Systems and methods for rapid, label-free imaging of biological tissues based on ultraviolet single-plane illumination microscopy

Abstract

A method and system (100) for rapid, label-free, non-destructive imaging of intact biological tissues using microscopy with ultraviolet single plane illumination (MUSI). The system (100) or method employs a selective biaxial planar illumination configuration that decouples the illumination light from the detection path, and utilizes the intrinsic fluorescence of several specific endogenous fluorophores from biological tissues as a natural source, compared to deep UV illumination sources. In contrast to images obtained by clinical standard methods (i.e., H&E staining of formalin-fixed, paraffin-embedded tissues), images obtained by MUSI exhibit similar or even superior diagnostic characteristics, making them a potentially valuable post- and intraoperative diagnostic tool for clinicians, pathologists, and surgeons.

Description

[Technical Field]

[0001] The present invention relates to a system and method for rapid, label-free, and non-destructive imaging of biological tissue using microscopy with ultraviolet single-plane illumination (MUSI).

[0002] [Citation of Related Applications] This application claims priority to U.S. Provisional Patent Application No. 63 / 417,682, filed October 19, 2022, which is incorporated by reference in its entirety. [Background technology]

[0003] For surgical margin assessment (SMA), histopathology has remained the gold standard for decades. However, conventional pathological examination based on formalin-fixed, paraffin-embedded (FFPE) tissue is labor-intensive, time-consuming, and provides no intraoperative guidance to surgeons. Frozen sections can serve as a rapid alternative to FFPE, but they still require a turnaround time of 20–30 minutes during surgery. Furthermore, frozen sections limit margin sampling and introduce freezing artifacts in fatty tissues, which negatively impact histopathological interpretation and diagnostic accuracy.

[0004] Recent technological advances in light microscopy have enabled rapid, nondestructive imaging of freshly cut tissues, holding great promise for streamlining current clinical practice in FFPE histology. Imaging modalities utilizing exogenous fluorophores, such as fluorescence confocal microscopy, light-sheet microscopy, structured illumination microscopy, and ultraviolet (UV) surface-excited microscopy, can provide sufficient sampling of large excision sections within a point-of-care timeframe, enabling highly specific cellular characterization for diagnosis. However, imaging modalities pose a threat to intraoperative surgical procedures, as some new fluorescent contrast agents can potentially cause toxicity to patients. Furthermore, the staining process can interfere with subsequent molecular assays, such as in situ hybridization and DNA / RNA sequencing.

[0005] Thus, imaging techniques that exploit intrinsic contrast mechanisms are advantageous in modern clinical settings. For example, in the fields of ophthalmology and dermatology, optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) have been used and successfully interpreted due to their deep penetration depth and noninvasive nature. However, the cellular content provided by these reflectance-based methods is relatively limited inside internal organs. Furthermore, photoacoustic microscopy (PAM) can spectrally probe distinct molecular targets with intrinsic absorption contrast and has shown promising results in breast cancer screening and vascular imaging. Furthermore, nonlinear microscopy techniques (NLM), such as coherent Raman scattering, multiphoton absorption, and second- and third-harmonic generation, can achieve high-resolution, label-free visualization of various biological processes in a nonperturbative and nondestructive manner, finding widespread application in oncological research, such as tumor invasion and proliferation. However, these techniques still face challenges in screening large surgical specimens within short diagnostic time frames due to the need for continuous beam scanning. The lasers used in PAM or NLM are typically bulky and expensive, thereby increasing facility requirements and costs.

[0006] A recently proposed method, computational high-throughput autofluorescence microscopy with patterned illumination (CHAMP), enables rapid and label-free histological imaging of thick, unprocessed tissues with micrometer-scale resolution, making it particularly advantageous for intraoperative SMP applications, where immediate feedback should be provided to surgeons for optimal adjuvant treatment. However, the depth of field (DOF) of CHAMP is limited to within 80 μm, which is insufficient to accommodate rough resection sections with significant surface irregularities, causing resection margins to move in and out of focus during imaging. Furthermore, CHAMP achieves optical sectioning by leveraging the shallow penetration depth of deep UV light, which can produce significant variations between different tissue types. Thus, CHAMP images may deviate from traditional slide-based FFPE histology techniques. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need for improved methods and systems that at least reduce or eliminate the above-mentioned disadvantages and problems. [Means for solving the problem]

[0008] Thus, a first aspect of the present invention provides an imaging system for histological analysis of biological tissues using ultraviolet single-plane illumination microscopy. The MUSI of the present invention is a dual-axis configuration that utilizes the intrinsic fluorescence from biological tissues as a deep-UV-excited contrast source, while decoupling the illumination light or radiation from the detection path to eliminate the inherent trade-off between long DOF and high spatial resolution.

[0009] Unlike some nondestructive optical imaging techniques, such as ultraviolet surface-excited microscopy (MUSE), structured illumination microscopy (SIM), and light-sheet microscopy (LIM), MUSI does not require fluorescent labeling before imaging. While some label-free microscopy techniques, such as ultraviolet photoacoustic microscopy (UV-PAM), multiphoton microscopy (MPM), stimulated Raman scattering microscopy (SRS), and / or nonlinear microscopy (NLM) including second- and third-harmonic generation (S / THG), can provide illumination light with distinct intrinsic molecular / cellular responses, such as absorption-induced thermoelastic expansion (UV-PAM), intrinsic autofluorescence (MPM), molecular vibrations (SRS), and noncentrosymmetric orientation (SHG), to achieve cell phenotyping / classification, these scanning-based techniques have limited imaging throughput. Reflectance-based imaging techniques, such as full-field optical coherence tomography (FF-OCT), reflectance confocal microscopy (RCM), and dark-field reflectance ultraviolet microscopy (DRUM), can detect backreflected light from structures with different refractive indices and generate rapid, label-free images. However, the important diagnostic features revealed by these techniques can differ significantly from clinically standard methods due to limitations in contrast, resolution, and signal-to-noise ratio. In particular, DRUM relies on the detection of diffuse reflectance, whereas the present invention, which utilizes MUSI, relies on the intrinsic absorption of light and uses fluorescence from the specimen as contrast. DRUM also has a shorter depth of field (DOF) than MUSI, making MUSI ideal for scanning irregular tissue surfaces. In addition, the optical sectioning intensity of DRUM is tissue-dependent, whereas MUSI does not have this limitation, resulting in robust performance across different tissue types. The present invention can also be used for both external and internal imaging of target tissue, whereas the DRUM can only be used for external imaging.

[0010] Thus, in a first aspect, the imaging system of the present invention comprises: a movable specimen holding platform having a specimen holder and a liquid holder, the liquid holder having a prism-like structure; a deep UV excitation source that provides deep UV illumination light (or deep ultraviolet light); a first plurality of optical elements for generating a light sheet from the deep UV illumination light provided by the deep UV excitation source and directing the light sheet toward a bottom side of the specimen holder through one of the lateral faces of the prismatic structure on the at least one bottom surface of the liquid holder and onto the bottom surface of the specimen at an angle of incidence relative to a vertical axis of the specimen holder; a second plurality of optical elements configured to receive, at a detection angle relative to a normal axis of the specimen holder, emissions comprising fluorescent signals emitted from the specimens under excitation by the light sheet; an optical detection unit that detects and processes the luminescence received by the second plurality of optical elements; The incident angle and the detection angle are substantially identical in magnitude, and the beam path of the light sheet is substantially perpendicular to (orthogonal to) the path of the emitted light from the specimen.

[0011] In certain specific embodiments, the movable specimen-holding platform is coupled to a three-dimensional (3D) translation stage.

[0012] In certain embodiments, the specimen holder has a membrane for supporting the specimen or sample.

[0013] In certain specific embodiments, the liquid in the liquid holder of the movable specimen holding platform comprises water or a mixture of water and a UV-transparent substance.

[0014] In some specific embodiments, the first plurality of optical elements includes a first filter, a pair of lenses, a slit aperture, and a cylindrical lens.

[0015] In some particular embodiments, the first filter is a bandpass filter.

[0016] In some particular embodiments, the pair of lenses is a pair of UV-rated convex lenses.

[0017] In certain particular embodiments, the slit aperture is an adjustable slit aperture.

[0018] In some particular embodiments, the cylindrical lens is a UV cylindrical lens that generates a Gaussian light sheet.

[0019] In some specific embodiments, the light sheet is directed through the prismatic structure of the liquid holder of the movable specimen holding platform onto the bottom surface of the specimen at a 45° angle of incidence relative to the vertical axis of the specimen holder, with an average energy fluence that complies with safe UV radiation thresholds regulated by the American Conference of Governmental Industrial Hygienists (ACGIH®).

[0020] In certain specific embodiments, the liquid holder is positioned below the specimen holder within a movable specimen holding platform.

[0021] In some particular embodiments, the specimen is supported by a UV-transparent membrane that is fixed at the base of the specimen holder so that the light sheet can reach the bottom surface of the specimen held in the specimen holder.

[0022] In some particular embodiments, the UV transparent film is made from a highly transparent thermoplastic resin, including but not limited to polyethylene or any UV transparent material.

[0023] In some specific embodiments, the prismatic structures of the movable specimen holding platform each have at least two UV-transmitting windows located on two opposite lateral faces of the prismatic structure that allow a light sheet generated from a deep UV excitation source to enter or allow luminescence, including an emitted fluorescent signal from the specimen after excitation by the light sheet, to exit the movable specimen holding platform.

[0024] In certain embodiments, the specimen is biological tissue, including normal and abnormal tissue, freshly excised from a living organism or located in vivo.

[0025] In certain embodiments, the biological tissue is rich in endogenous fluorophores, including, but not limited to, reduced nicotinamide adenine dinucleotide (NADH), structural proteins such as collagen and elastin, aromatic amino acids such as tryptophan and tyrosine, and heterocyclic compounds such as flavins and lipofuscin.

[0026] In some specific embodiments, the second plurality of optical elements includes at least a UV objective lens, a second filter, and an infinity corrector lens.

[0027] In certain particular embodiments, the UV objective is an achromatic UV objective.

[0028] In some particular embodiments, the second filter is a long-pass filter.

[0029] In some particular embodiments, the infinity corrected lens is an infinity corrected tube lens.

[0030] In some specific embodiments, the optical detection unit includes multiple CMOS image sensors.

[0031] In some specific embodiments, the plurality of CMOS image sensors of the optical detection unit include, but are not limited to, scientific complementary metal-oxide semiconductor (sCMOS) sensors.

[0032] In certain embodiments, the 3D translation stage provides at least two scan directions to the movable specimen holding platform to translate the specimen through a light sheet that passes through the UV-transmitting member and reaches the bottom surface of the specimen at a constant velocity along a first scan direction, and further translating the specimen toward a scan direction in addition to the first scan direction along an axis lateral to the first scan direction allows obtaining the surface topography of the specimen from a maximum surface area of ​​the specimen.

[0033] A second aspect of the present invention provides a method for imaging biological tissue in a label-free, unprocessed manner using an imaging system according to the first aspect and various embodiments described herein to output images for histology showing a two-dimensional or three-dimensional profile of the biological tissue, the method comprising at least the following steps: (1a) placing the biological tissue in a specimen holder of a movable specimen holding platform of an imaging system, with a relatively flat side of the biological tissue facing a UV-transparent membrane disposed at the bottom of the specimen holder; (1b) immersing the specimen holder held with the biological tissue into a liquid holder of a movable specimen holding platform filled with liquid; (1c) adjusting one or more of the first plurality of optical elements to orient the light sheet produced by the optical elements to strike the bottom surface of the specimen at an incident angle of 45° with respect to a vertical axis of the specimen holder or with the incident beam path of the light sheet substantially perpendicular to a UV-transmitting window provided in one lateral face of the prismatic structure at the bottom of the liquid holder; (1d) adjusting one or more of the second plurality of optical elements to receive luminescence comprising a fluorescent signal emitted from the specimen at a detection angle of 45° relative to a vertical axis of the specimen holder or orthogonal to an incident beam path of the light sheet; (1e) activating a deep UV excitation source to generate UV illumination light (ultraviolet light) toward a first plurality of optical elements, then modulating the UV illumination light by the first plurality of optical elements to generate a first spot on the specimen through one of the UV-transmitting windows of a prismatic structure at the bottom of the liquid holder, and directing a light sheet toward the first spot at an incident angle of 45° with respect to a vertical axis of the specimen holder; (1f) receiving luminescence comprising emitted fluorescent signals from analytes excited by the light sheet via a second plurality of optical elements; (1g) detecting the received luminescence including the fluorescent signals emitted from the second plurality of optical elements by an optical detection unit including a plurality of CMOS image sensors; (1h) moving a movable specimen-holding platform driven by a 3D translation stage in a primary scanning direction from the first spot to a next spot on the specimen that is coplanar, and repeating steps (1e) to (1g) within each of the next spots until the last spot on the specimen that is coplanar in the primary scanning direction is reached to form a first image stripe; (1i) moving the movable specimen-holding platform transversely to the first scanning direction in a secondary scanning direction, and repeating steps (1e) through (1h) to form a next image stripe adjacent to the first image stripe until the entire maximum surface area of ​​the biological tissue is scanned; (1j) processing the image data detected by the optical detection unit to reconstruct the geometry of the biological tissue by an image processing module, and outputting one or more images representing the overall geometry of the biological tissue after reconstruction.

[0034] In some particular embodiments, the light sheet has a wavelength of 266 nm.

[0035] In some particular embodiments, the light sheet has a penetration depth of up to about 30 μm from the bottom surface of the biological tissue.

[0036] In some specific embodiments, the specimen is translated into the light sheet along the first scan direction at a constant speed of 250 μm / s, and images of the specimen along the primary scan direction within each of the image stripes are recorded at the optical detection unit at 250 frames / s with a specimen sampling pitch of 1 μm / pixel.

[0037] In some specific embodiments, following the formation of a first image stripe along the primary scan direction, the movable specimen holding platform is moved transversely to the primary scan direction in a secondary scan direction so that a next image stripe is formed adjacent to the first image stripe along the primary scan direction, and the path of movement of the movable specimen holding platform to cover the entire maximum surface area of ​​the biological tissue forms a serpentine or spiral centering pattern.

[0038] Optionally, the path of movement of the movable specimen holding platform over the entire maximum surface area of ​​the biological specimen can be any other pattern, as long as the entire maximum surface area of ​​the biological specimen is imaged and is subject to the degrees of freedom of movement of the 3D translation stage.

[0039] In some specific embodiments, the result is approximately 10% or less of the features in overlap between each pair of adjacent image stripes.

[0040] In some specific embodiments, the image processing module may be a stand-alone computer processor or network coupled to the imaging system or part of the imaging system, and the image processing module includes one or more image processing algorithms that process a series of image stripes of the detected image data.

[0041] In certain embodiments, distortion of the original image stripe due to the detection angle not being parallel to the vertical axis of the specimen holder can be corrected by one of the image processing algorithms.

[0042] In some specific embodiments, at least surface features in the detected image data are extracted by other image processing algorithms until all of the image stripes have been processed.

[0043] In some specific embodiments, one of the image processing algorithms that corrects for distortions in the original image stripe due to the detection angle not being parallel to the vertical axis of the specimen holder is written in MATLAB®.

[0044] In some particular embodiments, one of the image processing algorithms for surface feature extraction is an extended DOF algorithm.

[0045] In some specific embodiments, the extended DOF algorithm is implemented in MATLAB® via a Fiji plug-in.

[0046] In some specific embodiments, another image processing algorithm that combines extracted surface features of one image stripe with those of its neighboring image stripe is implemented by the Fiji Grid Stitching Plugin.

[0047] A third aspect of the present invention provides a method for imaging biological tissue or structures in a subject using the imaging system of the present invention in vivo, the method comprising: (2a) positioning a subject under anesthesia on the inner surface of the transparent base of the specimen holder in a position such that the biological tissue of interest is positioned as close as possible to the UV-transmitting window of a prismatic structure of the movable specimen holding platform that allows the light beam of the light sheet to enter or the light beam of the luminescence comprising the emitted fluorescent signal of the specimen to exit the specimen holder at an incident or detection angle of 45° relative to the vertical axis of the specimen holder; (2b) adjusting one or more of the first plurality of optical elements so that a light beam generated by the optical element strikes a bottom surface of the specimen at an angle of incidence of 45° relative to a vertical axis of the specimen holder; (2c) adjusting one or more of the second plurality of optical elements so that the light beam produced by the optical element is detected at a detection angle of 45° relative to a vertical axis of the specimen holder or orthogonal to the incident beam path of the light sheet; (2d) adjusting one or more of the second plurality of optical elements to receive luminescence comprising a fluorescent signal emitted from the specimen at a detection angle of 45° relative to a vertical axis of the specimen holder or orthogonal to the incident beam path of the light sheet; (2e) activating a deep UV excitation source to generate UV illumination light (ultraviolet light) toward a first plurality of optical elements, then modulating the UV illumination light by the first plurality of optical elements to generate a first spot on the specimen through one of the UV-transmitting windows of the prismatic structure at the bottom of the liquid holder, and directing a light sheet toward the first spot at a 45° angle of incidence with respect to a vertical axis of the specimen holder; (2f) receiving luminescence comprising emitted fluorescent signals from analytes excited by the light sheet via a second plurality of optical elements; (2g) detecting the received luminescence, including the fluorescent signals, emitted from the second plurality of optical elements by an optical detection unit including a plurality of CMOS image sensors; (2h) moving a movable specimen-holding platform driven by a 3D translation stage in a primary scanning direction from the first spot to a next spot on the specimen that is coplanar, and repeating steps (2e)-(2g) within each of the next spots until the last spot is reached that is coplanar in the primary scanning direction to form a first image stripe; (2i) moving the movable specimen-holding platform transversely to the first scanning direction in a secondary scanning direction, and repeating steps (2e) through (2h) to form subsequent image stripes adjacent to the first image stripe until the entire maximum surface area of ​​the biological tissue is scanned; (2j) processing the image data detected by the optical detection unit to reconstruct the geometry of the biological tissue by an image processing module, and outputting one or more images representing the overall geometry of the biological tissue after reconstruction.

[0048] In certain specific embodiments, the movable specimen holding platform is of an open-top configuration to allow specimens of any size and thickness to be conveniently loaded from above.

[0049] In certain embodiments, subjects include humans and non-human animals.

[0050] In certain embodiments, the biological tissue or structure includes freshly excised or intact normal and abnormal tissue, as well as parts of internal organs or entire internal organs.

[0051] Other possible configurations that can be utilized as described in the second aspect may be various embodiments of the method in the third aspect.

[0052] This Summary is intended to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. The Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to aid in determining the scope of the claimed subject matter. Other aspects of the present invention will be disclosed as illustrated by the following embodiments.

[0053] The accompanying drawings include illustrations of certain specific embodiments, wherein like reference numerals indicate identical or functionally similar elements, to further describe and clarify these and other aspects, advantages, and features of the present invention. It will be appreciated that the drawings illustrate embodiments of the present invention and are not intended to limit its scope. The present invention will be described and explained with the aid of the accompanying drawings, which will reveal additional features and details. [Brief explanation of the drawings]

[0054] [Figure 1A] FIG. 1 shows a schematic diagram of a MUSI-based system of the present invention according to some specific embodiments. [Figure 1B] 1B is a diagram showing a schematic diagram of the structure and working mechanism of the system of the present invention as shown in FIG. 1A. [Figure 1C] FIG. 1B illustrates an example of an image processing method for raw images of a tissue specimen collected by the system of FIG. 1A. [Figure 1D] 1D shows an image resulting from processing multiple image stripes of an original image and an image of an extracted surface of a tissue specimen collected by the image processing method shown in FIG. 1C, according to some specific embodiments. [Figure 2A] 1A and 1B show side-view images of light sheet illumination produced by the system of the present invention in accordance with some specific embodiments. [Figure 2B] 2B shows the intensity distribution along the lateral direction at the focal point of the light sheet as shown in FIG. 2A. [Figure 2C] FIG. 2B illustrates the distribution of beam radii along the axial direction at the focal point of the light sheet as shown in FIG. 2A. [Figure 3A-3C] Figure 3A shows label-free in vitro imaging of freshly excised mouse tissue by a system of the invention according to certain embodiments, showing a MUSI image of a fresh mouse tissue and a photograph of the specimen in the lower left inset (bar is 1 mm). Figure 3B shows label-free in vitro imaging of freshly excised mouse tissue by a system of the invention according to certain embodiments, showing one of the enlarged areas shown in Figure 3A and its corresponding H&E stained image (scale bar is 100 μm). Figure 3C shows label-free in vitro imaging of freshly excised mouse tissue by a system of the invention according to certain embodiments, showing another enlarged area shown in Figure 3A and its corresponding H&E stained image (scale bar is 100 μm). [Figure 3D] FIG. 3C shows label-free in vitro imaging of freshly excised mouse tissue by a system of the present invention according to some specific embodiments. FIG. 3D shows the distribution of nuclear features extracted from FIG. 3C, where a Wilcoxon rank sum test was performed across groups with n=50 for nuclear distribution, with significance defined as p*≦0.05 in all cases. [Figure 3E-3G] Figure 3E shows label-free in vitro imaging of freshly excised mouse tissue by a system of the present invention according to certain embodiments. The image shows a MUSI image of a fresh mouse brain, with the bottom left illustration showing a photograph of the specimen (bar is 1 mm, GL indicates the granular layer, ML indicates the molecular layer, and WM indicates the white matter layer). Figure 3F shows label-free in vitro imaging of freshly excised mouse tissue by a system of the present invention according to certain embodiments, with one of the magnified regions shown in Figure 3E and its corresponding H&E stained image (bar is 100 μm, GL indicates the granular layer, ML indicates the molecular layer, and WM indicates the white matter layer). Figure 3G shows label-free in vitro imaging of freshly excised mouse tissue by a system of the present invention according to certain embodiments, with another magnified region shown in Figure 3E and its corresponding H&E stained image (scale bar is 100 μm, GL indicates the granular layer, ML indicates the molecular layer, and WM indicates the white matter layer). [Figure 3H-3J]Figure 3H shows label-free in vitro imaging of freshly excised mouse tissue by a system of the present invention according to certain embodiments. The image shows a MUSI image of a fresh mouse kidney, and the bottom left inset shows a photograph of the specimen (bar is 1 mm). Figure 3I shows label-free in vitro imaging of freshly excised mouse tissue by a system of the present invention according to certain embodiments. The image shows one of the magnified areas shown in Figure 3H and its corresponding H&E stained image (scale bar is 100 μm). Figure 3J shows label-free in vitro imaging of freshly excised mouse tissue by a system of the present invention according to certain embodiments. The image shows another magnified area shown in Figure 3H and its corresponding H&E stained image (scale bar is 100 μm). [Figure 4A] FIG. 1 shows label-free in vivo imaging of live, intact mouse tissue by the system of the present invention according to some specific embodiments. FIG. 2 shows MUSI organization of live brain tissue. [Figure 4B] FIG. 1 shows label-free in vivo imaging of live, intact mouse tissue by the system of the present invention according to some specific embodiments. FIG. 2 shows MUSI tissue of live kidney tissue. [Figure 5] FIG. 1 shows images of human lung adenocarcinoma tissue by the system of the present invention according to some specific embodiments. [Figures 6A-6D]Figure 6A shows imaging of human lung adenocarcinoma tissue by a system of the present invention according to certain embodiments. The image shows MUSI and H&E staining images of a lung specimen containing acinar-predominant adenocarcinoma, and the illustration at the bottom left shows a photograph of the specimen (scale bar is 1 mm). Figure 6B shows imaging of human lung adenocarcinoma tissue by a system of the present invention according to certain embodiments. The image shows one of the magnified areas shown in Figure 6A and its corresponding H&E staining image (scale bar in the small panel is 50 μm). Figure 6C shows imaging of human lung adenocarcinoma tissue by a system of the present invention according to certain embodiments. The image shows another magnified area as shown in Figure 6A and its corresponding H&E staining image (scale bar in the small panel is 50 μm). Figure 6D shows imaging of human lung adenocarcinoma tissue by a system of the present invention according to certain embodiments. The image shows another magnified area as shown in Figure 6A and its corresponding H&E staining image (scale bar in the small panel is 50 μm). [Figures 6E-6H]Figure 6E shows MUSI and H&E staining images of a lung specimen containing papillary-predominant lung adenocarcinoma, with the lower left illustration showing a photograph of the specimen (scale bar 1 mm). Figure 6F shows one of the magnified areas shown in Figure 6E and its corresponding H&E staining image (scale bar 50 μm in the small panel, TN indicates tumor cell nuclei). Figure 6G shows another magnified area shown in Figure 6E and its corresponding H&E staining image (scale bar 50 μm in the small panel, TN indicates tumor cell nuclei, FC indicates branching fibrovascular core) using a system of the present invention, according to certain embodiments. Figure 6H shows imaging of human lung adenocarcinoma tissue by the system of the present invention according to some specific embodiments, showing another enlarged area as shown in Figure 6E and its corresponding H&E stained image (scale bar in small panel is 50 μm, AS indicates alveoli). [Figures 6I-6L]Figure 6I shows imaging of human lung adenocarcinoma tissue by a system of the present invention according to certain embodiments, showing MUSI and H&E staining images of a lung specimen containing micropapillary-predominant adenocarcinoma, with the lower left illustration showing a photograph of the specimen (scale bar is 1 mm). Figure 6J shows imaging of human lung adenocarcinoma tissue by a system of the present invention according to certain embodiments, showing one of the magnified areas shown in Figure 6I and its corresponding H&E staining image (scale bar is 100 μm, MC indicates tumor cell clusters). Figure 6K shows imaging of human lung adenocarcinoma tissue by a system of the present invention according to certain embodiments, showing another magnified area shown in Figure 6I and its corresponding H&E staining image (scale bar is 100 μm, MC indicates tumor cell clusters). FIG. 6L shows imaging of human lung adenocarcinoma tissue by the system of the present invention according to some specific embodiments, showing another enlarged area as shown in FIG. 6I and its corresponding H&E stained image (scale bar is 100 μm, LN indicates tumor-infiltrating lymphatic vessels, and AP indicates pneumoconiosis pigment). [Figures 7A-7D] FIG. 7A illustrates imaging of human skin tissue with a system of the present invention, according to certain embodiments, showing a MUSI of a human skin tissue specimen and its corresponding H&E stained image (scale bar is 1 mm). FIG. 7B illustrates imaging of human skin tissue with a system of the present invention, according to certain embodiments, showing one of the magnified areas shown in FIG. 7A and its corresponding H&E stained image (scale bar is 100 μm). FIG. 7C illustrates imaging of human skin tissue with a system of the present invention, according to certain embodiments, showing another magnified area shown in FIG. 7A and its corresponding H&E stained image (scale bar is 100 μm). FIG. 7D illustrates imaging of human skin tissue with a system of the present invention, according to certain embodiments, showing another magnified area shown in FIG. 7A and its corresponding H&E stained image (scale bar is 100 μm). [Figure 8] 1 is a flow chart outlining the imaging method of the present invention for ex vivo and in vivo imaging of target tissue according to some specific embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0055] Those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and clarity of drawing and have not necessarily been drawn to scale.

[0056] It will be apparent to those skilled in the art that modifications, including additions and / or substitutions, can be made without departing from the spirit and scope of the invention. While certain details may be omitted so as not to obscure the invention, the disclosure is written to enable those skilled in the art to practice the teachings herein without undue experimentation.

[0057] The present invention provides a rapid, label-free, non-destructive method and corresponding system that allows imaging of unprocessed biological tissues in vitro, and even in vivo, to provide a real-time qualitative assessment tool for medical technicians to intraoperatively locate and determine the extent of tissue to be removed compared to the conventional clinical standard, i.e., conventional histological evaluation including H&E staining. The present invention also has promising potential as an adjunct diagnostic tool for pathologists or clinicians to evaluate the histological characteristics of biopsies alongside conventional FFPE methods to assess the disease prognosis of a subject.

[0058] The following examples provide details of some specific configurations of the imaging methods and corresponding systems of the present invention, and such details should not be construed as limiting the scope of the invention.

[0059] Example (A) Configuration of the open-top MUSI system and imaging parameters 1A and 1B, a schematic diagram of an open-top MUSI system according to some specific embodiments is shown. The system 100 includes a movable specimen-holding platform 110 including a specimen (or sample) holder and a liquid holder, the bottom side of which is provided with a prismatic structure. Other polyhedral shapes may be incorporated at the bottom of the liquid holder, allowing incident and detection (including emitted light) light beams to enter and exit the specimen-holding platform perpendicularly. As can be seen from the close-up view of the specimen holding platform shown in FIG. 1B , UV-transmitting quartz windows are provided on each of the two lateral faces of the prismatic structure, such that an incident beam of light sheet (sheet of light) from the deep-UV excitation source 120 and modulated by the first plurality of optical elements 130 enters the liquid holder of the specimen holding platform and reaches the tissue of interest held at the specimen holder, while emitted light, including fluorescent signals emitted by the tissue under excitation by the incident beam of light sheet from the deep-UV excitation source 120, exits the liquid holder and is then received by the second plurality of optical elements 140. The liquid-filled prismatic structure is configured to reduce imaging aberrations caused by the angle of incidence of the incident light beam from the light sheet and to be provided or partially positioned below the specimen holder to perform detection of emitted light, including fluorescent signals, at the air-tissue-specimen holder interface (as shown in the dotted outline in FIG. 1B ). To provide design flexibility for the prismatic structure, 3D printing may be used according to certain embodiments. Other conventional methods, such as injection molding, can also be used to fabricate the polyhedral structure together with the liquid holder so that the prismatic structure is incorporated as part of the base or bottom face of the liquid holder.

[0060] To allow the light sheet to reach the tissue specimen held at the specimen holder, the bottom of the specimen holder is made of a UV-transparent material, such as a UV-transparent polyethylene membrane. In some specific embodiments, the UV-transparent polyethylene membrane is about 50 μm thick. In exemplary embodiments, the liquid is water or a mixture of water and an agent that facilitates visualization of tissue architecture under excitation by the light sheet from a deep UV excitation source. In some specific embodiments, it may be a Gaussian light sheet. Figure 2A shows an experimental profile of an illuminated light sheet in a dilute solution of acridine orange, in which the light sheet generated by the system of the present invention is illuminated at an illumination wavelength (λ) of about 266 nm. illu ) and an effective numerical aperture (NA) of approximately 0.03 eff 2B and 2C show the normalized intensity and beam radius distribution of the light sheet at the focal point along the lateral and axial directions as shown in FIG. 2A. As can be seen in FIG. 2A, the waist radius (w0) is 2.8 μm, the DOF (2Z RA 3 mm wide Gaussian light sheet with a λ / D of approximately 200 μm can be generated based on the following parameters and conditions: within the first plurality of optical elements, UV illumination light generated by the deep UV excitation source 120 is spectrally filtered by a bandpass filter F1 (FF01-300 / SP-25, Semrock Inc.) and expanded by a pair of convex lenses L1, L2 (LA-4647-UV and LA4874UV, Thorlabs Inc.), with a reflector between L1 and L2 configured to direct the UV illumination light after being filtered by F1 and entering L1 towards the next optical element, including L2. Next, the beam propagates through an adjustable slit aperture SA (VA100C, Thorlabs Inc.) after expansion by L2 to output a 3 mm × 6 mm sized beam, which is then focused by a UV cylindrical lens CL (LJ4395RM, f = 100 mm, Thorlabs Inc.). Both the incident and detection (including emission) beam paths are vertically focused through two respective UV illumination light-transmitting windows (20 mm × 20 mm) provided on the two lateral faces of the water prism. After passing through the first UV-transmitting quartz window, the just-generated light sheet emits 2 mJ / cm. 2 The bottom surface of the specimen is illuminated through a water prism at a 45° angle of incidence (relative to the virtual vertical axis of the specimen holder) with an average energy fluence of 1000 uV, which is within the safe UV radiation threshold set by ACGIH®. Therefore, it is suggested that, prior to illumination, the first plurality of optical elements be adjusted to take the tissue specimen into account, resulting in a light sheet passing through the water prism at a 45° angle of incidence. The selection of a specific width for the light sheet is consistent with the field of view (FOV) of the system of the present invention. When the system of the present invention is used for in vivo imaging, the illumination energy must be well controlled to ensure that the UV illumination light is below the aforementioned threshold.

[0061] On the detection side, the second plurality of optical elements 140 are oriented to receive the maximum luminescence intensity from the tissue sample. In this regard, an achromatic UV objective lens OL (LMU-5X-NUV, NA = 0.12, Thorlabs, Inc.) is positioned parallel to the optical path of the detection beam from the tissue sample. A long-pass filter F2 (BLP01-325R-25, Semrock, Inc.) is positioned at the rear end of the OL, oriented in the same direction as the OL. The homogeneous beam filtered by F2 is then refocused by an infinity-correcting tube lens TL (TTL200-A, Thorlabs, Inc.) and finally imaged by an sCMOS camera 150 (PCO Edge 4.2, 2048 × 2048 pixels, PCO Inc.).

[0062] To enable scanning over the largest surface area of ​​the tissue specimen, the specimen-holding platform 110 is coupled to a 3D translation stage 160 (L-509.20SD00, PI miCos Gesellschaft mit Beschlenkter Haftung (PI miCos GmbH)), which allows for at least two-axis translation of the specimen-holding platform 110. In some specific embodiments, the largest surface area of ​​the tissue specimen to be scanned may be up to 5 cm × 5 cm. The specimen may be translated under exposure along the primary scanning direction, i.e., along the x-axis in FIGS. 1B and 1C, to a stationary light sheet at a constant speed of 250 μm / s. Images may be recorded at 250 frames / s by an sCMOS camera 150 with a sampling pitch of 1 μm / pixel. In the example shown in FIG. 1A, the image height (h) can be adjusted with a maximum tolerance of approximately 200 nm depending on the surface irregularities of the imaged specimen. The imaging speed in the case of in vitro imaging can be increased by at least one order of magnitude by a high-power UV source, which is advantageous when the intrinsic fluorescence signal is weak under relatively low-power UV radiation / excitation sources (less than 2 mJ / cm of UV radiation). 2The frame rate can be reduced to a few hundred milliseconds or less because the sCMOS camera can detect UV radiation (only 1000 Hz is used in this embodiment). Higher frame rates can be employed with sCMOS cameras because high-power UV radiation can produce images with sufficient signal-to-noise ratios with short exposure times.

[0063] Following scanning of the tissue specimen in the primary scanning direction (along the x-axis), the specimen is moved laterally along the y-axis at 1.8 mm intervals, as in Figure 1C, and scanned in a serpentine pattern, e.g., a vertical serpentine pattern. Multiple image stripes, as in Figure 1D, are stitched together using a corresponding algorithm for stitching image stripes or grids over a wide field of view (FOV), resulting in approximately 10% overlap between each pair of adjacent stripes. Raw images are stored at 16-bit depth and transferred at 2040 pixels (w) × 200 pixels (h) × 2 bytes × 250 frames / s = 0.2 GB / s through a Camera Link interface to a local workstation equipped with a high-speed solid-state disk (970 EVO Plus, Samsung Inc.). LabView (LabVIEW) automated design software is used to synchronize the acquired images and scanned area with the motion of the 3D translation stage. With the above settings, MUSI allows rapid imaging of freshly excited and intact tissue (either in vitro or in vivo) at 0.5 mm resolution with 1.5 μm in-plane resolution. 2 Scanning speeds of up to 1000 x 1000 can be achieved with up to 1000 x 1000 scans per second, resulting in histology-like images with sufficient diagnostic quality for medical technicians, surgeons, and pathologists who may have to make intraoperative, postoperative, or therapeutic decisions.

[0064] As shown in Figure 1C, raw images of the tissue specimen are initially captured in a linear scanning direction to form image stripes, which are then scanned in a serpentine pattern to cover the maximum surface area of ​​the tissue specimen, thereby generating multiple image stripes. Because the raw images are recorded at a 45° incidence angle relative to the tissue surface, this distorts the tissue geometry reconstructed by the raw data volume and loaded into MATLAB® (MathWorks, Inc.). To correct this distortion, the raw data volume is sheared by 45° in the x-z plane to produce a trapezoidal data volume, as shown in Figure 1C. Following distortion correction, an enhanced DOF algorithm is applied to the sheared data volume via a Fiji plugin that extracts the intact tissue surface of each image stripe (upper panel of Figure 1D). The extracted surfaces of adjacent image stripes are then stitched together using the aligned Fiji grid stitching plugin to form a large-area tissue image (lower panel of Figure 1D). In some specific embodiments, the shearing of raw images, correction of distortions due to shearing, extraction of intact surfaces from each just-generated image stripe, and alignment and stitching of multiple adjacent image stripes to form large-area images are all handled by microscripts in the MATLAB-Fiji interface. 2The processing time for a tissue surface is approximately 10 minutes using a workstation equipped with a Core i9-10980XE CPU @ 4.8 GHz, 8 × 32 GB RAM, and four NVIDIA GeForce RTX 3090 GPUs. In some specific embodiments, a digitally scanned light sheet system with a non-diffracting beam is used to adopt a long DOF to improve system tolerance for irregular tissue surfaces without compromising axial resolution. In some specific embodiments, some components of the second optical element at the detection side / arm of the system of the present invention can be modified, such as a fixed objective lens replaced with a switchable objective lens with different resolutions, to image tissue structures from submicron to macroscopic spatial scales.

[0065] To extract the distribution of nuclear features in the tissue of interest, the tissue image can be segmented using a Fiji plugin, such as Trainable Weka Segmentation (TWS), and then binarized and analyzed to collect the cross-sectional area and centroid of each nucleus. The nearest neighbor distance to a neighboring nucleus can be determined by the intercellular distance calculated based on the localized center position of the nucleus from the extracted nuclear features. In some embodiments, a deep learning neural network can be used to train a virtual staining model to generate pseudo-histologically stained MUSI images, thereby assisting medical technicians, such as pathologists or surgeons, in performing intraoperative or postoperative clinical evaluations more efficiently and accurately.

[0066] (B) Ex vivo imaging of mouse tissues Referring to Figures 3A-3D, ex vivo imaging of internal organs from mice using the system of the present invention according to some specific embodiments is compared with tissues prepared and imaged using traditional FFPE histology (hematoxylin-eosin (H&E) staining). Initially, internal organs, such as lungs, brain, and kidneys, are removed immediately after sacrificing a mouse (C57BL / 6). After removal, these organs are manually cut into 3-5 mm thick tissue slabs for imaging. After imaging, all specimens were processed histologically using standard protocols to obtain H&E stained images for comparison.

[0067] MUSI images of freshly excised mouse liver (Figures 3A-3C), brain (Figures 3E-3G), and kidney (Figures 3H-3J) tissue slabs, each sectioned at 5 mm thickness, compared with corresponding H&E-stained images (right panels in Figures 3B, 3C, 3E, 3F, 3I, and 3J, respectively), demonstrate that the light sheet generated by our system exhibits a UV penetration depth ranging from approximately 5 μm to approximately 30 μm, depending on the tissue microarchitecture, the degree of tissue scattering, and the density / distribution of several endogenous fluorophores in different tissue types / states. For example, UV penetrates variously into regions including densely packed cell nuclei (e.g., primary tumors), lipid droplets (e.g., subcutaneous tissue), or regions of large intercellular spaces (e.g., lung alveoli). Regarding normal mouse tissues, kidney and brain generally showed higher intrinsic fluorescence intensity than liver and lung tissues, pancreatic tissue emitted the least fluorescence among the five tissues after UV absorption, and the intensities of some metabolic enzymes, such as NADH and FAD, were generally higher in tumors than in normal tissues, making them promising tumor-specific biomarkers.

[0068] Notably, FFPE thin sections cannot accurately replicate the surface imaged by MUSI due to different imaging thicknesses and tissue deformation during processing. However, the cellular morphological features remain similar. The morphological features of blood cells in mouse liver were well characterized 10 nm below the tissue surface, showing high similarity to the H&E stained image, with the sole exception of sinusoidal capillaries (Figure 3B).

[0069] Nuclear features, such as cross-sectional area and intercellular distance, are extracted from both the MUSI and H&E-stained images for comparison. The statistical results (Figure 3D) were calculated from 50 blood cells selected from both the MUSI and H&E-stained images in Figure 3C, suggesting that the cellular features extracted by MUSI are in fairly good agreement with the clinical standard.

[0070] In the mouse cerebellum, a clear interface between the molecular and granular layers, including intermediate Purkinje cells, can be visualized in both MUSI and H&E-stained images (Figure 3F and Figure 3G). Similarly, in the mouse kidney, features of the renal tubules (Figure 3I) and glomerular capsule (Figure 3J) can be clearly identified in MUSI images, even though Bowman's space in the renal corpuscle is less visible compared to conventional FFPE histological features.

[0071] (C) Intravital imaging of mouse tissues In this example, for kidney imaging, a small incision was made on the left side of the abdomen to expose the kidney, which was then positioned flat against a holder for imaging from below. For brain imaging, the mouse was placed on its back against the holder, and a 5 mm × 5 mm cranial window was created in the skull to expose the target brain region for imaging. Figures 4A and 4B show MUSI images of in vivo brain tissue and in vivo kidney tissue, respectively. These magnified views reveal structural details, such as blood vessels, membrane lipids, and tubules, from the corresponding tissues. The microvascular architecture and cell nuclei located at the brain cortical surface in Figure 4A facilitate real-time detection of tumor margins during neurological surgery, and renal tubules are easily identified with a high signal-to-noise ratio (Figure 4B) due to highly fluorescent cytoplasmic lipofuscin and urinary cast material. Although the penetration depth of MUSI is limited by strong tissue scattering, the present invention still provides a nondestructive and rapid assessment tool for intact tissue, especially in cases where biopsy may be contraindicated.

[0072] (D) Ex vivo imaging of human tissues In this example, lung adenocarcinoma specimens were used to demonstrate the capabilities of the MUSI system of the present invention. Adenocarcinoma typically arises from mucous glands and is known to be the most common type of non-small cell adenocarcinoma occurring in the peripheral lung. To obtain freshly resected intact human lung cancer tissue, specimens (n ​​= 11) were obtained from lung cancer patients who underwent therapeutic lung cancer surgery with informed consent at Queen Mary Hospital. Following lobectomy, lung cancer tissue was sectioned from the resected lobe and imaged with the system of the present invention to obtain MUSI images. The lung cancer tissue was then formalin-fixed and subjected to standard imaging of H&E-stained slices for comparison. Figure 5 shows a collection of representative MUSI images of a human lung adenocarcinoma specimen obtained with the system of the present invention and their corresponding H&E images. Microstructures representative of different subtypes of lung adenocarcinoma, such as lepidic, acinar, papillary, micropapillary, and solid, were visible in some of the MUSI images. Figures 6A-6D show a specimen containing acinar-predominant adenocarcinoma, in which irregularly shaped glands in a fibrous stroma are easily identified by MUSI (Figures 6B and 6C). Figure 6C shows that some glands are arranged as solid clusters of tumor cells with lumens that are difficult to discern. Figure 6D shows that tissue fragments resulting from cell destruction are easily identified by MUSI, with significantly increased intrinsic fluorescence.

[0073] In comparison, a papillary-predominant lung adenocarcinoma specimen was imaged by MUSI, and the results are compared with H&E-stained images, a clinical standard, shown in Figures 6E–6H. A positive surgical margin, delineating a clear interface between normal and cancerous areas, is indicated by the dotted line in Figure 6E. Figure 6F shows that tumor cell nuclei (TN) can be clearly characterized by MUSI at a penetration depth of approximately 10 μm below the tissue specimen surface. Figure 6G shows that both MUSI and H&E-stained images reveal a finger-like papillary architecture in which tumor cells line the surface of a branching fibrovascular core (FC). Figure 6H shows that alveolar structures (AS) from normal areas have normal morphological characteristics with large air spaces.

[0074] Figures 6I-6K show images of a pathologically confirmed micropapillary-predominant adenocarcinoma specimen by MUSI and clinical standard (H&E staining). Tumor cell clusters (MCs) are seen floating and isolated within open spaces lacking a fibrous core (Figures 6J and 6K). Figure 6L shows an area containing numerous tumor-infiltrating lymphatic vessels (LNs) and pneumoconiotic pigments (APs), which are consistent with the H&E staining image.

[0075] Figures 7A-7D show images of human skin by MUSI and clinical standard methods (H&E staining), in which the microarchitecture, e.g., sweat glands (Figure 7B), red blood cell-filled arterial lumens (Figure 7C), and adipose tissue (Figure 7D), were simultaneously characterized by MUSI in a label-free and non-destructive manner, and again, the images were in good agreement with the H&E staining images.

[0076] FIG. 8 summarizes the imaging method of the present invention. Initially, a tissue of interest (or specimen) is placed on the inner surface of the base of a specimen holder, which in this case is formed by a UV-transparent membrane. The specimen holder is then immersed in the liquid holder of the movable specimen holding platform (801). Preferably, a relatively flat surface of the tissue of interest is positioned on the inner surface of the base of the specimen holder to result in a laser light scattering effect. In some specific embodiments, the tissue of interest is freshly excised from a subject for ex vivo imaging with MUSI, or the surface of the tissue of interest is directly exposed to a light sheet for in vivo imaging. In embodiments in which in vivo imaging of the tissue of interest is performed, the specimen holder and liquid holder of the movable specimen holding platform must be sterilized because the surface of the tissue of interest (e.g., the surface of an internal organ) and adjacent tissues are in direct contact with the inner surface of the base of the specimen holder. The specimen holder, along with the tissue of interest, is then immersed in a liquid held in a liquid holder of the movable specimen holding platform, resulting in an interface between the surface of the tissue of interest and the UV-transparent membrane forming the base of the specimen holder. In some specific embodiments, the liquid held in the liquid holder for in vitro or in vivo imaging with MUSI is selected from water or a mixture of water and UV-transparent materials. Other liquids with appropriate refractive index and high UV transparency, such as liquids with refractive indexes between 1.3 and 1.5, may be used to hold the tissue of interest and fill the interface between the tissue of interest surface and the specimen holder base. As in FIG. 1B, the system of the present invention, according to some specific embodiments, is an open-top light sheet configuration, where the light sheet can pass through the tissue of interest and exit the specimen holder through the open top of the movable specimen holding platform. A long depth of field of up to 200 μm from the surface of the tissue of interest (as in the illustration in Figure 1B) and a high spatial resolution of 1.5 μm (lateral) and 2.8 μm (axial) can be achieved by the selective biaxial planar illumination produced by MUSI, thereby enabling the identification of intracellular diagnostic features in surgical tissues with large irregular surfaces.The dual-axis planar illumination configuration separates the illumination light from the detection path.

[0077] To ensure that the incident and detection beam paths are orthogonal to each other and each lie below the specimen at a 45° angle relative to the vertical axis of the specimen holder, multiple optical elements in the illumination arm of the system of the present invention, including a reflector, a bandpass filter, a pair of convex lenses, an adjustable slit aperture, and a UV cylindrical lens, direct a deep UV excitation light beam generated by a nanosecond UV pulsed laser (wavelength of about 266 nm) toward the bottom of the tissue specimen held in the specimen holder at the aforementioned angles of incidence, modulating the light beam into a light sheet several millimeters wide (803), e.g., with a waist radius (w) of about 2.8 μm and a DOF (DOF) of about 200 nm. R ) of the light sheet (as shown in FIGS. 2A-2C). In some specific embodiments, the just-generated light sheet remains within the safe UV radiation thresholds set by ACGIH®. The beam path of the light sheet is configured to be substantially perpendicular to one of the UV-transmitting windows located at one of the lateral faces of the prism-like structure, which is partially at the bottom of the liquid holder. The two opposite lateral faces of the two UV-transmitting windows allow maximum transmission of the incident light beam of the light sheet and the light emission light beam, including the emitted fluorescent signal from the tissue specimen after excitation, and the detection light beam. In some specific embodiments, the at least two UV-transmitting windows are UV-transmitting quartz windows. Preferably, the light sheet passing through the prism-like structure at the bottom of the liquid holder has a fluence of 2 mJ / cm. 2 , which is sufficient to excite endogenous fluorophores in tissue to emit fluorescent signals as a contrast source with deep UV excitation.

[0078] Similarly, the second plurality of optical elements in the detection arm of the system are adjusted accordingly to receive a maximum level of the detection (including emission) beam of fluorescent signal from the tissue specimen at a detection angle of 45° relative to the vertical axis of the specimen holder (804), and this detection beam exits the liquid holder through another UV-transmitting quartz window on the lateral face of the prism-like structure opposite the lateral face through which the incident beam of the light sheet passes. The other UV-transmitting quartz window is configured to be substantially perpendicular to the path of the detection beam, such that the second plurality of optical elements receive a maximum emission fluorescent signal from the tissue specimen. In some specific embodiments, the second plurality of optical elements include, but are not limited to, an achromatic UV objective lens, a long-pass filter, an infinity-corrected tube lens, and a mirror. The optical emission received from the tissue specimen by the second plurality of optical elements is captured by an optical detection unit, for example, an sCMOS camera (805). The tissue specimen is translated by the coupled 3D translation stage in a primary scan direction from one edge of the tissue specimen to the opposite edge of the tissue specimen to obtain a stripe of raw image data, and then translated in a secondary scan direction transverse to the primary scan direction at a distance adjacent to the first stripe of raw image data in the primary scan direction, until multiple stripes of raw image data are obtained to cover substantially the entire bottom surface of the tissue specimen (806). In some specific embodiments, the multiple stripes of raw image data are obtained by scanning the bottom surface of the tissue specimen in a serpentine pattern between the primary and secondary scan directions. Other scan patterns are possible in the present invention, provided that the maximum surface area of ​​the tissue specimen can be scanned. A large image height may be employed to accommodate large surface irregularities, such as rough tissue including lung resection and lobectomy specimens. In some specific embodiments, two adjacent stripes of raw image data each have an overlap area of ​​approximately 10%.The multiple stripes of raw image data are separately stored in memory within the system or workstation of the present invention for subsequent image processing and stitching to form a wide field of view (FOV). After stitching the multiple image stripes, the stored raw image data is then loaded as a stack (data volume) into an image processing module with different plug-ins to reconstruct tissue geometry from the raw data volume and extract surface features from the large FOV (807). In some specific embodiments, the image processing module is a MATLAB®-Fiji interface with multiple plug-ins (algorithms) for shearing a raw data volume originally recorded at a 45° angle relative to the tissue surface (e.g., the x-z plane as shown in FIG. 1C ) to form a trapezoidal data volume to correct image distortion resulting from the raw image data obtained at a 45° detection angle, stitching the image stripes of raw data to form a wide FOV, and extracting nuclear features from the stitched images to train a deep learning model for the spatial distribution of cell nuclei. The reconstructed tissue image can be output directly 808 for interpretation by a medical technician or surgeon of the condition of the tissue specimen, or can be compared 809 with a corresponding H&E stained image, which is then interpreted by the medical technician or surgeon. In some specific embodiments, the deep learning model generates a pseudo-histologically stained MUSI image to aid the medical technician or surgeon in interpreting the condition of the tissue specimen for intraoperative decision making or postoperative clinical evaluation.

[0079] Although the present invention has been described with reference to certain specific embodiments, other embodiments apparent to those skilled in the art are also within the scope of the present invention, and the scope of the present invention is therefore to be determined solely by the scope of the claims that follow.

[0080] Industrial Applicability The present invention provides a platform and imaging method for freshly excised and live, intact tissue surfaces, and has great potential as a next-generation clinical standard and intraoperative diagnostic tool that can provide real-time guidance to surgeons or pathologists for clinical or medical decisions, such as the removal of suspicious tumors during cancer surgery. The accuracy and specificity of this label-free, non-destructive imaging method can be improved by utilizing a combination of MUSI and autofluorescence spectroscopy to facilitate in vivo characterization of functional, structural, and / or metabolic changes in tissues of interest. The incorporation of deep learning neural networks and models in identifying the presence and spatial distribution of distinct microarchitectures in tissues of interest also facilitates pathologists' interpretation of tissue status for disease diagnosis.

Claims

1. 1. An imaging system comprising: a movable specimen holding platform having a specimen holder and a liquid holder, the liquid holder having a prism-like structure; a deep UV excitation source that provides deep UV illumination light; a first plurality of optical elements for generating a light sheet from the deep UV illumination light provided by the deep UV excitation source and directing the light sheet toward a bottom side of the specimen holder through one of the lateral faces of the prismatic structures on the bottom surface of at least one of the liquid holders and onto the bottom surface of the specimen at an angle of incidence relative to a vertical axis of the specimen holder; a second plurality of optical elements configured to receive luminescence comprising a fluorescent signal emitted from the specimen under excitation by the light sheet at a detection angle relative to the normal axis of the specimen holder; an optical detection unit that detects and processes the emitted light received by the second plurality of optical elements; An imaging system wherein the angle of incidence and the angle of detection are substantially the same in magnitude and the beam path of the light sheet is substantially perpendicular to the path of the emission light from the specimen.

2. The imaging system of claim 1 , wherein the movable specimen-holding platform is of an open-top configuration.

3. The imaging system of claim 1 , wherein the movable specimen-holding platform is coupled to a three-dimensional (3D) translation stage.

4. The imaging system of claim 1 , wherein at least one surface of the specimen holder includes a membrane for supporting the specimen.

5. 10. The imaging system of claim 1, wherein the liquid in the liquid holder of the movable specimen holding platform comprises water or a mixture of water and a UV-transparent substance.

6. The imaging system of claim 1 , wherein the first plurality of optical elements comprises a first filter, a pair of lenses, a slit aperture, and a cylindrical lens.

7. The imaging system of claim 6 , wherein the first filter is a bandpass filter.

8. 7. The imaging system of claim 6, wherein said pair of lenses is a pair of UV-grade convex lenses.

9. The imaging system of claim 6 , wherein the slit aperture is an adjustable slit aperture.

10. The imaging system of claim 6 , wherein the cylindrical lens is a UV cylindrical lens that generates a Gaussian light sheet.

11. 2. The imaging system of claim 1, wherein the angle of incidence of the light sheet illuminating the bottom surface of the specimen is 45° relative to the vertical axis of the specimen holder, and the light sheet has an average energy fluence that complies with safety UV radiation thresholds regulated by the American Conference of Government Industrial Hygienists.

12. The imaging system of claim 1 , wherein the liquid holder is positioned below the specimen holder within the movable specimen holding platform.

13. 2. The imaging system of claim 1, wherein the specimen is supported by a UV-transparent film secured at the base of the specimen holder so that the light sheet can reach the bottom surface of the specimen held in the specimen holder.

14. 14. The imaging system of claim 13, wherein the UV transparent film is made of a highly transparent thermoplastic resin such as polyethylene or any UV transparent material.

15. 2. The imaging system of claim 1, wherein the prismatic structure of the movable specimen holding platform each has at least two UV-transmitting windows located on two opposite lateral faces of the prismatic structure to allow the light sheet generated from the deep UV excitation source to enter or the emitted light, including the emitted fluorescent signal from the specimen after excitation by the light sheet, to exit the movable specimen holding platform.

16. 10. The imaging system of claim 1, wherein the specimen is biological tissue, including normal and abnormal tissue, freshly excised from a living organism or located in vivo.

17. 17. The imaging system of claim 16, wherein the biological tissue is rich in endogenous fluorophores including reduced nicotinamide adenine dinucleotide (NADH), structural proteins, aromatic amino acids, and heterocyclic compounds.

18. The imaging system of claim 1 , wherein the second plurality of optical elements includes at least a UV objective lens, a second filter, and an infinity corrected lens.

19. 20. The imaging system of claim 18, wherein the UV objective lens is an achromatic UV objective lens.

20. 20. The imaging system of claim 18, wherein the second filter is a long-pass filter.

21. 20. The imaging system of claim 18, wherein the infinity corrected lens is an infinity corrected tube lens.

22. The imaging system of claim 1 , wherein the optical detection unit comprises a plurality of CMOS image sensors.

23. 23. The imaging system of claim 22, wherein the plurality of CMOS image sensors of the optical detection unit comprise scientific complementary metal-oxide semiconductor (sCMOS) sensors.

24. A method for imaging biological tissue in a flavor-free, unprocessed manner, said method comprising the step of using the imaging system according to any one of claims 1 to 23 to output a histological-like image showing a two-dimensional or three-dimensional profile of said biological tissue, said step of using the imaging system according to claim 1 comprising the following steps: (a) placing the biological tissue in the specimen holder of the movable specimen holding platform of the imaging system with a relatively flat side of the biological tissue facing a UV-transparent membrane located at the bottom of the specimen holder; (b) immersing the specimen holder held with the biological tissue in the liquid holder of the movable specimen holding platform filled with liquid; (c) adjusting one or more of the first plurality of optical elements to orient a light sheet produced by the optical elements to strike the bottom surface of the specimen at a 45° angle of incidence with respect to the vertical axis of the specimen holder or with an incident beam path of the light sheet substantially perpendicular to a UV-transmitting window provided in one lateral face of the prismatic structure at the bottom of the liquid holder; (d) adjusting one or more of the second plurality of optical elements to receive luminescence comprising a fluorescent signal emitted from the specimen at a detection angle of 45° relative to the normal axis of the specimen holder or orthogonal to the incident beam path of the light sheet; (e) activating the deep UV excitation source to produce UV illumination light toward the first plurality of optical elements, then modulating the UV illumination light by the first plurality of optical elements to generate a first spot on the specimen through one of the UV-transmitting windows of the prismatic structure at the bottom of the liquid holder, and directing the light sheet toward the first spot at the angle of incidence of 45° relative to the vertical axis of the specimen holder; (f) receiving luminescence comprising emitted fluorescent signals from the analytes excited by the light sheet via the second plurality of optical elements; (g) detecting the received luminescence comprising fluorescent signals emitted from the second plurality of optical elements by the optical detection unit comprising the plurality of CMOS image sensors; (h) moving the movable specimen-holding platform driven by the 3D translation stage in a primary scan direction from the first spot to a next spot on the specimen in the same plane, and repeating steps (e) to (g) within each of the next spots until the last spot on the same plane is reached in the primary scan direction to form a first image stripe; (i) moving the movable specimen-holding platform transversely to the first scan direction in a secondary scan direction, and repeating steps (e) through (h) to form subsequent image stripes adjacent to the first image stripe until a maximum overall surface area of ​​the biological tissue is scanned; (j) processing the image data detected by the optical detection unit to reconstruct the geometry of the biological tissue by an image processing module, and outputting one or more images representing the overall geometry of the biological tissue after reconstruction.

25. 25. The method of claim 24, wherein the generated light sheet has a wavelength of 266 nm.

26. 25. The method of claim 24, wherein the light sheet has a penetration depth of up to about 30 μm from the bottom surface of the biological tissue.

27. 25. The method of claim 24, wherein following formation of a first image stripe along the primary scan direction, the movable specimen holding platform is moved transversely to the primary scan direction toward the secondary scan direction so that a next image stripe is formed along the primary scan direction adjacent to the first image stripe, and the path of movement of the movable specimen holding platform to cover the entire maximum surface area of ​​the biological tissue forms a serpentine or spiral centering pattern.

28. 25. The method of claim 24, wherein the image processing module is a stand-alone computer processor or network coupled to the imaging system or a portion of the imaging system, and the image processing module includes one or more image processing algorithms that process a series of image stripes of detected image data.

29. 25. The method of claim 24, wherein distortions of the original image of the image stripe caused by the detection angle not being parallel to the vertical axis of the specimen holder are corrected by one of the image processing algorithms.

30. 25. The method of claim 24, wherein at least surface features in the detected image data are extracted by other image processing algorithms until all of the image stripes have been processed.

Images