Three-dimensional two-photon miniature microscope

Abstract

An implantable multi-photon optical probe includes a probe housing having a proximal end and a distal end implantable on a sample. The probe housing is adapted to provide excitation energy to the sample and collect emitted radiation from the sample. The probe housing includes scanning optics configured to receive excitation radiation and to scan the excitation radiation laterally and axially throughout a field of view of the optical probe. A scanning lens is disposed in the probe housing, configured to receive the excitation radiation from the scanning optics, and an objective lens shapes and focuses the excitation radiation. A focusing lens is disposed to receive the excitation radiation and focus the excitation radiation into the sample.

Description

Technical Field

[0001] The present disclosure relates generally to methods and systems for imaging tissue using optical instruments, and in particular, to methods and systems for performing high spatiotemporal multiphoton imaging using micro-electromechanical systems. Background Art

[0002] The background description provided herein is for the purpose of generally presenting the context of the present disclosure. The work of the inventors presently mentioned within the scope described in this background section and aspects of the description that may not otherwise qualify as prior art at the time of filing are neither explicitly nor implicitly admitted to be prior art with respect to the present disclosure.

[0003] The imaging of biological fluids, tissues and other soft biomaterials is important for basic scientific research, engineering applications and clinical diagnosis. Endoscopes are generally used to scan objects in a horizontal plane parallel to the surface of the tissue. However, the vertical plane (also referred to as the axial plane or the plane within the tissue in this article) may be more useful for imaging biological structures and processes that develop perpendicular to the surface of the tissue, such as normal epithelial development, stem cell migration, neural pathways and tumor invasion. The need to image the vertical plane of biological structures and processes in vivo has driven the development of many designs of micro-instruments including multiphoton microscopy devices. However, none of the previous designs provide the axial displacement and scanning speed required to generate real-time cross-sectional images of deep tissue neural pathways and connections in brain tissue, and none of the previous designs do this with a useful form factor that does not seriously damage the sample. For example, traditional endoscopes are bulky and cannot repeatedly enter areas to be imaged such as the brain.

[0004] Multiphoton microscopy provides high resolution and significant imaging depth using intense pulses of long wavelength light that are able to penetrate the image surface to excite shorter wavelength photons through nonlinear effects. Relative to other deep tissue optical imaging modalities, multiphoton microscopy has the benefits of reduced photobleaching and the ability to excite endogenous fluorescence, in addition to compatibility with a variety of targeted fluorescent biomarkers. Despite the many miniature multiphoton instruments, there is still a need for endoscopic microscopes that can image tissues in depth through axial beam scanning. Existing systems capable of deep imaging utilize traditional benchtop microscopes, thus increasing experimental complexity, study invasiveness, and biological behavior. In addition,

[0005] In order to best exploit multiphoton imaging capabilities in a small instrument, it is desirable to support fast axial (i.e., vertical or in-tissue) scanning of ultrafast lasers. Sufficiently fast axial scanning can support vertical optical sectioning in vivo, providing real-time cross-sectional images of tissues in the same plane that histologists use to diagnose and monitor diseases such as colon cancer or esophageal cancer. However, previous endoscopic instruments have provided limited support for changing the depth of focus during multiphoton imaging. Several multiphoton endoscopes have characterized out-of-plane or z-axis resolution, but only by physically moving the sample being imaged. When moving in the z-axis, it is particularly important to maintain a horizontal imaging plane to provide a clear image. In order to create an image in the vertical plane, a series of horizontal plane images are acquired and then reconstructed. This approach is typically slow and difficult to implement because vibrations in the sample can cause motion artifacts. In addition, some current three-dimensional imaging systems use fiber bundles and electrowetting lenses for imaging in three axes. Due to the physical limitations of fiber bundles and electrowetting optics, these systems have limited frame rates, limited spatial resolution, and limited field of view.

[0006] Current tools for tracking neuronal activity in moving animals lack the ability to track the same neuron over a sustained period of time in 3D space at milliseconds. Observing neuronal signaling events requires sub-millisecond temporal resolution and is therefore not achievable using current technology. For example, neural probes can track a large number of neurons, but cannot track the same group of neurons over multiple days. Optical microscopes used to observe moving or freely moving animals can typically only image one fixed 2D plane at a time. Some 3D imaging instruments use adjustable lenses, which are limited in response time, temporal resolution, and spatial resolution. Therefore, current imaging modalities cannot achieve cellular level resolution for observing 3D volumes of samples for observing cellular and subcellular level activity.

[0007] Miniaturized and implantable versions of such imaging tools can provide real-time and dynamic scanning images, allowing repeated examination of the same area without causing damage. Such systems can be used to monitor the progression of diseases and treatment outcomes as well as drug delivery monitoring. In addition, in neuroscience research, implantable, lightweight and compact versions can be installed on small, freely moving animals such as mice. Most implantable microscopes are limited in their working distance and field of view (FOV). In addition, most endoscopic systems use bulky fiber bundles to deliver and collect light, which limits the flexibility of the system and adds weight, which may harm or damage the animal or subject being imaged. Laser scanning at the distal optics, where a movable mirror is used to direct the laser close to the microscope objective, generally provides the largest FOV without excessive optical aberrations. Microelectromechanical system (MEMS) micromirrors can provide such scanning in small fiber-coupled microscopes. However, limitations on the range of motion and operating frequency of MEMS mirrors lead to additional compromises in overall instrument performance. Therefore, a lightweight, compact endoscopic system is needed for real-time axial and lateral imaging of freely moving animals and subjects. Summary of the invention

[0008] The present application describes a handheld optical device that can be used as a microscope system for real-time 3D optical imaging. The system for multiphoton endoscope microscopy utilizes a micro vertical actuator to provide fast axial scanning in addition to the large axial (i.e., vertical) displacement using a miniaturized device. Vertical actuation is obtained using an electrostatic actuator, a thin film piezoelectric actuator, or an electrothermal actuator in combination with a multiphoton imaging modality. Axial scanning is achieved based on the translation of a rigid mirror using a piezoelectrically actuated actuator, an electrothermally actuated actuator, or an electrostatic actuation under parametric resonance (each of which can achieve high-speed and large-displacement scanning through appropriate scanner design). In addition, this axial scanning (i.e., along the Z axis) can be combined with lateral scanning, which is also based on piezoelectric actuation or electrostatic actuation under parametric resonance, enabling the beam to scan over different planar axes (i.e., X-axis and Y-axis). In addition, this lateral scanning can be achieved at different axial scanning depths without changing the scanning performance. Using a different resonance mode for the axial scanner than for the lateral scanner, or using a DC mode when operating by piezoelectric or electrothermal actuation, further allows for better isolated control of scanning in different directions.

[0009] Compared to confocal imaging, multiphoton imaging offers many benefits for real-time in vivo imaging of epithelia and other regions. For example, multiphoton imaging can provide greater image depth, less photobleaching for long-term imaging studies, and can also provide increased image resolution or reduced scattering. Although dual-axis confocal microscopy has shown the potential to have comparable imaging depth and vertical slice resolution, a larger instrument diameter is required to obtain equivalent depth due to the need to provide light paths at off-axis angles. Because a single excitation wavelength can excite multiple fluorophores, multiphoton imaging is significantly easier to multiplex than other imaging modalities such as magnetic resonance imaging (MRI) and positron emission tomography (PET). In addition, multiphoton excitation produces reduced photodamage and out-of-plane photobleaching compared to other techniques. Multiphoton systems use a single optical path for incident and return light (although the paths can be separated for collection purposes) and, therefore, use a circular mirror geometry (as opposed to the general dog-bone geometry used for dual-axis) compared to dual-axis imaging systems. In addition, the lens arrangement in a dual-axis system requires precise alignment of the two collimating lenses using a mechanism such as a parabolic fixed reflector to ensure that the separate beam paths are aligned at the same point. Multiphoton arrangements do not have this alignment problem, although these systems require appropriate relay lens configurations to map changes in the axial displacement of the scanning mirror to displacements of the focal point in the tissue. One advantage of the present technology is that imaging can be performed without the need for fluorescent labeling using endogenous fluorescence of the tissue and / or cells.

[0010] The system of the present application provides real-time, axial or vertical scanning of tissue using a mirror scanning method. This axial movement of the mirror can be performed using a thin film piezoelectric actuator, an electrothermal actuator, and / or an electrostatic actuator, which can provide a considerable displacement (e.g., about 500 microns) while still having a small diameter (e.g., about 3 mm). Thin film piezoelectric actuators can produce larger axial translations at lower operating voltages and can individually address the actuated legs to compensate for uneven motion. Electrothermal actuators provide large displacements and improved linearity, but at lower scanning speeds. Electrostatic actuators are relatively simple to make and work at higher operating speeds. In addition, if the mirror surface is constructed monolithically (i.e., as a single component), the device will have better initial uniformity.

[0011] The present multiphoton imaging system uses the two-photon effect, which occurs when two lower energy (i.e., longer wavelength) photons simultaneously reach a biomolecule to excite fluorescence. The probability of absorbing two photons increases with the square of the intensity; therefore, a high numerical aperture objective in a single-axis configuration is used to maximize the intensity at the focus. Due to this physical principle, sensitivity to tissue scattering is lower and photobleaching is reduced when compared to single-photon fluorescence. In addition, the longer excitation wavelengths used provide deeper tissue penetration. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The drawings described below depict various aspects of the systems and methods disclosed herein. It should be understood that each of the drawings depicts an embodiment of a specific aspect of the disclosed systems and methods, and that each of the drawings in the drawings is intended to conform to one or more possible embodiments thereof. In addition, whenever possible, the following description refers to reference numerals included in the following drawings, wherein features depicted in multiple drawings are represented by consistent reference numerals.

[0013] Figure 1 is a schematic diagram of a multiphoton optical imaging system with implanted optics and a benchtop portion.

[0014] Figure 2A Is used to accommodate Figure 1 A perspective view of the probe housing of the implantable optical device.

[0015] Figure 2B is a photograph of a mouse with a portion of the brain exposed for imaging and the probe unit physically mounted on the mouse.

[0016] Figure 2C FIG. 1 is a diagram showing the mounting of the housing to a probe base to secure the housing to the probe base for imaging the brain of a rat. Figure 2A An image of the shell.

[0017] Figure 3 A lateral MEMS stage is shown having a mirror forming part of a lateral actuator system for scanning radiation across a sample.

[0018] Figure 4 The design of an axial MEMS stage for controlling the working distance of a compact microscope is presented in accordance with various embodiments.

[0019] Figure 5A is along the path of the exciting radiation passing through Figure 1 Ray diagram of the excitation radiation path of the implantable optical device.

[0020] Figure 5B are ray diagrams of the excitation radiation path with the axial MEMS stage at three different positions.

[0021] Figure 5C It is shown by Figure 5B Ray diagrams for three different imaging planes resulting from the axial MEMS stage position.

[0022] Figure 5D It passes through Figure 1 Ray diagram of the emitted radiation path of an implantable optical device.

[0023] Figure 6The Zemax ray simulation results of the implantable microscope are summarized.

[0024] Figure 7 Presented and demonstrated Figure 6 A series of ray spot diagrams of the spot sizes of the simulation results.

[0025] Figure 8 is a graph of the detected radiation intensity used to determine the lateral resolution by knife-edge testing.

[0026] Fig. 9 is a graph of detected radiation intensity used to determine the axial resolution of the implantable microscope.

[0027] Fig.10 Schematic diagram of implantable optical devices for a single MEMS multiphoton optical imaging system.

[0028] Fig.11 is an example block diagram illustrating various components for implementing an example embodiment of an implantable thin film piezoelectric multiphoton microscopy system.

[0029] Fig.12 is an example schematic of a miniaturized multiphoton optical imaging system with miniaturized remote z-scanning using implanted optics and a benchtop portion.

[0030] Fig.13A It shows Fig.12 A simulated ray diagram of the various elements of the implantable optical device 1205 and the optical path of the excitation radiation.

[0031] Fig. 13B is a graph showing the emitted fluorescence passing through Fig.12 A simulated ray diagram of the optical path of an implantable optical device.

[0032] Fig.14A is an example point spread function (PSF) of the excitation radiation at a working distance of 20 µm.

[0033] Fig. 14B is an example PSF of the excitation radiation at a working distance of 250 µm.

[0034] Fig. 14C is an example PSF of the excitation radiation at a working distance of 350 µm.

[0035] Fig.15A is an example spot pattern of the excitation radiation at a working distance of 20 µm.

[0036] Fig. 15B is an example spot pattern of the excitation radiation at a working distance of 250 µm.

[0037] Fig. 15C is an example spot pattern of the excitation radiation at a working distance of 350 µm.

[0038] Fig.16 Demonstrated assembly and test setup for a miniaturized two-photon three-dimensional (3D) imaging system.

[0039] Fig.17 Shown through Fig.16 Diagram of the optical pathways of the components of the miniaturized two-photon 3D imaging system.

[0040] Fig.18A is a simulated ray diagram of a focused beam using an axially scanning MEMS device.

[0041] Fig.18B is a simulated ray diagram of a focused beam using a side-scanning MEMS device.

[0042] Fig.18C is a simulated ray diagram of a focused beam using a combined axial-lateral scanning MEMS device.

[0043] Fig.19 is a table showing the working distance, numerical aperture, field of view, and scanner geometry of the axial z scanner at different axial scanner positionings.

[0044] Fig. 20A is a block diagram of an implantable optical device having excitation radiation provided by a Ti:sapphire laser and a single optical path utilizing a single optical fiber to provide the excitation radiation and collect the emitted radiation.

[0045] Fig. 20B yes Fig. 20A Simulated ray diagrams for the implantable optics objective and various lateral MEMS stage positions.

[0046] Fig. 20C yes Fig. 20A Simulated ray diagrams of the implantable optics objective and various axial MEMS stage positions.

[0047] Fig.20D It is presented Fig. 20A A table of lateral scanning angles with corresponding lateral focal lengths or coordinates and axial MEMS positions with corresponding working distances and numerical apertures for an implantable optical device.

[0048] Fig.21 Shown are multiple ex vivo images of long-range axial scans of a Thy1-YFP mouse sample tissue, where each image was taken at a different focal depth in the tissue sample.

[0049] Fig.22A Presented in Fig.21Volumetric image reconstruction of an axial imaging scan presented in multiple images.

[0050] Fig. 22B Presented Fig.22A Side view of the volume image reconstruction presented in .

[0051] Fig. 22C Presented Fig.22A A second side view of different vertical slices of the volumetric image reconstructed.

[0052] Fig.22D Presented Fig.22A Top view of the volume image reconstruction presented in .

[0053] Fig.23A Figure 2 is an example image of a brain tumor sample expressing green fluorescent protein (GFP) obtained using the multiphoton imaging system described in this article.

[0054] Fig. 23B is another example image of a brain tumor sample with green fluorescent protein (GFP) GFP expression.

[0055] Fig.23C is another example image of a brain tumor sample with green fluorescent protein (GFP) GFP expression.

[0056] Fig.23D is another example image of a brain tumor sample with green fluorescent protein (GFP) GFP expression.

[0057] FIG. 23F is another example image of a brain tumor sample having green fluorescent protein (GFP) GFP expression.

[0058] Fig.24 Shown are ex vivo images of confetti mouse oviduct animal samples obtained using excitation radiation at wavelengths of 1050 nm, 920 nm, 965 nm, and 837 nm.

[0059] Fig.25 is an example schematic diagram of another miniaturized multiphoton optical imaging system with miniaturized remote z-scanning using implanted optics and a tabletop portion, configured for air coupling. DETAILED DESCRIPTION

[0060] The present application provides techniques for endoscopic measurements using an implantable reflective confocal and / or multiphoton microscope with a long working distance. Confocal laser scanning microscopy is an optical imaging technique that uses a spatial pinhole to block out-of-focus light. Reflective confocal microscopy (RCM) is a non-invasive, label-free biomedical imaging technique that has attracted much attention due to its simplicity and low cost. RCM works based on the natural refractive properties of cells and subcellular structures. For example, in brain imaging, the phospholipids in the myelin sheath are brighter than the axons because they have a higher refractive index. Similarly, the mitochondria within the axons appear darker than the myelin covering. RCM applications include, but are not limited to, imaging of brain, skin, bone, teeth, and eye tissues.

[0061] For brain-related applications, RCM has demonstrated in vivo imaging of myelinated axons at a depth of approximately 400 μm in the 400-600 nm wavelength range. Therefore, the development of deep RCM for clinical and preclinical applications, such as monitoring metal nanoparticles in the skin and brain in drug delivery studies, is of interest. In addition, this technology can be applied to blood flow and blood cell monitoring and early cancer detection. The described device utilizes coupled microelectromechanical system (MEMS) mirrors to achieve diffraction-limited resolution throughout a wide field of view (FOV). The described system is capable of providing axial resolutions of 1.3 microns, 1.5 microns, and 6 microns, with a FOV of 500 microns by 500 microns, and a working distance of up to 550 microns. Previous 3D brain imaging systems utilize fiber optic bundles to deliver and collect light. Fiber bundles are rigid structures that restrict the movement of animals or living subjects. In addition, the image is degraded due to the honeycomb pixelation pattern of the fiber bundles. In addition, the FOV of such systems is limited by the number of optical fibers in the bundle, these systems exhibit low frame rates, and typically utilize large scanners with high power. Additionally, scanners are typically far away from the sample being imaged, which reduces temporal resolution and frame rate, and there can be large dispersion in the fiber bundles, further reducing resolution in space and time. The disclosed imaging system does not employ fiber bundles, and therefore overcomes all of these challenges, allowing for miniaturized 3D imaging in real time with higher spatial and temporal resolution.

[0062] The disclosed device includes a miniaturized microscope system for three-dimensional (3D) imaging of tissue in freely moving animals. The device provides high spatiotemporal images at a fast imaging rate, allowing real-time imaging of biological structures and processes. In addition, 500 × 500 × 550 µm 3High image sampling rates are performed at local areas within the sample volume. As described herein, the use of MEMS mirrors allows three-axis scanning with increased lateral and axial resolution compared to current technology. The three-axis scanning includes fast scanning in the axial directions for imaging over the entire volume, and random access control of all axes to select the imaging plane and increase the image sampling rate at any local area within the entire field of view (FOV).

[0063] The addition of random access control to the MEMS scanner allows for higher spatiotemporal resolution of arbitrary regions of interest (ROIs) within the entire volume. Random access control using a MEMS device allows cross-sectional imaging at multiple locations of the FOV in a single frame. However, it also allows the laser scanning to exceed the nominal mirror bandwidth, which results in a reduction in the FOV at higher imaging frame rates centered at an arbitrary point within the imaging space. For the fabricated device described herein, imaging of the full FOV can be performed at a nominal frame rate of 20 Hz to frame rates of 200-500 Hz at ROIs approximately 10-100 µm in size. Increasing the sampling rate in small regions allows for the observation of fast biological and electrical processes, such as action potential propagation across cortical layers in tethered animals, which has not been previously accomplished using other imaging systems.

[0064] The scanning method described ensures that multiphoton imaging can be performed across the fundamental imaging depth while maintaining cellular resolution. The ability to monitor the activity of neuronal populations across multiple layers almost simultaneously enables important new experiments that are currently not possible with existing imaging technologies. The use of MEMS scanning mirrors based on thin film piezoelectrics and folded optical paths allows the reduction of heavy components, resulting in an implantable device that does not harm or stress the animal or biological subject, and limits the scanner's sensitivity to motion artifacts from moving animals. Two-photon imaging is used in the described method to perform deep tissue imaging in order to perform in vivo non-invasive 3D brain imaging with cellular and subcellular resolution. The use of multiphoton imaging allows the use of separate optical fibers to provide light to the microscope and to collect light. Unlike other 3D imaging designs, the ability to use two different optical paths through separate optical fibers does not require special polarization-maintaining fibers to deliver the excitation light. Therefore, the separate optical paths for the excitation radiation and the emitted radiation further allow the system to be simplified and the size and overall cost to be reduced.

[0065] As described herein, implantable can describe the element or probe that is fully implanted in the interior of a subject (such as a human or animal) or fully implanted in an organ or tissue to image the tissue in a sample, an organ or tissue. Alternatively, the term "implantation" should also be understood to refer to an element or probe that is implanted on a subject. For example, an implantable probe can be a probe that is physically attached to a part of a subject (such as being attached to the skull of a mouse) to image the tissue of a subject. In addition, an implantable probe or microscope can include some components that are implanted in a subject, an organ or a tissue region, and other elements of the implantable probe are outside the subject, the organ or the tissue region.

[0066] Implantable microendoscopes can repeatedly image the same tissue at frequent intervals over an extended period of time. Such devices can be placed in a fixed position relative to the tumor, collect images in real time, and achieve cellular to subcellular resolution. For typical implantable microendoscopic systems, the field of view (FOV) is limited, and most microendoscopes can only image in a 2D horizontal plane. These systems usually require adjustment of the position of the optical fiber within the probe, which is very slow and performed at discrete intervals. In other approaches, electro-active lenses have been implemented to adjust the working distance (WD), but this results in increased optical aberrations and has a limited axial field of view. The proposed implantable imaging system provides an enhanced FOV throughout a large WD while also providing a compact implantable design.

[0067] The disclosed microendoscope design utilizes an objective with a large WD and a scanner placed nearby to maintain lateral and axial resolution at the cellular level, thereby achieving a wide lateral FOV and a large axial range (up to 650 × 500 × 400 μm). 3 ), with flexibility in selecting the nominal imaging depth in the distal optics. In an exemplary embodiment, the disclosed microendoscope uses a folded beam path where incident light from an optical fiber is reflected off an axial scanning mirror surface before being relayed to the objective. A single path endoscope design is also disclosed where scanning is performed using a single optical path aligned with a narrow GRIN lens. The single path endoscope design is capable of scanning over approximately 440 × 300 × 300 μm 3 The FOV images depths of 1.0 mm to 1.3 mm in mouse brain tissue with 1.0-2.0 μm lateral resolution and 9.0-12 μm axial resolution.

[0068] Figure 11 is a schematic diagram of a multiphoton optical imaging system 100 having an implantable optical device 105 and a table portion 110. The implantable optical device 105 is a component implanted into a biological subject to image tissue in the subject. For example, the subject can be a mouse, and the implantable optical device 105 can be physically positioned to image the brain tissue of the mouse. The implantable optical device 105 can be housed in a housing (not shown), which is housed and transmitted through a port in the proximal side 101a of the optical device 105, and is physically connected to the subject or mouse through the distal side 101b of the implantable optical device. As used herein, the term "downstream" indicates a further component along the optical path compared to an "upstream" component that propagates along the same radiation. The target for imaging will be referred to as a sample 103. The implantable optical device 105 includes an axial MEMS scanning unit or stage 130, a lateral MEMS scanning unit or stage 140, and an objective lens 145. The lateral scanning unit 140 is adapted to scan the output laser energy throughout the planar scanning area of ​​the sample 103 by moving the lateral reflector assembly, and the axial scanning stage 130 includes an axial reflector assembly and is adapted to scan the output laser energy throughout the imaging depth range of the sample. The axial MEMS stage 130 and the lateral MEMS stage 140 may be referred to as scanning optics together herein. The imaging depth range and the planar scanning area are combined to form a three-dimensional volume, also referred to as an imaging voxel. The imaging depth may also be referred to herein as the focal length, focal depth, range depth, working range, working depth or working distance of the imaging system 100. The axial scanning unit 140, the lateral scanning unit 130 and the objective lens 145 are all optically coupled to the laser and the light collection electronic device through the input and output fiber optic cables 102a, 102b. As shown, the input fiber optic cable 102a is a component further upstream of the implantable optical device, and the output fiber optic cable 102b is the most downstream component of the implantable optical device. Any number of these components may be at least partially housed in a single handheld probe housing frame (not shown), with the probe housing being implanted in a subject.

[0069] As used herein, "benchtop" (as in "benchtop portion") refers to a structure, component, assembly, or other that is positioned external to a subject and optically, electrically, or otherwise communicatively coupled to an implantable portion. For example, the positioning may include being placed on, mounted to, or integrated with any suitable working surface. The benchtop portion 110 includes a laser and light collection electronics, such as a Ti-sapphire laser 112 having an adjustable spectral range of approximately 690-1040 nm. This laser 112 emits excitation radiation 114 with a pulse width of approximately 100 fs at 80 MHz. A dispersion precompensation unit located inside the laser housing may be used to minimize pulse duration. A half-wave plate 106 is used with a linear polarizer 107 to adjust the laser power. A lens 109 couples the excitation radiation 114 into an optical fiber 102 to provide the excitation radiation 114 to the implantable optical device 105. The desktop portion 110 further includes a light collection optical device to receive light from the implantable optical device 105 and detect the light. The lens 110 collects the light from the output optical fiber 102b, the bandpass filter 111 filters the light to remove any noise, and then the detector 113 detects the light. The detector 113 can be a photomultiplier tube, a photodiode, or another detector capable of detecting optical radiation. Although a Ti-sapphire laser is used in the embodiment, other light sources and imaging processes can also be used. For example, a radiation source with an excitation radiation wavelength of up to 1200 nm or 1300 nm can be implemented as a laser 112 for performing three-photon imaging.

[0070] The table portion 110 includes additional hardware components for processing and displaying images. For example, the amplifier 115 may receive an electrical signal indicating an image or pixel from the detector 113. The amplifier 115 provides an amplified signal to an analog-to-digital converter 117 (ADC). The ADC 117 provides a digital signal to the image processing unit 120. The image processing unit 120 performs image processing to construct an image from the digital signal. The image processing unit 120 may provide image data to the display 122, and the display 122 may present the image to the user. The image processing unit 120 may also provide information to the MEMS actuator 125. Based on the received signal, the MEMS actuator may control the position and / or orientation of the axial MEMS stage 130 and the lateral MEMS stage 140.

[0071] Once the excitation radiation 114 has been coupled to the input fiber 102a, the excitation radiation 102a is provided to the axial MEMS stage 140 through an aperture in the mirror 132. The mirror 132 is included in the optical path so that the input fiber 102a can be attached to the subject or animal perpendicular to the subject (e.g., perpendicular to the surface of the subject's skin or bone, such as the surface of the skull). The vertical configuration of the input fiber 102a reduces the tension on the sleeve of the input fiber 102. The axial MEMS stage 130 reflects the excitation radiation 114 onto the mirror 132, and the first lens 135 performs full or partial collimation of the excitation radiation 114. The mirror 138 reflects the excitation radiation 114 to the lateral MEMS stage 140, and the lateral MEMS stage 140 reflects the excitation radiation 114 to the scan lens 139. The scan lens 139 focuses the excitation radiation 114 onto the dichroic mirror 140.

[0072] The composite objective 145 further collimates the excitation radiation 114 to a given spot size and focuses the excitation radiation 114 into the sample 103. In various instances, the composite objective 145 combines a gradient refractive index (GRIN) lens 146 with an aspheric focusing lens 147 to achieve a desired working distance, such as a working distance of 303 microns. The GRIN lens 146 provides a large numerical aperture and working distance over a wide range of lateral positions where the excitation radiation enters the GRIN lens 146. The aspheric lens 147 compensates for the spherical aberration of the GRIN lens while also further extending the working distance of the probe. The lateral MEMS stage has a ±5° deflection scan angle, which produces a field of view (FOV) of 400 × 400 µm. In an embodiment, the provided optical design can have a FOV of 500 × 500µm with a working distance of up to 550 µm. In an embodiment, the GRIN lens 146 has a diameter of 1.8 mm, a length of 4.31 mm, a numerical aperture of 0.52, and an effective focal length of 1.69 mm. The focal length of the GRIN lens 146 can be less than 1.5 mm, less than 1.7 mm, or less than 2 mm to ensure the compact size of the implantable optical device 105. In addition, the GRIN lens can be a dual-wavelength lens to provide similar optical transformations in more than one wavelength range. In an embodiment, the aspheric lens 147 has a diameter of 2.4 mm, a numerical aperture of 0.54, and an effective focal length of 1.45. The focal length of the aspheric lens can be shorter than 1.4 mm, 1.5 mm, or less than 1.8 mm. The GRIN lens 146 and the aspheric lens 147 can each have an anti-reflection coating, such as a visible light coating, a NIR coating, or a broadband anti-reflection (BBAR) coating to reduce light loss through the system.

[0073] Due to the short distances between components, the specific placement of the various optical devices in the implantable optical device 105 allows the probe housing 200 to be very compact. For example, in one manufacturing design, the propagation path length between the lateral MEMS stage 140 and the GRIN lens 146 is 6 mm, and the distance from the axial MEMS stage 130 to the GRIN lens 146 is 15 mm. This allows the housing 200 to have a height and width of 15 mm × 15 mm for each dimension at its widest cross-section. In addition, more or fewer reflectors can be used to increase or decrease the width and length of the probe housing 200 to include more propagation in one dimension or another. In an embodiment, the total size of the probe housing is less than 20 mm × 20 mm, less than 15 mm × 15 mm, or less than 12 mm × 12 mm.

[0074] The sample 103 includes a fluorophore, such as a fluorescent tag or fluorescent probe, which fluoresces in response to the presence of a biomolecule, such as a protein, an antibody, or an amino acid. The excitation radiation 114 provided causes the fluorescent tag to fluoresce, providing radiation 150 emitted from the sample 103 back to the implanted optical device 105. The emitted radiation 150 is provided to a lens 147, which collimates the emitted radiation 150 back into the objective lens 145. The emitted radiation 150 passes through the dichroic mirror 140, and the lens 152 focuses the emitted radiation 150 into the output optical fiber 102b. The output optical fiber 102b then provides the emitted radiation 150 to the lens 110 and other components of the table portion 110 to detect the emitted radiation 150 and generate an image of the sample 103. The input optical fiber 102a and the output optical fiber 102b can be single-mode optical fibers, multi-mode optical fibers, polarization-maintaining optical fibers, or another optical fiber capable of coupling light into and out of the implanted optical device 105.

[0075] Figure 2A Is used to accommodate Figure 116 is a perspective view of a probe housing 200 of an implantable optical device 105. The probe housing 200 has a proximal end 201a and a distal end 201b. During operation, the distal end 201b of the probe housing 200 is implanted into a subject so that the implantable optical device 105 is positioned to image a sample 103 tissue of the subject. The proximal end 201a of the probe housing 200 includes an input port 202a and an output port 202b for input optical fiber 102a and output optical fiber 102b, respectively. The input port 202a and the output port 202b are disposed in one or more walls of the housing 200 to allow radiation to enter and exit the housing 200. The path of the excitation radiation 114 is shown by the excitation path 165, and the path of the emitted radiation 150 is shown by the emission path 160. The input optical fiber 102a injects the excitation radiation into the housing 200 through the input port 202a and the aperture in the reflector 132. The excitation radiation is then reflected from axial mirror 130, mirror 132, and a first lens collimates the excitation radiation along excitation path 165. Axial mirror 130 may be axially translated along the propagation path of excitation radiation 114 to focus the excitation radiation at different working distances in sample 103.

[0076] The mirror 138 reflects the excitation radiation to the lateral MEMS stage 140, and the lateral MEMS stage 140 reflects the excitation radiation to the dichroic mirror 140, which reflects the excitation radiation to the objective lens 145. The lateral MEMS stage 140 is configured to tilt the reflective surface so that the excitation radiation is scanned throughout the FOV of the optical probe. The objective lens 145 shapes the excitation radiation and focuses the excitation radiation into an imaging volume 104 including at least a portion of the sample 103 to image the portion of the sample 103. In applications, the imaging volume 104 can be a single voxel for forming an image of the sample, the imaging volume 104 can be a region of interest within a larger field of view or working range of a microscope, or the imaging volume 104 can be the entirety of a sample or volume for imaging.

[0077] The excitation radiation excites the fluorescent tags in the sample 103, and the fluorescent tags emit radiation back into the lens 147 and into the housing 200 containing the implanted optical device 105. The emitted radiation propagates through the objective lens 145 and the dichroic mirror 140. And the coupling lens 152 focuses the emitted radiation into the output fiber 102b to provide radiation to the benchtop portion of the imaging system.

[0078] Housing 200 containing implantable optical device 105 is physically coupled to or implanted in a subject to image a volume of tissue of the subject. Figure 2Bis a photograph of a mouse 220, with a portion of the exposed brain 222 of the mouse being used as a sample for imaging. A probe base 225 is fixed to the skull of the mouse 220 by two mounting screws 227. The probe base 225 has a hole 229 through which the brain 222 of the mouse is exposed. Figure 2C is shown as reference Figure 2A An image of housing 200 is depicted, with the housing being secured to probe base 225 using nut 228 to mount housing 200 to probe base 225. Thus, implantable optical device 105 can be coupled and decoupled from a subject, or reused with a different subject at a different time.

[0079] Each of the lateral MEMS stage 140 and the axial MEMS stage 130 may include a high energy density PZT thin film to produce a MEMS scanning mirror with a large range of motion and addressable scanning motion at a high operating bandwidth. The MEMS stage is designed to achieve high-speed, large-displacement laser scanning in a small space, either by rotating the mirror for lateral scanning or by translating the mirror parallel to the propagation direction of the excitation radiation for axial scanning. In MEMS scanners, large-displacement scanning is usually only feasible at low speeds at resonance or through mechanisms such as electrothermal actuation. Thin-film PZT, a high-voltage electric coefficient ceramic material, provides a unique large driving force. This allows for substantial vertical translation of the axial scanner with high bandwidth, which is not possible with other MEMS scanners. Thin-film PZT scanners can quickly translate to a specified depth to provide imaging across a 3D volume, or perform vertical cross-sectional imaging. Vertical plane images can be directly acquired by continuous axial scanning while scanning with a complementary lateral axis. The high energy density film further allows for significant DC offsets in rotating scanning mirrors with high resonant frequencies, which allows video rate imaging across large fields of view. By manipulating the amplitude of the resonance while varying the quasi-static angular offset, the thin film PZT scanner can support random access scanning of regions of interest within a large imaging field at increased scanning frequencies and frame rates.

[0080] Based on the principle of parametric resonance, different electrostatic MEMS scanners can be provided for lateral and axial scanning. Large mechanical actuation can be achieved by driving the structure near 2ω0 / n, where ω0 is the natural frequency of the scanner and n is an integer ≥ 1. Figure 3A lateral MEMS stage 140 is shown with a mirror 342 that forms part of a lateral actuator system 340 of the lateral MEMS stage 140. Although the mirror 342 can take different shapes, in the illustrated example, the mirror 342 is a circular mirror. In the illustrated example, the diameter is 1.8 mm, and the mirror 342 is capable of accommodating an excitation beam that is 1.27 mm wide at normal incidence (compared to 45°). The front side of the lateral actuator system 340 is coated with aluminum to achieve a reflectivity greater than about 85% between about 200-900 nm wavelengths, thereby reflecting the wavelength range used for multiphoton excitation.

[0081] In the illustrated example, the lateral actuator system 340 includes two actuation axes about which the reflector 342 can be independently rotated. An X-axis is defined as shown, which is aligned with the inner leg or spring member of the inner reflector actuator. The Y-axis is defined as being aligned with the outer leg of the outer reflector actuator. Each leg is part of an inner and outer comb filter driver, respectively. The comb filter driver provides electrostatic actuation so that when driven by a drive signal, the reflector 342 rotates around an axis (X axis) and an outer axis (Y axis), respectively. Each of the inner comb filter driver and the outer comb filter driver can be operated at a resonant frequency, for example, a resonant frequency selected between about 1 kHz and about 4 kHz. In addition, the system 340 can be driven with a selected resonant frequency drive signal to each comb driver so that the reflector 342 undergoes a sinusoidal scan pattern. In the example, the system 340 is driven by the resonant frequency to image at ≥ 5 frames / second using a Lissajous scan pattern and scan at 400 × 400 pixels per frame.

[0082] Both the inner and outer axes of the lateral actuator system 340 can be actuated to image in the horizontal (XY) plane. The resonant frequencies of these axes are designed to be approximately 1 kHz or 4 kHz, respectively. In addition, by combining the actuation of the inner axis of the lateral scanner and the out-of-plane motion of the axial scanner, images can be produced in the vertical (XZ) plane. When scanning the object, a dense Lissajous scan pattern is formed, which repeats itself at a rate of 5 frames per second to generate a horizontal or vertical image with 100% coverage of 400 × 400 pixels or 400 × 320 pixels in size. The MEMS scanner is driven by custom software developed in LabView, which also reconstructs the image by remapping the time series signal to a 2D image using a calibrated motion profile from the scanner to generate a lookup table. In other words, by knowing where the laser is pointing at a specific time, the intensity of the returning laser can be mapped to form a 2D image. The relationship between the displacement of the axial MEMS stage 130 and the focus in the sample 103 is quantified by mounting the axial actuator on a motorized stage to precisely control the position. Advantageously, vertical segments can be collected at approximately 5 frames per second, compared to conventional imaging devices, which may require several minutes to acquire a stack of horizontal segments and reconstruct the corresponding vertical segments.

[0083] The lateral actuator system 340 can be formed on a chip with dimensions of 3 × 3 mm2. The mirror 342 is mounted on a gimbal 341 to minimize crosstalk between the axes (i.e., between the inner and outer axis drives). The lateral actuator system 340 operates at an increased resonant frequency to scan at a higher frame rate. An orthogonal set of electrostatic comb drive actuators 345 are coupled to the inner and outer torsion legs or springs 344a, 344b, and determine the resonant frequency of the scanner based on their shape and configuration. Large scan angles can be achieved by scanning down or scanning up, but larger deflection angles can be achieved by scanning down. For the X and Y axes, in the example shown, the lateral MEMS stage 140 can achieve a mechanical scan angle of > 5° at drive frequencies approaching 8.2 kHz and 2 kHz at 60 Vpp. Using drive frequencies of 8570 Hz and 2100 Hz yields actual tilt frequencies of 4285 Hz and 1050 Hz in the X and Y axes, respectively. The result is a dense Lissajous scan pattern that repeats itself at 5 Hz to contain an image of size 400 × 400 pixels with 100% coverage for a FOV of 250 × 250 μm2.

[0084] In some examples, the control system can be configured to implement Lissajous scanning. Lissajous scanning is used if the axial mirror scanning frequency is too close to the lateral mirror scanning frequency due to increased actuator motion uniformity. Fast Lissajous frame rates are obtained by adjusting the axial actuator design to operate at a specific frequency.

[0085] Figure 4 The design of an axial MEMS stage 430 is shown. The design can accommodate additional square actuators on the silicon wafer. The central mirror or mirror platform 432 is supported by four serpentine piezoelectric bending beams, legs or actuators 435, where the piezoelectric stack material varies to produce selective upward or downward bending motion in successive sections of the beam 435. This results in a well-defined vertical translation of the mirror surface 432, which is further enhanced by the symmetry of the structure under ideal conditions.

[0086] The overall dimensions of the axial MEMS stage 430 are approximately 3 mm × 3 mm × 0.5 mm for eventual integration into 5 mm diameter or smaller endoscopic microscope instruments, and the actuator 435 design includes four individual beams 1.2 mm long to produce a vertical (i.e., out-of-plane) displacement of approximately 400 µm along the optical axis to produce an image in a plane perpendicular to the tissue surface at a natural frequency of approximately 100 Hz. The actuator 435 operates at near resonance, with Lissajous scanning used to extract the image from the combined motion of the vertical piezoelectric actuator and the in-plane electrostatic scanning mirror. The use of resonant operation enables a large scan range at low voltage, as well as dynamic balancing of the mirror for uniform vertical motion even with only a single input to the four actuation legs.

[0087] The foregoing description demonstrates a novel multiphoton microscope that uses a remote scanning configuration and a MEMS scanner to provide real-time switchable XY / XZ imaging. The axial MEMS scanner operates in resonant mode at a mechanical resonance frequency of approximately 440 Hz, yielding a line scan rate of 880 Hz over a 200 um range. This high-speed axial scanning technique can be used to study fast biological processes such as action potentials between neurons in living animals. While ultrafast pulses were focused on the axial MEMS, no signs of damage to the mirror surface were observed after one hour of exposure to 50 mW laser power. The damage threshold can be further increased by changing the mirror coating from aluminum to gold. The lateral MEMS scanner also operates in resonant mode at resonant frequencies of approximately 4 kHz and 1 kHz for the inner and outer axes. Lissajous scanning is used for imaging in the horizontal or vertical plane with a FOV of 250 µm × 250 µm and 250 µm × 200 µm, respectively, and a frame rate of 5 Hz. These MEMS scanners are very compact, with a footprint of approximately 3 mm × 3 mm, while maintaining good mechanical properties. They are also highly reliable, low-cost, and easy to mass-produce. These MEMS scanners can be a good alternative to large actuators used in traditional microscopes. By using this scanning strategy, ultra-compact live microscopes with real-time horizontal and vertical slicing capabilities, or even micro-devices, can be developed. In addition, in some systems, electrothermal, electromagnetic, and / or other microactuators can be used in an arrangement with a geometry similar to the axial scanning actuator described herein, or electrostatic actuators with offset electrodes can be used to achieve low-frequency (DC) scanning to accomplish axial scanning.

[0088] In large displacement vertical stages that rely on a bent beam architecture, the center stage motion can be very sensitive to asymmetries in the individual legs, which can be due to local variations in residual stresses, lithographic misalignment, or other processing non-idealities. One way to handle non-uniform center stage motion is to calibrate and compensate for the asymmetry by applying different voltages to two or more legs. By identifying the frequencies at which the contributions of multiple vibration modes produce almost pure vertical translation, uniform vertical motion can be achieved, allowing balancing to be performed solely by the voltage input to the stage.

[0089] Figure 3 and Figure 4 The examples of MEMS devices shown in are two examples of potential devices for performing axial and lateral scanning as described herein. Figure 3 and Figure 4 MEMS devices and further examples of MEMS scanning devices are described in US Pat. No. 5,877,679, which are incorporated herein by reference in their entirety.

[0090] The optical design of the implantable optical device 105 maintains lateral and axial resolution at the cellular level with a relatively large lateral FOV and a large axial range of approximately 550 μm. Figure 1 As shown in , the microscope uses a folded beam path for axial scanning, where incident light from the input fiber 102a is reflected from the vertical mirror plane of the axial MEMS stage 130 before being relayed to the objective lens 145. The use of the vertical surface of the axial MEMS stage 130 allows the focal depth of imaging to be controlled while maintaining a small diameter, low inertial mass axial scanner. The proposed optical design achieves diffraction-limited performance over a large FOV area in a compact form factor (e.g., approximately 17 mm × 19 mm × 8 mm, 2.5 g). The use of a composite objective enables high-resolution (e.g., 0.9-1.9 µm lateral resolution, 3.5-8.5 μm axial resolution) imaging over a wide FOV in all three axes at a large working distance.

[0091] Figure 5A is a ray diagram of the implanted optical device 105 along the path of the excitation radiation 114. The excitation radiation 114 in the form of light from a femtosecond laser (SpectraPhysics MaiTai) is delivered by a photonic crystal fiber (PCF) as the input fiber 102a to the axial MEMS stage 130 scanner through a small aperture in a mirror 132. Changing the position of the axial MEMS stage 130 changes the optical path distance from the input fiber 102a to the series of lenses, which translates the focus at the objective lens 145. The excitation radiation 114 is reflected from the mirror 132 surrounding the ferrule of the input fiber 102a, and the excitation radiation is propagated to the objective lens 145 through a pair of aspheric lenses, namely a first lens 135 and a scanning lens 139. The objective lens 145 has a GRIN lens (Edmund Optics) 146 and an aspheric focusing lens 147, which are combined to act as a compound lens, thereby improving the resolution and providing a long imaging working distance. The numerical aperture of the imaging system ranges from 0.41-0.52 at working distance. A lateral MEMS stage 140 is positioned before (ie, upstream) the scan lens 139 to change the position of the incident beam on the GRIN lens, thereby translating the focal position in the transverse plane of the FOV.

[0092] Figure 5B 1 are ray diagrams of the mirror 132, the first lens 135, and the excitation radiation 114 reflected from the axial MEMS stage 130 at three different positions 130a, 130b, and 130c of the axial MEMS stage 130. The first axial MEMS stage position 130a provides the longest optical path for the excitation radiation 114, while the third MEMS stage position 130c provides the shortest optical path. Figure 5Cis a ray diagram showing three different imaging planes 148a, 148b, and 148c produced by axial MEMS stage positions 130a, 130b, and 130c, respectively. The first imaging plane position 130a has a working distance of 550 produced by the first axial MEMS stage position 130a. Similarly, the second imaging plane position 148b and the third imaging plane position 148c have working distances of 350 μm and 250 μm produced by the second axial MEMS stage position 130b and the third axial MEMS stage position 130c, respectively.

[0093] Figure 5D is a ray diagram of the implanted optics along the path (e.g., referred to as the collection path) of the emitted radiation 150. After the excitation radiation 114 is provided to the sample at a given working distance, the sample fluoresces and provides the emitted radiation 150 to the aspheric focusing lens 147. The emitted radiation 150 propagates through the objective lens (including the GRIN lens) 145, and the dichroic mirror 140 transmits the shorter wavelength emitted radiation 150 into the emitted radiation collection path, where it is focused by the aspheric lens 150 onto a multimode optical fiber (MMF) as the output fiber 102b for transmission to the photodetector, as shown in reference Figure 1 The desktop portion 110 is described.

[0094] The Zemax software was used to simulate Figures 5A-5D Each ray diagram of . Figure 6 The Zemax simulation results of the proposed microscope are summarized and Figure 7 Presented and demonstrated Figure 6 A series of ray spot diagrams for the spot sizes of the simulation results. The simulated lateral resolution varies from 0.68 μm at a working distance of 10 μm to 1.9 μm at a working distance of 550 μm. The simulated axial resolution varies from 4.5 μm to 8.5 μm over the same range of working distances. Diffraction-limited performance is maintained on-axis for all simulated working distances. The proposed optical design optimizes the performance of the microscope for working distances between 250 µm and 400 µm, for which diffraction-limited resolution is maintained over approximately the central 400 μm of the lateral FOV.

[0095] In addition to higher image resolution and a wider range of working distances, the proposed optical design eliminates the need for polarization-maintaining fibers as input fibers 102a or output fibers 102b, and eliminates the need for other associated dispersive and refractive optics for controlling the polarization of the excitation radiation 114 and / or emitted radiation 150, compared to other endoscopes and / or remote imaging devices. This polarization-independent property allows for a folded optical path design with fewer optical components, thereby reducing the overall form factor size and weight of the microscope.

[0096] A multiphoton imaging system was fabricated as described herein. Lateral and axial resolutions were measured to determine device performance. Figure 8 is a graph of the detected radiation intensity used to determine the lateral resolution by knife-edge testing. In the knife-edge test method, the 10-90% intensity transition on a sharp edge corresponds to the Rayleigh-limited resolution. To provide a sharp knife-edge, a standard USAF 1951 resolution target was used. The measured lateral resolution was determined to be 1.5 µm, which is in good agreement with the Zemax simulation. Fig. 9 is a graph of the detected radiation intensity used to determine the axial resolution of the microscope. The axial resolution was measured to be 6 µm at a working distance of 350 µm, which again agrees well with simulations of the fabricated system and working distance.

[0097] Although described above as having independent axial MEMS stages and lateral MEMS stages, Figure 1 The multiphoton optical system 100 can be fabricated using a single MEMS stage to perform axial and lateral scanning. Fig.10 is a schematic diagram of an implantable optical device 1005 for a single MEMS stage multiphoton optical imaging system. Fig.10 The implantable optical device 1005 can be used as Figure 1The implanted optical device in the system 100 of FIG. 1005. The single MEMS stage implanted optical device 1005 includes an input optical fiber 1002a that couples excitation radiation 1014 to the implanted optical device 1005. The collimating lens 1135 collimates the excitation radiation 1014, and the first reflector 1032 redirects the excitation radiation 1014 through the dichroic mirror, and the second lens 1138 focuses the excitation radiation 1014 onto the axial and lateral dual MEMS stage 1130. The dual MEMS stage 1130 enables both axial scanning of the excitation radiation to change the working distance of the microscope optical system and lateral scanning of the excitation radiation to image different pixels of the 2D image plane at a specific working distance. The dual MEMS stage 1130 reflects light into the GRIN lens 1145, which shapes the excitation radiation 1014 into the focusing lens 1147, which focuses the excitation radiation 1014 into the sample 1103 to image an area of ​​the sample 1103.

[0098] Fluorophores in the sample 1103 fluoresce and provide emitted radiation 1150 back to the focusing lens 1147. The GRIN lens 1145 focuses the emitted radiation 1150 onto the dual MEMS stage 1130, which reflects the emitted radiation back through the second lens 1138. In the two-photon emission process, the wavelength of the emitted radiation 1150 is shorter than the wavelength of the excitation radiation 1014. The dichroic mirror 1140 reflects the emitted radiation 1150, which is transparent to the excitation radiation 1014 due to the different wavelengths of the different radiation. The coupling lens 1152 couples the emitted radiation 1150 to the output fiber 1002b. The output fiber 1002b then couples the emitted radiation 1150 to the stage electronics and optical components to detect the emitted radiation 1150 and generate an image of the area of ​​the sample 1103.

[0099] Fig.111 is an example block diagram 1200 illustrating various components for implementing an example embodiment of a thin film piezoelectric multiphoton microscope system 1202 discussed herein. The implantable optical device 1205 discussed previously herein may be positioned adjacent to a sample 1203 or operably coupled to the sample, depending on the function of performing the disclosed embodiments. The system 1202 may have a controller 1204 operably connected to a database 1214 via a link 1222 connected to an input / output (I / O) circuit 1212. It should be noted that, although not shown, additional databases may be linked to the controller 1204 in a known manner. The controller 1204 includes a program memory 1206, a processor 1208 (which may be referred to as a microcontroller or microprocessor), a random access memory (RAM) 1210, and an input / output (I / O) circuit 1212, all interconnected via an address / data bus 1220. It should be appreciated that, although only one microprocessor 1208 is shown, the controller 1204 may include multiple microprocessors 1208. Similarly, the memory of the controller 1204 may include multiple RAMs 1210 and multiple program memories 1206. Although the I / O circuit 1212 is shown as a single block, it should be understood that the I / O circuit 1212 may include multiple different types of I / O circuits. For example, the RAM 1210 and the program memory 1206 may be implemented as semiconductor memory, magnetically readable memory, and / or optically readable memory. The link 1224 may operably connect the controller 1204 to the optical device 1205 through the I / O circuit 1212.

[0100] The program memory 1206 and / or the RAM 1210 may store various application programs (i.e., machine-readable instructions) for execution by the microprocessor 1208. For example, the operating system 1230 may generally control the operation of the endoscopic microscope system 1202 and provide a user interface to the test device to implement the processes described herein. The program memory 1206 and / or the RAM 1210 may also store various subroutines 1232 for accessing specific functions of the endoscopic microscope system 1202. By way of example and not limitation, the subroutines 1232 may include, among other items: a subroutine for controlling the operation of the optical device 1200 or other endoscopic device as described herein; a subroutine for capturing images with the optical device 1205 as described herein; and other subroutines, such as implementing software keyboard functions, interfacing with other hardware in the endoscopic microscope system 1202, etc. The program memory 1206 and / or the RAM 1210 may further store data related to the configuration and / or operation of the endoscopic microscope system 1202 and / or related to the operation of one or more subroutines. For example, the data may be data collected by the optics 1205, data determined and / or calculated by the processor 1208, and the like.

[0101] In addition to the controller 1204, the endoscopic microscope system 1202 may include other hardware resources. The endoscopic microscope system 1202 may also be coupled to various types of input / output hardware, such as a visual display 1226 and an input device 1228 (e.g., a keypad, keyboard, etc.) to fine-tune the actuation of the axial scanner and the lateral scanner. In one embodiment, the display 1226 is touch-sensitive and may cooperate with a software keyboard routine as one of the software routines 1232 to accept user input. The endoscopic microscope system 1202 may include a network interface 1234 for communicating with an external network 1236 to send or receive data and information on the external network 1236 or from the external network.

[0102] Fig.12 1 is a schematic diagram of a miniaturized multiphoton optical imaging system 1200 with miniaturized remote z-scanning using an implanted optical device 1205 and a table-top portion 1210. The implanted optical device 1205 includes a Z scanning mirror 1262 and a focal length f z The Z scan lens 1264 is a compact Z scan unit. Fig.12 The system can achieve precise and remote control of z-scan depth while maintaining aberration-reduced or aberration-free imaging of the subject's tissue. Fig.12 The System 1200 provides a compact, miniaturized laser scanning microscope system capable of long-range z-scanning with high precision and aberration-free performance. The System 1200 is capable of imaging at significant working distances and diffraction-limited imaging across a wide range of focal plane distances to image various cross-sections or depths of a sample or object. Aberration-free and diffraction-limited operation allows for high-resolution imaging, which permits imaging of smaller elements, more complex structures, and neural activity of tissues such as neurons and brain tissue. Precise imaging of multiple planes in a sample opens new possibilities for studying brain dynamics and function.

[0103] In an example, the implantable optical device 1205 is a component that is sized and positioned to be implanted in a biological subject to image tissue in the subject. For example, the subject can be a mouse, and the implantable optical device 1205 can be physically positioned in the mouse to image the brain tissue of the mouse. The implantable optical device 1205 can be housed in a housing (not shown), which is housed and transmitted by a port in the proximal side 1201a of the optical device 1205, and is physically connected to the subject or mouse by the distal side 1201b of the implantable optical device. An example target for imaging is a sample 1203. The implantable optical device 1205 includes a z scanning unit 1260 or stage, which includes a z scanning mirror 1262 and a z scanning lens 1264, a lateral MEMS scanning unit or stage 1240, and an objective lens 1245. The lateral scanning unit 1260 can be configured to scan the output laser energy throughout the planar scanning area of ​​the sample 1203 by moving the lateral reflector assembly, and the axial scanning stage 1260 can be adapted to scan the output laser energy throughout the imaging depth range of the sample. The axial MEMS stage 1260 and the lateral MEMS stage 1240 are also referred to as examples of scanning optical devices in this article. As in other examples in this article, the imaging depth range and the planar scanning area are combined to form a three-dimensional volume, also referred to as an imaging voxel. The imaging depth can also be referred to as the focal length, focal depth, range depth, working range, working depth or working distance of the imaging system 1200 in this article. The axial scanning unit 1260, the lateral scanning unit 1230 and the objective lens 1245 are all optically coupled to the laser and the light collection electronic device through the input and output fiber optic cables 1202a, 1202b. As shown in the figure, the input fiber optic cable 1202a is the most upstream component of the implantable optical device, and the output fiber optic cable 1202b is the most downstream component of the implantable optical device. Any number of these components may be at least partially housed in a single handheld probe housing frame (not shown), with the probe housing being implanted in a subject.

[0104] The table top portion 1210 includes a laser and light collection electronics, such as a Ti-sapphire laser 1212 with a tunable spectral range of about 690-1040 nm. In an example, this laser 1212 emits excitation radiation 1214 with a pulse width of about 100 fs at 80 MHz. A dispersion pre-compensation unit located inside the laser housing can be used to minimize the pulse duration. Lens 1209 couples the excitation radiation 1214 into the optical fiber 1202a to provide the excitation radiation 1214 to the implantable optical device 1205. The table top portion 1210 further includes light collection optics to receive light from the implantable optical device 1205 and detect the light. Lens 1210 collects light from the output optical fiber 1202b, bandpass filter 1211 filters the light to remove any noise, and then detector 113 detects the light. Detector 113 can be a photomultiplier tube, a photodiode, or another detector capable of detecting optical radiation. Although a Ti-sapphire laser is used in the embodiment, other light sources and imaging processes may also be used. For example, a radiation source with an excitation radiation wavelength up to 1200 nm or 1300 nm may be implemented as the laser 112 for performing three-photon imaging.

[0105] The table portion 1210 includes additional hardware components for processing and displaying images. For example, the amplifier 1215 can receive an electrical signal indicating an image or pixel from the detector 1213. The amplifier 1215 provides an amplified signal to the ADC 1217. The ADC 1217 provides a digital signal to the image processing unit 1220. The image processing unit 1220 performs image processing to construct an image from the digital signal. The image processing unit 1220 can provide image data to the display 1222, and the display 1222 can present the image to the user. The image processing unit 1220 can also provide information to the MEMS driver 1225. Based on the received signal, the MEMS driver can control the position and / or orientation of the axial MEMS stage 1260 and the lateral MEMS stage 1240.

[0106] Once the excitation radiation 1214 is coupled to the input fiber 1202a, the excitation radiation 1202a is provided to the collimating lens 1237 and is reflected from the polarization beam splitter (PBS) 1239 and passes through the quarter wave plate 1241 (QWP) through the z-scan lens 1264 to the z-scan mirror 1262 of the axial MEMS stage 1260. The input fiber 1202a is configured so that it can be fixed to the subject for imaging. The vertical configuration of the input fiber 1202a reduces the tension on the ferrule of the input fiber 1202. The z-scan MEMS stage 1260 reflects the excitation radiation 1214 back to the z-scan lens 1264, rotates through the QWP 1241 and transmits through the PBS 1239. The dichroic mirror 1238 reflects the excitation radiation 1214 to the lateral MEMS stage 1240, and the lateral MEMS stage 1240 reflects the excitation radiation 1214 to the scan lens 1232. Scan lens 1232 focuses the excitation radiation 1214 onto mirror 1242 .

[0107] The compound objective 1245 further collimates the excitation radiation 1214 to a given spot size and focuses the excitation radiation 1214 into the sample 1203. In various instances, the compound objective 1245 combines a GRIN lens 1246 with an aspheric focusing lens 1247 to achieve a desired working distance, such as a working distance of 350 microns. The GRIN lens 1246 provides a large numerical aperture and working distance over a wide range of lateral positions where the excitation radiation enters the GRIN lens 1246. The aspheric lens 1247 compensates for the spherical aberration of the GRIN lens while also further extending the working distance of the probe. The lateral MEMS stage has a ±5° deflection scan angle, which produces a field of view (FOV) of 400 × 400 µm. In an embodiment, the provided optical design can have a FOV of 500 × 500µm with a working distance of up to 550 µm. In an embodiment, the GRIN lens 1246 has a diameter of 1.8 mm, a length of 4.31 mm, a numerical aperture of 0.52, and an effective focal length of 1.69 mm. The focal length of the GRIN lens 1246 can be less than 1.5 mm, less than 1.7 mm, or less than 2 mm to ensure the compact size of the implantable optical device 105. In addition, the GRIN lens 1246 can be a dual-wavelength lens to provide similar optical transformations in more than one wavelength range. In an embodiment, the aspheric lens 1247 has a diameter of 2.4 mm, a numerical aperture of 0.54, and an effective focal length of 1.45. The focal length of the aspheric lens can be shorter than 1.4 mm, 1.5 mm, or less than 1.8 mm. The GRIN lens 1246 and the aspheric lens 1247 can each have an anti-reflection coating, such as a visible light coating, a NIR coating, or a broadband anti-reflection (BBAR) coating to reduce light loss through the system.

[0108] The specific placement of the various optical components in the implantable optical device 1205 allows the probe housing 1600 (in Fig.16 12 is a schematic diagram of a probe housing 1600 that is very compact. For example, in one fabricated design, the propagation path length between the lateral MEMS stage 1240 and the GRIN lens 1246 is 6 mm, and the distance from the axial MEMS stage 130 to the GRIN lens 146 is 15 mm. This allows the housing 1600 to have a height and width of 15 mm × 15 mm for each dimension at its widest cross-section. In addition, more or fewer mirrors can be used to increase or decrease the width and length of the probe housing 1600 to include more propagation in one dimension or another. In embodiments, the total size of the probe housing is less than 20 mm × 20 mm, less than 15 mm × 15 mm, or less than 12 mm × 12 mm.

[0109] The sample 1203 includes a fluorophore, such as a fluorescent tag or fluorescent probe, which fluoresces in response to the presence of a biomolecule, such as a protein, an antibody, or an amino acid. The excitation radiation 1214 provided causes the fluorescent tag to fluoresce, providing radiation 1250 emitted from the sample 1203 back to the implanted optical device 1205. The emitted radiation 1250 is provided to a lens 1247, which collimates the emitted radiation 1250 back into the objective lens 1245. The emitted radiation 1250 passes through the dichroic mirror 1238, and the lens 1252 focuses the emitted radiation 1250 into the output optical fiber 1202b. The output optical fiber 1202b then provides the emitted radiation 1250 to the lens 1210 and other components of the table portion 1210 to detect the emitted radiation 1250 and generate an image of the sample 1203. The input optical fiber 1202a and the output optical fiber 1202b can be single-mode optical fiber, multi-mode optical fiber, polarization-maintaining optical fiber, or another optical fiber capable of coupling light into and out of the implantable optical device 1205.

[0110] Fig.13A It shows Fig.12 A simulated ray diagram of various elements of implantable optical device 1205. The ray diagram shows the path of excitation radiation 1214, which reflects from PBS 1214, passes through QWP 1241, is focused by z-scan lens 1264, reflects from z-scan mirror 1262, is collimated by z-scan lens 1264, passes through QWP 1241, passes through PBS 1239, reflects from lateral MEMS stage 1240, is focused by scan lens 1232, reflects from mirror 1242, and is focused by GRIN lens 1246 and aspheric lens 1247. Fig. 13Bis a simulated ray diagram showing the path of emitted fluorescent 1250 light, which passes through the aspheric lens 1247, the GRIN lens 1246, the dichroic mirror 1238, through the lens 1252 and is focused into the collection fiber from various points where the beam can be scanned by the axial scanner 1262 and the lateral scanner 1240.

[0111] Fig.14A , 14B and 14C are example point spread functions (PSFs) of the focused excitation radiation at three different working distances: 20 µm, 250 µm, and 350 µm. Fig.15A , 15B 15C are example spot diagrams of the excitation radiation at the same three working distances of 20 µm, 250 µm, and 350 µm. The PSF and spot diagrams provide valuable insights into the performance of the imaging system. Notably, the diagrams show that the system maintains diffraction-limited resolution (i.e., on the order of hundreds of microns) throughout long working distances. This diffraction-limited resolution is demonstrated based on the PSF and spot size within the Airy disk. The PSF and spot diagrams provide a visual representation of the system's ability to achieve sharply focused imaging even at extended working distances, demonstrating its ability to capture high-precision detailed information.

[0112] Fig.16 An assembly and test setup 1600 of a two-photon three-dimensional (3D) imaging system is shown. The setup 1600 may be Fig.12 1200. The setup 1600 shows a housing 1602, a dichroic mirror 1238, a lateral MEMS stage 1240, a scanning mirror 1242, and an objective lens 1245 with an aspheric lens 1247. An additional mirror 1270 is shown to change the direction of the emitted radiation 1250 so as to focus the emitted radiation 1250 into a collection fiber 1202b. In some embodiments, the additional mirror 1270 can be omitted. The probe housing 1602 has a proximal end 1604a and a distal end 1604b. During operation, the distal end 1604b of the probe housing 1602 is implanted into a subject so that the implanted optical device 1205 is positioned to image a sample 1203 tissue of the subject. The proximal end 1604a of the probe housing 1602 includes an input port and an output port for the input fiber 1202a and the output fiber 1202b, respectively. Input ports and output ports are disposed in one or more walls of the housing 1604 to allow radiation to enter and exit the housing 1604 .

[0113] Fig.17 Shown through Fig.16FIG. 1 is a diagram of the assembly of a miniaturized two-photon 3D imaging system and the optical path of a test setup 1600. Excitation radiation 1214 enters the setup 1600 and is reflected from the PBS 1239 through the QWP 1241 and is focused by the z-scan lens 1264 onto the z-scan mirror 1262. The excitation radiation 1214 is then reflected back through the z-scan lens 1264, rotated by the QWP 1241 and passes through the PBS 1214. The lateral MEMS stage 1240 and the dichroic mirror 1238 then reflect the excitation radiation 1214 through the objective lens 1245 and focus the excitation radiation into the sample. The sample emits emitted radiation 1250, which passes through the objective lens 1245 and is transmitted through the dichroic mirror 1238. The emitted radiation 1250 is focused by the lens 1252 and is further coupled to the output fiber 1202b through the mirror 1270.

[0114] Figures 18A-18C Simulated ray diagrams of axial scanning, lateral scanning, and combined axial-lateral scanning MEMS devices, respectively. Figures 18A-18C The ray diagram shows a scanning lens 1138, a reflector 1140 and a GRIN lens 1145. Fig.10 MEMS device 1130 in an optical configuration. Fig.18A It is shown that z-axis translation of the axial scanning mirror also moves the focus of the radiation in a combined axial and lateral path or displacement. Fig. 23B It is shown that x or y rotation of the lateral MEMS stage 1240 moves the focus of the radiation primary in a lateral path in a plane along the axis of radiation propagation. Fig.23C It is shown that the combined motion of axial and lateral scanning can effectively scan a tilted FOV in a parallelogram in the lateral xy plane of the sample.

[0115] Fig.19 is a table showing the working distance, numerical aperture, field of view, and scanner geometry of an axial z scanner at different axial scanner positions. As the scanner is moved back or forward from its neutral position (i.e., left and right columns relative to the center column), the working distance and NA change. The bottom two rows are data that apply to all z scanning positions, including the total FOV and the requirements on the scanner to achieve the total FOV. Fig.19 As reported in , the total FOV is 300 um × 440 um × 210 um in the oblique plane. In an embodiment, a spacer, such as a glass spacer approximately 700 um thick, can be placed between the distal end of the probe and the sample so that the effective imaging depth inside the sample can be between about 0-217 um, while the total working distance of the optics is greater.

[0116] Fig. 20Ais a block diagram of an implantable optical device 2005 with excitation radiation 2003 provided by a Ti:sapphire laser and an input optical fiber 2002. Fig. 20A The configuration utilizes a single optical fiber 2002 to provide excitation radiation 2003 and collect the emitted radiation 2004 for further detection and analysis. The excitation radiation 2003 is collimated by lens 2010 and reflected from lateral MEMS stage 2012. The excitation radiation 2003 then passes through scan lens 2015 and is reflected from axial MEMS stage 2020 and is focused into sample 2050 through objective lens 2025. Fig. 20B and 20C yes Fig. 20A Simulated ray diagrams of the objective lens and MEMS stage of the implantable optical device 2005, showing various lateral MEMS stage 2021 positions and various axial MEMS stage 2020 positions, respectively.

[0117] In order to perform volumetric imaging in deeper brain regions, objective 2025 must be extended to a longer overall length while having a smaller diameter. Figures 20A-20C The optical setup shows an optical scanning geometry that achieves 300 × 440 × 300 μm 3 The FOV, combined with the use of a significantly reduced final objective lens diameter (< 1.0 mm), is suitable for insertion up to 1 mm into the mouse cortex without significant adverse effects on cortical function. This setup allows access to the interior of tumors for imaging and allows for imaging of tumors before they are large enough to extend into the upper layers of the brain. Figures 20A-20C The design presented in is based on a GRIN lens designed for deep tissue imaging. Coupled with a MEMS scanner introduced before the objective lens, this design allows 3-axis laser displacement on the lens for 3D imaging compared to fixed depth fiber bundles or scanning fibers, and the presented design produces a much smaller footprint than dedicated axial scanning modules. Relative to the surface of the GRIN lens, the simulated lateral resolution varies from 1 μm at a WD of 50 μm to 2 μm at a WD of 350 μm. Over the same WD range, the simulated axial resolution varies from 9 μm to 12 μm. Compared to long-range dual MEMS scanning, Figures 20A-20C The design provided in can be implemented without a polarization-maintaining PCF and associated dispersive and refractive optics. Instead, a single double-clad fiber 2002 can be used to deliver excitation light and collect fluorescence (i.e., excitation radiation 2004). Across the FOV, the NA can be approximately 0.39-0.45. Figures 20A-20C The design provided in can reduce system complexity and produce a smaller instrument size suitable for smaller diameter objectives. Fig.20D is a table of lateral scan angles with corresponding lateral focal lengths or coordinates in various examples. Fig.20DThe axial MEMS position of the implantable optical device 2005 of FIG. 20 is further presented along with the corresponding working distance and numerical aperture.

[0118] Fig.21 Multiple ex vivo images of long-range axial scans of Thy1-YFP mouse sample tissue are shown. Each image is taken at a different focal depth in the tissue sample (ranging from about 250 microns to about 300 microns) at a step of about 10 um each time. Images are collected plane by plane at different axial depth slices, and a 3D image volume is reconstructed from multiple images. Fig.22A Presented in Fig.21 Volumetric image reconstruction of an axial imaging scan presented in multiple images. Fig. 22B Presented Fig.22A Side view of a vertical slice reconstructed from the volume image presented in . Fig. 22C Presented Fig.22A A second side view of different vertical slices of the volumetric image reconstructed. Fig.22D The yz plane is shown Fig.22A Slices of the volume image reconstructed. Figures 22B-22D Demonstrates the ability to slice vertically in either the xz or yz plane. Fig.21 Volumetric image reconstruction is performed by aligning the various images and stacking the images to stack slices of the volume, thereby producing a comprehensive visualization of the axial image volume of the sample. This in vitro imaging method presented allows detailed examination of Thy1-YFP mouse samples at the cellular level. The reconstructed three-dimensional representation provides valuable insights into the spatial arrangement and distribution of YFP-labeled neurons within the sample. This information can be used to further understand and analyze neuronal morphology, connectivity, and organization.

[0119] Figures 23A-23E This is an image of a brain tumor sample expressing green fluorescent protein (GFP) obtained using the multiphoton imaging system described in this article. Figures 23A-23EThe images were acquired at a single depth at different lateral locations in the XY plane using the setup shown in Figure 22. The images were acquired from a post-mortem brain tissue sample with GFP expression in a brain tumor at different locations of the sample. The imaged samples enable multi-focal plane visualization and allow the properties of the tumor to be studied at a microscopic level. For example, post-mortem imaging allows for detailed examination of the spatial distribution of the tumor, cellular characteristics, and interactions within the surrounding brain tissue. Imaging GFP expression within the tumor provides researchers with insight into the tumor's extent, localization, and potential interactions with neighboring cells. This information helps to better understand tumor biology and aids in the development of targeted therapeutic strategies. Post-mortem imaging methods provide valuable tools for studying molecular and cellular aspects of brain tumors, paving the way for advances in tumor research and treatment.

[0120] Fig.24 Shown are ex vivo images of animal samples obtained using multi-wavelength excitation of the oviduct of a confetti mouse. Various excitation radiation wavelengths were used to excite different markers. The excitation wavelengths used were 1050 nm, 920 nm, 965 nm, and 837 nm. The use of the 1050 nm laser wavelength allows deep tissue penetration, enabling imaging at greater depths within the oviduct. The 1050 nm wavelength range is particularly advantageous for imaging thick samples or structures located deeper in the tissue. The 920 nm, 965 nm, and 837 nm laser wavelengths were used to selectively excite specific fluorophores within the oviduct of a confetti mouse. These different wavelengths enable different fluorescence patterns emitted by different cell populations labeled with different fluorophores to be discerned and visualized. Testing the system with multiple laser wavelengths on oviducts from confetti mice provides valuable insights into the multicolor imaging capabilities of the system and its ability to effectively visualize and distinguish different fluorophores within complex samples. Fig.24 The system demonstrates the ability to excite and operate efficiently over a wide range of laser wavelengths, as demonstrated by the images of Figure 5. Additional wavelengths and filtering can be added to the system to further improve image resolution, brightness, SNR, and clarity.

[0121] Fig.25 Another example miniaturized multiphoton optical imaging system 2100 is shown with miniaturized remote z-scanning using implanted optics 2102 and a table-top portion 2104. The imaging system 2100 is configured for air coupling and has a focusing lens for the emission path, wherein the emission path before the PMT does not pass through the scanning lens for the excitation path, nor does the emission path reflect from the MEMS mirror, thereby avoiding potential signal intensity degradation.

[0122] Throughout the entire specification, multiple instances can implement the parts, operations or structures described as a single instance. Although the individual operations of one or more methods are shown and described as separate operations, one or more of the individual operations can be performed simultaneously, and the operations do not need to be performed in the order shown. The structure and function presented as a separate component in the example configuration can be implemented as a combined structure or assembly. Similarly, the structure and function presented as a single component can be implemented as a separate assembly. These and other variations, modifications, additions and improvements all fall within the scope of this article's object.

[0123] In addition, some embodiments are described herein as including logic or multiple routines, subroutines, applications, or instructions. These may constitute software (e.g., code implemented on a non-transitory machine-readable medium) or hardware. In hardware, routines, etc. are tangible units that can perform certain operations and may be configured or arranged in a certain manner. In an example embodiment, one or more computer systems (e.g., independent client or server computer systems) or one or more hardware modules of a computer system (e.g., a processor or a processor group) may be configured by software (e.g., an application or an application portion) to be a hardware module that operates to perform certain operations as described herein.

[0124] In various embodiments, hardware modules may be implemented mechanically or electronically. For example, a hardware module may include dedicated circuits or logic that are permanently configured to perform certain operations (e.g., a dedicated processor such as a field programmable gate array (FPGA) or an application specific integrated circuit (ASIC)). A hardware module may also include programmable logic or circuits that are temporarily configured to perform certain operations by software (e.g., as contained in a dedicated processor or other programmable processor). It should be appreciated that the decision to implement a hardware module mechanically in a dedicated and permanently configured circuit or in a temporarily configured circuit (e.g., configured by software) may be driven by cost and time considerations.

[0125] Thus, the term "hardware module" should be understood to encompass tangible entities, referring to entities that are physically constructed, permanently configured (e.g., hardwired), or temporarily configured (e.g., programmed) to operate in a certain manner or to perform certain operations described herein. In view of embodiments in which hardware modules are temporarily configured (e.g., programmed), it is not necessary to configure or instantiate each hardware module at any one time. For example, in the case where the hardware modules include a general-purpose processor configured using software, the general-purpose processor can be configured into corresponding different hardware modules at different times. Thus, software can configure a processor, for example, to constitute a particular module at one time and to constitute a different module at a different time.

[0126] A hardware module can provide information to other hardware modules, or receive information from other hardware modules. Therefore, the described hardware modules can be considered to be communicatively coupled. In the case of multiple such hardware modules being present at the same time, communication can be achieved by signal transmission (e.g., through appropriate circuits and buses) connecting the hardware modules. In an embodiment in which multiple hardware modules are configured or instantiated at different times, communication between such hardware modules can be achieved, for example, by storing and retrieving information in a memory structure accessible to multiple hardware modules. For example, a hardware module can perform an operation and store the output of such operation in a memory device to which it is communicatively coupled. Then, another hardware module can access this memory device at a later time to retrieve and process the stored output. The hardware module can also initiate communication with an input or output device, and can operate on a resource (e.g., a collection of information).

[0127] The various operations of the example methods described herein may be performed, at least in part, by one or more processors that are temporarily configured (e.g., by software) or permanently configured to perform the relevant operations. Such processors, whether temporarily or permanently configured, may constitute processor-implemented modules that operate to perform one or more operations or functions. In some example embodiments, the modules referred to herein may include processor-implemented modules.

[0128] Similarly, the methods or routines described herein may be implemented at least in part by a processor. For example, at least some operations of a method may be performed by one or more processors or a hardware module implemented by a processor. The performance of certain operations may be distributed between one or more processors, not only residing in a single machine, but also deployed across multiple machines. In some example embodiments, a processor or multiple processors may be located in a single location (e.g., in a home environment, an office environment, or as a server farm), while in other embodiments, the processor may be distributed in multiple locations.

[0129] The performance of certain operations may be distributed among one or more processors, not only residing within a single machine, but also deployed across multiple machines. In some exemplary embodiments, one or more processors or processor-implemented modules may be located in a single geographic location (e.g., in a home environment, an office environment, or a server farm). In other exemplary embodiments, one or more processors or processor-implemented modules may be distributed in multiple geographic locations.

[0130] Unless expressly stated otherwise, discussions herein using terms such as "processing," "computing," "caculating," "determining," "presenting," "displaying," and the like may refer to the action or process of a machine (e.g., a computer) to manipulate or transform data represented as physical (e.g., electrical, magnetic, or optical) quantities in one or more memories (e.g., volatile memory, non-volatile memory, or a combination thereof), registers, or other machine components that receive, store, transmit, or display information.

[0131] As used herein, any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. Thus, the various places where the phrase "in one embodiment" appears in this specification do not necessarily refer to the same embodiment.

[0132] Some embodiments may be described using the expressions "coupled" and "connected" and their derivatives. For example, some embodiments may be described using the term "coupled" to indicate that two or more elements are in direct physical or electrical contact. However, the term "coupled" may also mean that two or more elements are not in direct contact with each other, but still cooperate or interact with each other. The embodiments are not limited in this context.

[0133] Those skilled in the art will appreciate that various modifications, changes and combinations may be made with respect to the above-described embodiments without departing from the scope of the present invention, and such modifications, changes and combinations will be deemed to be within the scope of the inventive concept.

[0134] Although the present invention has been described with reference to specific examples, the specific examples are intended to be illustrative rather than limiting, and it will be apparent to those skilled in the art that changes, additions and / or deletions may be made to the disclosed embodiments without departing from the spirit and scope of the present invention.

[0135] The foregoing description has been given for clarity of understanding; and no unnecessary limitations should be understood therefrom, since modifications within the scope of the invention may be obvious to one skilled in the art.

Claims

1. An implantable multiphoton optical probe comprising: a probe housing having a proximal end and a distal end implantable on a sample, the probe housing being configured to provide excitation radiation to the sample and to collect emitted radiation from the sample; an input fiber port located in a wall of the probe housing, the input fiber port having an input fiber disposed therein, wherein the input fiber is configured to direct the excitation radiation from outside the probe housing through the wall of the probe housing into the probe housing; scanning optics disposed in the probe housing, the scanning optics configured to receive the excitation radiation and (i) scan the excitation radiation laterally across a field of view of the optical probe and (ii) scan the excitation radiation axially across an imaging depth range of the optical probe; a scan lens disposed in the probe housing to receive the excitation radiation from the scanning optics, the scan lens being configured to collect the excitation radiation to focus the excitation radiation; as well as An objective lens is disposed in the probe housing to receive the excitation radiation from the scanning lens, the objective lens being disposed in the probe housing and configured to shape and focus the excitation radiation into the sample.

2. The optical probe of claim 1, wherein the objective lens comprises a gradient index lens.

3. The optical probe of claim 1, wherein the objective comprises an aspheric lens that corrects aberrations and further focuses the excitation radiation into the sample.

4. The optical probe of claim 1, wherein the scanning optics comprises: an axial scanning stage disposed in the probe housing, the axial scanning stage being configured to scan the excitation radiation throughout an imaging depth range of the sample; and A lateral scanning stage is disposed in the probe housing, the lateral scanning stage being suitable for scanning the excitation radiation across a planar scanning area of ​​the sample, wherein the imaging depth range and the planar scanning area form a three-dimensional volume, the three-dimensional volume being the field of view of the optical probe across multiple imaging depths.

5. The optical probe according to claim 4 further comprises a first lens, which is disposed between the axial scanning stage and the lateral scanning stage, and the first lens is configured to collimate the excitation radiation or partially collimate the excitation radiation within a collimation of 5 degrees or less, 10 degrees or less, or 20 degrees or less.

6. An optical probe according to claim 1, wherein the scanning optical device comprises a single lateral-axial scanning stage disposed in the probe housing, and the lateral-axial scanning stage is suitable for scanning the excitation radiation across a planar scanning area of ​​the sample and across a range depth of the sample, wherein the range depth and the planar scanning area form a three-dimensional volume, and the three-dimensional volume includes the field of view and imaging depth range of the optical probe.

7. The optical probe of claim 1, further comprising an output fiber port having an output fiber disposed therein, the output fiber being configured to direct the emitted radiation from the interior of the probe housing through the wall of the probe housing to the exterior of the probe housing.

8. The optical probe of claim 1, wherein a distance from the scanning optics to the objective lens is less than 20 mm.

9. The optical probe of claim 1, wherein a focusing lens comprises an aspheric lens that corrects aberrations caused by the objective lens.

10. The optical probe according to claim 1, wherein the objective lens comprises: a gradient index lens disposed between the scanning optics and the sample; and A focusing lens is disposed between the gradient index lens and the sample.

11. The optical probe according to claim 1, wherein the objective lens comprises: Gradient index lenses with an effective focal length shorter than 2 mm; and Aspheric lenses with effective focal lengths less than 1.5 mm.

12. The optical probe of claim 1, wherein the objective lens is disposed between the scanning optics and the sample.

13. The optical probe of claim 1, wherein at least one component of the objective lens has a visible wavelength anti-reflection coating.

14. The optical probe of claim 1, further comprising an axial scanning stage disposed in the probe housing, the axial scanning stage configured to scan the excitation radiation throughout an imaging depth range of the sample; a polarizing beam splitter disposed upstream of the axial scanning stage, the polarizing beam splitter being disposed and oriented to (i) receive the excitation radiation and (ii) provide the excitation radiation to the axial scanning stage; as well as A wave plate is disposed between the polarizing beam splitter and the axial scanning stage to rotate the polarization of the excitation radiation.

Images