detecting blood cells or blood cell types and related statistics. Blood cells and blood cell types that can be detected by the systems, devices, and methods disclosed herein include, but are not limited to, red blood cells, hemoglobin, white blood cells (including neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelets, reticulocytes, and nucleated red blood cells. Various measurements of different blood components can be performed, including, but not limited to, cell count, cell size, cell complexity, granularity, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. In some embodiments, the disclosed measurements can be performed using stain-independent methods in the absence of histological stains.

[0066] Embodiments of the systems, devices, and methods include extracting stain-independent features from a histologically stained specimen to determine a complete blood component analysis. Histologically stained specimens include biological samples and / or body fluids prepared with histological stains for analysis of cell morphology. In some cases, the histologically stained specimens include, but are not limited to, hematological samples prepared from blood containing cell types or elements that can be found in blood, including, but not limited to, nucleated blood cells, enucleated blood cells, white blood cells, nucleated red blood cells, red blood cells, giant platelets, leukocytes, basophils, eosinophils, lymphocytes, monocytes, neutrophils, platelets, mature or immature blood cells, malignant tumor or tumor cells, parasites, bacteria, etc.

[0067] In some cases, the methods described herein may include preparing a histologically stained specimen from a subject, where the specimen contains or is prepared to contain histologically stained cells. In other cases, the specimen may be previously prepared, and the method may include processing a digital image obtained from the histologically stained specimen from the subject.

[0068] As used herein, histological stains refer to stains used in the microscopic analysis of the cellular anatomy and / or morphology of cells obtained from multicellular organisms. Histological stains generally include at least one dye that stains one or more cell types and / or components of one or more cell types in a contrasting color. Histological stains may also include at least one counterstain that stains the remainder of one or more cells in a different color. Histological techniques, stains, and staining methods are well known and include, but are not limited to, those described in Kierman. Histological and histochemical methods: Theory and practice. Oxford: Butterworth / Heinemann, 1999, and Bancroft & Stevens. Theory and practice of histological techniques. New York, NY: Churchill Livingstone, 1996, the disclosures of which are incorporated herein by reference in their entireties.

[0069] Histological staining techniques can be specific, staining one or more particular cells in a specific manner, or non-specific, staining essentially all or most cells in the same or similar manner. Histological stains include, for example, Alcian blue stain, Aniline blue stain, Azan stain, Biebrich's acid fuchsin stain, carbolic acid fuchsin stain, chrome alum / hematoxylin stain, Congo red stain, crystal violet stain, Fast Red stain, hematoxylin and eosin (H&E) stain, iron hematoxylin stain, Isamine blue / eosin stain, Jenner stain, Mallory's phosphotungstic acid hematoxylin (PTAH) stain, Mallory's trichrome stain, Masson's stain, Malachite green stain, and methyl green- These include, but are not limited to, pyronin (MGP) stain, Nissl and methylene blue stain, Nissl stain, oil red O stain, orcein stain, osmic acid stain, osmium tetroxide stain, Papanicolaou stain, periodic acid Schiff (PAS) stain, reticulin stain, Romanowsky stain, Safranin O stain, silver stain, Sudan black and osmium stain, toluidine blue stain, Trichrome AB, Trichrome LG, trypan blue stain, Van Gieson stain, Verhof stain, and Weigel-tresorcin-fuchsin stain.

[0070] The dyes included in histological stains vary depending on the stain formulation and the desired staining results. In some cases, dyes useful in histological stains include, for example, acid fuchsin calcium salt, acid fuchsin, Alcian blue, Alizarin red, aniline blue, aniline blue diammonium salt, Auramine O dye, Azure, Azure A chloride, Azure B, basic fuchsin, Bismarck Brown Y, Brilliant Cresyl Blue, Brilliant Green, Carmine, Congo Red, Cresyl Violet Acetate, Crystal Violet, Darrow Red, Eosin, Eosin B, Eosin Y, Eosin Y disodium salt, Erythrosin B, Erythrosin Extra Blue, Ethyl Eosin, Fast Green FCF, Hematoxylin, Indigo Carmine, The dyes may include, but are not limited to, methyl blue, methyl green, methyl green zinc chloride, methyl orange, methyl violet 2B, methylene blue, methylene violet (Bernthsen), neutral red, nigrosine, nile blue A, oil red O, orange G, orange II sodium salt, orcein synthetic, phloxine B dye, pyronin B, pyronin G, pyronin Y, resazurin sodium salt, rose bengal sodium salt, safranine O, sudan black B, sudan III, sudan IV, thionin acetate, toluidine, and toluidine blue O.

[0071] Histological stains include Romanowsky stains. Romanowsky stains are generally neutral stains composed of various components, including, but not limited to, methylene blue (e.g., Azure B) and eosin (e.g., Eosin Y) dyes. Azure is a basic dye that binds acidic nuclei and produces a blue-purple color. Eosin is an acidic dye that adheres to alkaline cytoplasm, producing a red color. Romanowsky stains vary and include various formulations, including those containing various azure and eosin analogs. Romanowsky stains and their staining mechanisms are well known and are described, for example, in Horobin & Walter. Histochemistry (1987) 86:331-336, Marshall et al. J Clin Pathol (1978) 31(3):280-2, Marshall et al. J Clin Pathol. (1975) 28(11):920-3; J Clin Pathol (1975) 28(8):680-5, the disclosures of which are incorporated herein by reference.

[0072] Romanowsky stains include, but are not limited to, Giemsa stain, Wright stain, Wright-Giemsa stain, Jenner stain, Jenner-Giemsa stain, Leschmann stain, May-Grünwald stain, and May-Grünwald-Giemsa stain. Each Romanowsky stain can exist in a variety of formulations, either derived from a variety of different recipes or supplied by a variety of suppliers. Romanowsky stain formulations can include a variety of staining components, including, but not limited to, methylene blue, azure A, azure B, azure C, toluidine blue, thionin, methylene violet Berndtsen, methylthionorin, thionorin, eosin, eosin Y, tribromofluorescein, fluorescein, and thiazine dyes. The Romanowsky staining formulation may include various solvents for dissolving the staining components, including aqueous and organic solvents, such as, but not limited to, water and alcohol, such as, but not limited to, methanol, ethanol, isopropyl alcohol, and the like.

[0073] Histological stains and their components include those commercially available from suppliers, including, but not limited to, Sigma Aldrich, Thermo Fisher Scientific, Avantor Performance Materials, VWR International, and Polysciences Inc.

[0074] Subjects from which specimens may be obtained include, but are not limited to, human subjects, mammalian subjects (e.g., primates (such as apes, gorillas, monkeys, baboons, orangutans, etc.), ungulates (e.g., horses, cows, camelids, pigs, etc.), dogs, cats, rodents (such as mice and rats), etc. Specimens may include biological fluid samples and biological samples, which may be processed prior to imaging, for example, processed onto slides and stained histologically. In the case where the specimen is a blood sample, the sample may be processed into a blood smear and stained with a hematological stain.

[0075] Embodiments of the systems, devices, and methods disclosed herein, including those relating to blood flow or flow analysis, are capable of detecting structures within tissue, not limited to blood, as described above. Embodiments may be configured to detect changes in the underlying tissue, and may leverage the ability to detect changes in tissue to identify the underlying structure of the tissue, or to identify or distinguish underlying disease states, e.g., cancer cells. Embodiments may be configured to utilize any negative contrast images, e.g., negative contrast images collected from the multiphoton imaging techniques of the present disclosure, to identify substructures within such negative contrast images.

[0076] Upon completion of imaging the structure based on the detected light in step 108 , the process ends in step 110 .

[0077] FIG. 1B illustrates a flowchart 120 for imaging a structure through non-transparent tissue, e.g., ocular or periocular tissue, in accordance with another embodiment of the present invention. The embodiment of the present invention illustrated in FIG. 1B relates to imaging any convenient structure accessible through non-transparent tissue, e.g., ocular or periocular tissue, in accordance with the present techniques, as such may vary. Flowchart 120 is an exemplary embodiment of the present invention provided for illustrative purposes, and the applied structures and optical techniques may vary as desired in embodiments of the present invention. Certain steps illustrated in flowchart 120 are similar or identical to those illustrated in connection with the embodiment illustrated by flowchart 100 in FIG. 1A. The description of such similar or identical steps will not be repeated in connection with the discussion of FIG. 1B. Furthermore, flowchart 120 presents certain additional steps present in embodiments of the present invention. However, as will be apparent to one skilled in the art, in some embodiments of the present invention, certain of the steps presented in flowchart 120 need not be implemented.

[0078] Flowchart 120 begins at step 122. From starting step 122, the process then proceeds to step 124.

[0079] Fluorophores / dyes: In step 124, a fluorophore is introduced into the structure to be imaged. As described herein, in embodiments, introducing a fluorophore into the structure includes introducing a fluorescent dye into the structure to be imaged or labeling plasma present in the structure to be imaged with a fluorescent dye. Any convenient fluorescent dye may be applied, for example, a particle, e.g., a dye, that contains a fluorophore and emits stimulated light in response to receiving light transmitted from an excitation source. Any convenient commercially available fluorophore may be applied, and such may vary. Generally, introducing an exogenous fluorophore into the structure may be a technique used to increase light emitted from the structure via multiphoton excitation, so that such emitted or stimulated light can be transmitted through non-transparent tissue, e.g., ocular or periocular tissue, and thereby be detectable for purposes of imaging the structure. In other cases, endogenous fluorophores may be utilized to similar effect.

[0080] Upon completion of introducing the fluorophore into the structure in step 108 , the process then proceeds to step 126 .

[0081] The excitation source is deployed in step 126. Step 126 is identical to step 104 described above in connection with flowchart 100 of Figure 1A.

[0082] Upon completion of the deployment of the excitation source in step 126 , the process then proceeds to step 128 .

[0083] Light emitted from the structures is detected in step 128. More specifically, in embodiments, light emitted from at least the fluorophores present in the structures introduced in step 124 is detected. Such emitted or induced light may be detected in any convenient manner, for example, in those techniques and aspects described above in connection with step 106 of flowchart 100 in FIG. 1A.

[0084] In embodiments, instead of introducing a fluorophore in step 124, an endogenous fluorophore may instead be used in connection with imaging the structure. Endogenous fluorophore refers to any entity (e.g., a molecule, protein, or dye) capable of autofluorescence when exposed to an excitation source of the present invention. In embodiments, the endogenous fluorophore may be present in the structure to be imaged, and the excitation source may be configured to induce autofluorescence from such endogenous fluorophore in order to image the structure. That is, in embodiments, step 124 may include identifying an endogenous fluorophore present in the structure to be imaged and / or configuring the excitation source to emit light with specific characteristics that enable the endogenous fluorophore to initiate autofluorescence. Endogenous fluorophores of interest include, but are not limited to, flavins, i.e., derivatives of riboflavin, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), intracellular riboflavin, flavin coenzymes, and flavoproteins. Other endogenous fluorophores of interest include, but are not limited to, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), lipofuscin, elastin, and collagen.Background information, including that regarding endogenous fluorophores, is presented in Billington N, Knight AW. Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal Biochem. 2001 Apr 15;291(2):175-97. doi:10.1006 / abio.2000.5006. PMID:11401292, and Aubin JE. Autofluorescence of viable cultured mammalian cells. Journal of Histochemistry & Cytochemistry. 1979;27(1):36-43. doi:10.1177 / 27.1.220325, the disclosures of each of which are incorporated herein in their entireties.For background information, including on leveraging intrinsic contrast in the context of second harmonic generation, see Chen, X., Nadiarynkh, O., Plotnikov, S. et al. Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nat Protoc 7, 654-669 (2012). https: / / doi.org / 10.1038 / nprot.2012.009; Lim H. Harmonic Generation Microscopy 2.0: New Tricks Empowering Intravital Imaging for Neuroscience. Front Mol Biosci. 2019 Oct 9;6:99. doi:10.3389 / fmolb.2019.00099. PMID:31649934; PMCID:PMC6794408; and Campagnola PJ, Loew LM. Second-harmonic imaging microscopy for visualizing and biomolecular arrays in cells, tissues and organisms. Nat Biotechnol. 2003 Nov;21(11):1356-60. Doi:10.1038 / nbt894. PMID:14595363, the disclosures of each of which are incorporated herein in their entireties.For background information, including on exploiting intrinsic contrast in the context of third harmonic generation, see Rehberg M, Krombach F, Pohl U, Dietzel S (2011) Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy. PLoS ONE 6(11):e28237. https: / / doi.org / 10.1371 / journal.pone.0028237, H. Lim, et al. Label-free imaging of Schwann cell myelination by third harmonic generation microscopy. PNAS December 1, 2014 111(50)18025-18030 https: / / doi.org / 10.1073 / pnas.1417820111, and Yildirim, M., Sugihara, H., So, PTC et al. Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy. Nat Commun 10,177(2019).https: / / doi.org / 10.1038 / s41467-018-08179-6, the disclosures of each of which are incorporated herein in their entireties.

[0085] Embodiments of the present invention may utilize second harmonic generation (SHG), third harmonic generation (THG), or higher-order processes, such as fourth harmonic generation or harmonic generation greater than fourth harmonic generation. Generally, harmonic generation refers to a nonlinear optical process in which (i) several photons having the same frequency interact with a nonlinear material, (ii) such photons are "combined," and (iii) as a result of such combination, such photons generate new photons having a multiple of the energy of the initial photons, where such multiple corresponds to the number of interacting photons. For example, second harmonic generation is a nonlinear optical process in which two photons having the same frequency interact with a nonlinear material and "combine" to generate new photons having twice the energy of the initial photons, where the new photons preserve the coherence of the excitation.

[0086] Upon completion of detecting light emitted from the structure in step 128, the process then proceeds to step .

[0087] Adaptive Optics: In step 130, adaptive optics techniques are used for the light used for multiphoton excitation of the structures detected in step 128 in connection with imaging the structures. Any convenient adaptive optics technique may be used, and such may vary. For example, adaptive optics techniques may be used to reduce the effects of incident wavefront distortions associated with multiphoton excitation by the structures, for example.

[0088] In embodiments, adaptive optics is used to shape the wavefront of the excitation light, rather than the detected light. This allows for maximum efficiency of excitation at the smallest possible focal point, which in turn allows for improved efficiency of signal generation at the smallest point, which also maximizes resolution. While adaptive optics techniques may be used in conjunction with signal collection, in embodiments, adaptive optics techniques need not be used for image signal collection; in some cases, distortions do not interfere with image collection because embodiments only need to collect as much signal as possible. In embodiments, image formation is extrapolated from the known position of the laser focal point. Thus, in certain cases, step 130 may begin as part of or immediately thereafter step 126, in which the excitation source is deployed to emit excitation energy; in such cases, step 130 may be performed before step 128, in which signal light is detected from the structure.

[0089] In embodiments, adaptive optics techniques can be used to increase the efficiency of multiphoton excitation. Increasing the efficiency of multiphoton excitation means increasing the efficiency of light generation emitted by a structure via multiphoton excitation. For example, in embodiments, adaptive optics (AO) through direct sensing and correction of wavefront distortions can be used to further enhance the resolution of multiphoton microscopy deep within non-transparent and scattering tissues to achieve subcellular and diffraction-limited resolution. Further details are provided in Wang, K., et al., Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue. Nat Commun, 2015.6:p.7276, the disclosure of which is incorporated herein in its entirety. In some cases, adaptive optics techniques are further configured to improve the resolution of the imaged structure. In embodiments, the adaptive optics technique includes one or more of direct sensing, direct wavefront sensing, correction of wavefront distortion, image point spread function, laser guide star techniques, or indirect techniques. In such embodiments, direct wavefront sensing can include using a Shack-Hartmann sensor and a deformable mirror. Direct wavefront sensing can be combined with multiphoton imaging to improve morphological and functional imaging deep within highly scattering tissue. In embodiments, direct wavefront sensing is performed with a Shack-Hartmann sensor to measure the wavefront distortion of a fluorescent guide star created inside the specimen. This information can be conveyed to a deformable mirror to reshape the excitation wavefront, compensating for aberrations and achieving the narrowest possible excitation focus. In embodiments, indirect wavefront sensing is performed via a feedback loop, without the use of a point guide star or Shack-Hartmann sensor, which modulates the excitation wavefront to maximize emitted power.Further background information is provided in Jung, S., et al., Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol, 2000. 20(11): pp. 4106-14, the disclosure of which is incorporated herein in its entirety.

[0090] In some cases, the adaptive optics technique of interest involves an indirect (eg, algorithmic) technique for determining the optimal excitation wavefront. For further details, see Debarre D, Botcherby EJ, Watanabe T, Srinivas S, Booth MJ, Wilson T. Image-based adaptive optics for two-photon microscopy. Opt Lett. 2009 Aug 15;34(16):2495-7. doi:10.1364 / ol.34.002495. PMID:19684827; PMCID:PMC3320043, and Ji N, Milkie DE, Betzig E. Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat Methods. 2010 Feb;7(2):141-7. doi:10.1038 / nmeth.1411. Epub 2009 Dec 27. PMID:20037592, and Tang J, Germain RN, Cui M. Superpenetration optical microscopy by iterative multiphoton adaptive compensation technique. Proc Natl Acad Sci US A. 2012 May 29;109(22):8434-9. doi:10.1073 / pnas.1119590109. Epub 2012 May 14. PMID:22586078; PMCID:PMC3365222, the disclosures of each of which are incorporated herein by reference.

[0091] In other embodiments, adaptive optics techniques are configured to improve spatiotemporal resolution. With respect to guide star techniques, in embodiments, a guide star represents the image point spread function and is scanned along all positions in the image (i.e., according to a raster). The distortion / blurring of the guide star is used to extrapolate how the wavefront should be corrected to determine and achieve the best resolution via adaptive optics (AO) techniques.

[0092] For indirect approaches, the excitation wavefront can be modified through optimization of an objective function, the most successful of which segment the image at the target plane into independent subregions due to the spatially localized nature of the inhomogeneity to maximize either emission intensity, focal radius, or spatial frequency. Further details regarding indirect adaptive optics approaches, in which the optimal excitation wavefront is determined based on focal radius (less blurring is better), can be found in Booth, M. J., Wavefront sensorless adaptive optics for large aberrations. Opt. Lett. 32, 5-7 (2007), the disclosure of which is incorporated herein. Further details regarding the indirect adaptive optics approach, in which the objective function states "Living things are displayed here, not TV snow. The less high frequency garbage there is in the image, the better," can be found in Debarre, D., Booth, MJ & Wilson, T. Image based adaptive optics through optimisation of low spatial frequencies. Opt. Express 15, 8176-8190 (2007), the disclosure of which is incorporated herein.

[0093] Upon completion of using the adaptive optics technique in step 130, the process may then proceed to step 132 or may return to step 126. In the event that application of the adaptive optics technique results in a sufficient improvement in the ability to image the structure (e.g., a sufficient improvement in image resolution of the structure), then the process proceeds to step 132. In the event that application of the adaptive optics technique does not result in a sufficient improvement in the ability to image the structure (e.g., insufficient improvement in image resolution of the structure), the process may return to step 126, where changes to the light emitted by the excitation source or changes to the adaptive optics technique used may be made to achieve a sufficient improvement (e.g., a sufficient improvement in resolution of the structure). Such a return to step 126 may be applied iteratively, whereby, for example, iterative changes to the light emitted by the excitation source or the adaptive optics technique may be implemented.

[0094] Tissue operations: In step 132, the imaged structure is manipulated in connection with one or more of steps 124, 126, 128, and 130. As described above, embodiments of the present invention further include manipulating the imaged structure. In embodiments, manipulating the imaged structure includes manipulating the imaged structure using an excitation source, i.e., an excitation source such as those described above in connection with step 104 of flowchart 100 of FIG. 1A. In embodiments, the same excitation source used to image the structure may be used to manipulate the structure. That is, in such embodiments, a first excitation source may be used to image the structure, and this same first excitation source may also be used to manipulate the structure. In other embodiments, separate excitation sources may be used for imaging the structure and manipulating the structure. That is, in such embodiments, a first excitation source may be used to image the structure, and a second excitation source may be used to manipulate the structure.

[0095] In embodiments, manipulating the imaged structure includes using the excitation source for non-incisional therapy. Non-incisional therapy means applying therapy without making an incision, e.g., via a scalpel. In other embodiments, manipulating the imaged structure includes using the excitation source to disrupt tissue in the imaged structure. In other embodiments, manipulating the imaged structure includes using the excitation source for light-tissue interaction. In such cases, the light-tissue interaction may include laser-tissue interaction. In other cases, the laser-tissue interaction includes laser tissue perturbation. In certain embodiments, using the excitation source for light-tissue interaction includes configuring the excitation source for multiphoton-mediated damage, e.g., thermal damage. In other embodiments, using the excitation source for light-tissue interaction includes configuring the excitation source for photodisruption. In yet other embodiments, using the excitation source for light-tissue interaction includes configuring the excitation source for vascular coagulation. In embodiments, manipulating the imaged structure includes ablating the imaged structure.

[0096] In certain embodiments, manipulating the imaged structure includes treating glaucoma using the excitation source. In some cases, treating glaucoma using the excitation source includes reducing aqueous humor production in the ocular tissue using the excitation source. In such embodiments, reducing aqueous humor production in the ocular tissue using the excitation source may include damaging the ciliary body, for example, by thermally damaging the ciliary body or applying a photodisruption-mediated process to reduce aqueous humor production. In other embodiments, treating glaucoma using the excitation source includes increasing aqueous humor outflow. In such embodiments, increasing aqueous humor outflow using the excitation source may include performing laser trabeculotomy. In some cases, increasing aqueous humor outflow using the excitation source includes performing laser trabeculoplasty directly through non-translucent tissue of the eye. In contrast, conventional laser trabeculoplasty, i.e., one that does not have the benefit of the present invention, is performed exclusively through the cornea.

[0097] In embodiments, manipulating the imaged structure includes using the excitation source to prevent or treat a retinal hole, e.g., a retinal tear. In such embodiments, preventing or treating a retinal hole, e.g., a retinal tear, may include identifying a region of interest and providing photocoagulation therapy. Embodiments of the present invention are configured to prevent or treat retinal holes in the peripheral region of the retina. The peripheral region of the retina is typically the most vulnerable and prone to hole formation, and is also the most difficult part of the retina to access, and in some cases is not accessible using conventional techniques.

[0098] In other embodiments, using the excitation source for non-incisional therapy includes providing photocoagulation and thermal treatment to a tissue, such as a tumor located in an ocular or periocular tissue. For example, using the excitation source for non-incisional therapy includes providing photocoagulation or thermal treatment to a ciliary body tumor. In some cases, using the excitation source for non-incisional therapy includes providing photocoagulation or thermal treatment to a peripheral choroid tumor. In other examples, using the excitation source for non-incisional therapy includes providing photocoagulation or thermal treatment or ablation to cancer cells, including cancer cells located in dermatological tissue, such as melanoma.

[0099] In yet other embodiments, manipulating the imaged structure includes optically crosslinking scleral tissue using the excitation source. Such embodiments may include methods for preventing myopia, or methods for intervening in myopia or preventing the progression of myopia. In certain embodiments, manipulating the imaged structure includes visualizing and performing targeted modification of extraocular muscle function using the excitation source. In other embodiments, manipulating the imaged structure includes visualizing and performing targeted thermal or photocoagulation therapy of orbital fat using the excitation source. In embodiments, manipulating the imaged structure includes visualizing and performing targeted modification of eyelid tissue using the excitation source.

[0100] In embodiments, manipulating the imaged structure using an excitation source includes using one or more of the adaptive optics techniques described above. That is, the adaptive optics techniques described herein may be applied to the light energy emitted by the excitation source used to manipulate the tissue. Such adaptive optics techniques may be used to make the multiphoton excitation process used in connection with manipulating the tissue more efficient, more precise, or more localized, i.e., applied with finer granularity.

[0101] 1B shows that manipulating a structure in step 132 is performed after imaging of the structure, but as described herein, manipulating a structure may be performed without prior imaging of such structure. That is, embodiments of the present invention include manipulating a structure using only the techniques described herein, i.e., also without imaging such structure.

[0102] Upon completion of manipulating the imaged structure in step 132, the process may then proceed to step 134 where the process ends, or may return to step 126. In the event that it is desired to further image the structure manipulated in step 134 as described above, the process may return to step 126. As described above, in some cases, upon returning to step 126, changes to the light emitted by the excitation source or changes to the adaptive optics technique used may be made to achieve an improvement (e.g., a sufficient improvement in the resolution of the structure). Such returns to step 126 may be applied iteratively, whereby manipulating the structure as described in step 132 may be iterative and / or may alternate with imaging the structure as described in steps 126, 128, and 130.

[0103] Upon finally completing the manipulation of the imaged structure in step 132, the process ends in step 134.

[0104] FIG. 1C illustrates a flow chart 150 for imaging a structure through non-transparent tissue, e.g., ocular or periocular tissue, in accordance with another embodiment of the present invention. The embodiment of the present invention illustrated in FIG. 1C illustrates a method of delivering gene therapy, i.e., a method of using imaging through non-transparent tissue, e.g., ocular or periocular tissue, to guide the delivery of gene therapy to a structure. That is, flow chart 150 relates to the delivery of an active agent, e.g., a gene therapy, stem cell therapy, or engineered cell therapy technique, associated with implementing the gene therapy, stem cell therapy, or engineered cell therapy technique in the imaged structure through non-transparent tissue, e.g., ocular or periocular tissue. The gene therapy, stem cell therapy, or engineered cell therapy performed in association with flow chart 150 can be applied to any convenient structure, e.g., an ocular or periocular structure. Flow chart 150 is an exemplary embodiment of the present invention provided for illustrative purposes; the structure and optical technique applied, as well as the agent introduced in association with the gene therapy, stem cell therapy, or engineered cell therapy technique, can vary as desired in embodiments of the present invention. Certain steps shown in flowchart 150 are similar or identical to those shown in connection with the embodiments shown by flowchart 100 in Figure 1A and flowchart 120 in Figure 1B. A description of such similar or identical steps will not be repeated in connection with the discussion of Figure 1C.

[0105] Flowchart 150 begins at step 152. From starting step 152, the process then proceeds to step 154.

[0106] The excitation source is deployed in step 154. Step 154 ​​is identical to step 104 described above in connection with flowchart 100 of Figure 1A.

[0107] Upon completion of the deployment of the excitation source in step 154 ​​, the process then proceeds to step 156 .

[0108] In step 156, light emitted from the structure is detected. Step 156 is identical to step 106 described above in connection with flowchart 100 of FIG. 1A. Completion of steps 154 and 156 results in the generation of an image of the structure of interest. Such imaging is then used to guide the delivery of an agent to a desired location in steps 158 and 160. As a result of the imaging achieved in steps 158 and 160, delivery of the agent to the desired location can be achieved with a high degree of precision. Although steps 154 and 156 are shown as separate steps in FIG. 1C, such imaging of the structure of interest can begin in connection with steps 154 and 156 and continue over the course of completing steps 158 and 160, such that the structure of interest is continuously imaged as associated agents are introduced into the structure and the effects of such agents are evaluated, as described below.

[0109] Upon completion of detecting light emitted from the structure in step 156, the process then proceeds to step 158.

[0110] Gene or cell or drug therapy applications: In step 158, an agent is introduced into the structure, i.e., the structure of interest. Any convenient agent may be introduced into the structure, i.e., in connection with performing gene therapy, stem cell therapy, or engineered cell therapy in the structure, and such may vary. That is, the agent introduced into the structure may include any convenient agent capable of, for example, causing or facilitating genetic modification in the cells of the structure to produce a desired therapeutic effect, or treating a disease in the imaged structure by repairing or reconstructing defective genetic material. Delivery of the agent into the structure is guided, such that delivery can be precisely located with a high degree of granularity based on the image data collected in connection with completing steps 154 and 156 above. In some cases, the structure is continuously imaged over the course of delivering the agent to the structure of interest. For example, such imaging can be used to guide a needle capable of delivering the agent to the structure at a precise location.

[0111] In some cases, the agent introduced into the structure includes cells. In such cases, the cells may be stem cells or engineered cells, or a combination thereof. In other cases, the agent includes an active agent, such as a pharmaceutical or drug. In still other cases, the agent may include a virus, such as a recombinant virus, or a biological nanoparticle or viral vector. In still other cases, the specified agent includes a molecule. Molecules of interest include DNA, RNA, oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles or molecules involved in using CRISPR tools for gene editing, such as those known in the art. In some cases, one or more agents may be introduced as needed to perform gene therapy in the structure. In certain cases, introducing an agent into the imaged structure includes introducing the specified agent into one or more of the subretinal space, suprachoroidal space, subchoroidal space, or intravitreal space. In certain cases, introducing an agent into the imaged structure includes introducing the specified agent into one or more of the ciliary body or scleral stroma.

[0112] Embodiments of the present invention allow the location of delivery of such one or more agents to be imaged and carefully selected. For example, embodiments of the present invention may be applied to deliver one or more such agents to a precise location, for example, in the subretinal space, or the suprachoroidal space, or the subchoroidal space, or the intravitreal space. Embodiments of the present invention may include deploying a syringe, e.g., a needle syringe, to introduce one or more agents for use in connection with gene or cell or drug therapy, or any other therapy requiring the introduction of an agent, e.g., an active agent, into a structure. Embodiments of the present invention may further include visualizing aspects of the syringe, e.g., the tip of the needle syringe, in conjunction with imaging of the structure and delivering such agents.

[0113] Although not shown in FIG. 1C, in some embodiments, the method includes imaging the structure using the techniques of the present invention and subsequently, or substantially simultaneously, introducing an agent into the structure, whereby the agent can be introduced into a precise location of the structure.

[0114] Although not shown in FIG. 1C , in some embodiments, the method includes applying photocrosslinking to aspects of the imaged structure, such as aspects of the sclera. Such photocrosslinking of the sclera can be used, for example, to prevent the progression of myopia. Crosslinking agents used in existing techniques, absent the present invention, are applied to the cornea and penetrate the cornea but may not penetrate into the sclera. In cases where such crosslinking agents do not penetrate the sclera, embodiments of the present technique can be applied to inject agents directly into the sclera to improve the effectiveness of such photocrosslinking techniques and prevent the progression of myopia. Yet other embodiments of the present invention use intravascular chemicals photoactivated by the excitation source of the present invention, for example, a laser capable of penetrating non-transparent tissues, such as ocular or periocular tissues, to target a structure, such as the sclera, to achieve photocrosslinking of the sclera.

[0115] Upon completion of introducing the specified agent into the structure in step 158 , the process then proceeds to step 160 .

[0116] In step 160, one or more effects of introducing the agent into the structure are evaluated. In embodiments, in step 160, the continuous imaging of the structure initiated in steps 154 and 156 above may be used to assess the effect of the designated agent on the structure. That is, in embodiments, imaging the structure is used to evaluate whether the delivery of the agent is achieving the intended effect. Any convenient aspect of the image of the structure may be observed to evaluate or assess the effect of the designated agent on the structure, i.e., to evaluate whether the desired gene therapy technique is working or whether the agent has been delivered to the desired location. In other embodiments, techniques other than imaging the structure may be applied to evaluate the effectiveness of the delivery of the agent to the desired structure.

[0117] Upon completion of evaluating the effect of the agent on the structure, the process may then proceed to step 162 where the process ends, or may return to step 158. In the event that it is desired to introduce an additional or different agent, for example, an agent to perform gene therapy in the imaged structure, or to introduce an agent at a different location, for example, a different location in the imaged structure, the process may return to step 158. As noted above, in some cases, upon returning to step 158, an additional amount of the same or a different agent previously introduced into the structure may be further introduced into the structure. Continuing the flow chart after returning from step 160 to step 158 allows the opportunity to evaluate the introduction of an additional or different agent, or the introduction of an agent into another location within the structure, so that, for example, gene therapy or stem cell therapy or engineered cell therapy techniques may be applied under near-continuous observation to monitor and evaluate the effects of the introduction of one or more agents into the structure. That is, such a return to step 158 may be applied repeatedly, whereby assessing the effect of a drug as described in connection with step 160 may be alternated with introducing one or more drugs into the structure while continuing to image the structure as described in connection with steps 154, 156, and 158.

[0118] Upon finally completing the evaluation of the drug's effect on the structure in step 160, the process ends in step 162.

[0119] Flow analysis techniques: Embodiments of the present invention may further include advanced computational techniques capable of increasing the spatiotemporal resolution, execution speed, and dimensionality of flow analysis using multiphoton imaging. That is, embodiments of flow analysis techniques can increase not only execution speed but also the dimensionality of input and output data. Certain embodiments are configured to provide such flow analysis in real time, substantially in real time, or near real time.

[0120] Embodiments of the claimed invention include implementing a signal analysis algorithm that utilizes, for example, a multi-core processor to simultaneously process a series of negative contrast images, such as may be obtained utilizing multi-photon-based imaging according to the claimed invention. In embodiments, the signal analysis algorithm (e.g., one embodiment of such an algorithm according to the invention is the RBCPIV algorithm) includes two steps: (1) spatial object localization (e.g., by using an n-point discrete Fourier transform, i.e., a fast Fourier transform combined with curve fitting of a probability distribution to sub-pixel accuracy), and (2) physical displacement (velocity) of the object over time. In another embodiment, the signal analysis algorithm includes the following steps after a series of negative contrast images are obtained: (1) for any given pair of contrast images (a contrast image means, for example, a one- or two-dimensional frame, or a three-dimensional volume; in embodiments, the image is represented as an n-dimensional tensor), (2) an n-point discrete Fourier transform of each image is taken; (3) phase weights are computed; (4) the pair of images are cross-correlated in Fourier space; (5) a probability distribution (e.g., a Gaussian distribution) is fitted to each zero-frequency shift image by solving a nonlinear least-squares problem (e.g., via the Levenberg-Marquardt algorithm); (6) each fit is used to compute a displacement in physical space; and (7) velocity is calculated from the physical displacement using the time between images.Further details regarding aspects of the signal analysis algorithms of the present invention can be found in Levenberg, Kenneth (1944). "A Method for the Solution of Certain Non-Linear Problems in Least Squares." Quarterly of Applied Mathematics. 2(2):164-168. doi:10.1090 / qam / 10666, and Kim TN, Goodwill PW, Chen Y, Conolly SM, Schaffer CB, Liepmann D, Wang RA. Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals. PLoS One. 2012;7(6):e38590. doi:10.1371 / journal.pone.0038590. Epub 2012 Jun 26. PMID:22761686; PMCID:PMC3383695, in each case incorporated herein by reference. Further details regarding cross-correlation techniques specific to particle image velocimetry can be found in Keane, RD, Adrian, RJ Theory of cross-correlation analysis of PIV images. Applied Scientific Research 49, 191-215 (1992). https: / / doi.org / 10.1007 / BF00384623, the disclosure of which is incorporated herein by reference.

[0121] 1D-E illustrate aspects of a signal analysis algorithm according to an embodiment of the present invention. FIG. 1D illustrates an existing technique for a signal analysis algorithm for use in flow analysis, in which multiple velocity calculations using imaging data are performed sequentially. In FIG. 1D, reference to LS-PIV refers to the sequential calculation of velocities in only one dimension, as described in Kim TN, Goodwill PW, Chen Y, Conolly SM, Schaffer CB, Liepmann D, Wang RA. Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals. PLoS One. 2012;7(6):e38590. doi:10.1371 / journal.pone.0038590. Epub 2012 Jun 26. PMID:22761686; PMCID:PMC3383695, which is incorporated herein by reference.

[0122] FIG. 1E illustrates one embodiment of the present technique for a signal analysis algorithm for use in flow analysis, in which multiple velocity calculations using imaging data are performed simultaneously, enabling flow analysis results to be obtained substantially in real time. In FIG. 1E, RBC-PIV refers to an algorithm capable of parallelized execution of n-dimensional data obtained from any contrast modality, according to one embodiment of the present invention. An aspect of an embodiment of the present signal analysis algorithm for flow analysis is simultaneous processing. In a flow analysis algorithm (i.e., a signal-based analysis algorithm for flow analysis) embodiment of the present invention, flow analysis can be performed in parallel because each frame (i.e., image) contains information about the location of an object (e.g., a red blood cell or cluster of red blood cells, but the present invention is not so limited) in physical space. An aspect of such an algorithm embodiment, i.e., the RBCPIV aspect of locating an object in physical space, can do so simultaneously because each frame is essentially independent of the next frame. While velocity calculations require showing the same object displacing over time, simply locating an object in a frame does not require any information beyond that contained in the individual frames. Embodiments of the signal processing algorithm for flow analysis, i.e., embodiments of the RBCPIV algorithm, process a series of images independently of how the images were taken. That is, embodiments of the signal processing algorithm for flow analysis, i.e., embodiments of the RBCPIV algorithm, do not rely on specific techniques for collecting images and performing flow analysis based on such images, but can be used to analyze contrast signals obtained from any imaging modality, including, but not limited to, modalities such as micro-CT, second harmonic generation, third harmonic generation, two-photon and three-photon microscopy (or even higher-order processes), and confocal microscopy.Further details demonstrating the use of microCT in blood vessels can be found in Zagorchev L, Oses P, Zhuang ZW, Moody K, Mulligan-Kehoe MJ, Simons M, Couffinhal T. Micro computed tomography for vascular exploration. J Angiogenes Res. 2010 Mar 5;2:7. doi:10.1186 / 2040-2384-2-7. PMID:20298533; PMCID:PMC2841094, the disclosure of which is incorporated herein by reference.

[0123] In some cases, simultaneous processing may be achieved by leveraging any number of graphics processing units, such as commercially available graphics processing units, or other computer processing technologies capable of parallel processing. For example, the exemplary signal processing algorithm described above analyzes each pair of images simultaneously utilizing graphics processing unit (GPU) cores from a commercially available NVIDIA GPU, resulting in a substantial increase in execution speed.

[0124] Usage: As noted above, any convenient structure may be optionally imaged and / or manipulated in connection with the methods, adapters, and systems of the present invention, and such may vary. Furthermore, structures may be imaged and / or manipulated through any convenient non-transparent tissue, such as ocular or periocular tissue, and such may vary.

[0125] Thus, in some cases, the method of the present invention is a method of transscleral imaging. In other cases, the method of the present invention is a method of transconjunctival imaging. In still other cases, the method of the present invention is a method of trans-Tenon's capsule imaging. In still other cases, the method of the present invention is a method of extraocular muscle imaging. In certain cases, the method of the present invention is a method of imaging through outer eyelid tissue, including one or more of the dermis, muscle, or aponeurosis. In some cases, the method of the present invention is a method of imaging through inner eyelid tissue, including the conjunctiva, tarsal plate, meibomian gland, or muscle. In other cases, the method of the present invention is a method of transorbital septum imaging. In still other cases, the method of the present invention is a method of transcapsular parvebral fascial imaging. In still other cases, the method of the present invention is a method of transtarsal fascial imaging. In some cases, the method of the present invention is a method of transtarsal gland imaging. In other cases, the method of the present invention is a method of transperiocular adipose tissue imaging. In still other cases, the methods of the invention are methods of transcutaneous imaging. In still other cases, the methods of the invention are methods of imaging through pigmented uveal tissue. In some cases, the methods of the invention are methods of imaging through light-scattering tissue. In other cases, the methods of the invention are methods of imaging through light-absorbing tissue. In still other cases, the methods of the invention are methods of in vivo imaging. In still other cases, the methods of the invention are methods of quantifying fluid flow, e.g., blood flow, e.g., choroidal blood flow. In some cases, the methods of the invention are methods of quantifying retinal blood flow. In other cases, the methods of the invention are methods of quantifying ciliary body blood flow. In still other cases, the methods of the invention are methods of quantifying uveal blood flow. In still other cases, the methods of the invention are methods of quantifying conjunctival blood flow.

[0126] As described above, the present invention relates to the application of multiphoton excitation to microscopy. Thus, in some cases, the method of the present invention is a method of deploying multiphoton excitation microscopy on non-transparent tissue, such as ocular or periocular tissue. In other cases, the method of the present invention is a method of deploying multiphoton excitation microscopy through non-transparent tissue, such as periocular tissue. In such embodiments, the multiphoton excitation microscopy may include one or more of two-photon excitation fluorescence (2PEF), second harmonic generation (SHG), three-photon excitation fluorescence (3PEF), third harmonic generation (THG), four-photon excitation fluorescence, fourth harmonic generation, or other higher-order multiphoton processes.

[0127] As noted above, the present invention relates to imaging and / or manipulating any convenient structure, as the case may be. Thus, in some cases, the methods of the present invention are methods of imaging and / or manipulating living tissue. It is contemplated that embodiments of the present invention may be applied to any convenient living tissue. In some cases, the living tissue may be living ocular or living periocular tissue. In other cases, the living tissue may be dermatological tissue, or vascular tissue, or neural tissue.

[0128] Other potential uses of imaging and / or manipulating tissue with embodiments of the invention include, for example, imaging neural activity, imaging corneal nerves, imaging aspects of the central nervous system, utilizing an invasive probe to image aspects of the central nervous system, thinning a region of the skull to image aspects of the central nervous system, visualizing neural structures, utilizing imaged neural structures to alleviate pain, utilizing imaged structures to perform flow analysis, performing flow analysis in substantially real time, generating a velocity map of fluid flow within an imaged structure, diagnosing disease, diagnosing cancer, distinguishing between cancerous and non-cancerous tissue, distinguishing between cancerous and non-cancerous cells, preventing or treating retinal holes, providing non-thermal therapy, providing non-thermal therapy to tumors, visualizing orbital fat, manipulating dermatological tissue, imaging one or more of the epidermis, dermis, or subcutaneous tissue, manipulating tear ducts to facilitate fluid flow within the tear duct, reducing ocular tissue redness, delivering therapy to an imaged structure, intraocular imaging, and the like. guiding the positioning of an implant based on imaged structures, predicting the effective placement of an intraocular implant based on imaged structures, guiding the positioning of an intraocular implant based on imaged structures while implanting an intraocular implant, assessing the position of an implanted intraocular implant based on imaged structures, assessing the effective lens position (ELP) of an implanted intraocular implant based on imaged structures, detecting cancerous tissue, e.g., melanoma, providing cosmetic treatments, providing cosmetic dermatological treatments, providing cosmetic surgery, treating scars, removing scars, treating acne scars, removing acne scars, treating skin discolouration, removing birthmarks, removing port wine stains, treating skin discolouration disorders, removing tattoos, treating rosacea, providing controlled cutting of tissue, providing controlled cutting of dermatological tissue, performing a skin biopsy procedure, destroying cancerous tissue, destroying one or more cancer cells, ablating cancerous tissue, ablating one or more cancer cells, ablating melanoma, ablating cutaneous melanoma, removing hair, destroying hair follicles,These include, but are not limited to, ablation of follicular tissue, affecting tissue shape, affecting the shape of one or more fat deposits, reducing the volume of one or more fat deposits, reducing one or more subdural fat deposits, providing tissue sculpting, providing skin tightening, treating dry eye syndrome, reducing eye redness, providing pain management, or perforating tissue.

[0129] Imaging and / or Manipulation of Tissue of Interest: In some cases, the method of the present invention is a method of imaging and / or manipulating a subject's tissue, such as ocular or periocular tissue. In such embodiments, the subject may have glaucoma, a retinal hole, a choroidal tumor, or a similar condition or disease in the same tissue or a tissue adjacent thereto. In some cases, the method of the present invention is a method of imaging and / or manipulating a subject's dermatological tissue. In such embodiments, the subject may have a dermatological condition or feature, such as a skin discoloration condition, the presence of a birthmark, a scar, or a disease condition, such as melanoma or other cancerous tissue. In such embodiments, the subject may be seeking cosmetic treatment, such as skin tightening, or reduction in fat deposits, or tissue reshaping. In embodiments, the subject is a human, and may be male or female, of any age, with no specific medical or disease history or family history of disease. In embodiments, the subject is a human, and may have any degree of pigmentation in the ocular or periocular tissue or dermatological tissue, i.e., whereby the ocular or periocular tissue is opaque to any greater or lesser degree. In embodiments, the ocular or periocular tissue and / or imaged structure comprises living tissue. In embodiments, the subject is a living human.

[0130] Computer-Implemented Embodiments Certain of the method and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or a combination of both. To clearly illustrate this interchangeability of hardware and software, various exemplary steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends on the particular application and design constraints imposed on the overall system applying the methods of the present disclosure. The described functionality may be implemented in various ways for each particular application, and such implementation decisions should not be interpreted as causing a departure from the scope of the present disclosure.

[0131] Certain example steps, components, and computing systems (e.g., devices, databases, interfaces, and engines) described in connection with the embodiments disclosed herein may be implemented or performed by machines designed to perform the functions described herein, such as a general-purpose processor, a graphics processor unit, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof. A general-purpose processor may be a microprocessor, but in the alternative, the processor may be a controller, microcontroller, or state machine, or a combination thereof, or the like. A processor may also be implemented as a combination of computing devices, such as a combination of a DSP and a microprocessor, multiple microprocessors, one or more microprocessors with a DSP core, or any other such configuration. While described herein primarily with reference to digital technology, a processor may also include primarily analog components. The computing environment can include any type of computer system, including, but not limited to, computer systems based on computer computation engines in microprocessors, graphic processor units, mainframe computers, digital signal processors, portable computing devices, personal organizers, device controllers, and appliances, to name a few.

[0132] Certain steps of a method, process, or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or a combination of the two. The software modules, engines, and associated databases may reside in memory resources, such as RAM memory, FRAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, a hard disk, a removable disk, a CD-ROM, or any other form of non-transitory computer-readable storage medium, medium, or physical computer storage known in the art. An external storage medium may be coupled to the processor such that the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integrated into the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

[0133] Adapters for imaging and manipulating structures through non-transparent tissues Aspects of the present disclosure include adapters for use in practicing the methods of the present invention. In particular, the present disclosure includes adapters for coupling an optical system to non-transparent tissue, such as ocular or periocular tissue. The adapters of the present invention provide stabilization and physical coupling to the tissue, such as ocular or periocular tissue. Specifically, the present disclosure includes adapters for coupling an optical system to non-transparent tissue, such as ocular or periocular tissue, comprising: a first component configured to interface with an optical system configured to image or manipulate a structure through the non-transparent tissue; and a second component connected to the first component, the second component configured to interface with the non-transparent tissue. Embodiments of the adapters of the present invention are utilized in connection with imaging a structure. Embodiments of the adapters of the present invention are utilized in connection with manipulating a structure (e.g., to provide non-incisional therapy or light-tissue interaction, optionally including laser-tissue interaction or laser tissue perturbation, or multiphoton-mediated thermal damage, or non-thermal therapy, or photodisruption, or vascular coagulation, or ablation).

[0134] Further embodiments of the present invention include immersion media for biological imaging, including immersion gels. In some cases, the immersion gel comprises hyaluronan gel. In certain cases, the immersion gel comprises hyaluronic acid and deuterium oxide (heavy water). In other cases, the immersion gel is configured for imaging deep biological structures, such as tissue. In still other cases, the immersion gel is configured for use with long-wavelength (e.g., 1,700 nm) lasers. In some cases, the immersion gel is configured to be an adapter between an optical system and biological tissue (e.g., the immersion gel directly interfaces between an optical system and aspects of the biological tissue). The methods of the present invention described herein (i.e., methods for imaging structures and methods for manipulating structures) further include using an embodiment of such an immersion gel. The immersion gels of the present invention are described in more detail below.

[0135] Embodiments of the adapter include a coupling agent. Coupling agents of interest include transparent media, such as water or viscous gels. In some cases, the coupling agent is present between the optical system and the ocular or periocular tissue, e.g., between the first component and the ocular or periocular tissue. In some cases, the coupling agent includes a gel immersion medium configured to increase the stability and duration of the practice of the subject methods, e.g., imaging or manipulating a structure according to the methods of the invention. Embodiments of the invention further include creating and / or utilizing a viscoelastic gel containing heavy water. Such components are of interest in connection with embodiments for imaging at longer wavelengths (e.g., 1,700 nm), where regular water (but not heavy water) largely absorbs these wavelengths.

[0136] In embodiments, the lens used to focus the laser light into tissue can be air-immersed, water-immersed, oil-immersed, or gel-immersed. In certain embodiments, water-immersed lenses are preferred for multiphoton imaging. For example, for three-photon imaging at approximately 1,700 nm, water absorbs a significant amount of this light, and therefore, it may be preferable to use deuterium oxide (heavy water) as the immersion fluid at this wavelength. In certain cases, using a viscoelastic gel with a water-immersion objective lens may be preferable over the use of water in certain imaging contexts (e.g., the conventional use of water in certain imaging contexts is known in the art). In some cases, it is preferable to use a transparent gel for the immersion medium. In embodiments, a transparent gel can make the optical interface easier and much more stable, and in some cases, can completely avoid the need for a coupling device. Transparent gels of interest include viscoelastic gels, such as viscoelastic gels designed for intraocular surgery.

[0137] In embodiments of the adapter of the present invention, the second component is configured to interface with tissue, e.g., ocular or periocular tissue, using suction. In some cases, the second component comprises a suction mechanism for attaching the second component to the tissue, e.g., ocular or periocular tissue.

[0138] In embodiments, the second component of an adapter of the present invention includes a central portion. In some cases, the central portion is hollow. In some cases, the hollow central portion receives a fluid. In other cases, the central portion is solid. In some cases, the solid central portion is optically transparent. In some cases, the second component includes an interface surface that contacts non-transparent tissue, such as ocular or periocular tissue. In such cases, the interface surface can interface with the eye or ocular tissue. In some cases, the interface surface is shaped to contact the eye or ocular tissue. In some cases, the interface surface is shaped to be displaced relative to the cornea or sclerocorneal limbus. That is, a recess or cutout can be present in the interface surface to accommodate a raised corneal or sclerocorneal limbus shape. For example, in embodiments, the interface surface is shaped to accommodate placement on an area of ​​the cornea or sclerocorneal limbus. In other embodiments, an area of ​​the interface surface is recessed to accommodate placement on the cornea or sclerocorneal limbus. Embodiments of the present invention are not limited to imaging and / or manipulating structures exclusively through non-transparent tissue. In embodiments, the adapter may interface with the cornea, for example, may reside over the central cornea.

[0139] In embodiments, the second component is configured to interface with periocular tissue. In some cases, the second component interfaces with the conjunctival fornix. In other cases, the second component is shaped to interface with the conjunctival fornix. In some cases, the second component is shaped to fit between the eye and the lower eyelid of the eye. In other cases, the second component is shaped to fit between the eye and the upper eyelid of the eye.

[0140] In embodiments of the adapter of the present invention, the second component comprises a contact lens interface. In some cases, the first component translates relative to a non-transparent tissue, e.g., ocular or periocular tissue. In some cases, the first component allows the optical system to translate relative to the second component. In other cases, the first component rotates relative to a non-transparent tissue, e.g., ocular or periocular tissue. In some cases, the first component allows the optical system to rotate relative to a non-transparent tissue, e.g., ocular or periocular tissue. In some cases, the first component articulates relative to a non-transparent tissue, e.g., ocular or periocular tissue. In certain cases, the adapter further comprises a mechanism for controlling the translation, rotation, or articulation of the optical system relative to a non-transparent tissue, e.g., ocular or periocular tissue. Any convenient device capable of translation, rotation, or articulation may be applied. For example, a commercially available stepper motor or servo motor and controller may be applied. Embodiments of the adapter of the present invention are configured to interface with dermatological tissue, such as the surface of the skin. Embodiments of the adapter of the present invention are configured to interface with tissue such that the optical system can be configured to perform a dermatological procedure, or to provide a dermatological therapy, or to perform a cosmetic therapy or treatment, such as tissue reshaping, reducing fat deposits, skin tightening, or addressing skin pigmentation issues.

[0141] In some cases, the adapter of the present invention further comprises a bonding agent, which is present at the interface between the adapter and tissue, e.g., ocular or periocular tissue. Bonding agents of interest include any convenient biocompatible material, e.g., a liquid or gel. The adapter of the present invention may be sterile or may have a sterile surface. The adapter of the present invention may be disposable or reusable, in whole or in part.

[0142] In other embodiments of the adapters of the present invention, e.g., those for use in connection with gene or cell or drug therapy, the adapter may include a syringe for injecting a drug into a structure. In some cases, the adapters of the present invention include a needle syringe. In other cases, the adapters of the present invention include a defined path for positioning the syringe, e.g., a hole through the adapter or a cutout from the adapter, so that the location of the syringe injection site can be precisely known.

[0143] Exemplary embodiments of the adapter: FIG. 2A illustrates a first adapter 210 for coupling an optical system to non-transparent ocular tissue, according to one embodiment of the present invention, and a second adapter 220 for coupling an optical system to a different aspect of non-transparent ocular tissue, according to one embodiment of the present invention. The embodiments of the adapters 210 and 220 of the present invention illustrated in FIG. 2A relate to adapters for imaging or manipulating any convenient structure accessible through non-transparent ocular tissue, according to the present technique, and as such may vary. Similarly, the embodiments of the adapters 210 and 220 of the present invention illustrated in FIG. 2A relate to adapters for interfacing with any convenient optical system, and as such may vary. The adapters 210 and 220 are exemplary embodiments of the present invention provided for illustrative purposes, and aspects thereof may vary as desired in embodiments of the present invention.

[0144] As shown in FIG. 2A , the adapter 210 includes a second component 212 that interfaces directly with non-transparent ocular tissue 218. In particular, the second component 212 of the adapter 210 includes a surface shaped to interface directly with the ocular tissue surface 218, i.e., the surface of the eye. The second component 212 includes a cutout, recess, or depression 216 that is shaped to accommodate the shape of a raised cornea 219 on the ocular tissue surface 218. The adapter 210 also includes a first component 214 located proximal to the second component 212. The first component 214 is shaped to receive an optical system (not shown in FIG. 2A ) for imaging a structure (not shown in FIG. 2A ) through the non-transparent ocular tissue 218. The adapter 210 includes a central portion 212a that is optically transparent and solid.

[0145] As also shown in FIG. 2A , the adapter 220 includes a second component 222 that interfaces directly with non-transparent ocular tissue 228. In particular, the second component 222 of the adapter 220 includes a surface shaped to interface directly with an ocular tissue surface 228, i.e., the surface of the eye. Unlike the second component 212 of the adapter 210, the second component 222 is shaped to interface with an ocular tissue surface 228 that does not include a raised cornea 229, and thus the surface 226 of the second component 222 includes a smooth, continuous surface without cutouts, recesses, or depressions. The adapter 220 also includes a first component 224 located proximal to the second component 222. The first component 224 is shaped to receive an optical system (not shown in FIG. 2A ) for imaging a structure (not shown in FIG. 2A ) through the non-transparent ocular tissue 228. The adapter 220 includes a central portion 222a that is optically transparent and solid.

[0146] Sub-panel (a) of Figure 2B shows the periocular fat pad 236, which resides within the non-transparent periocular tissue 235, i.e., the lower eyelid 235, of an eye 237 having a raised cornea 238, carrying the under-eye capsule, resulting in the configuration of the third adapter 230 shown in sub-panel (b) of Figure 2B.

[0147] Subpanel (b) of Figure 2B illustrates a third adapter 230 for coupling an optical system to non-transparent periocular tissue 235, according to one embodiment of the present invention. The embodiment of the adapter 230 of the present invention illustrated in subpanel (b) of Figure 2B relates to an adapter for imaging any convenient structure accessible through non-transparent periocular tissue 235, according to the present technique, and as such may vary. Similarly, the embodiment of the adapter 230 of the present invention illustrated in subpanel (b) of Figure 2B relates to an adapter for interfacing with any convenient optical system, and as such may vary. The adapter 230 is an exemplary embodiment of the present invention provided for illustrative purposes, and its aspects may vary as desired in embodiments of the present invention.

[0148] As shown in subpanel (b) of FIG. 2B , the adapter 230 includes a second component 231 that interfaces directly with non-transparent periocular tissue 235. In the illustrated embodiment, the second component 231 is shaped to fit between the lower eyelids 235 of the eye 237. In particular, the second component 231 includes a wedge-like shape, with either side of the wedge contacting the periocular tissue, including the lower eyelid 235, and the eye 237. Furthermore, in the illustrated embodiment, the second component 231 is shaped to allow access to the fat pad 236 through the periocular tissue 235. In the illustrated embodiment, the optical system can be configured to image and manipulate such periocular tissue 235, for example, via non-incisional therapy, light-tissue interaction, or delivery of multiphoton-mediated damage, such as thermal damage, photodisruption, photocrosslinking, vascular coagulation, or tissue ablation. The second component 231 may, but need not, be provided with a surface having cutouts or recesses or indentations (not shown in sub-panel (a) or (b) of FIG. 2B ) shaped to accommodate the shape of a raised cornea 238 on the ocular tissue surface of the eye 237. The adapter 230 also includes a first component 232 located proximal to the second component 231. The first component 232 is shaped to receive an optical system (not shown in sub-panel (a) or (b) of FIG. 2B ) for imaging a structure, such as a fat pad 236, through non-transparent periocular tissue 235. The adapter 230 includes a central portion 233 that is optically transparent and solid.

[0149] FIG. 2C illustrates a fourth adapter 240 for coupling an optical system to non-transparent ocular tissue 250, according to one embodiment of the present invention. The fourth adapter 240 illustrates an embodiment including a needle syringe 245 for use with gene or cell or drug therapy. The embodiment of the adapter 240 of the present invention shown in FIG. 2C relates to an adapter for imaging and accessing any convenient structure accessible through non-transparent ocular tissue 250 via a needle syringe, according to the present technique, and as such may vary. Similarly, the embodiment of the adapter 240 of the present invention shown in FIG. 2C relates to an adapter for interfacing with any convenient optical system (not shown in FIG. 2C), and as such may vary. The adapter 240 is an exemplary embodiment of the present invention provided for illustrative purposes, and its aspects may vary as desired in embodiments of the present invention.

[0150] 2C , the adapter 240 includes a second component 241 that interfaces directly with non-transparent ocular tissue 250. In the illustrated embodiment, the second component 241 is shaped to interface with, i.e., rest on, the top surface of the ocular tissue 250, including the optic nerve 251, retina 252, iris 253, lens 254, pupil 255, and cornea 256. In particular, the interfacing surface of the second component 241 includes a substantially curved shape that corresponds to the curved shape of the ocular tissue 250, and in particular, the top surface of the ocular tissue 250. In the illustrated embodiment, the optical system can be configured to image aspects of the ocular tissue 250 and manipulate such ocular tissue 250, for example, through the provision of non-incisional therapy, light-tissue interaction, multiphoton-mediated damage, such as thermal damage, photodisruption, photocrosslinking, vascular coagulation, or tissue ablation. Second component 231 may, but need not, be provided with a surface (not shown in FIG. 2C ) having cutouts or recesses or indentations shaped to accommodate the shape of a raised cornea 256 on the surface of ocular tissue 250. Second component 241 comprises a contact lens 243 for interfacing with ocular tissue 250 and for optically conditioning light transmitted to or detected from non-transparent ocular tissue 250. Adapter 240 also comprises a first component 242 positioned proximal to second component 241. First component 242 is shaped to receive an optical system (not shown in FIG. 2C ) for imaging aspects of ocular tissue 250 through non-transparent ocular tissue 250, including aspects of a structure, e.g., optic nerve 251, retina 252, iris 253, lens 254, pupil 255, or cornea 256.

[0151] The adapter 240 further comprises a suction mechanism 244 for attaching the adapter 240 to the surface of the ocular tissue 250. The suction mechanism 244 comprises a hollow tube that fluidly connects the interface volume between the second component 241 and the surface of the ocular tissue 250, whereby applying a low pressure source to the suction mechanism 244 has the effect of drawing the adapter 240 to the ocular tissue 250 and sealing the adapter 240 thereto while the low pressure source is applied to the suction mechanism 244.

[0152] The adapter 240 further comprises a needle syringe 245 for use in connection with the application of gene, cell, or drug therapy. The needle syringe 245 comprises a needle 246 having an injection tip 247, i.e., the tip of the needle, through which a fluid can be delivered. The needle syringe 245 is integrated within the adapter 240, such that the needle 246 and injection tip 247 are positioned at one or more known locations relative to the imaged structure (i.e., one or more known locations of the imaging field of view of an optical system (not shown in FIG. 2C ) attached to the first component 242 of the adapter 240, e.g., the center of the field of view at various configurable depths from the center of the field of view). This needle syringe 245 resides at one or more fixed locations relative to the imaged structure, allowing delivery of one or more agents for gene, cell, or drug therapy to be delivered to precise locations in the ocular tissue 250, thereby facilitating more specific or targeted gene, cell, or drug therapy, i.e., whereby the agent is delivered to the desired location and substantially not elsewhere.

[0153] As noted above, embodiments of the present invention include utilizing an immersion medium in conjunction with an embodiment of an adapter. In embodiments, a viscoelastic gel may be used in place of water as a viscous substitute immersion medium to help reduce visual aberrations caused by motion artifacts. Furthermore, certain embodiments of the viscoelastic gel do not require an adapter (e.g., the adapter shown in FIGS. 2A-C) to remain between the sample and an aspect of the optical system, e.g., the objective lens (in some cases, such an element may be referred to as a reservoir, and in certain contexts, such an element is configured to function as a reservoir (a reservoir of immersion medium), i.e., whereby embodiments of the viscoelastic gel when used in optical systems, e.g., those described herein, do not require the use of such an adapter and / or reservoir), and may provide equivalent, if not superior, imaging quality for in vivo applications (i.e., compared to embodiments requiring an adapter and / or reservoir). Additionally, the viscoelastic gel, when applicable, can help stabilize aspects of the optical system, such as the focal point between the objective lens and the sample, i.e., the structure, during long-term imaging or manipulation of the structure, which allows for better image capture or manipulation of the structure in the context of time-lapse photography.

[0154] The transparent viscous gels of embodiments of the present invention can be used as an alternative immersion medium to water. Such viscous gels partially reduce distortion caused by motion artifacts, evaporate at a slower rate than water, do not require reservoirs like those found in cranial windows, and have a refractive index (RI 1.3365) sufficiently close to that of water (RI 1.333) to be compatible with water-immersion objective lenses. In embodiments with viscous gels having variable formulations of 0.5% to 4%, the refractive index is typically expected to be in the range of approximately 1.335 to 1.350. In embodiments, aspects of an optical system, such as an objective or lens, can be specifically designed for a desired immersion medium that is a viscous gel. That is, embodiments of the present invention include aspects of an optical system, such as a lens or objective, or other components of an optical system, configured for use with (e.g., adapted to compensate for the effects of) a viscous gel, such as the viscous gels described herein.

[0155] Viscoelastic gel embodiments of the present invention include hyaluronic acid. Certain viscoelastic gel embodiments of the present invention include hyaluronic acid and water. Hyaluronic acid is relatively inexpensive, and non-clinical versions of this gel may be sufficient. Other viscoelastic gel embodiments further include other ingredients, such as deuterium oxide (heavy water), chondroitin sulfate, and hydroxypropyl methylcellulose. Viscoelastic gel embodiments of the present invention are highly customizable in their formulations when using varying concentrations of hyaluronic acid or other viscosity-enhancing substances, such as chondroitin sulfate. Other viscoelastic gel embodiments of the present invention include aspects, i.e., components, that enhance viscosity and / or high-quality imaging. Modifying the gel composition may produce more viscous gels with improved utility in long-term imaging and surgical procedures with poor surgical and / or optical access. While these modifications may result in a greater difference in refractive index from water, embodiments of the present invention can compensate for this effect using aspects of the optical system, such as a correction collar that adjusts internal optical components of the objective lens. Additionally, other aspects of the optical system, such as new objective lenses, may be specially designed to optimize compatibility with viscoelastic gel embodiments and further improve imaging quality and stability. Embodiments utilizing three-photon imaging using water-immersion objective lenses may require heavy water (deuterium oxide) to avoid absorption that occurs at longer infrared laser wavelengths, typically above 1,700 nm. Because viscoelastic gel embodiments may be primarily composed of hyaluronic acid and water, heavy water may be used to replace regular water in new formulations, which may enable compatibility with both two- and three-photon laser microscopy.

[0156] Embodiments of viscoelastic gels containing hyaluronic acid (HA) optimized for imaging purposes may have a concentration of 0.1% to 10% (% weight / volume) in water. The molecular weight (MW) of the HA polymer can range from 100,000 Daltons to 20,000,000 Daltons. In embodiments, the concentration and MW can be adjusted to fine-tune the viscoelastic properties and improve their usefulness in various imaging contexts. Additionally, in embodiments, the inclusion of chondroitin sulfate (CS) with a MW of 1,000 Daltons to 1,000,000 Daltons at a concentration of 0.1 to 10% can be used to further modify the viscoelastic properties of such gels.

[0157] Figures 2D-H present a comparison of imaging quality when using water versus gel immersion media in accordance with one embodiment of the present invention in the context of a standard mouse cranial window. Figures 2D-E show 900 μm z-stacks taken with a 25x water immersion objective using water (Figure 2D) and gel (Figure 2E) from mouse cortical vasculature stained with Texas-Red. Figure 2F shows how the average signal-to-noise ratio (SNR) (calculated as 10*log(average signal / noise)) results tend to be roughly the same when imaging depths up to 900 μm using water or gel in accordance with one embodiment of the present invention. 2G-H present normalized cross-sections at a depth of 100 μm in sub-panels (a) of FIG. 2G and (a) of FIG. 2H, a depth of 300 μm in sub-panels (b) of FIG. 2G and (b) of FIG. 2H, a depth of 500 μm in sub-panels (c) of FIG. 2G and (c) of FIG. 2H, a depth of 700 μm in sub-panels (d) of FIG. 2G and (d) of FIG. 2H, and a depth of 900 μm in sub-panels (e) of FIG. 2G and (e) of FIG. 2H, which show approximately equivalent imaging quality in water (sub-panels (a)-(e) of FIG. 2G) and gel immersion medium (sub-panels (a)-(e) of FIG. 2H) according to one embodiment of the present invention.

[0158] Figures 2I-K present experimental results demonstrating improved stability of immersion media during long-term in vivo imaging when using a gel according to one embodiment of the present invention. In Figures 2I-K, microglia tagged with enhanced green fluorescent protein (eGFP) were imaged in mouse retina over a time-lapse period of at least 30 minutes. Figure 2I shows the initial image quality in subpanel (a) of Figure 2I using water, followed by the image quality after 10 minutes in subpanel (b). Figure 2I also shows how the image quality using water alone significantly deteriorates by 20 minutes (i.e., subpanel (c) of Figure 2I), until the signal is essentially absent by 30 minutes (i.e., subpanel (d) of Figure 2I). Figure 2J shows the initial image quality in subpanel (a) of Figure 2J using a gel according to one embodiment of the present invention, followed by the image quality after 10 minutes in subpanel (b) of Figure 2J. Figure 2J shows how image quality using a gel according to one embodiment of the present invention remains roughly constant for the first 20 minutes (i.e., subpanel (c) of Figure 2J), then deteriorates at a much slower rate, with subpanel (d) of Figure 2J showing image quality at 45 minutes, until it becomes unusable at approximately 60 minutes (i.e., subpanel (f) of Figure 2J). Figure 2K shows that the average SNR (calculated as 10*log(average signal / noise)) for the time-lapse images shown in Figures 2I-J can be seen to be initially lower for a gel according to one embodiment of the present invention, but is more stable over time with continuous in vivo imaging, and is superior to water after 15 minutes.

[0159] Figures 2L-N present results showing how the reduced image quality resulting from the use of a gel, an embodiment of the present invention, can be improved using an optical system equipped with a correction collar to adjust for refractive index differences. The correction collar settings are provided assuming a hypothetical glass coverslip placed between the sample (i.e., non-transparent tissue) and the optical system (i.e., the objective lens) has a thickness range of 0.01 mm to 0.17 mm, which can be corrected through adjustments to internal optical components. Figure 2L shows a Z-projection of several 100 nm fluorescent beads. Figure 2M shows the 3D radial point spread function (PSF) of the red round beads in Figure 2L. The point spread function (PSF) can be used to aid in measuring the imaging quality of an optical system by showing the 3D distribution of the signal generated by so-called "infinitely small points." In this case, the "infinitely small points" are several 100 nm fluorescent beads, each of which generates a signal approximately 1 μm wide in Figure 2L. The actual PSF is the 3D distribution shown in Figure 2M.

[0160] Figure 2N shows that five random beads were selected at evenly spaced correction collar settings from 0.01 mm to 0.17 mm, and their PSFs were analyzed to obtain the average resolution (calculated as the full width at half maximum (FWHM) in μm) at each correction collar setting. The full width at half maximum (FWHM) is the width of the PSF at half its maximum. This parameter is commonly used to describe spatial resolution, where a smaller FWHM indicates better resolution. Initially, water performed better than gel, but from 0.07 mm to 0.15 mm, the FWHM was lower when using gel, suggesting that any impairments in imaging quality when using gel can be corrected and even result in better resolution than water. Correction collar settings were provided for a range of thicknesses of glass coverslips placed between the sample and the objective lens, ranging from 0.00 mm (no coverslip) to 0.17 mm, which can be corrected through adjustments of internal optical components.

[0161] For the embodiments shown and / or reported in Figures 2D-N, the gel immersion medium embodiment used was a viscoelastic gel containing 1% sodium hyaluronate (sodium hyaluronate is hyaluronic acid in unhydrated / powdered form; i.e., sodium hyaluronate and hyaluronic acid are otherwise the same compound), having an average molecular weight of 2,500,000 daltons, dissolved in phosphate-buffered saline (PBS). Specifically, the formulation was 10 mg sodium hyaluronate, 0.45 mg sodium phosphate, and 7.5 mg sodium chloride, all dissolved in 1 mL of water. This formulation uses PBS as a base, which includes all of its salts, and is designed for procedures such as cataract surgery. However, in other embodiments, water alone can be used instead of PBS, and the formulation can be optimized for imaging (i.e., by using different concentrations, molecular weights, and other potential components, such as heavy water and chondroitin sulfate).

[0162] Further details regarding alternative formulations of viscoelastic gels can be found in Kaur K, Gurnani B. Viscoelastics. [Updated June 11, 2023]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available at: https: / / www.ncbi.nlm.nih.gov / books / NBK578189 / , which is incorporated herein by reference.

[0163] System for imaging and manipulating structures through non-transparent tissues - Patents.com Aspects of the present disclosure include systems for use in practicing the methods of the present invention. In particular, the present disclosure includes systems for imaging and / or manipulating a structure through non-transparent tissue, such as ocular or periocular tissue. Specifically, the present disclosure includes systems for imaging and / or manipulating a structure through non-transparent tissue, such as ocular or periocular tissue, comprising: an optical system configured to image and / or manipulate the structure through the non-transparent tissue; an adapter configured to couple the optical system to the non-transparent tissue, as described herein; a processor comprising a memory operably coupled to the processor, the memory comprising instructions stored in the memory, the instructions, when executed by the processor, causing the processor to direct the optical system to image the structure through the non-transparent tissue, receive information about light emitted from the structure from the optical system, and combine the information about the light emitted from the structure to generate an image of the structure; and an operative connection between the processor and the optical system. Alternatively, in some cases, the system comprises a processor, the processor comprising a memory operatively coupled to the processor, the memory comprising instructions stored in the memory that, when executed by the processor, cause the processor to direct the optical system to manipulate the structure through non-transparent tissue or to direct the optical system to image and manipulate the structure through non-transparent tissue.

[0164] In embodiments of the system of the present invention, any convenient commercially available optical system or aspects thereof may be used, such as a microscope equivalent to the Bergamo II series, ThorLabs, and / or a femtosecond laser for excitation equivalent to the Insight X3, Spectra-Physics. Aspects of the optical system of interest include: (1) longer wavelength applications (e.g., 1,700 nm) may require special coatings on the optics to efficiently direct light in this range; (2) embodiments employ novel high-speed laser scanning techniques to dramatically increase the speed of imaging; such aspects include modular modifications of multiphoton microscopes.

[0165] In certain embodiments, a "two-photon microscope" or "multiphoton microscope" may be used. Regarding the light source, e.g., a laser, in certain embodiments, commercially available femtosecond lasers, such as the Mira or Chameleon models from Coherent and the commercially available Octavius-2P laser from ThorLabs, may be used. In other cases, such lasers do not encompass the longer critical wavelengths required for three-photon imaging. An example of a laser that does not encompass such longer wavelengths is a Monaco 1035 (60 W) pumping an OPA. Such equipment is tunable from 650 to 2500 nm. In embodiments, a Monaco pump laser may be used for cauterization and / or cutting applications.

[0166] Desired aspects of commercially available lasers for use in embodiments utilizing three-photon imaging include those configured to emit light at longer wavelengths, such as 1,700 nm or 1,800 nm. Desired aspects of commercially available lasers for use in embodiments of the present invention include those described below with respect to the Monaco 1035-80-60 Series II pump laser. The Monaco 1035 is an industrial femtosecond laser with MOPA architecture. Designed for high uptime in 24 / 7 applications, the laser family delivers greater than 80 μJ / pulse at 1035 nm. Typical repetition rates of up to 50 MHz at 60 W enable current and future throughput requirements in materials processing and microelectronics applications. Homogeneous materials, such as glass and metals, as well as complex layered structures for the FPD and mobile markets, can be easily addressed with Monaco's sub-350 fs pulse width. Desired features of such an AOM include: 80 μJ / pulse for processing high ablation threshold materials; 60 W average power for high throughput; standard pulse width less than 350 fs; variable pulse width from less than 350 fs to more than 10 ps for process tailoring; repetition rate up to 50 MHz for fast processing in polygon scanners; seeder burst mode >320 μJ; compact single-box design for ease of integration; central wavelength: 1035 ± 5 nm; output power: 60 W; energy: 80 μJ (up to 750 kHz); seeder burst mode >320 μJ; repetition rate: single shot ~50 MHz; outbound AOM can pulse pick at single shot ~1 MHz; M <1.2

number

[0167] In some embodiments, the optical system comprises a multiphoton excitation microscopy system. In some cases, the multiphoton excitation microscopy system comprises an excitation source for emitting light energy and a detector for sensing light emitted from the structure via multiphoton excitation. That is, the optical system of interest comprises an excitation source, such as one or more lasers or other excitation sources described in detail herein, and a detector for use in detecting light induced in the structure being imaged, such as one or more photomultiplier tubes (PMTs) or silicon photomultipliers and avalanche photodiodes. The optical system may comprise one or more laser scanners for rapidly imaging or targeting lasers for tissue treatment or structure treatment. The optical system may comprise a tube lens or a scanning lens. Dispersion compensation may be used to correct for chromatic dispersion and increase the efficiency of signal generation or tissue treatment. Adaptive optics correction may be used to improve resolution and / or increase the efficiency of signal generation or tissue treatment. Power attenuation control may be used in several ways, such as by an acousto-optic modulator, a Pockels cell, or a pair of rotating polarizers. In embodiments, the excitation laser is focused into the tissue with a high numerical aperture optic or objective lens. In some cases, the optical system and / or adapter includes a translation stage controllable by a processor to move the excitation source and / or detector of the optical system over a specified region, volume, or direction to direct the light in the imaged structure over a desired region or volume. In certain embodiments, the optical system mechanically adjusts the focus in the Z-axis. For example, such an embodiment may use a translation stage or a piezoelectric mechanism, e.g., a piezoelectric actuator, to adjust the focus in the Z-axis. In other embodiments, the optical system may adjust the focus in the Z-axis using optical scanning in the Z-axis. Such an optical scanning approach may provide less latency in adjusting the focus, which may be preferred in certain clinical or experimental applications. In embodiments, commercially available components of the optical system may be used.For example, certain embodiments use one or more of a high numerical aperture objective, such as the N25X-APO-MP manufactured by Nikon and available from ThorLabs; a photomultiplier tube (PMT), such as the PMT2100 available from ThorLabs; a rotating polarizer, such as the BCM-PA variable attenuator available from ThorLabs; a Pockels cell, such as the BCM-PCA Pockels cell attenuator available from ThorLabs; or a piezoelectric actuator, such as the PFM450-E available from ThorLabs.

[0168] In some embodiments, the memory further comprises instructions that, when executed by the processor, cause the processor to direct the optical system to image the structure over a specified volume. In other cases, the memory further comprises instructions that, when executed by the processor, cause the processor to direct the optical system to translate, rotate, or articulate relative to non-transparent tissue, such as ocular or periocular tissue. In still other cases, the memory further comprises instructions that, when executed by the processor, cause the processor to direct the optical system to image the structure over a specified period of time. Any convenient time interval can be used, for example, from a moment to potentially several hours. In some cases, the structure is imaged for one or more seconds to an hour or more. In embodiments, the structural imaging and blood flow measurement are very fast (from a moment to a few seconds). In other embodiments, image guidance for surgical procedures can be continuous for several hours. In still other embodiments, additional time-lapse imaging of biological processes, for example, continuous imaging of aqueous humor flow changes before and after drug or surgical therapy, can take from minutes to several hours.

[0169] In embodiments, the memory further comprises instructions that, when executed by the processor, cause the processor to direct the optical system to manipulate the structure. In some cases, manipulating the structure includes using the excitation source for non-ablative therapy. In other cases, the non-ablative therapy includes multiphoton-mediated damage, such as one or more of thermal damage, photodisruption, photocrosslinking, vascular coagulation, or ablation of the structure.

[0170] In certain embodiments, manipulating the structure includes treating glaucoma using the excitation source. In some cases, treating glaucoma using the excitation source includes reducing aqueous humor production in the ocular tissue using the excitation source. In other cases, reducing aqueous humor production in the ocular tissue using the excitation source includes damaging the ciliary body, thermally damaging the ciliary body, or applying a photodisruption-mediated process, in each case reducing aqueous humor production. In still other cases, treating glaucoma using the excitation source includes increasing aqueous humor outflow using the excitation source. In embodiments, increasing aqueous humor outflow using the excitation source includes performing laser trabeculotomy through non-transparent tissue of the eye. In certain cases, performing laser trabeculoplasty includes performing laser trabeculoplasty directly through non-transparent tissue of the eye.

[0171] In embodiments, manipulating the structure includes using the excitation source to prevent or treat a retinal hole, e.g., a retinal break. In some cases, preventing or treating a retinal break includes identifying and providing photocoagulation therapy. In embodiments, the retinal hole or break is a peripheral retinal hole or break.

[0172] In other embodiments, manipulating the structure includes providing photocoagulation and thermal therapy to the structure, for example, a ciliary body tumor. In yet other embodiments, manipulating the structure includes providing photocoagulation and thermal therapy to a peripheral choroidal tumor. In certain embodiments, manipulating the structure includes optically crosslinking scleral tissue to prevent myopia. In embodiments, manipulating the structure includes visualizing and performing targeted modification of extraocular muscle function. In other embodiments, manipulating the structure includes visualizing and performing targeted thermal or photocoagulation therapy of orbital fat. In yet other embodiments, manipulating the structure includes visualizing and performing targeted modification of eyelid tissue.

[0173] In some cases, the processor and / or memory may be operably connected to the optical system, and in some cases, to the adapter. In embodiments, the processor and / or memory are operably connected to certain aspects of the optical system. Such operable connection may take any convenient form, whereby data and / or control signals generated by the processor, optical system, and / or adapter may be communicated therebetween by any convenient input / output technique, such as via a wired or wireless network connection, shared memory, a bus, or similar communication protocol, e.g., an Ethernet connection, or a Universal Serial Bus (USB) connection, or a portable memory device, etc.

[0174] Any convenient processor and memory may be used in embodiments of the subject systems. For example, any off-the-shelf, commercially available processor or memory, such as those discussed in detail above, may be used. In particular, in embodiments, the processor may comprise a general-purpose processor, or a plurality of multi-core processors or parallel processing units, such as a processor with a graphics processing unit, or other processor configured to support parallel processing operations, or a combination thereof. In some cases, the processor and memory are operatively connected to one another. Such operative connection may take any convenient form, whereby instructions and data may be obtained by the processor by any convenient input technique, for example, via a wired or wireless network connection, shared memory, a bus, or similar communications protocol.

[0175] Exemplary embodiments of the system of the present invention: 3 shows a schematic diagram of a system 300 for imaging a structure through non-transparent ocular or periocular tissue, according to one embodiment of the present invention. The embodiment of the system 300 of the present invention shown in FIG. 3 relates to a system for imaging any convenient structure accessible through non-transparent tissue, e.g., ocular or periocular tissue, according to the present technique, as such may vary. System 300 is an exemplary embodiment of the present invention provided for illustrative purposes, and aspects thereof may vary as desired in embodiments of the present invention.

[0176] As shown in FIG. 3 , system 300 includes an optical system 310 interfaced with each of a processor 330 and an adapter 320. In embodiments, optical system 310 may include both an excitation source for directing light at a structure and a detector for detecting the directed light resulting from the structure being imaged. In embodiments, adapter 320 may be shaped to receive optical system 310, e.g., by mechanically holding or supporting the optical system or aspects thereof. Either or both adapter 320 and optical system 310 may be capable of translating, rotating, or otherwise articulating relative to ocular or periocular tissue 340 ( FIG. 3 designates ocular or periocular tissue for illustrative purposes only; the present disclosure is not so limited). Optical system 310 is operably connected to processor 330 via operative connection 335 a, which may take the form of a wired or wireless connection, such as a universal serial bus (USB) connection or a Bluetooth connection. In system 300, processor 330 is operably connected to adapter 320 via operative connection 335b, which may take the form of a wired or wireless connection, such as a universal serial bus (USB) connection or a Bluetooth connection. Operable connections 335a, 335b may be configured to transmit data signals, e.g., data regarding light detected from the structure, and / or control signals, e.g., signals instructing the optical system to transmit specified light energy over a specified area or volume, etc. In the context of system 300, non-transparent tissue includes any convenient non-transparent ocular or periocular tissue, and such may vary (as noted above, embodiments of the inventive system are not limited to ocular or periocular tissue, but may include other tissues, e.g., dermatological tissue accessed in connection with a dermatological or cosmetic procedure, etc.).

[0177] 4A illustrates an exemplary system 400 for imaging a structure through non-transparent tissue, e.g., ocular or periocular tissue, in accordance with one embodiment of the present invention. The embodiment of the system 400 of the present invention illustrated in FIG. 4A relates to a system for imaging any convenient structure accessible through non-transparent tissue, e.g., ocular or periocular tissue, in accordance with the present technique, as such may vary. System 400 is an exemplary embodiment of the present invention provided for illustrative purposes, and its aspects may vary as desired in embodiments of the present invention. System 400 is used through the sclera of non-transparent ocular tissue 490.

[0178] As shown in FIG. 4A , system 400 includes an optical system 410 interfaced with each of processor 430 and adapter 420. In embodiments, optical system 410 may include both an excitation source 411 for directing light at structure 440 and first and second detectors 412a and 412b for detecting the directed light resulting from the structure being imaged. Optical system 410 further includes an intensity control element 413, which is an optical element used in conjunction with transmitting light energy to the structure through non-transparent tissue, such as ocular or periocular tissue; a scanning mirror 414 configured to move to adjust the position or angle of the light transmitted from excitation source 411; a scan lens 415; and a tube lens 416. In embodiments, the scan and tube lenses are electrostatic optics selected and / or configured to help define the size of the imaging field. In embodiments, such scan / tube lenses may be different for optimization for different objective lenses. Such is especially important when it is desired to scale up for the human eye and to image as much of the eye as possible in a single scan. In embodiments, different scan / tube lenses may be used for different setups or applications.

[0179] The optical system 410 further comprises the following optical elements used with light emitted from the structure via multi-photon excitation: dichroic 1 417a, dichroic 2 417b, first filter 418a, and second filter 418b. Dichroic 1 417a, dichroic 2 417b, first filter 418a, and second filter 418b are configured to reflect or allow light corresponding to a specific wavelength or range of wavelengths to pass. In embodiments, the filters and / or dichroics are custom made by companies via sputtered oxide thin film techniques. Wavelength specifications are highly variable across embodiments. Such are commercially available, for example, from Semrock or Chroma.

[0180] The optical system 410 further comprises an objective 419, i.e., an objective lens. As shown in the placement of the objective 419 within the optical system 410, both light transmitted from the excitation source 411 to the structure to be imaged present within the ocular tissue 440 and light collected from the structure to be imaged present within the ocular tissue 440 pass through the objective 419. That is, the same objective 419 is used for both delivery of light from the excitation source 411 and signal collection. Signal collection refers to collecting or detecting a signal, i.e., emitted or induced light generated by a structure present within the ocular tissue 440, at the focus of the laser.

[0181] In embodiments, the adapter 420 may be configured to receive the optical system 410, for example, by mechanically holding or supporting the optical system 410. The adapter 420 interfaces with the optical system 410 and with ocular tissue 440 or periocular tissue or other tissue (not shown in FIG. 2B ). The adapter 420 is shown to be capable of translating, rotating, or otherwise articulating relative to the ocular or periocular tissue 440. When the adapter 420 translates, rotates, or otherwise articulates relative to the ocular or periocular tissue 440, the adapter 420 may cause the optical system 410 to translate, rotate, or otherwise articulate correspondingly. Aspects of the optical system 410 are operably connected to the processor 430 via an operative connection 435 a, which may take the form of a wired or wireless connection, such as a universal serial bus (USB) connection or a Bluetooth connection. In system 400, processor 430 is shown operably connected to adapter 420 via operative connection 435b, which may take the form of a wired or wireless connection, such as a Universal Serial Bus (USB) connection or a Bluetooth connection. Operable connection 435a may be configured to transmit data signals, e.g., data regarding light detected from the structure, and / or control signals, e.g., signals instructing the optical system to transmit specified light energy over a specified area, etc. In the context of system 400, non-transparent ocular tissue includes the top surface of the eye.

[0182] 4B shows an exemplary alternative system 450 for imaging a structure through non-transparent ocular or periocular tissue, according to one embodiment of the present invention. System 450 illustrates additional optical components present in embodiments of the inventive system. While system 450 is also used through the sclera of non-transparent ocular tissue 490, system 450 further illustrates an alternative mechanism for sensing light emitted from the structure to be imaged, i.e., for detecting or collecting light for imaging the structure, which comprises a detector 470 positioned directly above the cornea 491 of ocular tissue 490.

[0183] The embodiment of the system 450 of the present invention shown in Figure 4B relates to a system for imaging any convenient structure accessible through non-transparent ocular or periocular tissue in accordance with the present technique, as such may vary. System 450 is an exemplary embodiment of the present invention provided for illustrative purposes, and its aspects may vary as desired in embodiments of the present invention.

[0184] As shown in FIG. 4B , system 450 includes a processor and memory 480 and an optical system 460 interfaced with an adapter (not shown in FIG. 4B ). In an embodiment, optical system 460 may include both an excitation source 461 for stimulating light at a structure and first and second detectors 462 a and 462 b for detecting the stimulated light resulting from the structure being imaged. In optical system 460, excitation source 461 includes a laser 461 a and a pulse compressor 461 b for adjusting, e.g., shortening, the pulse width of pulsed light energy generated by laser 461 a. Optical system 460 further includes additional optical elements used in conjunction with delivering light energy to a structure through non-transparent ocular or periocular tissue: an intensity control element 463; a scanning mirror 464 that can be moved to adjust the position or angle of light transmitted from excitation source 461, i.e., to adjust the focus of light transmitted from excitation source 461; a scanning lens 465; and a tube lens 466. The optical system 460 may include additional optical elements used in conjunction with transmitting light energy to the structure through non-transparent ocular or periocular tissue, such as a laser scanner (not shown in FIG. 4B) for moving the laser focal point across the image field in any desired scanning pattern or raster, a power attenuator (not shown in FIG. 4B), or adaptive optics for wavefront shaping (not shown in FIG. 4B). The optical system 460 further includes dichroic 1 467a, dichroic 2 467b, first filter 468a, and second filter 468b, which are optical elements used with light emitted from the structure via multiphoton excitation. Dichroic 1 467a, dichroic 2 467b, first filter 468a, and second filter 468b are configured to reflect or allow light corresponding to a specific wavelength or range of wavelengths to pass. The embodiment shown in FIGS. 4A-B includes two PMTs. This is a viable implementation. However, other embodiments may include additional PMTs to bring this to (for example) four PMTs or more (ie, including a third and fourth PMT).In such a case, there are additional dichroics that split the light between the first and second PMTS and the third and fourth PMTS, respectively.

[0185] The optical system 460 may interface with the eye tissue 490 via an adapter (not shown in FIG. 4B), which is configured to receive the optical system 460, for example, by mechanically holding or supporting the optical system 460.

[0186] Optical system 460 is operably connected to processor and memory 480 via operative connection 435a, which may take the form of a wired or wireless connection, such as a Universal Serial Bus (USB) connection or a Bluetooth connection. Operative connection 435a may be configured to transmit data signals, e.g., data regarding light detected from the structure, and / or control signals, e.g., signals directing optical system 460 to transmit specified light energy over a specified area, etc. In the context of system 450, non-transparent ocular tissue 490 includes the top surface of the eye.

[0187] Optical system 460 further comprises detector 470 positioned directly on the cornea 491 of ocular tissue 490. Detector 470 may be any convenient sensor, including, for example, a contact lens, a filter, a photomultiplier tube (PMT), and the like, positioned directly on the cornea 491 of ocular tissue 490. Detector 470 may be positioned to collect signal light emitted deep within ocular tissue 490, i.e., signal light that is backscattered and not collected by objective lens 469. In general, detector 470 may be positioned anywhere relative to ocular tissue 490 that is capable of transmitting light emitted from a structure through non-transparent ocular tissue 490. Detector 470 may be used in conjunction with, for example, first detector 462 a and second detector 462 b, or may be used separately from first detector 462 a and second detector 462 b. In some cases, first detector 462a and second detector 462b, or detector 470, may be better suited to collecting signals from a particular structure, or to collecting signals through a particular tissue, e.g., a particular ocular or periocular tissue, or to collecting signals characterized by a particular feature, e.g., a specified wavelength range. Generally, in an embodiment, the more signals are collected by optical system 460, i.e., by utilizing each of first detector 462a and second detector 462b and detector 470, the better the resulting image quality.

[0188] The pulse compressor 461b or dispersion compensation is achieved by using a set of prisms to correct for the chromatic dispersion of the laser 461a light as it travels to the focal point in the imaged structure, i.e., tissue. The goal of using the pulse compressor 461b or dispersion compensation is to have the narrowest possible pulse at the focal point in the imaged structure, i.e., tissue. This can achieve the highest efficiency of multiphoton excitation. The pulse compressor in question can be located within the laser system, the laser 461a, or in optical equipment located after (i.e., distal to) the laser 461a. Further information regarding pulse compression and dispersion compensation is provided in APE Angewandte Physik & Elektronik GmbH, Dispersion Compensation & Pulse Compression for Microscopy, https: / / www.ape-berlin.de / en / dispersion-compensation-pulse-compression / (last accessed October 25, 2022), the disclosure of which is incorporated herein by reference in its entirety.

[0189] Desired methods for achieving intensity control include deploying a pair of polarizers that rotate relative to each other, or a Pockels cell, or an acousto-optic modulator (AOM). Embodiments of the former approach are relatively inexpensive, while embodiments of the latter two approaches can be very fast, and even allow for varying the laser power in different locations, essentially as fast as the laser can be scanned.

[0190] Embodiments of the scanning mirror 464 in the XY axis can include galvanometer mirrors or resonator mirrors, or a combination of the two. Galvanometer mirrors allow for precise targeting of specific locations (i.e., for laser treatment). Resonator mirrors allow for relatively faster imaging. Rapid scanning in the Z axis can be more challenging and is typically achieved by one or more of the objective lens, a deformable focusing lens, or piezo movement of a deformable mirror. Further information regarding deformable mirrors is presented in KN Ito, K. Isobe & F. Osakada, "Fast z-focus controlling and multiplexing strategies for multiplane two-photon imaging of neural dynamics," Neuroscience Research, Volume 179, June 2022, pp. 15-23, available at: https: / / www.sciencedirect.com / science / article / pii / S0168010222000827, the disclosure of which is incorporated herein by reference in its entirety.

[0191] Utilities The subject methods, adapters, and systems find use in a variety of applications where it is desirable to image through non-transparent tissue, e.g., ocular or periocular tissue, i.e., to image structures through tissue at a resolution that is inaccessible with current imaging techniques. In some embodiments, the methods, adapters, and systems described herein find use in clinical situations, e.g., any clinical situation where there is a need to image structures through non-transparent tissue, e.g., ocular or periocular tissue, particularly a need to image structures that were previously inaccessible due to the presence of non-transparent tissue. Additionally, the subject methods, adapters, and systems find use in the treatment of conditions such as glaucoma, or the treatment and prevention of retinal detachment, or the treatment of ciliary body tumors or peripheral choroidal tumors. Furthermore, the subject methods, adapters, and systems find use in gene therapy applications, e.g., the application of gene therapy techniques to structures present in ocular or periocular tissue.

[0192] The subject methods, adapters, and systems find use as transscleral imaging tools for visualizing ocular structures at high resolution, including intraocular structures previously inaccessible by optical techniques, the previously inaccessible intraocular structures including the ciliary body, peripheral retina, and choroid. Embodiments of the methods, adapters, and systems according to the present invention can provide cellular resolution and functional imaging capabilities that exceed information obtainable by other transscleral imaging techniques, such as ultrasound and MRI.

[0193] The subject methods, adapters, and systems may find use as non-incisional therapeutic tools for performing precise photocoagulation and / or tissue ablation of the ciliary body to controllably and safely reduce aqueous humor production in the eye to treat glaucoma.

[0194] The subject methods, adapters, and systems may find use as non-incisional therapeutic tools for performing laser trabeculotomy and / or trabeculoplasty to increase aqueous humor outflow and treat glaucoma.

[0195] The subject methods, adapters, and systems may find use as non-incisional therapeutic tools for identifying and delivering photocoagulation therapy to peripheral retinal breaks to treat and prevent retinal detachment.

[0196] The subject methods, adapters, and systems may find use as non-incisional therapy tools for identifying and delivering photocoagulation and thermal treatment to ciliary body tumors and peripheral choroidal tumors.

[0197] The subject methods, adapters, and systems may find use as non-incisional therapeutic tools for scleral photocrosslinking.

[0198] The subject methods, adapters, and systems may find use in connection with the application of gene or cell or drug therapy treatments, particularly when applied to ocular or periocular tissues.

[0199] Embodiments of the present invention may find use in imaging and / or manipulating tissue in clinical or experimental settings, for example, this may include, for example, imaging nerve activity, imaging corneal nerves, imaging aspects of the central nervous system, utilizing an invasive probe to image aspects of the central nervous system, thinning a region of the skull to image aspects of the central nervous system, visualizing nerve structures, utilizing an imaged nerve structure to alleviate pain, utilizing an imaged structure to perform flow analysis, performing flow analysis in substantially real time, generating a velocity map of fluid flow within an imaged structure, diagnosing disease, diagnosing cancer, distinguishing between cancerous and non-cancerous tissue, distinguishing between cancerous and non-cancerous cells, preventing or treating retinal holes, providing non-thermal therapy, providing non-thermal therapy to tumors, visualizing orbital fat, manipulating dermatological tissue, imaging one or more of the epidermis, dermis, or subcutaneous tissue, manipulating tear ducts to facilitate fluid flow within the tear duct, reducing ocular tissue redness, administering therapy, and the like. delivering to an imaged structure, guiding positioning of an intraocular implant based on the imaged structure, predicting effective placement of an intraocular implant based on the imaged structure, guiding positioning of an intraocular implant based on the imaged structure while implanting the intraocular implant, assessing the position of an implanted intraocular implant based on the imaged structure, assessing effective lens position (ELP) of an implanted intraocular implant based on the imaged structure, detecting cancerous tissue, e.g., melanoma, providing cosmetic treatments, providing cosmetic dermatological treatments, providing cosmetic surgery, treating scars, removing scars, treating acne scars, removing acne scars, treating skin discoloration, removing birthmarks, removing port wine stains, treating skin discoloration disorders, removing tattoos, treating rosacea, providing controlled cutting of tissue, providing controlled cutting of dermatological tissue, performing a skin biopsy procedure, destroying cancerous tissue, destroying one or more cancer cells, ablating cancerous tissue, ablating one or more cancer cells, ablating melanoma,including those associated with ablating cutaneous melanoma, removing hair, destroying hair follicles, ablating follicular tissue, affecting tissue shape, affecting the shape of one or more fat deposits, reducing the volume of one or more fat deposits, reducing one or more subdural fat deposits, providing tissue sculpting, providing skin tightening, treating dry eye syndrome, reducing eye redness, providing pain management, or perforating tissue.

[0200] The following are offered by way of example and not by way of limitation.

[0201] experiment Figures 5A-F show the results of utilizing an embodiment of the present invention for 3PEF transscleral imaging to visualize the chorioretinal vasculature and chorioretinal anastomoses. Figure 5A shows a three-dimensional projection of 3PEF data acquired through the intact sclera in a whole-fixed pigmented mouse eye (Ch refers to choroidal vessels, and Rt refers to retinal vessels). Figure 5B shows in vivo 2PEF imaging and projection of the choroidal vasculature, where plasma is labeled with a red fluorescent dye. Figure 5C shows in vivo imaging and full-thickness projection of the retinal vasculature directly beneath the choroid in Figure 5B. Figure 5D shows chorioretinal anastomoses in a mouse model of neovascular age-related macular degeneration (AMD). Figure 5E shows the development of chorioretinal anastomoses arising from the retinal vasculature. Figure 5F shows the development of chorioretinal anastomoses arising from the choroidal vasculature. Figures 5A-F demonstrate investigations into the developmental mechanisms and molecular identity of chorioretinal neovascularization and anastomosis formation, further elucidating the role of arterial-venous identity in a subset of diseases resistant to existing therapies. Figures 5A-F demonstrate that multiphoton microscopy, as utilized in embodiments of the present invention, is a powerful technique for in vivo imaging deep within tissues with subcellular resolution. Transpupillary two-photon excitation fluorescence (2PEF) microscopy, combined with dispersion compensation and adaptive optics (AO) correction, overcomes corneal and lens aberrations to provide superior imaging of the retina and photoreceptors. Further details are provided in Palczewska, G., et al., Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye. Nat Med, 2014. 20(7): pp. 785-9, and Palczewska, G., T. S. Kern, and K. Palczewski, Noninvasive Two-Photon Microscopy Imaging of Mouse Retina and Retinal Pigment Epithelium. Methods Mol Biol, 2019. 1834: pp. 333-343, the disclosures of which are incorporated herein in their entireties.However, to our knowledge, current 2PEF techniques are unable to image within the choroid due to the inherent optical constraints imposed by transpupillary imaging and the high optical absorption of the RPE and choroid. As demonstrated in Figures 5A-F, embodiments of the present invention can successfully achieve transscleral imaging of the chorioretinal vasculature in intact C57BL / 6 mouse eyes. Embodiments of the present invention enable chorioretinal imaging in albino and pigmented eyes using conventional 2PEF, extended infrared 2PEF, 3PEF, 2HG, 3HG, and AO correction. Imaging using embodiments of the present invention is facilitated, in part, because transscleral imaging is not subject to the same focusing constraints as transpupillary imaging. Therefore, embodiments of the present invention utilizing transscleral multiphoton microscopy are capable of improved spatial resolution (most notably, axial resolution) for multiphoton imaging of the retina. To our knowledge, quantification of blood flow in the choroid has not previously been achieved at the microvascular level. Furthermore, combining transscleral imaging with robust analytical methods can quantify blood flow in the choroid down to the micron scale. Further details are provided in Kim, TN, et al., Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals. PLoS One, 2012.7(6):p.e38590, the disclosure of which is incorporated herein in its entirety.

[0202] Figure 6 shows the results of utilizing one embodiment of the present invention for 3PEF transscleral imaging to visualize the retinal pigment epithelium. Specifically, Figure 6 shows the results of transscleral 3PEF imaging of the RPE in a pigmented mouse eye, resolving nuclei, melanosome granules, and cell boundaries.

[0203] 7A-B show the results of utilizing an embodiment of the present invention for 3PEF transscleral imaging of the ciliary body. FIG. 7A shows a three-dimensional projection of the ciliary body from image data acquired through an intact sclera. FIG. 7A is an approximately 45-degree three-dimensional projection of the ciliary body in a live mouse eye. FIG. 7B shows one cross-sectional slice from the three-dimensional data in FIG. 7A, which demonstrates the ciliary body processes with cellular resolution and distinction of the epithelium involved in the production of aqueous humor in the eye.

[0204] Figures 8A-E show results of utilizing embodiments of the present invention for in vivo cellular imaging in living eyes. In particular, Figures 8A-E show imaging of cellular structure and dynamics deep within the eye. Figures 8A-C show results of imaging chorioretinal microglia, specifically green fluorescent protein (GFP) expression, in cells from transgenic mice. Figure 8D shows results of corneal imaging, specifically second harmonic generation (SHG) imaging of the cornea. Figure 8E shows results of imaging of the retinal pigment epithelium (RPE).

[0205] Figure 9 shows the results of utilizing one embodiment of the present invention for imaging chorioretinal vascular dynamics. Specifically, Figure 9 shows imaging results of chorioretinal anastomoses. The structural and imaging results in Figure 9 demonstrate how spontaneous chorioretinal neovascularization occurs in a model of neovascular AMD, and further demonstrate how chorioretinal anastomoses can form and mature into large vascular connections.

[0206] 10A-F show results of utilizing embodiments of the present invention for imaging choroidal blood flow and reconstruction. FIG. 10A shows the results of imaging choroidal blood flow. FIG. 10B shows the results of imaging flow velocity at high temporal resolution and the analysis applied to such imaging results. FIG. 10C shows the results of imaging the choroid in a model of retinitis pigmentosa. FIG. 10D shows the results of imaging normal choroid. FIG. 10E shows blood velocity data in different vascular structures obtained using the imaging data. FIG. 10F shows the fluid flow profile in blood at high spatial resolution obtained using the imaging data.

[0207] As described herein, embodiments of the present invention include utilizing a multi-core processor, e.g., a graphics processing unit, in conjunction with performing flow analysis, e.g., analysis of fluid flow through the choroid, which results in, for example, a high spatial resolution analysis, e.g., that shown in Figure 10F. Such embodiments utilizing a multi-core processor can provide real-time flow analysis, whereas single processing unit techniques may not be able to provide real-time results.

[0208] For example, one embodiment of the present invention utilizing a single processing unit, e.g., a single CPU, used in conjunction with a 5.0-second acquisition at a 100 KHz sampling rate results in a total analysis time of 3,418.41 seconds with an analysis-to-acquisition ratio of 683.74. This embodiment corresponds to the real-time acquisition limit of 146 Hz. Such results are not feasible for use in real-time flow analysis. In embodiments, analysis-to-acquisition refers to the ratio of the elapsed real-time time (e.g., seconds) it takes to analyze a given amount of data to the elapsed real-time time (e.g., seconds) it takes to acquire this amount of data. In our example, this metric is useful because (1) the metric is independent of the time scale (e.g., elapsed real-time can be milliseconds or minutes), and (2) the real-time analysis-to-acquisition ratio is intuitively less than or equal to 1. This 683.74 means that no matter how long you spend acquiring data, you must spend 683.74 times as long to analyze it as you spent acquiring it.

[0209] In contrast to the above, for example, one embodiment of the present invention utilizing a graphics processing unit with multiple multi-core processors used in conjunction with a 5.0-second acquisition at a 100 KHz sampling rate results in a total analysis time of 1.76 seconds with an analysis-to-acquisition ratio of 0.35. This embodiment corresponds to a real-time acquisition limit of 283,959 Hz. In contrast to single-CPU embodiments, such results are viable for use in real-time flow analysis. In connection with the embodiments described and reported herein, the GPU comprises an NVIDIA Amphere or Ada Lovelace Architecture Card; generally, other commercially available GPUs, such as those from AMD, may be used.

[0210] The embodiment of the invention used to capture the images shown in Figures 5-9 includes a Bergamo Series II multiphoton microscope system used with a Spectra-Physics InSight X3 tunable femtosecond laser; commercially available high numerical aperture objectives N25X-APO-MP from Nikon and ThorLabs; a detector used that includes a photomultiplier tube (PMT2100 from ThorLabs); and one or more fluorophores used to generate such images, including, but not limited to, dextran, Texas Red from Invitrogen, TRITC-dextran (tetramethylrhodamine isothiocyanate-dextran) from Sigma Aldrich, FITC-dextran (fluorescein isothiocyanate-dextran), eGFP (enhanced green fluorescent protein), and tdTomato (bright red fluorescent protein). The embodiment of the present invention used in connection with the three-photon images of corneal collagen fibers and retinal pigment epithelium (Figures 8D-E) includes a similar microscope but with appropriate lens / mirror coatings to transmit 1,700 nm light. The laser used for these two images (Figures 8D-E) comprises a Coherent Monaco, as described herein. The signal for corneal collagen is second harmonic generation, and for RPE is third harmonic generation (both are intrinsic signals from the tissue without the use of exogenous fluorophores).

[0211] 11 illustrates an exemplary quantitative analysis technique for blood flow analysis based on imaging data, according to an embodiment of the present invention. Embodiments of such modeling techniques facilitate quantitative blood flow analysis in tissues, such as choroidal blood flow analysis when combined with transscleral imaging, i.e., the multiphoton imaging techniques described herein.

[0212] 12A-D show hemodynamic analysis by line-scanning particle image velocimetry according to an embodiment of the present invention. + / H2B-eGFPFigure 12 shows 2PEF imaging of the vasculature in a 3D model, where nuclear GFP distinguishes arterial from venous endothelial cells. Figure 12B shows a 2PEF image of the arteriole from the white box in Figure 12A. Figure 12C shows line scan data, where each successive line scan appears below the previous one, forming a spatiotemporal image in which dark stripes represent moving RBCs. Figure 12D shows an analysis of central vascular velocity across the cardiac cycle. As mentioned above, Figure 10F also shows a cross-sectional analysis of the flow profile (maximum, average, minimum) across the vascular lumen during the cardiac cycle.

[0213] The choroid is a unique, poorly understood vascular bed with variable flow and substantial contractility from nonvascular smooth muscle. Further information can be found in Poukens, V., BJ Glasgow, and JL Demer, "Nonvascular contractile cells in the sclera and choroid of humans and monkeys." Invest Ophthalmol Vis Sci, 1998. 39(10): pp. 1765-74, and May, CA, "Nonvascular smooth muscle alpha-actin positive cells in the choroid of higher primates." Curr Eye Res, 2003. 27(1): pp. 1-6, the disclosures of each of which are incorporated herein in their entirety. Using in vivo transscleral multiphoton microscopy according to embodiments of the present invention, high-resolution imaging of the choroid can be achieved ( FIG. 12B ), and blood flow data at the microvascular scale can be collected. In embodiments, transscleral multiphoton microscopy combined with line-scanning particle image velocimetry (LS-PIV) allows for quantification of choroidal blood flow with high spatiotemporal resolution. LS-PIV is a robust analytical method for analyzing blood flow data generated by multiphoton imaging (e.g., as shown in Figures 12A-D and 10F), which finds use in research and clinical contexts in a diverse range of systems, including the hindlimb and spinal cord.Further information regarding such techniques is provided in Kim, T. N., et al., Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals. PLoS One, 2012. 7(6): p. e38590, and Lasch, M., et al., Estimating hemodynamic shear stress in murine peripheral collateral arteries by two-photon line scanning. Mol Cell Biochem, 2019. 453(1-2): p. 41-51, and Chen, C., et al., An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury. J Vis Exp, 2017(130), the disclosures of each of which are incorporated herein in their entirety. Embodiments of the present invention use LS-PIV to analyze blood flow along vascular segments of the choroid and choriocapillaris, including capillaries, venules, veins, arterioles, and arteries. Because the morphology of the choroidal vasculature is unique, hemodynamic analysis can be performed using ephrin-B2. + / H2B-eGFPThis can be performed in a subset of mice in which nuclear GFP is expressed under the ephrin-B2 promoter, allowing blood vessels to be distinguished by arterial molecular identity (e.g., as shown in Figure 12A). Further information is provided in Davy, A., JOBush, and P. Soriano, Inhibition of gap junction communication at ectopic Eph / ephrin boundaries underlies craniofrontonasal syndrome. PLoS Biol, 2006. 4(10): p. e315, and Murphy, PA, et al., Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels. Proc Natl Acad Sci USA, 2014. 111(50): p. 18007-12, and Murphy, PA, et al., Notch4 normalization reduces blood vessel size in arteriovenous malformations. Sci Transl Med, 2012. 4(117): p. 117ra8, the disclosures of each of which are incorporated herein in their entirety.

[0214] An exemplary embodiment of the system according to the present invention includes an optimized microscope for highly efficient collection and detection of fluorescence, constructed to allow excitation wavelengths up to 1.7 μm. The primary excitation source is an advanced solid-state ytterbium-doped fiber laser with a usable power bandwidth of 350 nm to 5.0 μm, e.g., 680 nm to 1.3 μm, and dispersion compensation to further improve excitation efficiency. The secondary excitation source is a fixed 1,045 nm laser with a high power of over 3.5 W for additional imaging or photodisruption applications. Photodisruption refers to tissue vaporization achieved via higher-order processes, e.g., three-, four-, or five-photon mechanisms. The 1.7 μm tertiary excitation source utilizes the Raman shift of 1.5 μm light in a large-mode-area photonic crystal rod for excitation wavelengths of 1.3 to 3.6 μm. The microscope has the ability to articulate around a subject, e.g., an animal model, e.g., a mouse, in an imaging plane that is not parallel to the floor, maximizing the area of ​​the subject's eye that can be imaged in a single session. The system has a multimodal laser scanner that can switch between resonant imaging or patterned point scanning, which are necessary for studying fast cellular dynamics (e.g., neuronal activity with calcium indicators) or measuring hemodynamics, respectively.

[0215] 13A-C present an overview of ophthalmic imaging and therapy applications of embodiments of the present invention. In particular, FIGURES 13A-C show steps of a flowchart 1300 for utilizing embodiments for imaging and optical manipulation for glaucoma analysis and therapy, according to one embodiment of the present invention.

[0216] Glaucoma is a progressive optic neuropathy and a leading cause of irreversible blindness worldwide. See Quigley, H.A. and A.T. Broman, "The number of people with glaucoma worldwide in 2010 and 2020." Br J Ophthalmol, 2006. 90(3):262-7, which is incorporated herein by reference. The only approach available through existing techniques to slow disease progression is reducing intraocular pressure (IOP), which is regulated by aqueous humor production from the ciliary body and drainage via the outflow pathway. See Weinreb, R.N., T. Aung, and F.A. Medeiros, "The pathophysiology and treatment of glaucoma: a review." JAMA, 2014. 311(18):1901-11, which is incorporated herein by reference. Transscleral laser-induced ciliary body disruption is a class of non-incisional interventions in which the ciliary body is damaged, inhibiting aqueous humor production. In existing techniques, this approach is reserved for late-stage therapy due to potential complications, including pain, vision loss, and tabes. See Dastiridou, AI, et al., Cyclodestructive Procedures in Glaucoma: A Review of Current and Emerging Options. Adv Ther, 2018. 35(12): pp. 2103-2127; Quigley, HA, Histological and physiological studies of cyclocryotherapy in primate and human eyes. Am J Ophthalmol, 1976. 82(5): pp. 722-32, which are incorporated herein by reference. Recent improvements in the delivery of laser energy to the ciliary body have increased safety, but at the expense of a sustained IOP-lowering response.Tan,AM,et al.,Micropulse transscleral diode laser cyclophotocoagulation in the treatment of refractory glaucoma.Clin Exp Ophthalmol,2010.38(3):p.266-72,Garcia,GA,et al.,Micropulse Transscleral Diode Laser cyclophotocoagulation in Refractory Glaucoma:Short-Term Efficacy,Safety,and Impact of Surgical History on Outcomes.Ophthalmol Glaucoma,2019.2(6):p.402-412, Varikuti,VNV,et al.,Outcomes of Micropulse Transscleral Cyclophotocoagulation in Eyes With Good Central Vision.J Glaucoma,2019.28(10):p.901-905,Zaarour,K.,et al.,Outcomes of Micropulse Transscleral Cyclophotocoagulation in Uncontrolled See Glaucoma Patients. J Glaucoma, 2019. 28(3):270-275, which are incorporated herein by reference. The mechanism of this treatment failure is unclear, but previous studies have correlated ciliary body regeneration with an increase in IOP.

[0217] Embodiments of the present invention provide a novel therapy for the safe, highly effective, and sustained treatment of the ciliary body in patients with glaucoma. One particular embodiment, termed transscleral multiphoton image-guided laser therapy (TMILT), enables detailed imaging of the ciliary body at cellular resolution and equally precise laser treatment. Embodiments involving TMILT do not require contrast or photosensitizing agents and can leverage intrinsic signals from tissue, including multiphoton-excited autofluorescence and second- and third-harmonic generation. Embodiments involving TMILT can precisely target the ciliary body while avoiding damage to adjacent tissue, resulting in a sustained IOP-lowering response. Such embodiments address the significant need to reduce the surgical burden on glaucoma patients with treatments that are less invasive, safe, effective, and sustained.

[0218] For example, embodiments of the present invention induce precise multiphoton-mediated thermal damage to 180 degrees, 270 degrees, or 360 degrees of the ciliary body while preventing visible laser-mediated damage to the ciliary body and avoiding adjacent structures. Other embodiments include in vivo imaging to observe the cellular dynamics contributing to ciliary body regeneration over consecutive time points in the same eye.

[0219] As described herein, multiphoton microscopy allows for in vivo imaging deep into tissues with subcellular resolution and is a powerful tool for studying cell biology in living systems. See Helmchen, F. and W. Denk, Deep tissue two-photon microscopy. Nat Methods, 2005. 2(12): pp. 932-40; Zipfel, W.R., R.M. Williams, and W.W. Webb, Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol, 2003. 21(11): pp. 1369-77, which are incorporated herein by reference. The depth of multiphoton imaging is inherently limited to approximately five times the attenuation distance of the excitation wavelength in tissue, making imaging through highly scattering tissues, such as bone or sclera, challenging. Embodiments of the present invention use next-generation two-photon excitation (2PE), three-photon excitation (3PE), and adaptive optics correction to visualize underlying structures with subcellular resolution directly through the sclera. Such embodiments also enable precise imaging and targeting of tissue for multiphoton-mediated damage, i.e., structural manipulation.

[0220] Further embodiments use such imaging techniques in conjunction with multiphoton-excited aqueous humor velocimetry (MAF), which relies on time-lapse image data of the aqueous humor outflow (AHO) pathway, which is combined with computational analysis, as described herein, to generate flow velocimetry maps. Yet further embodiments include using transscleral multiphoton imaging to characterize the conventional AHO pathway in three dimensions at high resolution by injecting fluorescein into the anterior chamber and mapping the outflow pathway, including the anterior chamber, trabecular meshwork, Schlemm's canal, collecting duct, aqueous veins, and episcleral veins, to create a detailed atlas of AHO anatomy to use as a guide for subsequent applications, e.g., clinical or experimental use. Huang,AS,et al.,Aqueous Angiography:Aqueous Humor Outflow Imaging in Live Human Subjects.Ophthalmology,2017.124(8):p.1249-1251, Saraswathy,S.,et al.,Aqueous Angiography:Real-Time and Physiologic Aqueous Humor Outflow Imaging.PLoS One,2016.11(1):p.e0147176, Helmchen,F.and W.Denk,Deep tissue two-photon microscopy.Nat Methods,2005.2(12):p.932-40, Zipfel,WR,RMWilliams,and WWWebb,Nonlinear magic:multiphoton microscopy in the biosciences.Nat Biotechnol, 2003.21(11):p.1369-77, Wang, T., et al. al.,Three-photon imaging of mouse brain structure and function through the intact skull.Nat Methods,2018.15(10):p.789-792, Ouzounov,DG,et al.,In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain.Nat Methods,2017.14(4):p.388-390, Yildirim,M.,et al.,Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy.Nat Commun,2019.10(1):p.177, Masihzadeh,O.,et al.,Third harmonic generation microscopy of a mouse retina.Mol Vis,2015.21:p.538-47,Kim,TN,et al.,Line-scanning particle image velocimetry:an optical approach for quantifying a wide range of blood flow speeds in live animals.PLoS One,2012.7(6):p.e38590, Kazemi,A.,et al.,Effect of Timolol on See Aqueous Humor Outflow Facility in Healthy Human Eyes. Am J Ophthalmol, 2019, 202: pp. 126-132; Larsson, LI, Aqueous humor flow in normal human eyes treated with brimonidine and timolol, alone and in combination. Arch Ophthalmol, 2001, 119(4): pp. 492-5, which are incorporated herein by reference. Also see H. Yamashita, MS, Proof that the Ciliary Epithelium can Regenerate. Exp. Eye Res., 1978, 27: pp. 199-213; Kuwahara, A., et al.,Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue.Nat Commun,2015.6:p.6286、Cicero,S.A.,et al.,Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.Proc Natl Acad Sci U S A,2009.106(16):p.6685-90、Del Debbio,C.B.,et al.,Rho GTPases control ciliary epithelium cells proliferation and progenitor profile induction in vivo.Invest Ophthalmol Vis Sci,2014.55(4):p.2631-41、Gualdoni,S.,et al.,Adult ciliary epithelial cells,previously identified as retinal stem cells with potential for retinal repair,fail to differentiate into new rod photoreceptors.Stem Cells,2010.28(6):p.1048-59、Barker,N.,et al.,Lgr5(+ve)stem / progenitor cells contribute to nephron formation during kidney development.Cell Rep,2012.2(3):p.540-52、Humphreys,B.D.and D.P.DiRocco,Lineage-tracing methods and the kidney.Kidney Int,2014.86(3):p.481-8、Park,M.,et al., Peripheral (not central) corneal epithelia contribute to the closure of an annular debridement injury. Proc Natl Acad Sci USA, 2019. 116(52): pp. 26633-26643; Ventura, A., et al., Restoration of p53 function leads to tumor regression in vivo. Nature, 2007. 445(7128): pp. 661-5; Moussa, K., et al., Histologic Changes Following Continuous Wave and Micropulse Transscleral Cyclophotocoagulation: A Randomized Comparative Study. Transl Vis Sci Technol, 2020. 9(5): p. 22, which are incorporated herein by reference.

[0221] In step 1301 of FIG. 13A , preoperative imaging of the angle and outflow vessels of ocular tissue is obtained utilizing imaging techniques according to the present invention. Such imaging in step 1301 includes identifying ocular and / or periocular anatomical structures within non-translucent tissue 1340 associated with glaucoma, such as the cornea, trabecular meshwork, and Schlemm's canal, particularly those associated with causing glaucoma symptoms, such as aqueous veins, and analyzing such associated anatomical structures, e.g., to determine fluid flow velocities, which together identify and determine one or more sources of flow resistance within the associated anatomical structures that may be related to the underlying glaucoma and its symptomatic conditions. The image showing the aqueous veins in FIG. 13A is the result of a transscleral 2PEF imaging of 2D blood flow data in the episcleral veins. Such imaging is analyzed to produce a two-dimensional blood flow analysis showing a mean velocity map of RBC displacement within the blood flow.

[0222] From step 1301 in FIG. 13A, the flowchart 1300 then proceeds to step 1302 in FIG. 13B.

[0223] In step 1302 in FIG. 13B , intraoperative image-guided therapy is applied to the relevant anatomical structures visualized and analyzed in step 1301 that reside within the non-transparent tissue 1340. Image-guided therapy involves applying an embodiment of the invention with optical system 1310, which includes element 1370 used to apply aspects of therapy (i.e., tissue manipulation) to the non-transparent ocular tissue 1340. In some cases, element 1370 is a cable configured to transmit light energy, e.g., a fiber optic cable. In such cases, element 1370 may be configured to deliver therapeutic light to the non-transparent ocular tissue 1340, e.g., in cases where imaging is used to guide the application of therapy. In other cases, element 1370 may comprise other aspects of applying therapy, such as a needle for delivering a drug to the non-transparent ocular tissue 1340 (e.g., in connection with delivering stem cell therapy to the non-transparent ocular tissue 1340), in cases where imaging is used to guide needle position and injection placement. (In many embodiments, the therapeutic light is delivered via the same optics as the imaging light, but in other embodiments, e.g., embodiments configured for a form of cyclophotocoagulation, the therapeutic light may be delivered separately, e.g., by fiber optics, to a specific location in the eye, e.g., as shown with respect to element 1370.) (In yet other embodiments, element 1370 is configured to provide suction between adapter 1320 and ocular tissue 1340.) Optical system 1310 is used to image and manipulate the anatomical structures identified in step 1301, including, for example, using optical system 1310 in connection with performing transscleral laser trabeculotomy and / or trabeculoplasty and / or sclerotomy in connection with treating underlying glaucoma, e.g., to increase aqueous humor outflow via conventional drainage pathways. Element 1370 is delivered via adapter 1320, so that element 1370 can be positioned over non-transparent ocular tissue 1340.

[0224] From step 1302 in Figure 13B, the flowchart 1300 then proceeds to step 1303 in Figure 13C.

[0225] Step 1303 in FIG. 13C involves post-operative evaluation of the imaging and manipulation (e.g., laser trabeculotomy or trabeculoplasty) applied in step 1302 of FIG. 13B. In FIG. 13C, the outflow tracts within ocular tissue 1340 have been opened or dilated, thereby restoring the natural aqueous humor outflow process within ocular tissue 1340. Restoring the natural aqueous humor outflow process in step 1303 allows for relief of intraocular pressure, thereby normalizing the pressure, potentially alleviating corresponding glaucoma symptoms, and halting or slowing disease progression. Embodiments of the present invention can be used to quantify regional aqueous humor outflow at a level of detail not available in existing techniques. Thus, embodiments can use such data to guide and target surgical or laser intervention to specific anatomical regions, e.g., to maximize therapeutic efficacy and changes in aqueous humor outflow. For example, laser drilling drainage holes with the lowest outflow will result in the greatest therapeutic response.

[0226] 14A-C present an overview of another ophthalmic imaging and therapy application of an embodiment of the present invention. In particular, FIGURES 14A-C illustrate steps of a flowchart 1400 for utilizing an embodiment for cataract lens planning, i.e., imaging and optical manipulation related to intraocular lens (IOL) positioning, placement, and evaluation, according to an embodiment of the present invention.

[0227] Existing cataract surgery techniques are typically able to achieve a desired refractive outcome within ±0.5 D at best. Such limitations stem from the inability to predict the final location of an intraocular lens (IOL), i.e., the IOL's effective lens position. Utilizing embodiments of the present invention for imaging one or more potential placement zones for an intraocular lens (IOL) and its supporting structures, i.e., the IOL's connected haptics, within ocular tissue 1440 allows for determining a correlation between the underlying anatomical structure of ocular tissue 1440 and the post-surgical effective lens portion (ELP) of the IOL, including the IOL's haptics, to further eliminate discrepancies in achieving a desired refractive outcome.

[0228] 14A, pre-operative imaging of anatomical structures relevant to cataract lens procedures, particularly intraocular lens (IOL) positioning, such as the sulcus and capsular bag regions of ocular tissue 1440, is obtained utilizing imaging techniques in accordance with the present invention. Such imaging in step 1401 identifies ocular and / or periocular anatomical structures within non-transparent tissue 1440 relevant to cataract lens procedures, such as the cornea, iris, and lens, and particularly those relevant to cataract lens procedures involving an intraocular lens (IOL), such as the ciliary sulcus and capsular bag regions.

[0229] From step 1401 in FIG. 14A, the flowchart 1400 then proceeds to step 1402 in FIG. 14B.

[0230] In step 1402 in Figure 14B, intraoperative imaging is applied to the relevant anatomical structures visualized in step 1401 that reside within non-transparent tissue 1440 to provide intraoperative positioning guidance for positioning of an intraocular lens (IOL), including the lens and its haptics. An embodiment of the present invention, including an optical system for imaging ocular tissue, is used to image the anatomical structures identified in step 1401 and their relevant features and orientations to better characterize the position of the IOL.

[0231] From step 1402 in Figure 14B, the flowchart 1400 then proceeds to step 1403 in Figure 14C.

[0232] Step 1403 in FIG. 14C involves post-operative assessment of the position of the implanted intraocular lens (IOL) 1441 and assessment of the effective lens position (ELP) of the lens 1441 .

[0233] Certain embodiments include performing at least a subset of the steps presented in Figures 14A-B after the lens is already in place. That is, embodiments may apply thermal laser treatment to the anatomy to modify the position of the existing lens (e.g., shrinking tissue to move the lens slightly forward or backward, thereby changing its effective power and focal position relative to the retina). Such embodiments provide a way to make small corrections to aspects of the implanted lens in cases where lens selection is poor.

[0234] Other embodiments use laser therapy to relevant anatomical structures, in some cases including the sclera, to restore the eye's reading / focusing ability (which is lost later in life). Certain such embodiments apply laser therapy to the ciliary muscles and adjacent structures to "soften" these stiff tissue structures, restoring some compliance / flexibility and allowing accommodation to return.

[0235] 15A-B provide a schematic illustration of another ophthalmic imaging and therapy application of an embodiment of the present invention. In particular, FIGURES 15A-B illustrate the utilization of an embodiment for transscleral imaging and therapy, i.e., in connection with repairing or mitigating a retinal hole, in accordance with an embodiment of the present invention.

[0236] Figure 15A illustrates an existing technique in which laser light is focused onto ocular tissue 1540 to repair a retinal hole 1541. However, the peripheral retina 1542 is the most vulnerable tissue with respect to the retinal hole and is also the least accessible for repair using existing techniques, as highlighted in the close-up view in Figure 15A.

[0237] FIG. 15B illustrates the utilization of one embodiment of the optical system of the present invention for use in repairing a retinal hole. FIG. 15B illustrates the application of one embodiment of the present invention with optical system 1510, where at least some therapeutic light is transmitted to non-transparent ocular tissue 1540 via element 1570, thereby accessing peripheral retina 1542 through non-transparent ocular tissue 1540. In embodiments, therapeutic light may also be applied, partially or entirely, through the objective lens (i.e., the objective lens used for imaging) of optical system 1510. Optical system 1510 may be used to image and manipulate retinal hole 1542, including, for example, repairing the retinal hole by providing therapy, such as photocoagulation therapy, to retinal hole 1542 using certain therapeutic light 1570. Unlike the existing technique shown in FIG. 15A, the embodiment shown in FIG. 15B is not limited to accessing the retinal hole 1541 through transparent tissue, but instead can access the retinal hole through non-transparent tissue, making it well suited for high-resolution imaging and treatment of the retinal hole, for example, transscleral repair of the retinal hole 1541.

[0238] 16 shows an overview of a potential configuration 1600 for use with embodiments of the present invention relating to imaging of ocular tissue 1640 of a subject 1699. An optical system 1610 and an adapter 1620, in each case according to an embodiment of the present invention, may be secured to a support structure 1697, allowing a clinician 1698 to visualize the ocular tissue 1640 in real time, including using a control device 1696 to manipulate the focus of the optical system 1610, thereby allowing different aspects of the ocular tissue 1640 to be visualized.

[0239] 17 shows an overview of a potential structure 1700 for use with embodiments of the present invention relating to imaging of ocular tissue 1740. An optical system 1710 and an adapter 1720, in each case according to one embodiment of the present invention, may be integrated into structure 1700 at location 1799 such that a subject may be positioned below location 1799, which allows access so that adapter 1720 may interface with ocular tissue 1740. Optical system 1710 transmits light via element 1770 to non-transparent ocular or periocular tissue 1740 so that structures present therein may be imaged or manipulated as described herein.

[0240] Figure 18 illustrates the aspects of three-photon excited fluorescence (3PEF) versus two-photon excited fluorescence (2PEF). In 2PEF excitation 1801, excitation light containing two photons, first and second photons 1802a and 1802b, having wavelengths between 600 and 1,800 nm, are "combined" to produce a new photon 1803. The arrow representing new photon 1803 is shown as "shorter" than the sum of the lengths of the arrows representing photons 1802a and 1802b, which corresponds to the aspect of the valence electron diagram, indicating that the amount of energy of photons 1802a and 1802b is not contributed to new photon 1803 but is lost due to thermal emission. In 3PEF excitation 1811, excitation light comprising three photons, first, second, and third photons 1812a, 1812b, and 1812c, having wavelengths between 800 and 3,500 nm, are "combined" to produce new photon 1813. The arrow representing new photon 1813 is shown as being "shorter" than the sum of the lengths of the arrows representing photons 1812a, 1812b, and 1812c, which corresponds to the aspect of the valence electron diagram, indicating that the amount of energy of photons 1812a, 1812b, and 1812c is not contributed to new photon 1813 but is lost due to thermal emission. In embodiments utilizing second harmonic generation (SHG) or third harmonic generation (THG), energy is not lost to thermal emission (as shown in FIG. 18); i.e., in embodiments utilizing SHG or THG, the length of the arrow representing the new photon is the same length as the arrow of the photons that "combined" to produce the new photon. Although 3PEF and 2PEF are described in connection with FIG. 18 , embodiments of the present invention are not so limited. For example, embodiments of the present invention further include other higher order processes, such as four-photon excitation. Other contrast mechanisms used in embodiments include second harmonic generation (also a two-photon process such as 2PEF) and third harmonic generation (also a three-photon process such as 3PEF). Aspects of optical system 1820 according to one embodiment of the present invention are used to focus excitation light 1821 to a focal point 1823 using objective lens 1822. The excitation light may be any convenient light capable of producing three-photon excited fluorescence (3PEF) or two-photon excited fluorescence (2PEF) or second harmonic generation (SHG) or third harmonic generation (THG), and may include, for example, pulsed light.In some cases, the excitation light may include light having a wavelength of 600-1,800 nm, associated with two-photon excited fluorescence (2PEF), or light having a wavelength of 800-3,500 nm, associated with three-photon excited fluorescence (3PEF). Optical system 1820 is configured such that excitation light 1821 is focused using objective lens 1822, thereby allowing three photons to be "combined" (as shown in 3PEF diagram 1811), resulting in three-photon excited fluorescence (3PEF) at focal point 1823. Aspects of the invention include multiphoton laser therapy (i.e., energy deposition for thermal or injury), which may also include multiphoton processes. Certain such embodiments do not require the collection of an image signal.

[0241] As described herein, embodiments of the present invention include imaging utilizing three-photon excited fluorescence (3PEF) and two-photon excited fluorescence (2PEF). In some cases, imaging of structures using two-photon excited fluorescence (2PEF) can be further enhanced by utilizing second harmonic generation (SHG) information. Similarly, in some cases, imaging of structures using three-photon excited fluorescence (3PEF) can be further enhanced by utilizing third harmonic generation (THG) information. Further details, including those regarding two-photon excited fluorescence (2PEF), second harmonic generation (SHG), three-photon excited fluorescence (3PEF), and third harmonic generation (THG), can be found in Zipfel WR, Williams RM, Webb WW. Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol. 2003 Nov;21(11):1369-77. doi:10.1038 / nbt899. PMID:14595365, the disclosure of which is incorporated herein in its entirety. Certain embodiments may further include even higher order processes, such as four-photon excited fluorescence (4PEF) or fourth harmonic generation (FHG).

[0242] 19A-B provide an overview of another ophthalmic imaging and treatment application of an embodiment of the present invention. In particular, FIGS. 19A-B illustrate measurement of one or more of the angle, sulcus, and zonular insertion. "Angle," in embodiments, refers to the anterior chamber angle, i.e., the anatomical region where the iris and cornea meet, the trabecular meshwork is located, and aqueous humor drainage exits the anterior chamber. "Zonular insertion," in embodiments, refers to where the zonular fibers are positioned and attached (these fibers suspend the intraocular lens insert within their anchor point along the ciliary body). Using existing techniques, it is not possible to image or measure such aspects, and therefore this results in some approximation and / or guesswork in calculating the position of the intraocular lens. Measuring such aspects accurately (i.e., using embodiments of the present invention) allows for more precise calculation of the position of the intraocular lens implant after surgery, significantly reducing the errors inherent in calculations using existing techniques.

[0243] Figure 19A shows an optical system 1910 according to one embodiment of the present invention being used in conjunction with an adapter 1920 for interfacing with ocular tissue 1940 to visualize anatomical aspects of the ocular tissue 1940, for example, at certain insertion zones corresponding to different regions of the surface of the ocular tissue 1940. Figure 19B shows imaging results from use of the optical system 1910 and adapter 1940 of an embodiment of the present invention in connection with imaging relevant anatomical structures. Figure 19B shows a three-dimensional projection of the anterior segment of the eye, demonstrating the cornea with corneal nerves, the anterior chamber, and the iris.

[0244] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses.

[0245] 1. A method for imaging a structure through non-transparent tissue, comprising: deploying an excitation source to transmit light energy through non-transparent tissue to the structure; detecting light emitted from the structure via multiphoton excitation through the non-transparent tissue; and imaging the structure based on the detected light.

[0246] 2. A method of manipulating a structure through a non-transparent tissue, comprising: deploying an excitation source to transmit light energy through non-transparent tissue to the structure; manipulating the structure via optical energy transmitted through non-transparent tissue to the structure using multi-photon excitation.

[0247] 3. The method of any one of the preceding clauses, wherein the non-transparent tissue comprises non-transparent ocular or periocular tissue.

[0248] 4. The method of any one of the preceding clauses, wherein the non-transparent tissue comprises one or more of scleral tissue, retinal pigment epithelium (RPE), uvea, conjunctiva, Tenon's capsule, ocular muscles, ciliary body, palpebral conjunctiva, orbital septum, capsulopalveolar fascia, tarsal plate, tarsal gland, periocular adipose tissue, or dermis.

[0249] 5. The method of any one of the preceding clauses, wherein the non-transparent tissue comprises a light-scattering tissue or a light-absorbing tissue.

[0250] 6. The method of any one of the preceding clauses, wherein the non-transparent tissue comprises pigmented uveal tissue.

[0251] 7. The method of any one of the preceding clauses, wherein the structure comprises one or more of scleral tissue, corneal tissue, ocular vasculature, suprachoroidal space, choroid, chorioretinal vasculature, retinal pigment epithelium (RPE), photoreceptors, conjunctiva, Tenon's capsule, ocular muscles, ciliary body, peripheral retina, orbital fat, eyelid tissue, dermatological tissue optionally including one or more of epidermis, dermis, or subcutaneous tissue, lacrimal duct, sulcus, capsular bag region, extraocular muscles, collagen, dermal collagen, vascular tissue.

[0252] 8. The method of any one of the preceding clauses, wherein the structure comprises one or more of the following: palpebral conjunctiva, orbital septum, capsulopalveolar fascia, tarsal plate, tarsal gland, periocular adipose tissue or dermis, capillaries, venules, veins, arterioles or arteries, optionally those of the choroid, circulating cells.

[0253] 9. The method of any one of the preceding clauses, wherein the structure is in front of (relative to the excitation source) the retinal pigment epithelium (RPE).

[0254] 10. The method of any one of clauses 1 to 8, wherein the structure is located after (relative to the excitation source) the retinal pigment epithelium (RPE).

[0255] 11. The method of any one of the preceding clauses, wherein the structure comprises a light-scattering structure.

[0256] 12. The method of any one of the preceding clauses, wherein the structure comprises a light-absorbing structure.

[0257] 13. The method of any one of the preceding clauses, wherein the method further comprises introducing a fluorescent dye into the structure.

[0258] 14. The method of any one of the preceding clauses, wherein the method further comprises labeling the plasma present in the structure with a fluorescent dye.

[0259] 15. The method of any one of the preceding clauses, further comprising imaging the structure over a specified volume.

[0260] 16. The method of clause 15, wherein imaging the structure over a specified volume includes accumulating imaging data at a specified spatial resolution.

[0261] 17. The method of any one of the preceding clauses, further comprising imaging the structure for a specified period of time.

[0262] 18. The method of clause 17, wherein imaging the structure over a specified period of time includes accumulating imaging data at a specified pulse repetition rate.

[0263] 19. The method of claim 18, wherein the pulse repetition rate is between 1 Hz and 1,000 MHz.

[0264] 20. The method of claim 19, wherein the pulse repetition rate is between 1 kHz and 1,000 kHz.

[0265] 21. The method of any one of the preceding clauses, wherein the method is a method of imaging cell dynamics, or imaging neuronal activity, or imaging corneal nerves, or imaging aspects of the central nervous system.

[0266] 22. The method of any one of the preceding clauses, wherein the method further comprises utilizing an invasive probe to image aspects of the central nervous system, and wherein imaging optionally further comprises thinning a region of the skull to image aspects of the central nervous system.

[0267] 23. The method of any one of the preceding clauses, wherein the method further comprises visualizing nerve structures or pain receptors, and optionally utilizing the imaged nerve structures or pain receptors to reduce pain.

[0268] 24. A method according to any one of the preceding clauses, wherein the method is a method of imaging neuronal activity, optionally with a calcium indicator, or a method of imaging hemodynamics, or a method of imaging blood flow in the microvasculature.

[0269] 25. The method of any one of the preceding clauses, wherein the excitation source comprises one or more laser systems.

[0270] 26. The method of any one of the preceding clauses, wherein the excitation source comprises one or more laser systems configured to emit optical energy having an average power output of greater than 100 watts.

[0271] 27. The method of any one of the preceding clauses, wherein the pump source comprises one or more laser systems utilizing dispersion compensation.

[0272] 28. A method according to any one of the preceding clauses, wherein the excitation source comprises one or more laser systems and includes a usable power band from 350 nm to 5,000 nm.

[0273] 29. The method of any one of the preceding clauses, wherein the excitation source comprises a first laser.

[0274] 30. The method of clause 29, wherein the first laser is a solid-state laser.

[0275] 31. The method of clause 29 or 30, wherein the first laser is a fiber laser.

[0276] 32. The method of any one of clauses 29-31, wherein the first laser is an ytterbium-doped fiber laser.

[0277] 33. The method of any one of clauses 29 to 32, wherein the first laser is configured to generate optical energy having a wavelength in the range of 350 nm to 5.0 μm, a pulse duration of 1 to 1,000 femtoseconds, and a maximum power output of greater than 100 W.

[0278] 34. The method of any one of clauses 29-33, wherein the first laser is configured to emit light energy having an average power output of greater than 100 watts.

[0279] 35. The method of any one of clauses 29-34, wherein the first laser includes dispersion compensation.

[0280] 36. The method of any one of the preceding clauses, wherein the excitation source comprises a second laser.

[0281] 37. The method of clause 36, wherein the second laser is a titanium-doped sapphire laser.

[0282] 38. The method of clause 36 or 37, wherein the second laser is a fixed 1,045 nm laser.

[0283] 39. The method of any one of clauses 36-38, wherein the second laser is configured to generate optical energy having a maximum power output of greater than 100 W.

[0284] 40. The method of any one of clauses 36 to 39, wherein the second laser comprises a usable power band of 350 nm to 5,000 nm.

[0285] 41. The method of clause 40, wherein the second laser comprises a usable power band of 690 to 970 nm.

[0286] 42. The method of any one of the preceding clauses, wherein the excitation source comprises a third component.

[0287] 43. The method of clause 42, wherein the third component comprises a 1.7 μm excitation source.

[0288] 44. The method of clause 42 or 43, wherein the third component comprises a Raman shift of light at 1.5 μm in a large mode area photonic crystal rod.

[0289] 45. The method of any one of clauses 42 to 44, wherein the third component emits an excitation wavelength between 350 nm and 5 μm.

[0290] 46. ​​The method of clause 45, wherein the third component emits an excitation wavelength between 550 nm and 3.6 μm.

[0291] 47. The method of any one of the preceding clauses, wherein deploying the excitation source includes translating the excitation source across a predetermined region.

[0292] 48. The method of any one of the preceding clauses, wherein deploying the excitation source includes articulating the excitation source around the non-transparent tissue.

[0293] 49. The method of any one of the preceding clauses, wherein the excitation source comprises a pulsed laser.

[0294] 50. The method of clause 49, wherein the pulsed laser generates light energy having a pulse duration lasting from 1 to 1,000 femtoseconds.

[0295] 51. The method of clause 49 or 50, wherein the pulsed laser generates optical energy having a pulse repetition rate in the range of 1 Hz to 1,000 MHz.

[0296] 52. The method of clause 51, wherein the pulsed laser generates optical energy having a pulse repetition rate in the range of 1 Hz to 65 MHz.

[0297] 53. The method of any one of clauses 49-52, wherein the pulsed laser produces light having an average power output of greater than 100 watts.

[0298] 54. The method of any one of the preceding clauses, wherein deploying an excitation source includes deploying one or more of the following optical components: a laser scanner, a pulse compressor, a power attenuator, or adaptive optics for wavefront shaping.

[0299] 55. The method of any one of the preceding clauses, wherein the light emitted from the structure via multi-photon excitation comprises stimulated light.

[0300] 56. The method of clause 55, wherein the guided light is excited via a high-order nonlinear process.

[0301] 57. The method of clause 56, wherein the higher-order nonlinear process comprises one or more of two-photon excited fluorescence (2PEF), or second harmonic generation (SHG), or three-photon excited fluorescence (3PEF), or third harmonic generation (THG).

[0302] 58. The method of any one of the preceding clauses, wherein the light emitted via multi-photon excitation comprises light emitted from an endogenous fluorophore present in the structure or an exogenous fluorophore present in the structure.

[0303] 59. The method of any one of the preceding clauses, wherein the endogenous or exogenous fluorophore present in the structure emits one or more of ultraviolet, blue, green, red, or far-red light.

[0304] 60. The method of any one of the preceding clauses, wherein the light emitted from the structure comprises light emitted via two-photon excitation (2PEF) and further comprises a second harmonic generation (SHG) signal.

[0305] 61. The method of any one of the preceding clauses, wherein the light emitted from the structure comprises light emitted via three-photon excitation (3PEF) and further comprises a third harmonic generation (THG) signal.

[0306] 62. The method of any one of the preceding clauses, wherein deploying the excitation source to transmit light energy to the structure comprises spatially guiding the excitation source to transmit light energy through non-transparent tissue.

[0307] 63. The method of clause 62, wherein spatially guiding the excitation source to transmit optical energy to the structure comprises guiding the excitation source using a multimodal laser scanner.

[0308] 64. The method of clause 63, wherein the multimodal laser scanner is switchable between resonant imaging and patterned point scanning.

[0309] 65. The method of any one of the preceding clauses, further comprising using adaptive optics techniques.

[0310] 66. The method of any one of the preceding clauses, further comprising using adaptive optics techniques applied to light transmitted from the excitation source.

[0311] 67. The method of any one of the preceding clauses, further comprising using adaptive optics techniques configured to increase the efficiency of multiphoton excitation.

[0312] 68. The method of any one of the preceding clauses, further comprising using adaptive optics techniques configured to improve resolution of the imaged structures.

[0313] 69. A method according to any one of the preceding clauses, wherein the method further comprises using adaptive optics techniques including one or more of direct sensing, or direct wavefront sensing, or correction of wavefront distortion, or image point spread function, or laser guide star techniques, or indirect (algorithmic) adaptive optics techniques, wherein the indirect adaptive optics techniques optionally include determining an optimal excitation wavefront.

[0314] 70. The method of any one of the preceding clauses, wherein the method further comprises using adaptive optics techniques including direct wavefront sensing, wherein the direct wavefront sensing comprises using a Shack-Hartmann sensor and a deformable mirror.

[0315] 71. The method of any one of the preceding clauses, further comprising using adaptive optics techniques configured to improve spatiotemporal resolution.

[0316] 72. The method of any one of the preceding clauses, further comprising using particle tracking and velocity analysis by cross-correlation analysis.

[0317] 73. The method of any one of the preceding clauses, wherein the method further includes using particle tracking and velocity analysis by cross-correlation analysis, wherein using includes measuring blood flow, and wherein measuring blood flow optionally includes measuring choroidal blood flow.

[0318] 74. The method of any one of the preceding clauses, wherein the method further comprises velocity analysis by particle tracking and cross-correlation analysis, and the velocity analysis comprises using line-scanning particle image velocimetry (LS-PIV).

[0319] 75. The method of any one of the preceding clauses, further comprising utilizing the imaged structures to perform flow analysis.

[0320] 76. The method of any one of the preceding clauses, wherein the method further comprises performing a flow analysis, the flow analysis comprising an analysis of fluid flow within the structure.

[0321] 77. A method according to any one of the preceding clauses, wherein the method comprises utilizing a multi-core processor or a parallel processing unit to perform the flow analysis, the multi-core processor or the parallel processing unit optionally comprising a graphics processing unit (GPU).

[0322] 78. The method of any one of the preceding clauses, further comprising performing the flow analysis substantially in real time.

[0323] 79. A method according to any one of the preceding clauses, wherein the method further comprises performing a flow analysis, the flow analysis comprising generating a velocity map of fluid flow within the structure.

[0324] 80. The method of any one of the preceding clauses, further comprising deploying a sensor configured to collect backscattered light emitted from the structure through non-transparent tissue via multiphoton excitation.

[0325] 81. The method of any one of the preceding clauses, further comprising deploying a sensor configured to collect light other than backscattered light emitted from the structure via multiphoton excitation through non-transparent tissue.

[0326] 82. The method of clause 81, wherein collecting light other than backscattered light includes collecting light emitted deep within the tissue.

[0327] 83. The method of clause 81 or 82, wherein collecting light other than backscattered light includes positioning a sensor directly above the cornea of ​​the eye to capture light emitted from inside the eye.

[0328] 84. The method of any one of the preceding clauses, further comprising imaging with subcellular resolution.

[0329] 85. The method of any one of the preceding clauses, further comprising using imaging of the structure to detect or diagnose disease, or to detect or diagnose cancer or cancerous tissue, or to detect or diagnose melanoma, or to distinguish between cancerous and non-cancerous tissue, or to distinguish between cancerous and non-cancerous cells.

[0330] 86. The method of any one of the preceding clauses, further comprising manipulating the imaged structure.

[0331] 87. The method of any one of the preceding clauses, wherein the method further comprises manipulating the imaged structure, and wherein manipulating comprises manipulating the structure using an excitation source.

[0332] 88. The method of any one of the preceding clauses, wherein the method further comprises manipulating the structure, and wherein manipulating the structure comprises using the excitation source for one or more of non-incisional therapy, or light-tissue interaction, optionally including laser-tissue interaction or laser tissue perturbation, or multiphoton-mediated thermal damage, or non-thermal therapy, or photodisruption, or vascular coagulation, or ablation.

[0333] 89. A method according to any one of the preceding clauses, wherein manipulating the structure, optionally by using an excitation source for light-tissue interaction, includes providing treatment of tumor or cancerous tissue or cancer cells.

[0334] 90. A method according to any one of the preceding clauses, wherein manipulating the structure, optionally by using an excitation source for light-tissue interaction, comprises configuring the excitation source for photodisruption, or configuring the excitation source for non-thermal damage, or configuring the excitation source for multiphoton-mediated non-thermal damage, or configuring the excitation source for vascular coagulation, or configuring the excitation source to cause vascular coagulation, or configuring the excitation source to disrupt vascular coagulation.

[0335] 91. The method of any one of the preceding clauses, wherein manipulating the imaged structure includes ablatating the structure, or using an excitation source to treat glaucoma, or using an excitation source to reduce aqueous humor production in ocular tissue, or damaging the ciliary body, optionally by thermally damaging the ciliary body or applying a photodisruption-mediated process to reduce aqueous humor production.

[0336] 92. The method of any one of the preceding clauses, wherein the method further comprises treating glaucoma using the excitation source, and wherein treating optionally comprises increasing aqueous humor outflow.

[0337] 93. The method of clause 92, wherein increasing aqueous humor outflow using an excitation source comprises performing laser trabeculotomy through non-translucent tissue of the eye.

[0338] 94. The method of clause 92, wherein increasing aqueous humor outflow using an excitation source comprises performing laser trabeculoplasty directly through non-translucent tissue of the eye.

[0339] 95. A method according to any one of the preceding clauses, wherein the method further comprises manipulating the imaged structure using an excitation source to prevent or treat a retinal hole, which is optionally a peripheral retinal hole.

[0340] 96. The method of any one of the preceding clauses, wherein the method further comprises using the excitation source for non-incisional therapy, wherein using comprises using the excitation source to prevent or treat a retinal hole, and the retinal hole is optionally a peripheral retinal hole.

[0341] 97. The method of any one of the preceding clauses, wherein the method further comprises preventing or treating a retinal hole, and wherein the preventing or treating comprises transscleral imaging or transscleral treatment.

[0342] 98. The method of any one of the preceding clauses, wherein the method further comprises preventing or treating a retinal hole, wherein preventing or treating comprises identifying and providing photocoagulation therapy to the retina.

[0343] 99. The method of any one of the preceding clauses, wherein the method further comprises using the excitation source for non-incisional therapy, wherein using comprises providing photocoagulation and thermal therapy, optionally to the tumor or cancerous tissue or cancer cells, or ciliary body tumor, or peripheral choroidal tumor, or optionally to provide non-thermal therapy, to the tumor or cancerous tissue or cancer cells, or ciliary body tumor, or peripheral choroidal tumor.

[0344] 100. The method of any one of the preceding clauses, further comprising manipulating the imaged structures using an excitation source to optically crosslink scleral tissue.

[0345] 101. The method according to any one of the preceding clauses, wherein the method is a method for the prevention of myopia.

[0346] 102. The method of any one of the preceding clauses, further comprising manipulating the imaged structure using an excitation source to visualize extraocular muscle function.

[0347] 103. The method of any one of the preceding clauses, further comprising performing targeted alternation of extraocular muscle function.

[0348] 104. The method of any one of the preceding clauses, further comprising visualizing orbital fat.

[0349] 105. The method of any one of the preceding clauses, further comprising performing targeted thermal or photocoagulation therapy of orbital fat.

[0350] 106. The method of any one of the preceding clauses, further comprising visualizing eyelid tissue.

[0351] 107. The method of any one of the preceding clauses, further comprising manipulating the imaged structure by performing targeted alteration of eyelid tissue.

[0352] 108. The method of any one of the preceding clauses, further comprising using an excitation source to manipulate the structure using adaptive optics techniques.

[0353] 109. A method according to any one of the preceding clauses, wherein the method further comprises using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques being configured to increase the efficiency of manipulating the imaged structure using the excitation source.

[0354] 110. The method of any one of the preceding clauses, wherein the method further comprises using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques configured to improve accuracy of the excitation source.

[0355] 111. A method according to any one of the preceding clauses, wherein the method further comprises using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques being configured to improve the accuracy of the excitation source with respect to aspects of the structure.

[0356] 112. A method according to any one of the preceding clauses, wherein the method further comprises using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques being configured to improve the accuracy of manipulation of the imaged structure.

[0357] 113. The method of any one of the preceding clauses, wherein the method further comprises using an excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques comprising one or more of direct sensing, or direct wavefront sensing, or correction of wavefront distortion, or image point spread function, or laser guide star techniques, and the direct wavefront sensing optionally comprises using a Shack-Hartmann sensor and a deformable mirror.

[0358] 114. A method according to any one of the preceding clauses, wherein the method further comprises using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques being configured to improve spatiotemporal resolution.

[0359] 115. The method of any one of the preceding clauses, wherein the method further comprises manipulating dermatological tissue using the excitation source, the dermatological tissue optionally comprising one or more of the epidermis, dermis, or subcutaneous tissue.

[0360] 116. The method of any one of the preceding clauses, further comprising using an excitation source to manipulate the lacrimal duct, optionally facilitating fluid flow within the lacrimal duct.

[0361] 117. The method of any one of the preceding clauses, further comprising using an excitation source to manipulate the structure to reduce ocular tissue redness.

[0362] 118. The method of any one of the preceding clauses, further comprising using an excitation source to manipulate the structure to deliver therapy to the structure.

[0363] 119. The method of any one of the preceding clauses, wherein the method is a method for guiding the delivery of gene therapy.

[0364] 120. The method of clause 119, further comprising introducing a specified agent into the structure.

[0365] 121. The method of clause 120, wherein the specified agent comprises a cell.

[0366] 122. The method of clause 121, wherein the cells comprise stem cells or engineered cells.

[0367] 123. The method of clause 120, wherein the specified agent comprises an active agent.

[0368] 124. The method of clause 120, wherein the specified agent comprises a molecule.

[0369] 125. The method of clause 120, wherein introducing the specified agent into the structure includes introducing the specified agent into one or more of the subretinal space, or the suprachoroidal space, or the subchoroidal space, or the intravitreal space, or the ciliary body, or the stroma of the sclera.

[0370] 126. The method of any one of clauses 120-125, further comprising assessing the effect of the specified agent on the structure based on imaging the structure.

[0371] 127. The method of any one of the preceding clauses, further comprising guiding the positioning of the intraocular implant based on imaging the structure.

[0372] 128. The method of any one of the preceding clauses, further comprising predicting effective placement of an intraocular implant based on imaging the structure.

[0373] 129. The method of any one of the preceding clauses, further comprising imaging one or more of the sulcus or capsular bag region.

[0374] 130. The method of any one of the preceding clauses, further comprising guiding the positioning of the intraocular implant based on imaging the structure while implanting the intraocular implant.

[0375] 131. The method of any one of the preceding clauses, further comprising assessing the position of an implanted intraocular implant based on imaging the structure.

[0376] 132. The method of any one of the preceding clauses, further comprising assessing the effective lens position (ELP) of an implanted intraocular implant based on imaging the structure.

[0377] 133. The method according to any one of clauses 127 to 132, wherein the intraocular implant is an intraocular lens (IOL).

[0378] 134. The method of any one of the preceding clauses, wherein the method is a method of transscleral imaging, or transconjunctival imaging, or transTenon's capsule imaging, or extraocular muscle imaging, or imaging through outer eyelid tissue, optionally including imaging through one or more of the dermis, muscle, or aponeurosis, or imaging through inner eyelid tissue, optionally including one or more of the conjunctiva, or tarsus, or meibomian gland, or muscle, or transorbital septum imaging, or transcapsular parvebral fascial imaging, or transtarsal fascial imaging, or transtarsal gland imaging, or transperiocular adipose tissue imaging, or transcutaneous imaging, or imaging through pigmented uveal tissue.

[0379] 135. A method according to any one of the preceding clauses, wherein the method is a method of imaging through light-scattering or light-absorbing tissue.

[0380] 136. A method according to any one of the preceding clauses, wherein the method is a method for quantifying blood flow, and the blood flow optionally includes one or more of choroidal blood flow, or retinal blood flow, or ciliary blood flow, or uveal blood flow, or conjunctival blood flow.

[0381] 137. A method according to any one of the preceding clauses, wherein the method is a method of deploying multiphoton excitation microscopy on non-transparent tissue, which optionally includes ocular or periocular tissue.

[0382] 138. A method according to any one of the preceding clauses, wherein the method is a method of deploying multiphoton excitation microscopy through non-transparent tissue, which optionally includes ocular or periocular tissue.

[0383] 139. A method according to any one of the preceding clauses, wherein the method is a method of deploying multi-photon excitation microscopy, and the multi-photon excitation microscopy comprises one or more of two-photon excited fluorescence (2PEF), or second harmonic generation (SHG), or three-photon excited fluorescence (3PEF), or third harmonic generation (THG).

[0384] 140. A method according to any one of the preceding clauses, wherein the method is a method of imaging living tissue, the living tissue optionally comprising ocular or periocular tissue.

[0385] 141. A method according to any one of the preceding clauses, wherein the method is a method for imaging a subject.

[0386] 142. The method of any one of the preceding clauses, wherein the method is a method of imaging a subject having one or more of glaucoma, a retinal hole, a choroidal tumor, and myopia.

[0387] 143. The method according to any one of the preceding clauses, wherein the method is a method for imaging a human subject.

[0388] 144. The method of any one of the preceding clauses, wherein the method is a method of delivering a therapy.

[0389] 145. A method according to any one of the preceding clauses, wherein the method is a method for providing a cosmetic treatment.

[0390] 146. The method of any one of the preceding clauses, wherein the method is a method of providing cosmetic dermatological treatment, and the method of providing cosmetic dermatological treatment optionally includes one or more of providing cosmetic surgery, treating scars, scar removal, treating acne scars, removing acne scars, treating skin discoloration, treating skin discoloration disorders, removing birthmarks, removing port wine stains, removing tattoos, treating rosacea, removing hair, destroying hair follicles, stimulating hair follicles, cauterizing follicular tissue, and skin tightening.

[0391] 147. A method according to any one of the preceding clauses, wherein the method further comprises manipulating the structure by performing controlled cutting of the tissue, the tissue optionally being dermatological tissue.

[0392] 148. A method according to any one of the preceding clauses, wherein the method is a method for performing a skin biopsy procedure.

[0393] 149. The method of any one of the preceding clauses, further comprising manipulating the structure by destroying cancerous tissue or one or more cancerous cells, or ablating cancerous tissue, or ablating one or more cancerous cells, or ablating melanoma, or ablating cutaneous melanoma.

[0394] 150. A method according to any one of the preceding clauses, wherein the method is a method of affecting tissue shape, and the method of affecting tissue shape optionally includes reducing the shape of one or more fat deposits, or the volume of one or more fat deposits, or reducing one or more subdural fat deposits.

[0395] 151. The method of any one of the preceding clauses, wherein the method is a method of tissue sculpting, or treating dry eye syndrome, or applying thermal energy to a structure, or pain management.

[0396] 152. The method of any one of the preceding clauses, further comprising using an excitation source to perforate the structure, optionally in conjunction with providing thermal treatment to the tissue.

[0397] 153. The method of any one of the preceding clauses, further comprising deploying an adapter for coupling the optical system to non-transparent tissue.

[0398] 154. The method of any one of the preceding clauses, further comprising deploying a system for imaging structures through non-transparent tissue.

[0399] 155. The system an optical system configured to image the structure through a non-transparent tissue; an adapter configured to couple the optical system to non-transparent tissue; 1. A processor comprising: a memory operatively coupled to the processor, the memory comprising instructions stored in the memory, the instructions, when executed by the processor, causing the processor to: directing the optical system to image the structure through non-transparent tissue; receiving information about light emitted from the structure from an optical system; a processor that combines information about the light emitted from the structure to generate an image of the structure; 155. The method of claim 154, comprising: an operable connection between the processor and the optical system.

[0400] 156. An adapter for coupling an optical system to non-transparent tissue, comprising: a first component configured to interface with an optical system configured to image or manipulate structures through non-transparent tissue; a second component connected to the first component, the second component configured to interface with non-transparent tissue.

[0401] 157. The adapter according to clause 156, wherein the non-transparent tissue comprises ocular or periocular tissue.

[0402] 158. The adapter of clause 156, further comprising a bonding agent.

[0403] 159. The binder comprises a transparent medium; the transparent medium is optionally configured to increase stability or duration of use of the optical system, the transparent medium optionally configured for use with imaging at longer wavelengths; The adapter of clause 158, wherein the transparent medium optionally comprises one or more of water or a viscous gel, and the viscous gel optionally comprises sodium hyaluronate (optionally comprising a molecular weight of 100,000 to 20,000,000 daltons) or chondroitin sulfate (optionally comprising a molecular weight of 1,000 to 1,000,000 daltons).

[0404] 160. An adapter according to any one of clauses 156 to 159, wherein a coupling agent is present between the optical system and the tissue.

[0405] 161. The adapter according to clause 160, wherein a bonding agent is present between the first component and the tissue.

[0406] 162. The adapter of any one of clauses 156 to 161, further comprising an immersion medium.

[0407] 163. An adapter according to clause 162, in which a lens for focusing light into the tissue is present in the immersion medium.

[0408] 164. The adapter of clause 163, wherein the immersion medium comprises one or more of air, water-oil, or gel.

[0409] 165. The adapter according to clause 164, wherein the water is deuterium oxide (heavy water).

[0410] 166. The adapter according to clause 165, wherein the gel is a viscoelastic gel.

[0411] 167. The adapter according to clause 166, wherein the viscoelastic gel is composed of deuterium oxide (heavy water).

[0412] 168. The adapter of any one of clauses 156-167, wherein the second component is configured to interface with tissue using suction.

[0413] 169. The adapter according to clause 168, wherein the second component comprises a suction mechanism for attaching the second component to tissue.

[0414] 170. An adapter according to any one of clauses 156 to 169, wherein the second component comprises a central portion.

[0415] 171. An adapter according to clause 170, wherein the central portion is hollow.

[0416] 172. An adapter according to clause 171, wherein the hollow central portion receives a fluid.

[0417] 173. An adapter according to clause 170, wherein the central portion is solid.

[0418] 174. An adapter according to clause 173, wherein the solid central portion is optically transparent.

[0419] 175. An adapter according to any one of clauses 156-174, wherein the second component comprises an interface surface, the interface surface contacting the non-transparent tissue.

[0420] 176. The adapter of clause 175, wherein the interface surface interfaces with ocular tissue.

[0421] 177. An adapter according to clause 176, wherein the interface surface is shaped to contact ocular tissue.

[0422] 178. An adapter according to clause 177, wherein the interface surface is shaped to be displaced relative to the cornea or sclerocorneal limbus.

[0423] 179. An adapter according to any one of clauses 176 to 178, wherein the interface surface is shaped to accommodate placement on the cornea or sclerocorneal limbus area.

[0424] 180. An adapter according to any one of clauses 176 to 179, wherein an area of ​​the interface surface is recessed to accommodate placement on the cornea or sclerocorneal limbus.

[0425] 181. The adapter of any one of clauses 156-180, wherein the second component interfaces with the conjunctival fornix.

[0426] 182. The adapter according to clause 181, wherein the second component is shaped to interface with the conjunctival fornix.

[0427] 183. An adapter according to any one of clauses 156 to 182, wherein the second component is shaped to be fitted between the eye and the lower eyelid of the eye.

[0428] 184. An adapter according to any one of clauses 156 to 183, wherein the second component is shaped to be fitted between the eye and the upper eyelid of the eye.

[0429] 185. An adapter according to any one of clauses 156 to 184, wherein the second component comprises a contact lens interface.

[0430] 186. The adapter of any one of clauses 156-185, wherein the first component translates relative to non-transparent ocular or periocular tissue.

[0431] 187. An adapter according to any one of clauses 156 to 185, wherein the first component allows the optical system to translate relative to the second component.

[0432] 188. An adapter according to any one of clauses 156-187, wherein the first component rotates relative to the non-transparent tissue.

[0433] 189. An adapter according to any one of clauses 156-188, wherein the first component allows the optical system to rotate relative to the non-transparent tissue.

[0434] 190. The adapter of any one of clauses 156-189, wherein the first component articulates against non-transparent tissue.

[0435] 191. An adapter according to any one of clauses 156-190, wherein the first component allows the optical system to articulate relative to non-transparent tissue.

[0436] 192. An adapter according to any one of clauses 156 to 191, wherein the adapter further comprises a mechanism for controlling translation or rotation or articulation of the optical system relative to non-transparent tissue.

[0437] 193. An adapter according to any one of clauses 156 to 192, configured to perform any one of the methods described in clauses 1 to 155.

[0438] 194. An immersion medium for biological imaging, the immersion medium comprising an immersion gel.

[0439] 195. The immersion gel according to clause 194, wherein the immersion gel comprises a hyaluronan gel.

[0440] 196. The immersion gel according to clause 194 or 195, wherein the immersion gel comprises hyaluronic acid and deuterium oxide (heavy water).

[0441] 197. An immersion gel according to any one of clauses 194 to 196, wherein the immersion gel is configured for imaging of deep biological structures.

[0442] 198. An immersion gel according to any one of clauses 194 to 197, wherein the immersion gel is configured for use with a long wavelength laser.

[0443] 199. The immersion gel according to clause 198, wherein the long wavelength laser emits light having a wavelength of 1,700 nm or greater.

[0444] 200. An immersion gel according to any one of clauses 194 to 199, wherein the immersion gel is an adapter between an optical system and biological tissue.

[0445] 201. The method of any one of clauses 1 to 155, further comprising using an immersion gel of any one of clauses 194 to 199.

[0446] 202. An adapter according to any one of clauses 156 to 193, further comprising an immersion gel according to any one of clauses 194 to 199.

[0447] 203. A system for imaging a structure through non-transparent tissue, comprising: an optical system configured to image the structure through a non-transparent tissue; An adapter according to any one of clauses 156 to 193 configured to couple an optical system to non-transparent tissue; 1. A processor comprising: a memory operatively coupled to the processor, the memory comprising instructions stored in the memory, the instructions, when executed by the processor, causing the processor to: directing the optical system to image the structure through non-transparent tissue; receiving information about light emitted from the structure from an optical system; a processor that combines information about the light emitted from the structure to generate an image of the structure; an operative connection between the processor and the optical system.

[0448] 204. A system for manipulating structures through non-transparent tissue, comprising: an optical system configured to manipulate the structure through the non-transparent tissue; An adapter according to any one of clauses 156 to 193 configured to couple an optical system to non-transparent tissue; a processor comprising a memory operatively coupled to the processor, the memory comprising instructions stored in the memory that, when executed by the processor, cause the processor to direct an optical system to manipulate a structure through a non-transparent tissue; an operative connection between the processor and the optical system.

[0449] 205. The system of clause 203 or 204, wherein the non-transparent tissue comprises ocular or periocular tissue.

[0450] 206. The system of any one of clauses 203 to 205, wherein the optical system comprises a multiphoton excitation microscopy system.

[0451] 207. A multiphoton excitation microscopy system an excitation source for emitting light energy; and a detector for sensing light emitted from the structure via multi-photon excitation.

[0452] 208. The system of clause 207, wherein the detector collects backscattered light.

[0453] 209. The system of clause 208, wherein the detector collects light other than backscattered light.

[0454] 210. The system of clause 209, wherein the detector collects light emitted deep within the tissue.

[0455] 211. A system according to any one of clauses 207 to 210, wherein the detector is positioned directly above the cornea.

[0456] 212. A system according to any one of clauses 203 to 211, wherein the optical system comprises a laser scanner.

[0457] 213. The system of any one of clauses 203-212, wherein the optical system comprises one or more of a tube lens or a scan lens.

[0458] 214. The system of any one of clauses 203-213, wherein the optical system applies dispersion compensation.

[0459] 215. A system according to any one of clauses 203 to 214, wherein the optical system applies adaptive optics correction.

[0460] 216. A system according to any one of clauses 203 to 214, wherein the optical system applies power attenuation control.

[0461] 217. The system of clause 216, wherein the power attenuation control is applied by one or more of an acousto-optic modulator, or a Pockels cell, or a pair and a rotating polarizer.

[0462] 218. A system according to any one of clauses 203 to 217, wherein the optical system comprises a high numerical aperture optic or objective lens.

[0463] 219. A system according to any one of clauses 203 to 218, wherein the optical system comprises a translation stage.

[0464] 220. A system according to any one of clauses 203 to 219, wherein the optical system adjusts the focus using optical scanning.

[0465] 221. The system of any one of clauses 203 to 220, further comprising a mechanical component for adjusting the focus in the Z axis.

[0466] 222. The system of clause 221, wherein the mechanical component comprises one or more of a translation stage or a piezoelectric mechanism.

[0467] 223. A system according to any one of clauses 203 to 222, wherein the optical system adjusts the focus in the Z axis using optical scanning.

[0468] 224. The memory further comprises instructions, which when executed by the processor, cause the processor to: A system described in any one of clauses 203 to 223, instructing the optical system to image the structure over a specified volume.

[0469] 225. The memory further comprises instructions, which, when executed by the processor, cause the processor to: A system described in any one of clauses 203 to 224, wherein the optical system is instructed to translate, rotate or articulate relative to non-transparent tissue.

[0470] 226. The memory further comprises instructions, which, when executed by the processor, cause the processor to: A system described in any one of clauses 203 to 225, wherein the optical system is instructed to image the structure for a specified period of time.

[0471] 227. The memory further comprises instructions, which when executed by the processor, cause the processor to: A system according to any one of clauses 203 to 226, instructing an optical system to manipulate the structure.

[0472] 228. The system of any one of clauses 203-227, wherein manipulating the imaged structure includes using the excitation source for non-incisional therapy.

[0473] 229. The system of clause 228, wherein the non-incisional therapy comprises one or more of multiphoton-mediated thermal damage, photodisruption, photocrosslinking, vascular coagulation, and ablation of a structure.

[0474] 230. The system of any one of clauses 203-229, wherein manipulating the structure includes using an excitation source to treat glaucoma.

[0475] 231. The system of clause 230, wherein treating glaucoma using the excitation source includes reducing aqueous humor production in ocular tissue using the excitation source.

[0476] 232. The system of clause 231, wherein using the excitation source to reduce aqueous humor production in ocular tissue includes damaging the ciliary body to reduce aqueous humor production.

[0477] 233. The system of clause 232, wherein damaging the ciliary body to reduce aqueous humor production includes one or more of thermally damaging the ciliary body or applying a photodisruption-mediated process.

[0478] 234. The system of clause 233, wherein treating glaucoma using the excitation source includes increasing aqueous humor outflow using the excitation source.

[0479] 235. The system of clause 234, wherein increasing aqueous humor outflow using the excitation source includes performing laser trabeculotomy through non-translucent tissue of the eye.

[0480] 236. The system of clause 235, wherein performing laser trabeculoplasty includes performing laser trabeculoplasty directly through non-transparent tissue of the eye.

[0481] 237. A system described in any one of clauses 203 to 236, wherein manipulating the imaged structure includes using an excitation source to prevent or treat a retinal hole, and the retinal hole is optionally a peripheral retinal hole.

[0482] 238. The system of clause 237, wherein preventing or treating a retinal hole includes identifying and delivering photocoagulation therapy to the retina.

[0483] 239. The system of any one of clauses 203-238, wherein manipulating the structure includes providing photocoagulation and thermal therapy to ciliary body tumors or peripheral choroidal tumors.

[0484] 240. The system of any one of clauses 203-239, wherein manipulating the structure includes optically cross-linking scleral tissue to prevent myopia.

[0485] 241. A system described in any one of clauses 203 to 240, wherein manipulating the structure includes visualizing and performing targeted alteration of extraocular muscle function.

[0486] 242. A system described in any one of clauses 203 to 241, wherein manipulating the structure includes visualizing and performing targeted thermal or photocoagulation therapy of orbital fat.

[0487] 243. The system of any one of clauses 203-242, wherein manipulating the structure includes visualizing and performing targeted modification of eyelid tissue.

[0488] 244. The system of any one of clauses 203 to 243, wherein the processor comprises one or more multi-core processors or parallel processing units, and the multi-core processors or parallel processing units optionally comprise a graphics processing unit (GPU).

[0489] 245. The system of clause 244, wherein the multi-core processor or parallel processing unit is configured to perform the flow analysis, and optionally the flow analysis is performed substantially in real time.

[0490] 246. A system according to any one of clauses 203 to 245, configured to perform any of the methods described in clauses 1 to 155.

[0491] 247. The system of any one of clauses 203-246, further comprising an immersion gel of any one of clauses 194-199.

[0492] 248. A kit for imaging or manipulating structures through non-transparent tissue, comprising: An adapter according to any one of clauses 156 to 193; and packaging for the adapter.

[0493] 249. The kit of clause 248, further comprising a binding agent.

[0494] 250. A kit, a binder; and and packaging for the binding agent.

[0495] 251. 249. The kit of clause 248, further comprising an immersion medium.

[0496] 252. A kit, an immersion medium; and packaging for the immersion medium.

[0497] 253. A kit for imaging or manipulating structures through non-transparent tissue, comprising: A system according to any one of clauses 203 to 247; and packaging for the system.

[0498] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art, in light of the teachings of this invention, that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0499] Accordingly, the foregoing merely illustrates the principles of the present invention. Those skilled in the art will recognize that, although not explicitly described or shown herein, they can devise various configurations which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to further the art, and should not be construed as limitations on the examples and conditions specifically recited. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly recited in the claims.

[0500] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. For the purposes of the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) are expressly defined to apply to a limitation in a claim only if the exact phrase "means for" or the exact phrase "step for" appears at the beginning of such limitation in the claim. If such exact phrases are not used in a limitation in a claim, then neither 35 U.S.C. 112(f) nor 35 U.S.C. 112(6) applies.

Claims

1. 1. A method for imaging a structure through non-transparent tissue, comprising: deploying an excitation source to transmit light energy through non-transparent tissue to the structure; detecting light emitted from the structure via multiphoton excitation through the non-transparent tissue; imaging the structure based on the detected light.

2. 1. A method of manipulating a structure through a non-transparent tissue, comprising: deploying an excitation source to transmit light energy through non-transparent tissue to the structure; manipulating said structure via light energy transmitted through said non-transparent tissue to said structure using multi-photon excitation.

3. 10. The method of any one of the preceding claims, wherein the non-transparent tissue comprises non-transparent ocular or periocular tissue.

4. 10. The method of any one of the preceding claims, wherein the non-transparent tissue comprises one or more of scleral tissue, retinal pigment epithelium (RPE), uvea, conjunctiva, Tenon's capsule, ocular muscles, ciliary body, palpebral conjunctiva, orbital septum, capsulopalveolar fascia, tarsal plate, tarsal gland, periocular adipose tissue, or dermis.

5. 10. The method of any one of the preceding claims, wherein the non-transparent tissue comprises a light-scattering or light-absorbing tissue.

6. 10. The method of any one of the preceding claims, wherein the non-transparent tissue comprises pigmented uveal tissue.

7. 10. The method of any one of the preceding claims, wherein the structure comprises one or more of the following: scleral tissue, corneal tissue, ocular vasculature, suprachoroidal space, choroid, chorioretinal vasculature, retinal pigment epithelium (RPE), photoreceptors, conjunctiva, Tenon's capsule, ocular muscles, ciliary body, peripheral retina, orbital fat, eyelid tissue, dermatological tissue optionally including one or more of the epidermis, dermis, or subcutaneous tissue, lacrimal duct, sulcus, capsular bag region, extraocular muscles, collagen, dermal collagen, vascular tissue.

8. 10. The method of any one of the preceding claims, wherein the structures comprise one or more of the following: palpebral conjunctiva, orbital septum, capsulopalvebral fascia, tarsal plate, tarsal gland, periocular adipose tissue or dermis, capillaries, venules, veins, arterioles or arteries, optionally of the choroid, circulating cells.

9. 10. A method according to any one of the preceding claims, wherein the structure is in front of (relative to the excitation source) a retinal pigment epithelium (RPE).

10. The method of any one of claims 1 to 8, wherein the structure is located after (relative to the excitation source) the retinal pigment epithelium (RPE).

11. 10. The method of any one of the preceding claims, wherein the structure comprises a light scattering texture.

12. 10. The method of any one of the preceding claims, wherein the structure comprises a light-absorbing tissue.

13. 10. The method of any one of the preceding claims, wherein the method further comprises introducing a fluorescent dye into the structure.

14. 10. The method of any one of the preceding claims, wherein the method further comprises labeling the plasma present in the structure with a fluorescent dye.

15. 10. The method of any one of the preceding claims, further comprising imaging the structure over a specified volume.

16. The method of claim 15 , wherein imaging the structure over a specified volume comprises accumulating imaging data at a specified spatial resolution.

17. 10. The method of any one of the preceding claims, further comprising imaging the structure for a specified period of time.

18. 20. The method of claim 17, wherein imaging the structure for a specified period of time comprises accumulating imaging data at a specified pulse repetition rate.

19. 19. The method of claim 18, wherein the pulse repetition rate is between 1 Hz and 1,000 MHz.

20. 20. The method of claim 19, wherein the pulse repetition rate is between 1 kHz and 1,000 kHz.

21. 10. The method according to any one of the preceding claims, wherein the method is a method of imaging cell dynamics, or imaging neuronal activity, or imaging corneal nerves, or imaging aspects of the central nervous system.

22. 10. The method of any one of the preceding claims, wherein the method further comprises imaging aspects of the central nervous system with the aid of an invasive probe, and wherein the imaging optionally further comprises thinning a region of the skull to image aspects of the central nervous system.

23. 10. The method of any one of the preceding claims, wherein the method further comprises visualizing nerve structures or pain receptors, and optionally utilizing the imaged nerve structures or pain receptors to reduce pain.

24. 10. The method according to any one of the preceding claims, wherein the method is a method for imaging neuronal activity, optionally with a calcium indicator, or a method for imaging hemodynamics, or a method for imaging blood flow in the microvasculature.

25. 10. The method of any one of the preceding claims, wherein the excitation source comprises one or more laser systems.

26. 10. The method of any one of the preceding claims, wherein the excitation source comprises one or more laser systems configured to emit optical energy having an average power output of greater than 100 watts.

27. 10. A method according to any one of the preceding claims, wherein the pump source comprises one or more laser systems utilising dispersion compensation.

28. 10. A method according to any one of the preceding claims, wherein the excitation source comprises one or more laser systems and includes a usable power band from 350 nm to 5,000 nm.

29. 10. The method of any one of the preceding claims, wherein the excitation source comprises a first laser.

30. 30. The method of claim 29, wherein the first laser is a solid state laser.

31. 31. The method of claim 29 or 30, wherein the first laser is a fiber laser.

32. The method of any one of claims 29 to 31, wherein the first laser is an ytterbium-doped fiber laser.

33. 33. The method of any one of claims 29 to 32, wherein the first laser is configured to generate optical energy having a wavelength in the range of 350 nm to 5.0 μm, a pulse duration of 1 to 1,000 femtoseconds, and a maximum power output of greater than 100 W.

34. 34. The method of any one of claims 29 to 33, wherein the first laser is configured to emit optical energy having an average power output of greater than 100 watts.

35. The method of any one of claims 29 to 34, wherein the first laser includes dispersion compensation.

36. 10. The method of any one of the preceding claims, wherein the excitation source comprises a second laser.

37. 37. The method of claim 36, wherein the second laser is a titanium-doped sapphire laser.

38. 38. The method of claim 36 or 37, wherein the second laser is a fixed 1,045 nm laser.

39. 39. The method of any one of claims 36 to 38, wherein the second laser is configured to generate optical energy having a maximum power output of greater than 100 W.

40. 40. The method of any one of claims 36 to 39, wherein the second laser comprises a usable power band from 350 nm to 5,000 nm.

41. 41. The method of claim 40, wherein the second laser includes a usable power band from 690 to 970 nm.

42. 10. The method of any one of the preceding claims, wherein the excitation source comprises a third component.

43. 43. The method of claim 42, wherein the third component comprises a 1.7 μm excitation source.

44. 44. The method of claim 42 or 43, wherein the third component comprises a Raman shift of light at 1.5 μm in a large mode area photonic crystal rod.

45. 45. The method of any one of claims 42 to 44, wherein the third component emits an excitation wavelength between 350 nm and 5 μm.

46. 46. ​​The method of claim 45, wherein the third component emits excitation wavelengths between 550 nm and 3.6 μm.

47. 10. The method of any one of the preceding claims, wherein deploying the excitation source comprises translating the excitation source over a predetermined region.

48. 10. The method of any one of the preceding claims, wherein deploying an excitation source comprises articulating the excitation source around the non-transparent tissue.

49. 10. The method of any one of the preceding claims, wherein the excitation source comprises a pulsed laser.

50. 50. The method of claim 49, wherein the pulsed laser produces light energy having a pulse duration lasting between 1 and 1,000 femtoseconds.

51. 51. The method of claim 49 or 50, wherein the pulsed laser produces optical energy having a pulse repetition rate in the range of 1 Hz to 1,000 MHz.

52. 52. The method of claim 51, wherein the pulsed laser produces optical energy having a pulse repetition rate in the range of 1 Hz to 65 MHz.

53. 53. The method of any one of claims 49 to 52, wherein the pulsed laser produces light having an average power output of greater than 100 watts.

54. 10. The method of any one of the preceding claims, wherein deploying an excitation source comprises deploying one or more of the following optical components: a laser scanner, a pulse compressor, a power attenuator, or adaptive optics for wavefront shaping.

55. 10. The method of any one of the preceding claims, wherein the light emitted from the structure via multi-photon excitation comprises stimulated light.

56. 56. The method of claim 55, wherein the guided light is excited via a high-order nonlinear process.

57. 57. The method of claim 56, wherein the higher-order nonlinear process comprises one or more of two-photon excited fluorescence (2PEF), or second harmonic generation (SHG), or three-photon excited fluorescence (3PEF), or third harmonic generation (THG).

58. 10. The method of any one of the preceding claims, wherein the light emitted via multi-photon excitation comprises light emitted from endogenous fluorophores present in the structure or exogenous fluorophores present in the structure.

59. 10. The method of any one of the preceding claims, wherein the endogenous or exogenous fluorophores present in the structure emit one or more of ultraviolet, blue, green, red, or far-red light.

60. 10. The method of any one of the preceding claims, wherein the light emitted from the structure comprises light emitted via two-photon excitation (2PEF) and further comprises a second harmonic generation (SHG) signal.

61. 10. The method of any one of the preceding claims, wherein the light emitted from the structure comprises light emitted via three-photon excitation (3PEF) and further comprises a third harmonic generation (THG) signal.

62. 10. The method of any one of the preceding claims, wherein deploying an excitation source to deliver light energy to a structure comprises spatially guiding the excitation source to deliver light energy through the non-transparent tissue.

63. 63. The method of claim 62, wherein spatially guiding the excitation source to transmit optical energy to the structure comprises guiding the excitation source using a multimodal laser scanner.

64. 64. The method of claim 63, wherein the multimodal laser scanner is switchable between resonant imaging and patterned point scanning.

65. 10. The method of any one of the preceding claims, further comprising using adaptive optics techniques.

66. 10. The method of any one of the preceding claims, further comprising using adaptive optics techniques applied to light transmitted from the excitation source.

67. 10. The method of any one of the preceding claims, further comprising using adaptive optics techniques configured to increase the efficiency of multi-photon excitation.

68. 10. The method of any one of the preceding claims, further comprising using adaptive optics techniques configured to improve the resolution of the imaged structures.

69. 10. The method of any one of the preceding claims, further comprising using adaptive optics techniques including one or more of direct sensing, or direct wavefront sensing, or correction of wavefront distortion, or image point spread function, or laser guide star techniques, or indirect (algorithmic) adaptive optics techniques, wherein the indirect adaptive optics techniques optionally include determining an optimal excitation wavefront.

70. 10. The method of any one of the preceding claims, wherein the method further comprises using adaptive optics techniques including direct wavefront sensing, the direct wavefront sensing comprising using a Shack-Hartmann sensor and a deformable mirror.

71. 10. The method of any one of the preceding claims, further comprising using adaptive optics techniques configured to improve spatiotemporal resolution.

72. 10. The method of any one of the preceding claims, further comprising using particle tracking and velocity analysis by cross-correlation analysis.

73. 10. The method of claim 1, further comprising using particle tracking and cross-correlation velocity analysis, wherein the using comprises measuring blood flow, and wherein measuring blood flow optionally comprises measuring choroidal blood flow.

74. 10. The method of any one of the preceding claims, wherein the method further comprises velocity analysis by particle tracking and cross-correlation analysis, wherein the velocity analysis comprises using line-scanning particle image velocimetry (LS-PIV).

75. 10. The method of any one of the preceding claims, further comprising performing flow analysis with the aid of the imaged structures.

76. 10. The method of any one of the preceding claims, wherein the method further comprises performing a flow analysis, the flow analysis comprising an analysis of fluid flow within the structure.

77. 10. A method according to any one of the preceding claims, wherein the method comprises utilizing a multi-core processor or a parallel processing unit to perform the flow analysis, the multi-core processor or parallel processing unit optionally comprising a graphics processing unit (GPU).

78. 10. A method according to any one of the preceding claims, further comprising performing the flow analysis substantially in real time.

79. 10. The method of any one of the preceding claims, wherein the method further comprises performing a flow analysis, the flow analysis comprising generating a velocity map of fluid flow within the structure.

80. 10. The method of any one of the preceding claims, further comprising deploying a sensor configured to collect backscattered light emitted from the structure through the non-transparent tissue via multi-photon excitation.

81. 10. The method of any one of the preceding claims, further comprising deploying a sensor configured to collect light other than backscattered light emitted from the structure via multiphoton excitation through the non-transparent tissue.

82. 82. The method of claim 81, wherein collecting light other than backscattered light comprises collecting light emitted deep within the tissue.

83. 83. The method of claim 81 or 82, wherein collecting light other than backscattered light comprises positioning a sensor directly over the cornea of ​​the eye to capture light emitted from within the eye.

84. 10. The method of any one of the preceding claims, further comprising imaging with subcellular resolution.

85. 10. The method of any one of the preceding claims, further comprising using imaging of said structure to detect or diagnose disease, or to detect or diagnose cancer or cancerous tissue, or to detect or diagnose melanoma, or to distinguish between cancerous and non-cancerous tissue, or to distinguish between cancerous and non-cancerous cells.

86. 10. The method of any one of the preceding claims, further comprising manipulating the imaged structures.

87. 10. The method of any one of the preceding claims, wherein the method further comprises manipulating the imaged structure, said manipulating comprising manipulating the structure using the excitation source.

88. 10. The method of any one of the preceding claims, further comprising manipulating the structure, wherein manipulating the structure comprises using the excitation source for one or more of non-ablative therapy, or light-tissue interaction, optionally including laser-tissue interaction or laser tissue perturbation, or multiphoton mediated thermal damage, or non-thermal therapy, or photodisruption, or vascular coagulation, or ablation.

89. 10. The method of any one of the preceding claims, wherein manipulating a structure, optionally by using said excitation source for light-tissue interaction, comprises providing treatment of tumor or cancerous tissue or cells.

90. 10. The method of any one of the preceding claims, wherein manipulating the structure, optionally by using the excitation source for light-tissue interaction, comprises configuring the excitation source for photodisruption, or configuring the excitation source for non-thermal damage, or configuring the excitation source for multiphoton mediated non-thermal damage, or configuring the excitation source for vasocoagulation, or configuring the excitation source to cause vasocoagulation, or configuring the excitation source to disrupt vasocoagulation.

91. 10. The method of any one of the preceding claims, wherein manipulating the imaged structure comprises ablatating the structure, or using the excitation source to treat glaucoma, or using the excitation source to reduce aqueous humor production in an ocular tissue, or damaging the ciliary body, optionally by thermally damaging the ciliary body or applying a photodisruption mediated process to reduce aqueous humor production.

92. 10. The method of any one of the preceding claims, wherein the method further comprises treating glaucoma using the excitation source, wherein the treating optionally comprises increasing aqueous humor outflow.

93. 93. The method of claim 92, wherein increasing aqueous humor outflow using the excitation source comprises performing laser trabeculotomy through non-translucent tissue of the eye.

94. 93. The method of claim 92, wherein increasing aqueous humor outflow using the excitation source comprises performing laser trabeculoplasty directly through the non-translucent tissue of the eye.

95. 10. The method of any one of the preceding claims, wherein the method further comprises manipulating the imaged structure using the excitation source to prevent or treat a retinal hole, optionally a peripheral retinal hole.

96. 10. The method of any one of the preceding claims, wherein the method further comprises using the excitation source for non-incisional therapy, wherein the using comprises using the excitation source to prevent or treat a retinal hole, optionally a peripheral retinal hole.

97. 10. The method of any one of the preceding claims, wherein the method further comprises preventing or treating a retinal hole, wherein the preventing or treating comprises transscleral imaging or transscleral treatment.

98. 10. The method of any one of the preceding claims, wherein the method further comprises preventing or treating a retinal hole, wherein the preventing or treating comprises identifying and providing photocoagulation therapy to the retina.

99. 10. The method of any one of the preceding claims, further comprising using the excitation source for non-incisional therapy, wherein the using comprises providing photocoagulation and thermal therapy, optionally to a tumor or cancerous tissue or cancer cells, or a ciliary body tumor, or a peripheral choroidal tumor, or optionally to a non-thermal therapy, to a tumor or cancerous tissue or cancer cells, or a ciliary body tumor, or a peripheral choroidal tumor.

100. 10. The method of any one of the preceding claims, further comprising manipulating the imaged structures using the excitation source to optically crosslink scleral tissue.

101. 10. The method according to any one of the preceding claims, wherein the method is a method for the prevention of myopia.

102. 10. The method of any one of the preceding claims, further comprising manipulating the imaged structures using the excitation source to visualize extraocular muscle function.

103. 10. The method of any one of the preceding claims, further comprising performing targeted alterations of extraocular muscle function.

104. 10. The method of any one of the preceding claims, further comprising visualizing orbital fat.

105. 10. The method of any one of the preceding claims, further comprising performing targeted thermal or photocoagulation therapy of orbital fat.

106. 10. The method of any one of the preceding claims, further comprising visualizing eyelid tissue.

107. 10. The method of any one of the preceding claims, further comprising manipulating the imaged structures by performing targeted alternation of eyelid tissue.

108. 10. The method of any one of the preceding claims, further comprising using the excitation source to manipulate the structure using adaptive optics techniques.

109. 10. The method of any one of the preceding claims, further comprising manipulating the structure using the excitation source using adaptive optics techniques, the adaptive optics techniques configured to increase the efficiency of manipulating the imaged structure using the excitation source.

110. 10. The method of any one of the preceding claims, wherein the method further comprises using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques being configured to improve accuracy of the excitation source.

111. 10. The method of any one of the preceding claims, further comprising using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques being configured to improve the accuracy of the excitation source with respect to an aspect of the structure.

112. 10. The method of any one of the preceding claims, wherein the method further comprises using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques configured to improve accuracy of the manipulation of the imaged structure.

113. 10. The method of any one of the preceding claims, further comprising using the excitation source to manipulate the structure using adaptive optics techniques, the adaptive optics techniques comprising one or more of direct sensing, or direct wavefront sensing, or correction of wavefront distortion, or image point spread function, or laser guide star techniques, and wherein direct wavefront sensing optionally comprises using a Shack-Hartmann sensor and a deformable mirror.

114. 10. The method of any one of the preceding claims, wherein the method further comprises using the excitation source to manipulate the structure with adaptive optics techniques, the adaptive optics techniques being configured to improve spatiotemporal resolution.

115. 10. The method of any one of the preceding claims, wherein the method further comprises manipulating dermatological tissue using the excitation source, the dermatological tissue optionally comprising one or more of the epidermis, dermis, or subcutaneous tissue.

116. 10. The method of any one of the preceding claims, further comprising using the excitation source to manipulate a tear duct, optionally facilitating fluid flow within the tear duct.

117. 10. The method of any one of the preceding claims, further comprising using the excitation source to manipulate the structure to reduce ocular tissue redness.

118. 10. The method of any one of the preceding claims, further comprising using the excitation source to manipulate the structure to deliver therapy to the structure.

119. 10. The method of any one of the preceding claims, wherein the method is a method for guiding the delivery of gene therapy.

120. 120. The method of claim 119, further comprising introducing a designated agent into the structure.

121. 121. The method of claim 120, wherein the specified agent comprises a cell.

122. 122. The method of claim 121, wherein the cells comprise stem cells or engineered cells.

123. 121. The method of claim 120, wherein the designated agent comprises an active agent.

124. 121. The method of claim 120, wherein the designated agent comprises a molecule.

125. 121. The method of claim 120, wherein introducing the designated agent into the structure comprises introducing the designated agent into one or more of the subretinal space, or the suprachoroidal space, or the subchoroidal space, or the intravitreal space, or the ciliary body, or the stroma of the sclera.

126. 126. The method of any one of claims 120 to 125, further comprising assessing the effect of the specified agent on the structure based on imaging the structure.

127. 10. The method of any one of the preceding claims, further comprising guiding the positioning of an intraocular implant based on imaging the structure.

128. 10. The method of any one of the preceding claims, further comprising predicting effective placement of an intraocular implant based on imaging the structure.

129. 10. The method of any one of the preceding claims, further comprising imaging one or more of the sulcus or capsular bag region.

130. 10. The method of any one of the preceding claims, further comprising guiding the positioning of an intraocular implant based on imaging the structure while implanting the intraocular implant.

131. 10. The method of any one of the preceding claims, further comprising assessing the position of an implanted intraocular implant based on imaging said structure.

132. 10. The method of any one of the preceding claims, further comprising assessing an effective lens position (ELP) of an implanted intraocular implant based on imaging said structure.

133. 133. The method of any one of claims 127 to 132, wherein the intraocular implant is an intraocular lens (IOL).

134. 10. The method of any one of the preceding claims, wherein the method is a method of transscleral imaging, or transconjunctival imaging, or transTenon's capsule imaging, or extraocular muscle imaging, or imaging through outer eyelid tissue, optionally including imaging through one or more of the dermis, muscle, or aponeurosis, or imaging through inner eyelid tissue, optionally including one or more of the conjunctiva, tarsus, or meibomian glands, or muscle, or transorbital septum imaging, or transcapsular parvebral fascial imaging, or transtarsal fascial imaging, or transtarsal gland imaging, or transperiocular adipose tissue imaging, or transcutaneous imaging, or imaging through pigmented uveal tissue.

135. 10. The method according to any one of the preceding claims, wherein the method is a method for imaging through light-scattering or light-absorbing tissue.

136. 10. The method of any one of the preceding claims, wherein the method is a method for quantifying blood flow, and the blood flow optionally comprises one or more of choroidal blood flow, or retinal blood flow, or ciliary blood flow, or uveal blood flow, or conjunctival blood flow.

137. 10. The method of any one of the preceding claims, wherein the method is a method of deploying multiphoton excitation microscopy on non-transparent tissue, said non-transparent tissue optionally comprising ocular or periocular tissue.

138. 10. The method of any one of the preceding claims, wherein the method is a method of deploying multiphoton excitation microscopy through non-transparent tissue, said non-transparent tissue optionally comprising ocular or periocular tissue.

139. 10. The method of any one of the preceding claims, wherein the method is a method of deploying multi-photon excitation microscopy, the multi-photon excitation microscopy comprising one or more of two-photon excited fluorescence (2PEF), or second harmonic generation (SHG), or three-photon excited fluorescence (3PEF), or third harmonic generation (THG).

140. 10. A method according to any one of the preceding claims, wherein the method is a method of imaging living tissue, said living tissue optionally comprising ocular or periocular tissue.

141. 10. The method according to any one of the preceding claims, wherein the method is a method for imaging an object.

142. 10. The method of any one of the preceding claims, wherein the method is a method of imaging a subject having one or more of the following: glaucoma, a retinal hole, a choroidal tumor, or myopia.

143. 10. The method according to any one of the preceding claims, wherein the method is a method for imaging a human subject.

144. 10. The method of any one of the preceding claims, wherein the method is a method for delivering a therapy.

145. 10. A method according to any one of the preceding claims, wherein the method is a method for providing a cosmetic treatment.

146. 10. The method of any one of the preceding claims, wherein the method is a method of providing cosmetic dermatological treatment, and the method of providing cosmetic dermatological treatment optionally comprises one or more of providing cosmetic surgery, treating scars, scar removal, treatment of acne scars, removal of acne scars, treatment of skin discolouration, treatment of skin discolouration disorders, birthmark removal, port wine stain removal, tattoo removal, treatment of rosacea, hair removal, destruction of hair follicles, stimulation of hair follicles, ablation of follicular tissue, and skin tightening.

147. 10. The method of any one of the preceding claims, wherein the method further comprises manipulating the structure by performing controlled cutting of tissue, the tissue optionally being dermatological tissue.

148. 10. The method according to any one of the preceding claims, wherein the method is a method for performing a skin biopsy procedure.

149. 10. The method of any one of the preceding claims, further comprising manipulating the structure by destroying cancerous tissue or one or more cancerous cells, or ablating cancerous tissue, or ablating one or more cancerous cells, or ablating melanoma, or ablating cutaneous melanoma.

150. 10. A method according to any one of the preceding claims, wherein the method is a method of affecting tissue shape, the method of affecting tissue shape optionally comprising reducing the shape of one or more fat deposits, or the volume of one or more fat deposits, or reducing one or more subdural fat deposits.

151. 10. The method of any one of the preceding claims, wherein the method is a method of tissue sculpting, or treating dry eye syndrome, or applying thermal energy to the structure, or pain management.

152. 10. The method of any one of the preceding claims, further comprising using the excitation source to perforate the structure, optionally in conjunction with providing thermal therapy to tissue.

153. 10. The method of any one of the preceding claims, further comprising deploying an adapter for coupling the optical system to non-transparent tissue.

154. 10. The method of any one of the preceding claims, further comprising deploying a system for imaging structures through non-transparent tissue.

155. The system comprises: an optical system configured to image the structure through a non-transparent tissue; an adapter configured to couple the optical system to the non-transparent tissue; 1. A processor comprising: a memory operatively coupled to the processor, the memory comprising instructions stored in the memory, the instructions, when executed by the processor, causing the processor to: directing the optical system to image a structure through the non-transparent tissue; receiving information from the optical system regarding light emitted from the structure; a processor that combines information about the light emitted from the structures to generate an image of the structures; and an operable connection between the processor and the optical system.

156. 1. An adapter for coupling an optical system to non-transparent tissue, comprising: a first component configured to interface with an optical system configured to image or manipulate structures through non-transparent tissue; a second component connected to the first component, the second component configured to interface with non-transparent tissue.

157. 157. The adapter of claim 156, wherein the non-transparent tissue comprises ocular or periocular tissue.

158. 157. The adapter of claim 156, further comprising a bonding agent.

159. the binder comprises a transparent medium; the transparent medium is optionally configured to increase stability or duration of use of the optical system, the transparent medium optionally configured for use with imaging at longer wavelengths; 159. The adapter of claim 158, wherein the transparent medium optionally comprises one or more of water or a viscous gel, and the viscous gel optionally comprises sodium hyaluronate (optionally comprising a molecular weight of 100,000 to 20,000,000 Daltons) or chondroitin sulfate (optionally comprising a molecular weight of 1,000 to 1,000,000 Daltons).

160. An adapter according to any one of claims 156 to 159, wherein the coupling agent is present between the optical system and the tissue.

161. 161. The adapter of claim 160, wherein the bonding agent is present between the first component and the tissue.

162. An adapter according to any one of claims 156 to 161, further comprising an immersion medium.

163. 163. The adapter of claim 162, wherein a lens is present within the immersion medium to focus light into the tissue.

164. 164. The adapter of claim 163, wherein the immersion medium comprises one or more of air, water oil, or gel.

165. 165. The adapter of claim 164, wherein the water is deuterium oxide (heavy water).

166. 166. The adapter of claim 165, wherein the gel is a viscoelastic gel.

167. 167. The adapter of claim 166, wherein the viscoelastic gel is composed of deuterium oxide (heavy water).

168. The adapter of any one of claims 156 to 167, wherein the second component is configured to interface with tissue using suction.

169. 169. The adapter of claim 168, wherein the second component comprises a suction mechanism for attaching the second component to the tissue.

170. An adapter according to any one of claims 156 to 169, wherein the second component comprises a central portion.

171. 171. The adapter of claim 170, wherein the central portion is hollow.

172. 172. The adapter of claim 171, wherein the hollow central portion receives a fluid.

173. 171. The adapter of claim 170, wherein the central portion is solid.

174. 174. The adapter of claim 173, wherein the solid central portion is optically transparent.

175. An adapter according to any one of claims 156 to 174, wherein the second component comprises an interface surface, the interface surface contacting the non-transparent tissue.

176. 176. The adapter of claim 175, wherein the interface surface interfaces with ocular tissue.

177. 177. The adapter of claim 176, wherein the interface surface is shaped to contact ocular tissue.

178. 178. The adapter of claim 177, wherein the interface surface is shaped to displace relative to the cornea or sclerocorneal limbus.

179. An adapter according to any one of claims 176 to 178, wherein the interface surface is shaped to accommodate placement on the cornea or limbus area.

180. An adapter according to any one of claims 176 to 179, wherein an area of ​​the interface surface is recessed to accommodate placement on the cornea or sclerocorneal limbus.

181. 181. An adapter according to any one of claims 156 to 180, wherein the second component interfaces with the conjunctival fornix.

182. 182. The adapter of claim 181, wherein the second component is shaped to interface with the conjunctival fornix.

183. An adapter according to any one of claims 156 to 182, wherein the second component is shaped to be fitted between the eye and the lower eyelid of the eye.

184. An adapter according to any one of claims 156 to 183, wherein the second component is shaped to be fitted between the eye and the upper eyelid of the eye.

185. An adapter according to any one of claims 156 to 184, wherein the second component comprises a contact lens interface.

186. An adapter according to any one of claims 156 to 185, wherein the first component translates relative to non-transparent ocular or periocular tissue.

187. An adapter according to any one of claims 156 to 185, wherein the first component allows the optical system to translate relative to the second component.

188. The adapter of any one of claims 156 to 187, wherein the first component rotates relative to the non-transparent tissue.

189. An adapter according to any one of claims 156 to 188, wherein the first component allows the optical system to rotate relative to the non-transparent tissue.

190. The adapter of any one of claims 156 to 189, wherein the first component articulates against the non-transparent tissue.

191. An adapter according to any one of claims 156 to 190, wherein the first component allows the optical system to articulate relative to the non-transparent tissue.

192. An adapter according to any one of claims 156 to 191, wherein the adapter further comprises a mechanism for controlling translation or rotation or articulation of the optical system relative to the non-transparent tissue.

193. An adapter according to any one of claims 156 to 192, configured to perform any of the methods according to claims 1 to 155.

194. An immersion medium for biological imaging, the immersion medium comprising an immersion gel.

195. 195. The immersed gel of claim 194, wherein the immersed gel comprises a hyaluronan gel.

196. 196. The immersed gel of claim 194 or 195, wherein the immersed gel comprises hyaluronic acid and deuterium oxide (heavy water).

197. 197. The immersion gel of any one of claims 194 to 196, wherein the immersion gel is configured for imaging of deep biological structures.

198. 198. The immersion gel of any one of claims 194 to 197, wherein the immersion gel is configured for use with a long wavelength laser.

199. 200. The immersed gel of claim 198, wherein the long wavelength laser emits light having a wavelength of 1,700 nm or greater.

200. 200. An immersion gel according to any one of claims 194 to 199, wherein the immersion gel is an adapter between an optical system and biological tissue.

201. 200. The method of any one of claims 1 to 155, further comprising using an immersion gel of any one of claims 194 to 199.

202. 200. An adapter according to any one of claims 156 to 193, further comprising an immersion gel according to any one of claims 194 to 199.

203. 1. A system for imaging a structure through non-transparent tissue, comprising: an optical system configured to image the structure through a non-transparent tissue; An adapter according to any one of claims 156 to 193, configured to couple the optical system to the non-transparent tissue; 1. A processor comprising: a memory operatively coupled to the processor, the memory comprising instructions stored in the memory, the instructions, when executed by the processor, causing the processor to: directing the optical system to image a structure through the non-transparent tissue; receiving information from the optical system regarding light emitted from the structure; a processor that combines information about the light emitted from the structures to generate an image of the structures; an operable connection between the processor and the optical system.

204. 1. A system for manipulating a structure through a non-transparent tissue, comprising: an optical system configured to manipulate the structure through the non-transparent tissue; An adapter according to any one of claims 156 to 193, configured to couple the optical system to the non-transparent tissue; a processor comprising a memory operatively coupled to the processor, the memory comprising instructions stored in the memory that, when executed by the processor, cause the processor to direct the optical system to manipulate the structure through the non-transparent tissue; an operable connection between the processor and the optical system.

205. 205. The system of claim 203 or 204, wherein the non-transparent tissue comprises ocular or periocular tissue.

206. A system according to any one of claims 203 to 205, wherein the optical system comprises a multi-photon excitation microscopy system.

207. the multiphoton excitation microscopy system comprises: an excitation source for emitting light energy; 207. The system of claim 206, further comprising: a detector for sensing light emitted from the structure via multi-photon excitation.

208. 208. The system of claim 207, wherein the detector collects backscattered light.

209. 209. The system of claim 208, wherein the detector collects light other than backscattered light.

210. 210. The system of claim 209, wherein the detector collects light emitted deep within the tissue.

211. A system according to any one of claims 207 to 210, wherein the detector is positioned directly above the cornea.

212. A system according to any one of claims 203 to 211, wherein the optical system comprises a laser scanner.

213. The system of any one of claims 203 to 212, wherein the optical system comprises one or more of a tube lens or a scan lens.

214. A system according to any one of claims 203 to 213, wherein the optical system applies dispersion compensation.

215. A system according to any one of claims 203 to 214, wherein the optical system applies adaptive optics correction.

216. A system according to any one of claims 203 to 214, wherein the optical system applies power attenuation control.

217. 217. The system of claim 216, wherein the power attenuation control is applied by one or more of an acousto-optic modulator, or a Pockels cell, or a pair and a rotating polarizer.

218. A system according to any one of claims 203 to 217, wherein the optical system comprises a high numerical aperture optic or objective lens.

219. A system according to any one of claims 203 to 218, wherein the optical system comprises a translation stage.

220. A system according to any one of claims 203 to 219, wherein the optical system adjusts focus using optical scanning.

221. A system according to any one of claims 203 to 220, further comprising a mechanical component for adjusting the focus in the Z axis.

222. 222. The system of claim 221, wherein the mechanical component comprises one or more of a translation stage or a piezoelectric mechanism.

223. A system according to any one of claims 203 to 222, wherein the optical system adjusts the focus in the Z axis using optical scanning.

224. The memory further comprises instructions that, when executed by the processor, cause the processor to: A system according to any one of claims 203 to 223, wherein the optical system is directed to image the structure over a specified volume.

225. The memory further comprises instructions that, when executed by the processor, cause the processor to:

225. The system of any one of claims 203 to 224, wherein the optical system is instructed to translate, rotate or articulate relative to the non-transparent tissue.

226. The memory further comprises instructions that, when executed by the processor, cause the processor to: A system according to any one of claims 203 to 225, wherein the optical system is instructed to image a structure for a specified period of time.

227. The memory further comprises instructions that, when executed by the processor, cause the processor to: A system according to any one of claims 203 to 226, wherein the optical system is instructed to manipulate the structure.

228. The system of any one of claims 203 to 227, wherein manipulating the imaged structure comprises using the excitation source for non-incisional therapy.

229. The system of claim 228, wherein the non-incisional therapy comprises one or more of multiphoton-mediated thermal damage, photodisruption, photocrosslinking, vascular coagulation, and ablation of the structure.

230. The system of any one of claims 203 to 229, wherein manipulating the structure comprises using the excitation source to treat glaucoma.

231. 231. The system of claim 230, wherein treating glaucoma using the excitation source includes using the excitation source to reduce aqueous humor production in ocular tissue.

232. 232. The system of claim 231, wherein using the excitation source to reduce aqueous humor production in ocular tissue includes damaging the ciliary body to reduce aqueous humor production.

233. The system of claim 232, wherein damaging the ciliary body to reduce aqueous humor production comprises one or more of thermally damaging the ciliary body or applying a photodisruption-mediated process.

234. 234. The system of claim 233, wherein treating glaucoma using the excitation source includes increasing aqueous humor outflow using the excitation source.

235. 235. The system of claim 234, wherein increasing aqueous humor outflow using the excitation source comprises performing laser trabeculotomy through the non-transparent tissue of the eye.

236. 236. The system of claim 235, wherein performing laser trabeculoplasty includes performing laser trabeculoplasty directly through the non-transparent tissue of the eye.

237. The system of any one of claims 203 to 236, wherein manipulating the imaged structure includes using the excitation source to prevent or treat a retinal hole, which is optionally a peripheral retinal hole.

238. The system of claim 237, wherein preventing or treating a retinal hole includes identifying and delivering photocoagulation therapy to the retina.

239. The system of any one of claims 203 to 238, wherein manipulating the structure comprises providing photocoagulation and thermal therapy to ciliary body tumors or peripheral choroidal tumors.

240. The system of any one of claims 203 to 239, wherein manipulating the structure comprises optically cross-linking scleral tissue to prevent myopia.

241. The system of any one of claims 203 to 240, wherein manipulating the structure includes visualizing and performing targeted alteration of extraocular muscle function.

242. 242. The system of any one of claims 203 to 241, wherein manipulating the structure comprises visualizing and performing targeted thermal or photocoagulation therapy of orbital fat.

243. The system of any one of claims 203 to 242, wherein manipulating the structure includes visualizing and performing targeted alteration of eyelid tissue.

244. 244. The system of any one of claims 203 to 243, wherein the processor comprises one or more multi-core processors or parallel processing units, optionally comprising a graphics processing unit (GPU).

245. 245. The system of claim 244, wherein the multi-core processor or parallel processing unit is configured to perform flow analysis, and optionally, the flow analysis is performed substantially in real time.

246. A system according to any one of claims 203 to 245, configured to perform any of the methods according to claims 1 to 155.

247. 247. A system according to any one of claims 203 to 246, further comprising an immersion gel according to any one of claims 194 to 199.

248. 1. A kit for imaging or manipulating structures through non-transparent tissue, comprising: An adapter according to any one of claims 156 to 193; and packaging for the adapter.

249. The kit of claim 248, further comprising a binding agent.

250. A kit comprising: a binder; and and packaging for the binding agent.

251. 249. The kit of claim 248, further comprising an immersion medium.

252. A kit comprising: an immersion medium; and packaging for the immersion medium.

253. 1. A kit for imaging or manipulating structures through non-transparent tissue, comprising: A system according to any one of claims 203 to 247; and packaging for the system.

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