Optical clearing of tissues and organs

Abstract

In one aspect, the disclosure relates to clearing compounds capable of rendering at least one tissue transparent for a period of time, compositions comprising the same, pendant molecules bonded to the compounds capable of covalently or non-covalently binding to tissue such as collagen mimetic peptide, and macromolecules derivatized with the same. In another aspect, the compositions can be useful in clinic for visualizing internal anatomical structures and / or for clearing opacities of the eye. In one aspect, the compositions are topical, e.g., eye drops, lotions, or creams, or are injectable. In some aspects, an injection is performed directly into opaque tissue such as neural tissue, connective tissue, internal organs, ocular tissue, or the like. In other aspects, the clearing compound is injected adjacent to the target structure. The clearing compounds can be colorless across the entire visible spectrum, are non-toxic, and are excreted by the subject after visualization of tissue structures.

Description

ATTORNEY DOCKET NO. 221910-2030OPTICAL CLEARING OF TISSUES AND ORGANSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 720,810 filed on November 15, 2024, and of U.S. Provisional Application No. 63 / 766,569 filed on March 4, 2025, each of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Effective tissue clearing is vital for enabling deep, high-resolution imaging of internal biological structures, directly impacting the precision of diagnostics, therapeutic interventions, and advanced imaging techniques. Traditional clearing methods, including hydrophobic, hydrophilic, and hydrogel-based approaches, can effectively reduce scattering but are unsuitable for live tissues due to their toxicity or disruption of essential biological components, thus precluding their use in live organisms. Thus, they are incompatible with real-time, in vivo imaging of dynamic physiological processes.

[0003] Biomedical imaging plays a central role in clinical analysis and medical intervention while allowing for non-invasive studies of complex biological processes. However, optical imaging of biological tissues is fundamentally limited by scattering and absorption of light. In most tissues, the scattering coefficient is 10-1000 times larger than the absorption coefficient; thus, scattering processes can severely limit the imaging depth and spatial resolution in conventional microscopy. For this reason, the ability to achieve significant reductions in light scattering holds promise for transforming brightfield, fluorescence, nonlinear, and super-resolution imaging techniques.

[0004] Light scattering in tissue originates from the optical contrast between low refractive index (Rl) aqueous-based components (e.g., the interstitial fluid and cytosol) and high Rl lipid-based components (e.g., the plasma membrane, myelin, and myofibrils). Existing methods to reduce optical contrast usually replace water with high-RI chemicals or remove lipids to yield an all-aqueous environment. Despite their success, these approaches are seldom employed in live tissues as they involve the use of toxic substances (e.g., tetrahydrofuran and acrylamide) and removal of molecules vital to sustaining life (e.g., water and lipids).

[0005] Major advances in medicine and biology have been driven by imaging techniques such as microscopy, X-ray, and Magnetic Resonance Imaging (MRI). While methods like MRI and X-ray provide indirect measurements of tissue function, they are limited in capturing changes atATTORNEY DOCKET NO. 221910-2030biological timescales. Current non-invasive optical imaging techniques offer rapid assessment of tissue function and structure but are hindered by strong light scattering in biological tissues, restricting penetration depth and resolution.

[0006] Previous approaches to achieving optical transparency in vivo rely on dye molecules with major absorption peaks in the visible spectrum, such as tartrazine, which features peak absorption at 428 nm and lingering absorption up to 600 nm. Consequently, optical transparency is limited to wavelengths above 600 nm, restricting the application of this approach for imaging shorter-wavelength fluorophores, such as commonly used green and yellow fluorescent proteins (GFP and YFP, respectively).

[0007] Opacities of the eye such as corneal scars and cataracts are responsible for vision loss and can make inspection of the eye by medical personnel more difficult. Current treatments for blindness and vision loss induced by eye opacities is surgical in nature. However, even surgical treatment of corneal opacities is limited due to a lack of available tissue for transplantation. No non-surgical means are available for treating corneal scars or cataracts, although as many as 90% of people in the United States will develop cataracts by age 65, and many of these individuals wish to avoid the time, expense, and recovery time associated with surgery. Even temporary clearing of the cornea or cataract may have diagnostic benefits as it would allow health care providers to visualize or image the posterior structures of the eye for pre-operative planning purposes. Moreover, the ability to optically clear the normally opaque sclera opens up new diagnostic and adjunctive therapeutic opportunities to manage eye disease. Conferring a temporary “window” to the inner structures and layers of the eye could have benefit in the monitoring and / or treatment of ocular conditions, and may also enable better visualization during the creation of a surgical incision (e.g. to drain fluid or blood or to access a tumor) or during the placement of an instrument of device in the suprachoroidal space, for instance.

[0008] It would be desirable to develop non-invasive optical imaging techniques for the purpose of assessing tissue function and structure through the use of clearing compounds and / or compositions that reduce light scattering in biological tissues, including for the purpose of treating corneal opacities and diagnosing disorders of the eye and other organs. It would further be desirable if use of the clearing compounds enabled greater penetration depth and resolution than current methods. Additionally, it would be desirable for clinicians to be able to visualize certain structures of the body that are otherwise difficult to observe by temporarily clearing otherwise opaque tissue using biomicroscopy, optical coherence tomography, or other imaging modalities.ATTORNEY DOCKET NO. 221910-2030It would further be desirable if compounds and / or compositions enabling the clearing were nontoxic, biocompatible, and inexpensive, and allowed tissue transparency across the entire visual spectrum. These needs and other needs are satisfied by the present disclosure.SUMMARY

[0009] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to clearing compounds including, but not limited to, amino acids and vitamins capable of rendering at least one tissue transparent for a period of time, compositions comprising the same, pendant molecules bonded to the compounds capable of covalently or non-covalently binding to tissue including, but not limited to, collagen mimetic peptide (CMP), and macromolecules including, but not limited to, glycosaminoglycans derivatized with the same. In another aspect, the disclosed compositions can be useful in clinic for visualizing internal anatomical structures and / or for clearing opacities of the eye. In one aspect, the disclosed compositions can be topical, such as, for example, eye drops, lotions, or creams, or can be injectable. In some aspects, an injection is performed directly into the opaque tissue including, but not limited to, neural tissue, connective tissue, internal organs, ocular tissue, or the like. In other aspects, the clearing compound is injected adjacent to the target structure to render the target structure transparent. In some aspects, the clearing compounds are colorless across the entire visible spectrum, are non-toxic, and are excreted by the subject after visualization of tissue structures is completed.

[0010] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover,ATTORNEY DOCKET NO. 221910-2030in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0012] FIG. 1 shows change over time in rabbit sclera samples immersed in different clearing solutions as disclosed herein, with a control sample in the right-hand column using phosphate buffered saline (PBS) exhibiting no change.

[0013] FIG. 2 shows a time series of immersion of an excised cornea into two different disclosed solutions (top and bottom, respectively).

[0014] FIG. 3 shows whole eyeball immersion for PBS control and two different disclosed solutions.

[0015] FIG. 4 shows optical coherence tomography (OCT) of the sclera plus the macula for a PBS control and treated sclera and macula using two different disclosed solutions.

[0016] FIG. 5 shows scleral injection of a disclosed solution.

[0017] FIG. 6 shows the results of scleral injection of a disclosed solution over time. High scleral transparency develops at 15-20 minutes with a return to initial state after dipping in PBS.

[0018] FIG. 7 shows the effects of whole eyeball soaking in a disclosed solution for one day. Sclera is effectively made transparent after injection or immersion, but melanin pigment in the choroid is not made transparent.

[0019] FIG. 8 shows an experimental (top row) and control (PBS solution) treatment of corneal alkali burn over time.

[0020] FIG. 9 shows OCT images of treatment of corneal alkali burns with a disclosed solution.

[0021] FIG. 10 shows OCT images of treatment of corneal alkali burns with a disclosed solution.

[0022] FIG. 11 shows treatment of corneal alkali burn with a disclosed solution before treatment and after 40 min.

[0023] FIG. 12 shows the opacity change from soaking a pair of lenses in balanced salt solution (BSS) or an experimental solution over time.

[0024] FIGs. 13A-13D show representative optical photographs of (FIG. 13A) domestic pigmented pig eyeballs ex vivo, and (FIG. 13B) New Zealand White rabbit (NZW) and (FIG. 13C) Brown Norway (BN) rat eyeballs in vivo, after subconjunctival injection of 2 L arginine. The initially opaque conjunctiva and sclera became transparent, creating a window (blue arrows) thatATTORNEY DOCKET NO. 221910-2030allowed visualization of the inner layers. (FIG. 13D) In vivo optical clearing in NZW rabbits (n = 3) before and after subconjunctival injection of arginine or isosmotic saline control. Optical coherence tomography (OCT) images show the structural features of the conjunctiva, subconjunctival space, and sclera. Scale bars= (FIGs. 13A-13C) 5 mm; (FIG. 13D) 1 mm.

[0025] FIG. 14 shows representative light microscopy images of conjunctival / scleral tissue of New Zealand White rabbit after 30 minute immersion in arginine or hypertonic saline. Arginine increased tissue transparency, allowing clear visualization of conjunctival microvasculature, whereas in the opaque sclera, The internal structure could not be visualized due to insufficient light transmission. Scale bar= 20 pm.

[0026] FIG. 15 shows optical coherence tomography (OCT) images of New Zealand White rabbit eyeballs before and after 30-minute immersion in arginine, showing structural changes in the conjunctiva and sclera. Arginine-induced optical clearing of the sclera is accompanied by a reduction in opacity in the OCT images, indicating decreased light scattering. The persistent opaque line at the periphery of the sclera may correspond to fibroblasts. Scale bar = 1 mm.

[0027] FIG. 16 shows New Zealand White rabbit eyeballs were incubated for 30 minutes in hypertonic saline, arginine, glutamic acid, lysine, thiamine, or glycerol with same osmotic pressure to evaluate ex vivo optical clearing. Notable transparency was observed with arginine and thiamine, as well as with glycerol, a previously established optical clearing agent.

[0028] FIG. 17A shows mouse skin prior to application of L-arginine hydrochloride and FIG. 17B shows the same mouse skin after application of L-arginine hydrochloride. Dosage: 2.5 g / kg of body weight.

[0029] FIG. 18A shows a live mouse prior to application of L-arginine hydrochloride and FIG. 18B shows the same live mouse after application of L-arginine hydrochloride. Dosage: 2.5 g / kg of body weight.

[0030] FIGs. 19A-19C show optical absorption spectra of (FIG. 19A) weakly absorbing amino acids, (FIG. 19B) moderately absorbing amino acids and (FIG. 19C) strongly absorbing amino acids. Spectra were measured at 0.5 mg / mL.

[0031] FIGs.20A-20B show (FIG. 20A) Rl change per molar concentration versus -log([H+]+[OFT ]) for twenty amino acids. (FIG. 20B) Rl change per mass concentration versus -log([H+]+[OH']) for twenty amino acids.

[0032] FIGs. 21A-21I show (FIG. 21 A) transmittance T of Cys-Na+solution with an optical pathATTORNEY DOCKET NO. 221910-2030length of 10 pm measured at different concentrations, showing the transmission window across the entire visible region. (FIG. 21 B) Absorption of Cys-Na+solution at different concentrations.(FIG.21 C) Dependence of n' on molar concentrations of Cys Na+, Gly Na+, NaCI and NaOH. (FIG.21 D) Transmittance T of Arg-HCI solution with an optical path length of 10 pm measured at different concentrations, showing the transmission window across the entire visible region. (FIG.21 E) Absorption of Arg-HCI solution at different concentrations. (FIG. 21F) Dependence of n' on molar concentrations of Arg-HCI, Gly-HCI, NaCI and HCI. (FIG. 21G) Molar absorption coefficient of Cys-Na+, Arg-HCI, Cys-HCI, Gly-Na+and Gly-HCI. (FIG. 21 H) Dependence of OD on molar concentrations of Cys-Na+, Arg-HCI, Cys-HCI, Gly-Na+and Gly-HCI. (FIG. 211) An' and An" between Cys-Na+and Cys-HCI, along with a simulation of An" from An' and Kramers-Kronig relations.

[0033] FIGs. 22A-22C show brightfield transmission images of the mouse skin taken at 0 min, 30 min, 1 h, 3 h and 10 h after soaking in a (FIG. 22A) 2.5 M Cys-Na+ solution, (FIG. 22B) 2 M Arg-HCI solution, and (FIG. 22C) 2.5 M osmolarity-matched NaCI solution. Scale bars: 5 mm.

[0034] FIGs. 23A-23D show. (FIG. 23A) bright field image of an anesthetized mouse in a supine position after hair removal and preprocessing, prepared for treatment in (FIG. 23B). (FIG. 23B) Visualization of abdominal organs through the transparency window enabled by Cys-Na+. (FIG.23C) Bright field image of an anesthetized mouse in a supine position after hair removal and preprocessing, prepared for treatment in (FIG.23D). (FIG.23D) Visualization of abdominal organs through the transparency window enabled by Arg-HCI.

[0035] FIGs. 24A-24B show UV-vis absorption spectra of three FDA-approved compounds having Enax of from about 104to about 105M’1cm’1: FIG. 24A methylene blue; FIG. 24C, indocyanine green.

[0036] FIGs. 25A-25B show overlaid simulated real and imaginary parts of the refractive index for (FIG. 25A) methylene blue and (FIG. 25B) indocyanine green.

[0037] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.ATTORNEY DOCKET NO. 221910-2030DETAILED DESCRIPTION

[0038] Disclosed herein are compounds that, when applied to tissues of the eye, render opacities transparent. In one aspect, opacities include corneal scars, cataracts (e.g. nuclear sclerosis, cortical opacities, and / or both anterior and posterior capsular opacities), as well as the sclera itself. In a further aspect, the compounds that have this effect include certain amino acids, certain vitamins, and derivatives (including oligomers, multimers, and polymers of these including, but not limited to, oligopeptides and polypeptides, where the oligopeptides and polypeptides can include all of the same amino acid or different amino acids) and combinations thereof. Also disclosed are alternative approaches including coupling or tethering the disclosed compounds to macromolecules to confer longer or permanent residence time within target tissues, and / or coupling of the disclosed compounds (and their derivatives) directly to the target tissue to make the effect long-lasting or permanent. An example of such a derivative is one in which the clearing compound is conjugated with a collagen mimetic peptide, which is able to non-covalently bind to collagen in tissues. In another example, the clearing compound is derivatized with an active ester group that can covalently react with functional groups such as amines in tissues. In another example, the clearing compound is conjugated with a functional group capable of undergoing a chemical reaction with another functional group either naturally presented or added to tissue; such reactions include but are not limited to click chemistry reactions, disulfide reactions, ring-opening reactions, and combinations thereof. In an aspect, resulting interactions between the clearing compound and the tissue can be covalent or non-covalent (e.g. hydrogen bonds, van der Waals interactions, host-guest interactions, supramolecular interactions or other types of non-covalent interactions) as well as reversible interactions. In another aspect, a molecule conjugated to the clearing compound undergoes a polymerization reaction that causes the resulting macromolecule to become entangled with the extracellular matrix of the tissue, thus entrapping the clearing compound within the tissue. In one aspect, the agents can be delivered topically, by injection (e.g. subcutaneous, subconjunctival, interscleral, suprachoroidal, subretinal, intrastromal, or the like), via a drug delivery modality such as a therapeutic contact lens or depot / implant, or some combination of these.Useful Clearing Compounds

[0039] In one aspect, compounds useful herein include natural amino acids, salts of natural amino acids, vitamins or salts thereof, sugar alcohols, dyes, or any combination thereof. In one aspect, the natural amino acid can be selected from alanine, cysteine, aspartic acid, glutamicATTORNEY DOCKET NO. 221910-2030acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, or any combination thereof. In a further aspect, the salt of the natural amino acid can be a hydrochloride salt or a sodium salt such as, for example, arginine-HCI or cysteine-Na. In one aspect, the vitamin or salt thereof can be sodium ascorbate or niacinamide. In a further aspect, the sugar alcohol can be sorbitol. In another aspect, the dye can be selected from methylene blue, indocyanine green, or any combination thereof. In an aspect, the dye is not tartrazine, ampyrone, or sunset yellow.

[0040] In one aspect, a clearing compound is biocompatible. In another aspect, a clearing compound absorbs UV light and / or light in the violet / blue region of the visible spectrum to be highly refractive in the visible spectrum. In many aspects, it is desired for the clearing compound to be colorless, since application of colored compounds from the above list may produce acceptable clearing results, but would impart undesired color to the vision of a subject to whom the treatment was applied. However, in some aspects, when the desired length of time for clearing is short or temporary, a colored compound may be used.

[0041] In any of these aspects, the clearing compound or salt thereof can be soluble in water or a saline solution for ease of administration to the aqueous environment of the eye and / or other bodily tissues.Length of Treatment

[0042] In one aspect, the clearing result may be temporary; for example, to facilitate an eye exam or as a way of visualizing a tumor or other material (such as blood) internal to the eye, such as, for example, in the suprachoroidal space prior to conducting a surgical treatment or to assess the effectiveness of chemotherapy. In another aspect, the desired clearing may be longer-term, such as, for example, to clear a corneal burn or cataract. Further in this aspect, the clearing compound may be conjugated to a larger polymer such as, for example, polyethylene glycol, hyaluronic acid (HA) dextrose, cellulose, collagen, oligopeptides or polypeptides and / or derivatives thereof, to enable the clearing compound to persist in the ocular tissue for longer due to much slower diffusion times. In an aspect, the oligopeptides and / or polypeptides can include a single amino acid or a combination of amino acids. In one aspect, longer tissue persistence enables production and use of an eye drop that can be administered at home, by a patient, once or several times per day. In another aspect, the disclosed compounds and derivatives can be administered in a therapeutic contact lens (either by soaking the lens in the solution or conjugating the clearing compound to the surface of the lens), by injection, by microneedle-based delivery, byATTORNEY DOCKET NO. 221910-2030implantation, or another means such as iontophoresis, negative or positive pressure, or any combination thereof.

[0043] In another aspect, the treatment can last on the order of days, weeks, or months. In yet another aspect, the treatment can be permanent.Theoretical Background

[0044] Disclosed herein is a strategy for tissue clearing that enables effective modulation of the refractive index of a specific ocular tissue component by tuning its absorbance. This approach leverages the Kramers-Kronig (K-K) relation, which causally connects the real and imaginary components of the refractive index of a material. Specifically, by increasing the absorbance of the low-index components in the tissue (usually containing more water), its Rl is modulated to match that of high-index components (usually containing less water) to reduce scattering at their interfaces. Since Rl modulation occurs at a different wavelength than the absorption peak, this approach yields the counterintuitive effect that greater absorption leads to an increase in transmission.Processes for Tissue Transparency

[0045] In one aspect, existing methods for tissue transparency require toxic organic solvents having a high refractive index to reduce the refractive index mismatch between scatterers and aqueous background, or to remove scatterers inside biological tissues. In a further aspect, existing methods can only be applied to achieve optical transparency in fixed tissues from specific organs of interest, for example in post mortem examination or post-surgical histological examination. In some aspects, existing methods involve the replacement of original tissue components with exogenous chemicals, including, but not limited to, replacement of cellular lipids with hydrogels. In a further aspect, existing methods may further involve electrophoresing an excised specimen, exposing the specimen to hydrodynamic pressure, microwave radiation, or ultrasonic vibration.

[0046] In still another aspect, existing methods may be able to preserve three-dimensional structures of tissues, but the tissues must still be removed from the body of a subject and, for example, directly contacted with an exogenous component or composition such as, for example, a tissue clearing composition, a surfactant (e.g. a non-ionic surfactant such as a saponin), a buffer, an enzyme, an anticoagulant, a solvent (e.g. acetone), a non-ionic density gradient medium (e.g. a phthalimide), or any combination thereof. In one aspect, existing methodsATTORNEY DOCKET NO. 221910-2030employing such exogenous components as listed herein still require tissue removal from the subject for achieving optical transparency. In a further aspect, the present methods do not require use of some or all of the above-listed components.

[0047] By contrast, in one aspect, the disclosed method is based on refractive index modulation of existing tissue components using the K-K relation, thus enabling minimally invasive tissue transparency in the eye of live subjects. In another aspect, the disclosed method does not require performing a surgical procedure on a living subject to access tissue. In still another aspect, the disclosed method does not require use of toxic solvents, fixatives, or the like, in order to visualize tissues.Optical Clearing in Tissues

[0048] In an aspect, light refraction and reflection occur at interfaces when refractive indices change. In another aspect, biological systems such as tissues are inhomogeneous media with different length scales and refractive indices. In still another aspect, reducing the refractive index mismatch between scatterers and background inside tissues can increase light transmission.

[0049] In another aspect, the pigments and non-pigment compounds useful in the disclosed methods are minimally toxic, have good water solubility, and are safe for topical administration. In another aspect, the pigments can diffuse or be injected into the biological tissues. In still another aspect, the disclosed method enables a significant increase in optical transmission in otherwise turbid biological tissue. In one aspect, the method can be conducted in live subjects, achieving transmission in various tissues of the eye without the need for invasive surgical procedures. Further in this aspect, complete recovery of the subjects after performing the method is observed.Modulation of Refractive Index

[0050] In one aspect, the real part (n) and imaginary part (k) of the refractive index of a material are related by the Kramers-Kronig (K-K) relation. In another aspect, in the frequency domain, the Kramers-Kronig relationship can be represented by the following equation:>

[0051] In another aspect, the Kramers-Kronig relation can be rewritten in the wavelength domain as follows:ATTORNEY DOCKET NO. 221910-2030

[0052] In still another aspect, by increasing the imaginary part of the refractive index (absorption of the material) the real part of the refractive index will have a nonlinear change in the neighboring wavelength. Further in this aspect, the refractive index in the longer wavelength will increase.

[0053] In one aspect, the real part and imaginary part of refractive indices of dye solutions and other clearing compound solutions can be measured by any technique known in the art, such as using an ellipsometer. In another aspect, the real part of the refractive index can be modulated by increasing the imaginary part accordingly.Method for Imaging an Organ or Tissue in a Subject

[0054] In one aspect, disclosed herein is a method for imaging an organ or tissue in a subject, the method including at least the steps of (a) administering a composition comprising a clearing compound to the subject, wherein an interaction between the clearing compound and at least one ocular tissue in the subject creates a transparent spectral window in the at least one ocular tissue; and (b) visualizing the organ or tissue through the at least one overlying tissue. In another aspect, the transparent spectral window is in a UV-Visible region of the electromagnetic spectrum, such as, for example, from about 400 nm to about 800 nm or from about 380 nm to about 780 nm, or the entire visible spectrum.

[0055] In one aspect, the organ or tissue can be selected from the brain, the gastrointestinal tract, muscle, connective tissue, bones, blood vessels, the liver, the bladder, a tumor, the spinal cord, the heart, the liver, the pancreas, the spleen, the lungs, the trachea, the kidneys, lymph nodes, the thymus, the ovaries, the testes, the uterus, the prostate, an ocular tissue, or any combination thereof.

[0056] In one aspect, the composition can be administered topically or by injection. In a further aspect, the at least one overlying tissue can be or include skin, muscle, epithelial tissue, connective tissue, or any combination thereof. Further in this aspect, the composition can be administered to the subject by injection, intravenously, subcutaneously, topically, as a depot preparation, as an implant, or any combination thereof.

[0057] In another aspect, the organ or tissue can be an ocular tissue such as, for example, the sclera, the cornea, the lens, or any combination thereof. In another aspect, when the organ orATTORNEY DOCKET NO. 221910-2030tissue is an ocular tissue, the composition can be administered topically, intracorneally, intrasclerally, intracamerally, intravitreally, subconjunctivally, intracamerally, by sub-Tenon administration, suprachoroidally, subcapsularly, intralenticularly, subretinally, by peribulbar administration, in therapeutic contact lenses, or by retrobulbar administration.

[0058] In one aspect, the disclosed interaction can be a covalent interaction or non-covalent interaction. In another aspect, the non-covalent interaction can be hydrogen bonding, hydrophobic interactions, van der Wais interactions, host-guest interactions, supramolecular interactions, or any combination thereof, while the covalent interaction includes but is not limited to an active ester group that reacts with an amine functional group in tissue, aldehyde-mediated crosslinking, click chemistry, disulfide bond formation, a ring-opening reaction, or any combination thereof.

[0059] In one aspect, the subject is a mammal, a bird, a reptile, an amphibian, a fish, an arthropod, a mollusk, a cnidarian, an echinoderm, an annelid, a flatworm, a nematode, ora plant. In a further aspect, the mammal is a human, rat, mouse, rabbit, vole, tree shrew, guinea pig, hamster, cat, dog, pig, sheep, cow, horse, or non-human primate. In still another aspect, the bird is a chicken, turkey, duck, parrot, or finch.

[0060] In any of these aspects, the organ or tissue can be visualized in situ in the subject. In another aspect, performing step (a) reduces light scattering between two or more tissue components, the tissue components having different refractive indices. In a further aspect, performing step (a) increases light transmittance through the at least one overlying tissue by at least 50-fold compared to light transmittance through the at least one overlying tissue before performing the method.

[0061] In an aspect, performing the method allows visualization of at least one feature in the subject at least 200 pm below a skin or other external surface of the subject. In another aspect, from about 0.1 mg to about 20 g, from about 0.1 mg to about 10 g, about 0.1 mg to about 1 g, about 0.1 mg to about 5 mg, or about 0.1, 0.5, 1, 25, 50, 100, 250, 500, or 750 mg, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 g of the clearing compound are administered per kg of body weight of the subject. In one aspect, performing the method results in a local concentration of the clearing compound in the at least one overlying tissue of from about 1 pM to about 1 M, or from about 1 pM to about 0.75 M, or about 1 mm to about 0.65 M, or from about 0.16 M to about 0.62 M, or about 1, 5, 10, 25, 50, 75, 100, 250, 500, or 750 pM, 1, 5, 10, 25, 50, 75, 100, 250, 500, or 750 mM, or about 1 M.ATTORNEY DOCKET NO. 221910-2030

[0062] In any of these aspects, the clearing compound can be non-toxic. In another aspect, following visualizing, the compound is excreted by the subject. In one aspect, the clearing compound is excreted in less than about 10 hours.

[0063] In still another aspect, visualizing can be accomplished using reflectance imaging, fluorescence imaging, laser speckle imaging, two-photon excitation spectroscopy, optical coherence tomography (OCT), light sheet microscopy, super-resolution microscopy, epifluorescence microscopy, fluorescence mediated tomography, photoacoustic tomography, three-photon microscopy, Brillouin microscopy, Raman microscopy, confocal microscopy, TIRF microscopy, brightfield / darkfield microscopy, DIC microscopy, structured illumination microscopy, or a combination thereof.

[0064] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0065] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0066] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0067] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaningATTORNEY DOCKET NO. 221910-2030derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0068] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to 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 herein can be different from the actual publication dates, which can require independent confirmation.

[0069] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0070] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0071] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0072] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to includeATTORNEY DOCKET NO. 221910-2030examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0073] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a vitamin,” “a salt solution,” or “an amino acid,” include, but are not limited to, mixtures or combinations of two or more such vitamins, salt solutions, or amino acids, and the like.

[0074] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0075] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, 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 disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. 'about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, 'about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0076] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitlyATTORNEY DOCKET NO. 221910-2030recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0077] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0078] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a compound refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. achieving the desired level of transparency of a given eye structure. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the type of eye condition, degree of opacity resulting from the eye condition, desired length of time of transparency, and any other concurrent or subsequent treatments to be performed.

[0079] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0080] A “clearing compound” as used herein refers to a compound that, when administered to the eye of a subject, renders at least one ocular tissue transparent for a period of time. In anATTORNEY DOCKET NO. 221910-2030aspect, clearing compounds as disclosed herein include vitamins, amino acids, and biocompatible dyes as described above. In some aspects, the singular “clearing compound” refers to one compound but, in other aspects, “clearing compound” can refer to two or more vitamins, amino acids, a mixture of a vitamin and an amino acid, or any combination of disclosed clearing compounds.

[0081] As used herein, “overlying tissue” refers to a tissue in the body of a subject positioned over an organ or tissue that is desired to be visualized. The overlying tissue will typically have a different refractive index from the organ or tissue to be visualized and this can create scattering when standard visualization methods are attempted. In one aspect, application of a disclosed clearing compound or composition to the overlying tissue can create a transparent spectral window in the overlying tissue, allowing visualization of structures beneath. In one exemplary aspect, an overlying tissue could be skin, through which muscle is visualized, or an at least one overlying tissue could include both skin and muscle, through which bone, blood vessels, or neural tissue could be visualized. In any of these aspects, the overlying tissue can remain in place while visualization occurs, without the need for surgical intervention, laparoscopy, or the like.

[0082] As used herein, “administering” can refer to an administration that is topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intradermal, or any other useful means for clearing tissues and / or organs in an area of interest.

[0083] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g., human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0084] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed clearing compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0085] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).Pharmaceutical Compositions

[0086] In various aspects, the present disclosure relates to pharmaceutical compositions comprising an effective amount of at least one disclosed clearing compound or a pharmaceuticallyATTORNEY DOCKET NO. 221910-2030acceptable salt thereof. As used herein, “pharmaceutically-acceptable carriers” means one or more of a pharmaceutically acceptable diluents, preservatives, antioxidants, solubilizers, emulsifiers, coloring agents, releasing agents, coating agents, and adjuvants. The disclosed pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy and pharmaceutical sciences.

[0087] The clearing compounds described herein are typically to be administered in admixture with suitable pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The deliverable compound will be in a form suitable for topical administration, intravenous injection, or parenteral administration. Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used. The clearing compounds may be administered as a dosage that has a known quantity of the clearing compounds.

[0088] Pharmaceutical compositions of the present disclosure can be in a form suitable for topical administration. As used herein, the phrase “topical application” means administration onto a biological surface, whereby the biological surface includes, for example, a skin area (e.g., hands, forearms, elbows, legs, face, nails, anus, and genital areas) or a mucosal membrane including, but not limited to, the eye of a subject. By selecting the appropriate carrier and optionally other ingredients that can be included in the composition, as is detailed herein below, the compositions of the present invention may be formulated into any form typically employed for topical application. A topical pharmaceutical composition can be in a form of a cream, an ointment, a paste, a gel, a lotion, milk, a suspension, an aerosol, a spray, foam, an eye drop, a dusting powder, a pad, and a patch. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the present disclosure, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the clearing compound, to produce a cream or ointment having a desired consistency.

[0089] In one aspect, the clearing compound can also be delivered within nanoparticles, liposomes, liposomal nanoparticles, and other drug delivery modalities. In a further aspect, the clearing compound can also be stored and released from a hydrogel sheet or dressing and placed over the tissue to be cleared (e.g. the skin, or the surface of an internal organ), or in the case of the eye, a hydrogel contact lens. In one aspect, a hydrogel contact lens that releases the clearingATTORNEY DOCKET NO. 221910-2030agent is itself transparent, so both the contact lens and the underlying tissue are transparent during the treatment period. In some aspects, the tissue remains transparent for an extended period or time and in another embodiment, it is permanently cleared. In some aspects an energy source such as light can be used to render a longer lasting effect by causing a reaction that makes the clearing agent persist within the extracellular matrix of the tissue.

[0090] In another aspect, the disclosed compounds and derivatives can be administered by microneedle-based delivery, by implantation, or another means such as iontophoresis, negative or positive pressure, or any combination thereof.

[0091] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., one atmosphere).Applications of the Disclosed Compositions and Methods

[0092] The scope of the invention encompasses various methods of use of the compositions disclosed herein. The scope of the invention encompasses a method of achieving optical transparency in a selected tissue of a subject by the use of or more optical clearing compositions disclosed herein. In various embodiments, the method of use may be performed in the context of research, performance of a diagnostic procedure, or to aid in the administration of a treatment or intervention, e.g. performance of a surgical treatment or the placement or guidance of an implant, endoscopic instrument, injection, or device.

[0093] The subject of the method may be a live animal, for example a human patient, or a nonhuman animal such as a research animal (e.g. mouse, rat, or non-human primate), a pet (e.g. dog or housecat), and other animals including, but not limited to, livestock (e.g. cow, pig, chicken, or horse), exotic species, and wildlife. In other embodiments, the subject of the method may be a cadaver, an explanted body part, organ or tissue, or an organoid.

[0094] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.EXAMPLES

[0095] The following examples are put forth so as to provide those of ordinary skill in the art withATTORNEY DOCKET NO. 221910-2030a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.Example 1: Ocular Tissue Experiments

[0096] In the Examples that follow, the following solutions were used:Rabbit Lenses

[0097] 7 mm diameter circular sclera samples were immersed in sample or control solutions.FIG. 1 shows change over time in rabbit sclera samples immersed in different clearing solutions as disclosed herein, with a control sample in the right-hand column using phosphate buffered saline (PBS) and exhibiting no change. Solutions 2 and 4 worked well with solution 2 showing the fastest clearing speed.

[0098] Sample solutions were injected into the sclera of a whole eyeball. The clearing effect of solutions 2 and 4 was particularly good. A single injection made a wide area of the sclera transparent. After 24 hours of refrigeration, the sclera returns to the original state.

[0099] FIG. 2 shows time series of immersion of an excised cornea into two different disclosed solutions (top and bottom, respectively). Alkali burn was introduced using 2N NaOH exposure for 10 s in a model with an opaque cornea. Solutions 2 and 4 were tested on 5 mm diameter circles excited from corneas. The clearing effect of sample 2 was faster, even under saturation conditions.

[0100] FIG. 3 shows different views of whole eyeball immersion for PBS control and two different disclosed solutions. Solutions 2 and 4 were tested.ATTORNEY DOCKET NO. 221910-2030

[0101] FIG. 4 shows optical coherence tomography (OCT) of the sclera plus the macula for a PBS control and treated sclera and macula using two different disclosed solutions.

[0102] Overall, solutions 2 and 4 showed good clearing effects, with solution 2 having the fastest clearing rate. Eyeballs injected with solutions 1 and 3 did not show significant clearing effects. Furthermore, eyeballs injected with solutions 2 and 4 showed an increase in the cleared area over time.

[0103] When PBS was injected into the corneal stroma, the structures of the collagen lamellae were distorted and became opaque. Corneal opacity due to alkali burn could only be cleared by immersing the cut cornea in the solution. Whole eyeball immersion was ineffective.

[0104] Overall, it was difficult to achieve penetration or diffusion of the disclosed solutions into the cornea. Exposing the cornea to the solutions as cut tissue was more effective. Corneal penetration ability can be improved; in the case of the sclera, clearing agents are relatively easily absorbed and diffused.Pig Lenses

[0105] FIG. 5 shows scleral injection of a disclosed solution. In a typical experiment, 0.1 mL of a disclosed solution was injected into the suprachoroidal space. Low scleral transparency is observed initially, with high scleral transparency observed after about 20 min. FIG. 6 shows the results of scleral injection of a disclosed solution over time. High scleral transparency develops at 15-20 minutes with a return to initial state after dipping in PBS.

[0106] FIG. 7 shows the effects of whole eyeball soaking in a disclosed solution for one day. Sclera is effectively made transparent after injection or immersion, but melanin pigment in the choroid cannot be made transparent. When the eyeball is immersed in PBS, the transparent ex vivo sclera reverts to an opaque state within 8 min. When dried, it can remain transparent for over an hour.

[0107] A procedure for inducing alkali burn in the cornea is as follows: 50 pL of 2N NaOH is applied to the cornea for about 30 s. The cornea is then irrigated with PBS and allowed to rest for 10 min. For experiments where clearing agent is used, 50 pL of clearing agent are then applied and observation occurs for a period of about 20 min.

[0108] In an experimental (top row) and control (PBS solution) treatment of corneal alkali burn over time, dipping in PBS after one day returns the cornea to the initial state with slightly decreased opacity. FIG. 8 shows an experimental (top row) and control (PBS solution) treatmentATTORNEY DOCKET NO. 221910-2030of corneal alkali burn over time. Different effects can be observed with a typical phone camera versus a polarized lens camera (FIG. 8). Typically, a cornea remains transparent for about 20 min and then gradually returns to an opaque state. FIG. 9 shows OCT images of treatment of corneal alkali burns with a disclosed solution.

[0109] In the chronic model of corneal alkali burn, 50 pL of 2N NaOH is applied to the cornea for about 30 s. The cornea is then irrigated with PBS and allowed to rest for one day. For experiments where clearing agent is used, 50 pL of clearing agent are then applied and observation occurs for a period of about 60 min. Compared to the 10 min waiting period, the effect of the clearing agent appears slowly with this extended wait time. FIG. 10 shows OCT images of treatment of corneal alkali burns with a disclosed solution.

[0110] FIG. 11 shows treatment of treatment of corneal alkali burn with solution 5 before treatment and after 40 min. In this experiment, PBS irrigation was not performed, the sample was rested for 10 min before applying solution 5, and observation was carried out for about 40 min.Lenses and Eves from Additional Species

[0111] The weight of a lens immersed in solution 2 decreased over time. It is believed the clearing agent did not diffuse into the lens, but rather that liquid inside the lens diffused out due to osmotic pressure. Density of the lenses also differed; the lens floated slightly in solution 2 but sank in PBS.

[0112] FIG. 12 shows the opacity change from soaking a pair of lenses in balanced salt solution (BSS) or an experimental solution over time.

[0113] Similar experiments were performed in rat eyeballs and with rat lenses, with comparable results.In Vivo Studies: Methods

[0114] Animals’. New Zealand rabbits and Brown Norway rats (Rattus norvegicus) were purchased from Western Oregon Rabbit Co. and Charles River Laboratories, respectively. A total of n = 3 female rabbits per group, aged 4-8 months, n = 5 female rats per group, aged 8-10 weeks, were randomly assigned to this study. All procedures were reviewed and approved by the Stanford University Institutional Animal Care and Use Committee (APLAC protocol No. APLAC 33413 and 32765). Anesthesia in rats was induced by intramuscular injection of ketamine, xylazine, and distilled water mixed at a 2: 1 :3 volume ratio, administered at 1.68 mL / kg. Rats were housed in standard cages (two per cage) under a 12 / 12h light / dark cycle, with free access to foodATTORNEY DOCKET NO. 221910-2030and water. Ambient temperature and humidity were maintained at standard laboratory levels. Rats showing any signs of pre-existing glaucoma prior to drug administration were excluded from the study. All procedures involving anesthesia, postoperative monitoring, and euthanasia (intravenous administration of pentobarbital, 100 mg / kg) in rabbits were carried out by the Veterinary Service Center (VSC) at Stanford University. All experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

[0115] In vivo optical clearing agent treatment: For all procedures and follow-up examinations, additional topical anesthesia was administered using 0.5% proparacaine hydrochloride ophthalmic solution (Bausch and Lomb, Laval, Canada). A 30-gauge needle was used to deliver a 2 pL subconjunctival injection of the in vivo optical clearing agent or an equimolar hypertonic saline solution into the scleral region where the underlying pigmented choroid was not visible near the cornea. The morphology and thickness of the sclera and adjacent tissues were assessed using optical coherence tomography (OCT; Heidelberg Engineering, Heidelberg, Germany), and scleral clearing was monitored every 5 minutes using a digital portable slit lamp (LED16, Digital Eye Center, Miami, USA). Upon completion of all procedures and imaging, 0.3% ofloxacin ophthalmic solution (Bausch and Lomb) was applied to prevent bacterial infection. Any animals deemed moribund or dead by laboratory or animal care staff according to pre-established protocol criteria was euthanized and recorded as deceased. At 7 days post-injection, the ocular condition was evaluated, after which the animals were euthanized and the eyes were collected for further analysis.

[0116] Ocular histopathology and imaging: Following enucleation, the eyes from each rat were marked with tissue marking dye at the site of injection and fixed in 10% formalin for at least 24 hours. The tissues were subsequently dehydrated through graded alcohol series, cleared in xylene, and embedded in paraffin under standard conditions. Paraffin blocks were serially sectioned along the cornea-optic nerve plane perpendicular to the injection site at a thickness of 5 pm. Every fifth section was stained with hematoxylin and eosin (H&E) for histopathological evaluation. Histological assessment included examination of the conjunctiva, sclera, cornea, iris, choroid, retina, and optic nerve. Specific attention was paid to structural alterations such as cell necrosis, inflammatory cell infiltration, tissue edema, vascular dilation, and hemorrhage that could be associated with exposure to high-concentration solutions. Slides were digitized using an Axio Scan.ZI slide scanner (Zeiss, Germany), and qualitative analyses were performed with QuPath software.ATTORNEY DOCKET NO. 221910-2030

[0117] Cytotoxicity: The cytotoxicity of arginine was evaluated by analyzing the viability of induced corneal endothelial cells (iCECs) exposed to arginine-containing culture medium. iCECs (5 x 105cells) were seeded into culture dishes and allowed to adhere and incubate for 24 hours. The culture medium was then replaced with medium containing arginine at various concentrations, and cells were incubated for 0.5, 1 , 4, 8, or 24 hours. Cell viability was assessed using the Live / Dead Viability / Cytotoxicity Kit (Invitrogen, MA, USA), staining live and dead cells. Images were captured using a confocal microscope (Olympus, Japan), and cell counts in each image were quantified using ImageJ software.In Vivo Studies: Results and Discussion

[0118] Evaluation of ocular safety of in vivo optical clearing agent: Ocular toxicity potentially associated with arginine-based clearing agents can arise via two major mechanisms: inflammation and tissue deformation caused by hyperosmotic high-concentration solutions, and oxidative stress or neurotoxicity due to excessive nitric oxide (NO) production induced by arginine. Our in vitro results have shown that corneal epithelial cells exposed to optical clearing agents for prolonged periods undergo cell death due to rapid osmotic changes. To evaluate whether such effects are relevant to ocular histopathology in vivo, we examined ocular tissues one week after subconjunctival injection of the agent. The eye is a highly perfused organ with active fluid exchange, which allows rapid dilution and clearance of injected solutions compared with solid organs such as subcutaneous tissues. Conjunctival and scleral tissues, rich in lymphatic and vascular networks, exhibit fast clearance in drug delivery systems. The shorter in vivo duration of scleral transparency compared with ex vivo conditions is also caused by this rapid circulation. The regions of the conjunctiva and sclera directly exposed to high-concentration solutions represent the areas with the longest exposure time. Examination of tissues near the cornea-conjunctiva-sclera interface revealed normal morphology and structure, comparable to untreated or positive control tissues. The conjunctival epithelium maintained a thickness of approximately 20 pm with a three-layered epithelial structure, and no inflammation or vesicular cell changes were observed sclera integrity was also similar to that of controls. As transient intraocular pressure changes can occur during dilution of subconjunctivally injected solutions, we assessed the eye for signs of tissue contraction or hypertrophy. Mild hypertrophic changes were observed in some collagen regions lacking cells; however, the morphology and cellular appearance of the cornea, iris, and retina / choroid remained comparable to normal and control tissues. In the optic nerve, no axonal or glial cell degeneration or shrinkage was detected, and no inflammatory cell infiltration was observed across all ocular tissues.ATTORNEY DOCKET NO. 221910-2030

[0119] In vivo and ex vivo optical clearing of sclera: In mammals, the sclera exhibits lower collagen fiber density than the adjacent cornea, but its randomly arranged fibers induce strong light scattering, rendering the tissue opaque. Immersion in a 2.6 M arginine solution reduces both the dissociation of collagen fibers and the refractive index mismatch between collagen and body fluids, thereby decreasing light scattering and achieving scleral transparency. This effect was demonstrated in domestic pig eyeballs, New Zealand White (NZW) rabbits, and Brown Norway (BN) rats. Following subconjunctival injection of arginine, the surrounding sclera became transparent within 5 minutes, creating a localized optical window that revealed the underlying ocular tissue. The duration of transparency is dictated by physiological dilution. In ex vivo pig eyeballs, where intraocular fluid circulation is halted, the transparent window persisted for over 20 minutes (FIG. 13A). In vivo, the scleral window in rabbits and rats gradually diminished from 10 minutes post-injection and returned to initial state by 20 minutes (FIGs. 13B-13C).

[0120] Structural changes in the surrounding tissue induced by arginine were monitored using optical coherence tomography (OCT). Diffusion of arginine into adjacent tissue induced temporary structural alteration of collagen fibers (FIG. 13D). Beneath the expanded subconjunctival space by injection, the sclera exhibited reduced opacity in OCT, contrasting with the unchanged opacity in saline controls, indicating that arginine specifically decreases light scattering within collagen fibers. The conjunctiva, where light scattering is affected by blood vessels, lymphatics, and cells (FIG. 14), remained unaffected, showing that the clearing effect is restricted to collagen fibers. This effect was most pronounced in ex vivo eyeballs, where fluid circulation is absent. Notably, OCT imaging of ex vivo rabbit sclera cleared with arginine still revealed distinct scleral boundaries, demonstrating that surface tissue and fibroblast scattering are not reduced (FIG. 15). The clearing capability of arginine is comparable to established optical clearing agents such as glycerol and the recently reported tartrazine (FIG. 16). We further observed tissue-clearing effects with thiamine and niacinamide. Importantly, arginine solutions at 550 mg / mL (refractive index, Rl: 1.41) and 300 mg / mL (Rl: 1.37) were both effective in rendering scleral tissue transparent, suggesting that arginine-mediated clearing involves mechanisms beyond simple refractive index matching.

[0121] Cytotoxicity of in vivo optical clearing agent: Although arginine is a biocompatible amino acid, exposure to high concentrations can induce cytotoxicity. To determine appropriate concentrations and exposure durations, live / dead assays were performed using induced corneal endothelial cells (iCECs). Cells (5 x 1 o5) were seeded in culture dishes and incubated for 24 hours, after which the medium was replaced with medium containing arginine at concentrations of 100, 300, or 500 mg / mL. Cells were further incubated for 0.5, 1, 4, 8, and 24 hours, and liveATTORNEY DOCKET NO. 221910-2030and dead cells were stained with Calcein-AM and ethidium homodimer-1, respectively. Cell viability (%) was calculated as the proportion of live cells relative to the total cell count. At all tested concentrations, cytotoxicity became significant after 4 hours, with viability dropping below 60%, and most cells were dead by 8 hours. However, cells exposed for short periods of up to 30 minutes maintained high viability, with 99.8 ± 1.3%, 99.5 ± 1.5%, and 94.1 ± 1.2% survival for 100, 300, and 500 mg / mL, respectively. Extending the exposure to 1 hour, cells exposed to 300 mg / mL, which is the minimum concentration required for optical clearing, remained largely viable (97.8 ± 2.1%), whereas 500 mg / mL resulted in 47.6 ± 3.5% survival. These results indicate that if high-concentration arginine is diluted within 30 minutes in vivo, any cytotoxic effects such as osmotic stress, reactive oxygen species generation, and pH disturbance are likely negligible. In addition to cytotoxicity, subconjunctival administration of arginine may induce transient tissue swelling; however, as the agent is absorbed and diluted within the tissue, OCT images at week 1 post-injection showed normal tissue morphology and structure.Example 2: Full Visible Spectrum ExperimentsMaterials and Methods

[0122] Chemicals'. L-Asparagine (>98%), L-Alanine (>98.5%), L-Arginine (>99.5%), L-Cysteine (97%), L-Glutamic acid (>99%), L-Glutamine (>98.5%), L-H istidine (>99%), L-lsoleucine (>99%), L-Methionine (>98%), L-Proline (>99%), L-Phenylalanine (>98.5%), L-Serine (>98.5%), L-Threonine (>98%), L-Valine (>98.5%), L-Lysine (>98%) and L-Aspartic acid (>99%) were purchased from Sigma-Aldrich Inc. L-Tyrosine (99%) was purchased from Alfa Aesar. Sodium Chloride (99%) and Sodium hydroxide (97%) were purchased from Thermo Fisher Scientific. Glycine, Tryptophan and Leucine were purchased from Bulk Supplement. Hydrochloric acid (HCI, 36.5 to 38.0 w / w %) was purchased from Fisher Scientific Company (Pittsburgh, Pennsylvania, USA). All chemicals were used as purchased without further purification. The 1X phosphate-buffered saline (PBS) was purchased from Gibco (Waltham, Massachusetts, USA). Water was purified with a Millipore Milli-Q Integral 10 water purification system.

[0123] Preparation of 3M Sodium Salt Solutions of Amino Acids’. A 100 mL stock solution of 10 M sodium hydroxide was prepared by dissolving 40 g of NaOH in 60 mL of deionized water in a beaker. After complete dissolution of the NaOH and cooling of the solution to room temperature, the mixture was transferred to a 100 mL volumetric flask. The beaker was rinsed with deionized water, and the rinsings were added to the volumetric flask to ensure complete transfer. Deionized water was then carefully added to the flask until the bottom of the meniscus was aligned with theATTORNEY DOCKET NO. 221910-2030100 mL calibration mark. The flask was capped and inverted several times to ensure thorough mixing of the solution.

[0124] Next, 3 mL of the 10 M NaOH solution was mixed with 3 mL of deionized water and added to 30 mmol of the amino acid in a vial. Ultrasonic treatment was applied to facilitate the complete dissolution of the amino acid. Once fully dissolved, the mixture was transferred to a 10 mL volumetric flask, and the final volume was adjusted to 10 mL with deionized water.

[0125] Preparation of 3M Hydrochloride Salt Solutions of Amino Acids: 2.5 mL of a 12 M HCI solution was mixed with 3 mL of deionized water and added to 30 mmol of the amino acid in a vial. Ultrasonic treatment was applied to facilitate the complete dissolution of the amino acid. Once fully dissolved, the mixture was transferred to a 10 mL volumetric flask, and the final volume was adjusted to 10 mL with deionized water.

[0126] UV-Vis Absorption and Transmission Spectroscopy: All UV-vis absorption spectra of diluted amino acid solutions were measured on a Thermo Fisher Scientific Evolution 350 UV-vis spectrophotometer. Transmission and absorption measurements of concentrated amino acid solutions (i.e., 50 mM to 3 M) were measured on an Agilent Cary 6000i UV / Vis / NIR spectrophotometer.

[0127] For absorption and transmission measurements, a 1 mm optical quartz cuvette (high precision, two polished sides; Science Outlet, Weifang, China) and a 0.01 mm optical quartz cuvette were used (Starna Cells Inc, Atascadero, CA, USA).

[0128] Refractometry Measurements: An Abbe refractometer was used to measure the average Rl of amino acid solutions under ambient light conditions. Before each measurement, the illuminating and refracting prisms were thoroughly cleaned with deionized water and dried. The prisms were then locked in place, and 50 pL of the amino acid solution was applied to the refracting prism, forming a thin film. Ambient light was directed into the refractometer, and the compensator was adjusted via the eyepiece to align the light-dark boundary with the crosshairs. The Rl was read from the scale.

[0129] Ellipsometry Measurements of the Rl of Amino Acid Solutions: A Horiba Jobin Yvon UVISEL ellipsometer was used to measure the real and imaginary components of the refractive index of solutions containing amino acid molecules. To prepare the sample, 320-grit sandpaper was attached to the bottom of the cryomold using double-sided tape. Subsequently, 2.5 mL of the solution was added to the mold, forming a flat, reflective air-liquid interface. The real andATTORNEY DOCKET NO. 221910-2030imaginary Rl spectra of the amino acid solution were measured in the wavelength range of 250 nm to 850 nm, with a reflection angle of 69.85°, a step size of 2 nm, and a dwell time of 200 ms.

[0130] Achieving Optical Transparency in Dissected Tissues: Mice were euthanized using carbon dioxide via inhalation, followed by cervical dislocation to ensure death. Nair (Church & Dwight, Ewing Township, New Jersey, USA) was applied generously to the abdominal area and allowed to remain for 5 minutes before being wiped off with alcohol pads to depilate the skin. A 10 mm x 20 mm rectangle of abdominal skin was excised using surgical scissors. The excised skin was placed in a Petri dish, and its corners were adhered to the bottom of the dish using Max Bond Super Glue (Krazy Glue). The lid of the Petri dish was then closed. The Petri dish was positioned on a transparency film with a 1 x 1 mm grid pattern, which was placed on an LED light board inside a 37 °C orbital shaker. A color charge-coupled device (CCD) camera (DCU224C, Thorlabs) with an array size of 1280 x 1024 and a sensitivity range from 400 to 700 nm was set up. The camera was outfitted with a Xenon variable aperture f / 0.95 lens (25 mm focal length). The camera was positioned to look down at the Petri dish, ensuring the plane of the grid pattern was in focus. The camera was configured to record at 15 frames per second, with a 14 ms exposure time. The amino acid solution (2 M Arg-HCI, 2.5 M Cys-Na+, or 2.5 M NaCI) was carefully added to the Petri dish until it covered the excised mouse skin by at least 1 mm. Images were acquired using the camera at 0 min, 30 min, 1 h, 2 h and 10 h of soaking to document any changes in skin appearance over time.

[0131] Bright Field Imaging of Abdominal Organs in Live Mice: The hair on the mouse’s abdomen was removed with Nair (Church & Dwight, Ewing Township, New Jersey, USA) before the topical application of the amino acid solutions. In this study, both Arg-HCI, 2.5 M Cys-Na+have demonstrated their ability to achieve abdominal transparency. The mouse was then exposed to white light illumination during imaging. Brightfield images were captured by using a home-built imaging system equipped with a color charge-coupled device (CCD) camera (DCU224C, Thorlabs). The CCD camera has an array size of 1280 by 1024 and a sensitivity range from 400 to 700 nm, where the quantum efficiency is above 50%. The camera was outfitted with a Schneider Xenon 25 mm f / 0.95 Lens to capture the mouse abdominal images. No filters were used for imaging. An exposure time of 50 ms was used for taking brightfield images, and a frame rate of 15 fps was used for recording videos in the brightfield mode.Results

[0132] Biological components with high refractive indices (ranging from 1.4 to 1.5) are prevalentATTORNEY DOCKET NO. 221910-2030in living organisms, serving critical structural and functional roles, such as in the lens of the eye, the sarcomeres of muscle fibers, and collagen in the skin. These components are predominantly composed of proteins with intrinsically high refractive indices, arising from their densely packed molecular arrangements and the optical properties of their constituent amino acids. The twenty standard amino acids, as building blocks of these proteins, contribute to the precise organization and function of these biomaterials, influencing their interaction with light and their role in various physiological processes. This raises an intriguing question: which types of amino acid molecules contribute most significantly to the high Rl of these proteins? The refractive index and absorption coefficient, representing the real and imaginary components of the complex refractive index, are interconnected through the Kramers-Kronig relations. This relationship provides a framework for analyzing and predicting light-matter interactions in biological systems. Recent research demonstrates the Rl of an aqueous medium can be manipulated by tuning the absorption properties of solutes.

[0133] As indicated by the Kramers-Kronig relations, the absorption profiles of absorbing molecules in the short-wavelength range all contribute to the increase in the Rl at longer wavelengths of the aqueous medium upon dissolution. The three key criteria for the molecular engineering of an efficient optical clearing agent in the visible region are as follows: (1) to achieve higher Rl enhancement, molecules with stronger absorption peaks — thus higher molar refractivity — are preferred; (2) the Rl perturbation induced by an absorption peak decreases with increasing wavelength following wavelength-dependent dispersion, but broader absorption peaks, for a given intensity, slow this decay and maintain greater Rl enhancement across a wider spectral range; and (3) to ensure optical transparency in the visible region, the absorption peaks of these molecules must lie in the ultraviolet range, thereby avoiding unwanted absorption within the visible spectrum.

[0134] In accordance with these physical principles, an ideal candidate for achieving the disclosed imaging purposes should exhibit strong absorption near the short-wavelength edge of the visible spectrum. To identify potential candidates for Rl enhancement, the UV-vis absorption spectra of twenty common amino acids were measured. As illustrated in FIG. 19A, ten out of the twenty amino acids exhibited extremely weak absorption profiles within the spectral range of 190 nm-750 nm. This weak absorption can be explained by the nature of the chromophores present in their side chains, which primarily consist of aliphatic groups, hydroxyl groups and carboxyl groups. The absorption peaks of these functional groups are usually between 150 nm and 170 nm, which is far from the visible region, making them least favored for Rl enhancement. InATTORNEY DOCKET NO. 221910-2030contrast, the second group of amino acids (FIG. 19B), comprising seven members, exhibited stronger and red-shifted absorption profiles due to the presence of stronger UV chromophores on their side chains, such as guanidine, sulfur, amine, amide, and imidazole groups. Amino acids in this group show promise for enhancing the Rl of the aqueous medium. The third group (FIG. 19C) consists of three amino acids with aromatic side chains, among which tryptophan stands out due to the strong TT-TT* transitions of its indolyl group, making it the most effective UV absorber and the most promising candidate for Rl enhancement.

[0135] To validate these assumptions based on UV absorption profiles, the RIs of amino acid solutions were measured to access their Rl-enhancing capabilities. Reducing light scattering by lipids and proteins, which have RIs between 1.4 and 1.5, requires increasing the Rl of the aqueous medium from 1.33 (the Rl of water) to at least 1.4. Achieving this level of Rl enhancement demands a highly concentrated amino acid solution, typically several molar (M). However, most amino acids have limited solubility at such concentrations. To address this, hydrochloric acid and sodium hydroxide were used to convert amino acids into their hydrochloride and sodium salt forms, respectively, as detailed in the Materials and Methods section, thereby improving their solubility. Most amino acids were able to form a 3 M solution in their sodium salt form, while fewer could form a 3 M solution in their hydrochloride form (Table 2). Of note, tyrosine was the only amino acid that could not be dissolved to achieve a 3 M solution in either form.ATTORNEY DOCKET NO. 221910-2030>

[0136] The RIs of 3 M amino acid hydrochloride and sodium salt solutions were measured using an Abbe refractometer, with the results summarized in Table 2. For practical applications in live animals or humans, the Rl matching solution should have a neutral pH to prevent any potential harm to biological tissues. Therefore, the pH of the prepared solutions was also measured and is included in Table 2. Among the twenty amino acids, tryptophan exhibited the highest Rl in its sodium salt form, aligning with predictions based on its absorption spectra. Interestingly, when amino acids were soluble in both hydrochloride and sodium salt forms, the hydrochloride solutions consistently showed higher Rl than the sodium salt solutions. This difference arises from the higher polarizability of chloride anion compared to sodium cation, which slightly increases the overall Rl of the solution. Furthermore, the RIs of the amino acid hydrochloride and sodium salt solutions across the 250-750 nm range were measured using an ellipsometer, yielding results consistent with those obtained from the refractometer.

[0137] To systematically evaluate the efficiency of amino acid in Rl enhancement and their suitability for practical applications, two key parameters were defined: An' / Ac', representing the molar Rl increment, and -log([H+]+[OH']), which describes the combined concentration of [H+] and [OH-] in the solution. A higher An' / Ac' value indicates greater molar efficiency for increasing the Rl, making the amino acid a better candidate. A -log([H+]+[OH']) value close to 7 suggests a more neutral pH, which is preferable for the Rl matching solutions. As shown in FIG. 20A, most of the amino acids cluster in the bottom-left corner of the graph, indicating their limited capability to enhance Rl and the extreme acidic or alkaline conditions of their aqueous solution. Trp Na+, Phe-Na+and Arg-HCI emerge as the top three candidates for Rl enhancement. However, the -l°g([H+]+[OH']) values for both Trp-Na+ and Phe-Na+ are low, making Arg-HCI the most promising amino acid from a molar concentration perspective. Of note, tryptophan remains a compelling candidate due to its strong Rl enhancement potential, though it requires further molecular engineering to improve its solubility and mitigate the extreme pH of its solutions.ATTORNEY DOCKET NO. 221910-2030

[0138] Furthermore, a parameter, mass refractive index increment was defined: An / Am to evaluate the mass efficiency of amino acid molecules. This parameter addresses the limitations of molar concentration, which can be influenced by the use of larger or polymer molecules, potentially leading to a false impression of efficiency. In contrast, mass concentration avoids this issue, providing a more reliable measure of efficiency. To calculate the mass, the weight of HCI or Na' is added to the molecular weight of the amino acid. FIG. 20B illustrated the relationship between An' Am and -log([H+]+[OH’]) for twenty amino acids. Notably, Cys-Na+exhibits a high An / Am. value of 0.023 while maintaining a near-neutral pH. This value is approximately double those of traditional optical clearing agents, such as propylene glycol (0.011), glycerol (0.014), glucose (0.014), and sucrose (0.013). It is also comparable to the previously reported efficiency of tartrazine, which can increase the Rl of water by -0.028 per 100 mg / mL. While Trp Na+achieves a close value as Cys-Na+, the resulting pH is not suitable for practical use. Additionally, the Rl enhancement capability of Cys-Na+is significantly higher than Cys-HCI, despite HCI contributing more increase in Rl than Na+. The underlying reasons for this observation will be explored in the following sections.

[0139] The optical properties of the top two candidates from this screening, Cys-Na* and Arg-HCI, were further examined. As shown in FIGs. 21 A and 21 D, both solutions exhibit complete transparency across the visible spectrum. Upon increasing concentration, the 100% transmittance edge of Cys-Na+remains nearly constant, whereas Arg-HCI demonstrates a noticeable red-shift, suggesting stronger intermolecular interactions in the Arg-HCI solution. The UV-vis spectra (FIG. 21 B) of Cys-Na+reveals a major absorption peak at 231 nm, with the absorption profile remaining consistent across varying concentrations. For Arg-HCI, the guanidino group of arginine in diluted solutions typically shows an intense absorption peak centered at 178 nm. Although a slight red-shift is observed as the concentration of Arg-HCI increases, it remains insufficient to shift the peak into the measurable range of the instrument (FIG. 21 E). Furthermore, the molar absorption coefficients of Cys-Na', Arg-HCI, Cys-HCI, Gly-Na+and Gly-HCI (FIG. 21G) were measured at 500 mM. The molar absorption coefficient of Arg-HCI, Cys-HCI, Gly-Na* and Gly-HCI are consistent with the dilute solution of their original formula while the strong absorption peak (E = 7350 M’1-crrr1) at 231 nm of Cys-Na+is absent in the absorption spectra of Cys and Cys-HCI. The pKa value of thiol group for Cys is around 8, hence at a pH of 7 to 8, partial of the thiol group will be deprotonated, leading to an electron enriched thiolate group and yielding the strong absorption peak at 231 nm.ATTORNEY DOCKET NO. 221910-2030

[0140] The concentration dependent Rl behavior (FIGs. 21 C and 21 F) was measured for both Cys-Na+and Arg-HCI, taking Gly-Na+, Gly-HCI, NaCI, NaOH and HCI as control groups. NaCI and NaOH demonstrate almost identical Rl enhancement capabilities, while NaCI shows a higher Rl enhancement than HCI. Gly-Na+and Gly-HCI exhibit slightly higher Rl enhancement than NaCI, NaOH, and HCI, indicating that the amino acid backbone has limited efficiency in Rl enhancement. This confirms that the primary contributions of Cys-Na" and Arg-HCI to Rl enhancement arise from their respective side chain groups. Interestingly, an excellent linear relationship is observed across all measured samples, suggesting the intermolecular interactions between solute molecules or between solute and solvent are weak. While the intermolecular interactions in Arg-HCI and Cys-Na+solutions are relatively stronger than those in other solutions, they remain insufficient to induce non-linear behavior, such as positive or negative deviations. Additionally, several other amino acid solutions containing aromatic rings or charged side chains were tested, yet a linear Rl-concentration dependence was consistently observed. When plotting the absorption at 201 nm for the five amino acid salts as a function of concentration (FIG. 21 H), each sample again exhibits a linear relationship consistent with the concentration-dependent Rl measurements. Finally, the difference between the measured molar absorption spectra of Cys-Na+and Cys-HCI as calculated. Using the equation Jn''=2.3031£cA / 47r, An" was derived and Kramers-Kronig relations were applied to simulate the corresponding An'. Interestingly, the simulated An' matches well with the experimentally measured An", extracted from ellipsometry data (FIG. 211). This finding suggests that the higher Rl of Cys-Na+compared to Cys-HCI can be primarily attributed to the absorption peak at 231 nm.

[0141] The feasibility of using Cys-Na+and Arg-HCI solutions to induce optical transparency was then validated in excised mouse abdominal skin. To ensure a valid comparison, the RIs of both Cys-Na* and Arg-HCI solutions were standardized to 1.41, which corresponds to 2.5 M Cys-Na and 2.0 M Arg-HCI. A piece of mouse skin (approximately 10 mm x 20 mm) was glued to the bottom of a Petri dish. The experimental groups were treated with the respective amino acid solutions, while a 2.5 M osmolarity-matched NaCI solution served as the control. As shown in FIGs. 22A-22C, both Cys-Na+and Arg-HCI solutions are capable of inducing optical transparency of mouse skin within a short period of time while no obvious improvement can be seen when the osmolarity-matched NaCI solution was applied. Cys-Na+rendered the skin completely transparent within 3 hours, whereas Arg-HCI required approximately 10 hours to achieve the same effect, highlighting the significantly faster diffusion rate of Cys-Na* in excised skin.ATTORNEY DOCKET NO. 221910-2030

[0142] It was next sought to demonstrate the ability of amino acid solutions to achieve tissue transparency in the mouse abdomen. To achieve optical transparency in live mice using amino acid solutions, 3- to 4-week-old C57BI / 6 mice (9-15 g) were anesthetized with isoflurane and positioned on a heated pad. Their abdominal fur was removed using Nair, with a second application enhancing skin permeability by exfoliating the epidermis. A freshly prepared amino acid solution was applied topically using a cotton applicator, massaged into the depilated skin for 10 minutes to ensure penetration. As shown in FIGs. 23A-23B, treatment with a 2.5 M Cys Na* solution rendered the abdominal region of the mouse transparent, indicating a suppression of light scattering caused by Rl mismatch. Unlike previous reports where tartrazine-induced transparency resulted in an orange-to-red skin coloration, the skin retained its natural color, confirming transparency across the entire visible spectrum. Through the abdominal skin, internal organs such as the liver, cecum, spleen, small intestine, and bladder were clearly visible. Similarly, as shown in FIGs. 23C-23D, a 2 M Arg-HCI solution achieved a comparable effect, producing a transparent abdomen where the liver, cecum, small intestine, and bladder could be distinctly identified. Notably, while Arg HCI required slightly more time than Cys Na+to achieve transparency, a clear optical window was successfully established within 20 minutes of topical application.Conclusion

[0143] It was demonstrated that amino acids, as the building block to proteins, exhibit strong UV absorption and, according to the Kramers-Kronig relation, can modulate the Rl of the aqueous medium in the visible region. Among the twenty common amino acids, Cys Na+and Arg HCI were identified as having both exceptional Rl-enhancing capabilities and excellent biocompatibility. By matching the Rl differences, intrinsic light scattering within biological tissues can be significantly reduced, thereby achieving transparency in excised mouse skin. Furthermore, topical application of these amino acid solutions to the abdomen of live mice can reversibly transform opaque tissues into a transparent medium. This “transparent abdomen,” effective across the entire visible spectrum, enables direct observation of internal organs and their dynamic movements and activities in live animals. One potential concern with the use of high-concentration amino acid solutions is the introduction of hypertonic osmolarity, which can lead to tissue dehydration. Nevertheless, such limitation could be addressed by synthesizing polypeptides or proteins composed of high Rl amino acids. Notably, arginine, which favors a protonated form, and cysteine, which is more efficient in its deprotonated form, may interact to form an internal zwitterionic salt. This interaction could neutralize charges, lower the osmolarity, and mitigate adverse effects on tissues. Arginine has been identified as one of the most abundant amino acidsATTORNEY DOCKET NO. 221910-2030in human y-crystallin, a protein highly concentrated in the lens nucleus that exhibits the highest Rl among all crystallins. This natural example highlights how evolution has optimized proteins to achieve both high RIs and biocompatibility in the transparent lens. Inspired by this, these findings suggest that engineering high-RI proteins primarily composed of arginine and cysteine could mimic this biological design. Such synthetic proteins may hold great potential for applications in tissue-clearing technologies, where reducing light scattering through Rl matching is essential. The deliberate selection and combination of these amino acids could provide a scalable strategy for developing biocompatible, transparentizing agents for both research and medical applications, opening new avenues for optical imaging and diagnostics in live tissues.Example 3: Optical Clearing with FDA-Approved Dyes

[0144] To overcome light scattering in biological tissues — manifesting as reduced clarity, limited depth penetration, and compromised resolution — the disclosed strategy applies the Lorentz oscillator model and Kramers-Kronig relations to incorporate FDA-approved dyes such as methylene blue and indocyanine green (ICG) into the aqueous compartments of tissue, precisely tuning their refractive index to match protein and lipid-rich structures.

[0145] Methylene blue (FIG. 24A), authorized for the treatment of methemoglobinemia and utilized in sentinel lymph node mapping, displays prominent absorption bands centered at approximately 615 nm and 665 nm within the orange-red region. Indocyanine green (ICG, FIG.24B), extensively employed for monitoring cardiac output and hepatic function, demonstrates a sharp absorption peak in the near-infrared region around 780-800 nm. All three molecules exhibit high molar absorption coefficients (on the order of 104-105M-1crrr1), making them efficient agents for refractive index manipulation in the red and near-infrared region.

[0146] Simulations (FIGs. 25A-25B) based on the Kramers-Kronig relation are made for methylene blue and indocyanine green, suggesting both molecules can efficiently increase the refractive index of aqueous medium from red to the near-infrared region.

[0147] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the abovedescribed embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

ATTORNEY DOCKET NO. 221910-2030CLAIMSWhat is claimed is:

1. A method for imaging an organ or tissue in a subject, the method comprising:(a) administering a composition comprising a clearing compound to the subject, wherein an interaction between the clearing compound and at least one overlying tissue in the subject creates a transparent spectral window in the at least one overlying tissue; and(b) visualizing the organ or tissue through the at least one overlying tissue.

2. The method of claim 1 , wherein the transparent spectral window comprises the entire visible spectrum.

3. The method of claim 1, wherein the transparent spectral window is from about 380 nm to about 780 nm.

4. The method of claim 1 , wherein the clearing compound comprises a natural amino acid, a salt of a natural amino acid, a vitamin or a salt thereof, a sugar alcohol, a dye, or any combination thereof.

5. The method of claim 4, wherein the natural amino acid comprises alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, or any combination thereof.

6. The method of claim 4, wherein the salt of the natural amino acid comprises a hydrochloride salt or a sodium salt.

7. The method of claim 6, wherein the salt of the natural amino acid comprises arginine-HCI or cysteine- Na.

8. The method of claim 4, wherein the vitamin or salt thereof comprises sodium ascorbate or niacinamide.

9. The method of claim 4, wherein the sugar alcohol comprises sorbitol.

10. The method of claim 4, wherein the dye comprises methylene blue, indocyanine green, or any combination thereof.

11. The method of claim 1, wherein the organ or tissue comprises the brain, the gastrointestinal tract, muscle, connective tissue, bones, blood vessels, the liver, the bladder, a tumor, theATTORNEY DOCKET NO. 221910-2030spinal cord, the heart, the liver, the pancreas, the spleen, the lungs, the trachea, the kidneys, lymph nodes, the thymus, the ovaries, the testes, the uterus, the prostate, an ocular tissue, or any combination thereof.

12. The method of claim 1, wherein the composition is administered topically or by injection.

13. The method of claim 1, wherein the at least one overlying tissue comprises skin, muscle, epithelial tissue, connective tissue, or any combination thereof.

14. The method of claim 1 , wherein the organ or tissue comprises an ocular tissue.

15. The method of claim 14, wherein the ocular tissue comprises a sclera, a cornea, a lens, or any combination thereof.

16. The method of claim 14, wherein the composition is administered topically, intracorneally, intrasclerally, intravitreally, subconjunctivally, intracamerally, by sub-Tenon administration, suprachoroidally, subcapsularly, intralenticularly, subretinally, by peribulbar administration, via therapeutic contact lenses, or by retrobulbar administration.

17. The method of claim 13, wherein the composition is administered to the subject by injection, intravenously, subcutaneously, topically, as a depot preparation, as an implant, or any combination thereof.

18. The method of claim 1, wherein the interaction comprises a covalent interaction or non- covalent interaction.

19. The method of claim 18, wherein the non-covalent interaction comprises hydrogen bonding, hydrophobic interactions, host-guest interactions, or any combination thereof.

20. The method of claim 18, wherein the covalent interaction comprises an active ester group that reacts with an amine functional group in tissue, click chemistry, disulfide bond formation, a ring-opening reaction, or any combination thereof.

21. The method of claim 1, wherein the subject is a mammal, a bird, a reptile, an amphibian, a fish, an arthropod, a mollusk, a cnidarian, an echinoderm, an annelid, a flatworm, a nematode, or a plant.

22. The method of claim 21 , wherein the mammal is a human, rat, mouse, rabbit, vole, tree shrew, guinea pig, hamster, cat, dog, pig, sheep, cow, horse, or non-human primate.

23. The method of claim 21, wherein the bird is a chicken, turkey, duck, parrot, or finch.

24. The method of claim 1 , wherein the organ or tissue is visualized in situ in the subject.ATTORNEY DOCKET NO. 221910-203025. The method of claim 1, wherein performing step (a) reduces light scattering between two or more tissue components, the tissue components having different refractive indices.

26. The method of claim 1, wherein performing step (a) increases light transmittance through the at least one overlying tissue by at least 50-fold compared to light transmittance through the at least one overlying tissue before performing the method.

27. The method of claim 1, wherein performing the method allows visualization of at least one feature in the subject at least 200 pm below a skin surface of the subject.

28. The method of claim 1, wherein from about 0.1 mg to about 20 g of the clearing compound are administered per kg of body weight of the subject.

29. The method of claim 1 , wherein performing the method results in a local concentration of the clearing compound in the at least one overlying tissue of from about 1 pM to about 1 M.

30. The method of claim 1 , wherein the clearing compound is non-toxic.

31. The method of claim 1 , wherein following visualizing, the compound is excreted by the subject.

32. The method of claim 31, wherein the clearing compound is excreted in less than about 10 hours.

33. The method of claim 1, wherein visualizing is accomplished using reflectance imaging, fluorescence imaging, laser speckle imaging, two-photon excitation spectroscopy, optical coherence tomography (OCT), light sheet microscopy, super-resolution microscopy, epifluorescence microscopy, fluorescence mediated tomography, photoacoustic tomography, three-photon microscopy, Brillouin microscopy, Raman microscopy, confocal microscopy, TIRE microscopy, brightfield / darkfield microscopy, DIC microscopy, structured illumination microscopy, or a combination thereof.

34. A pharmaceutical composition comprising a clearing compound in an aqueous carrier.

35. The pharmaceutical composition of claim 34, wherein the pharmaceutical composition is formulated as an eye drop, an injectable formulation, or a topical composition.

36. The pharmaceutical composition of claim 35, wherein the clearing compound comprises a natural amino acid, a salt of a natural amino acid, a vitamin or a salt thereof, a sugar alcohol, a dye, or any combination thereof.

37. The pharmaceutical composition of claim 36, wherein the natural amino acid comprises alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine,ATTORNEY DOCKET NO. 221910-2030lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, or any combination thereof.

38. The pharmaceutical composition of claim 36, wherein the salt of the natural amino acid comprises a hydrochloride salt or a sodium salt.

39. The pharmaceutical composition of claim 38, wherein the salt of the natural amino acid comprises arginine-HCI or cysteine-Na.

40. The pharmaceutical composition of claim 36, wherein the vitamin or salt thereof comprises sodium ascorbate or niacinamide.

41. The pharmaceutical composition of claim 36, wherein the sugar alcohol comprises sorbitol.

42. The pharmaceutical composition of claim 36, wherein the dye comprises methylene blue, indocyanine green, or any combination thereof.

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