Methods and compositions for rapid tissue clearing and 3D imaging

Abstract

The present disclosure provides a treatment solution for rendering a biological sample optically transparent. The treatment solution comprises a contrast agent, a carbohydrate, a nitrogen-containing compound, and an antioxidant. The treatment solution renders the biological sample optically transparent while preserving fluorescent properties of the biological sample. Methods for processing biological samples using the treatment solution and related compositions are also provided.

Description

METHODS AND COMPOSITIONS FOR RAPID TISSUE CLEARING AND 3D IMAGINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 673,826 filed July 22, 2024, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under DK 130660, Al 163064, AI168044, HL007081, AG078106 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.FIELD OF INVENTION

[0003] The present disclosure relates to methods and compositions for tissue clearing and three-dimensional imaging, and more particularly to a rapid aqueous-based tissue clearing protocol that preserves tissue morphology and fluorescent signals while enabling high-resolution three-dimensional visualization across multiple tissue types and species.BACKGROUND

[0004] Over the last decade, interest in three-dimensional (3D) fluorescence tissue imaging has flourished, accompanied by advances in protocols to refine approaches. While some new imaging modalities like light-sheet microscopy have contributed to these advances, there have been many refinements in tissue processing to optimize image acquisition using established modalities like tile-scanning confocal microscopy. These efforts have focused on improving light penetration and reducing light diffraction through tissues to enable deeper and more detailed 3D imaging of biological structures.

[0005] A primary challenge in this field is achieving optical transparency of intact tissue samples while preserving their structural integrity and molecular information. Current methods often involve lengthy protocols that may prioritize either preserving fluorescent reporters or enabling immunolabeling, but struggle to accomplish both simultaneously. Additionally, manyexisting techniques can cause tissue shrinkage or distortion, limiting the accuracy of subsequent 3D reconstructions. The use of organic solvents in some clearing methods can denature fluorescent molecules, hindering certain applications and reducing the overall effectiveness of the imaging process.

[0006] Another significant hurdle is the time required for sample preparation and image acquisition. Existing protocols can take several days for the refractive index matching step alone, not including additional time for staining or other preparatory steps. This extended timeline limits the applicability of these techniques in time-sensitive scenarios, such as clinical diagnostics or rapid research workflows. Furthermore, the specialized equipment and expertise required for some advanced imaging modalities, like light-sheet microscopy, can restrict widespread adoption of 3D tissue imaging techniques in research and clinical settings. These limitations collectively impede the broader application and accessibility of high-quality 3D tissue imaging across various biological and medical fields.SUMMARY

[0007] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] In one aspect, the present invention provides a treatment solution for rendering a biological sample optically transparent. The treatment solution comprises a contrast agent, a carbohydrate, a nitrogen-containing compound, and an antioxidant. The treatment solution renders the biological sample optically transparent while preserving fluorescent properties of the biological sample.

[0009] In another aspect, the present invention provides a method for processing a biological sample. The method comprises treating the biological sample with a treatment solution comprising a contrast agent, a carbohydrate, a nitrogen-containing compound, and an antioxidant, wherein the treatment solution renders the biological sample optically transparent while preserving fluorescent properties of the biological sample. The method further comprises acquiring an image of the optically transparent biological sample and processing the acquired image to generate a three-dimensional visualization of the biological sample.

[0010] In yet another aspect, the present invention provides a method to optically clear and three-dimensionally image an intact tissue or whole organ while preserving tissue morphology. The method comprises treating the intact tissue or whole organ with at least one of a fixative, a decolorization buffer, a delipidation buffer, a refractive index matching (RIM) solution, and a decalcification solution. The method further comprises acquiring image(s) of the treated intact tissue or whole organ using microscopy and deconvolution of the acquired image(s).

[0011] In a further aspect, the present invention provides a composition of a refractive index matching (RIM) solution for use in imaging optically cleared three-dimensional images. The composition comprises an X-ray contrast reagent, a sugar, an amine, an inhibitor of Maillard- reactive browning, and an antioxidant.

[0012] The foregoing general description of the illustrative cases and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive.BRIEF DESCRIPTION OF FIGURES

[0013] Non-limiting and non-exhaustive examples are described with reference to the following figures.

[0014] FIG. 1 shows ADAPT-3D tissue processing and refractive index matching renders tissue optically transparent without shrinkage. (A) Comparison of fluorescent intensities of CD1 Ic-eYFP follicles in Peyer’s patches of mouse ileum following different fixative conditions (n = 4-5 follicles per condition, two-way ANOVA followed by Tukey test, *p-value < 0.05, Upvalue <0.01) and captured by stereomicroscopy. (B) Full thickness cross sections of fixed human colon untreated (top) or incubated in ADAPT :RI for 10 minutes (bottom) without decolorization or delipidation. (C) Luminal side of fixed mouse colons decolorized overnight (left) or left unprocessed (right) shown before (top) and after (bottom) a 60-minute incubation in ADAPT:RI. (D) Fixed mouse and piglet colon incubated for 60 minutes in ADAPT:DC, 60 minutes in ADAPT:PDL (left) and then incubated in ADAPERI for 30 or 60 minutes. (E) Fixed 1 mm brain section from a LysM-TdTomato mouse before ADAPT-3D processing. (F) The same section after 6 hours of ADAPT :DC followed by 5 hours of ADAPT :RI but without delipidation or (G) another section treated the same except for the addition of a 3 -hour incubation in ADAPT:PDL before viewing immersed in ADAPT:RI. (H) Tiled confocal image of a singleplane from a 1mm section with a LysM-TdTomato reporter (red) and nuclei stained with antihistone antibody (cyan). (I) Confocal acquired fluorescent z-stacks of the brain stem and (J) cerebral cortex from the 1mm section shown in F without ADAPT :PDL treatment (left) or from the 1mm section shown in G with 3 hours of ADAPT:PDL treatment. (K) Before and after top- down stereoscope image of fixed whole mouse brain and corresponding quantification of the brain area for duplicate samples following the iDISCO+ method including 4 hours of RIM in ethyl cinnamate or (L) following ADAPT-3D tissue processing consisting of 48 hours ADAPT:DC, 36 hours ADAPT:PDL, and 4 hours adapt RE Dashed lines on graph in K and L link paired samples before and after. ** in J indicates the location of the corpus callosum.

[0015] FIG. 2 shows AD APT-3 D maintains tissue integrity and fiuorescene intensity for accurate 3D imaging without excessive lipid removal. (A) Stereoscope images of 1 mm fixed brain section from a LysM-TdTomato mouse before treatment (top), immediately after 12hrs in CUBIC L at 37°C (middle), and after washing out of CUBIC-L buffer (bottom). (B) Matched brain section from the contralateral hemisphere to that in A before treatment (top), captured immediately after 12 hours in ADAPUPDL (middle), and after washing out of ADAPUPDL (bottom). (C) Stereoscope image of CUBIC processed 1 mm section from A after immersion first in CUBIC-R+(M) diluted to 50% in water and finally in full strength CUBIC-R+(M) for 4 hours (left) and the ADAPT-3D processed 1mm section from B after immersion first in 50% ADAPT:RI diluted in PBS and finally in full strength ADAP RI for 4 hours (right). (D) Tile scanned confocal image of the first in focus plane of the CUBIC processed section (left) and of the ADAPT-3D processed section (right) acquired with matched acquisition settings and displayed with equivalent scaling. (E) The area of CUBIC and ADAPT-3D processed sections measured from tiled confocal fluorescent images using image! software. (F) The mean fluorescence intensity of anti-Histone-atto488 and (G) of endogenous TdTomato in tiled confocal images acquired from sections processed with either CUBIC or ADAPT-3D and measured using image! after outlining the section borders (two-tailed unpaired T test, standard error of mean bars, *p-value < 0.05, *** p-value < 0.001, ****p-value <0.0001). (H) Zoomed in view of the lateral and 3rdventricle in the CUBIC processed section from D that was increased in brightness to be visable and (I) of the choroid plexus in the lateral ventricle of the ADAPT-3D processed section from D. (I) The leptomeninges on the cortical edge of a section processed with CUBIC vs (K) a section processed with ADAPT-3D. (L) Stereoscope image of white matter inmatched sections where one was treated for 3 hours with CUBIC-L at 37°C (top) and the other with ADAPT:PDL for 3 hours (bottom) that were washed out of their respective delipidation buffers. (M) Fixed peritoneal fluid cells from LysMcre-ABCAlfl / flABCGlfl / flmice untreated (left) or incubated in ADAPT:PDL for 15 minutes (right) and stained with LipidSpot 610. (N) Representative image of CellBrite Orange staining of peritoneal fluid cells after incubation in ADAPT :PDL for 15 minutes. (O) Representative image of EEA1 staining for endosomes in fixed cells from peritoneal fluid without delipidation (left) or after a 15 minute incubation in ADAPT:PDL (right) where combined endosomal and nuclear staining is shown as an inset.

[0016] FIG. 3 shows Light sheet imaging of the whole brain and of connections at the skullbrain interface visualized using endogenous fluorophores preserved by ADAPT-3D. (A) 3D whole-mount projection of the brain from a ChAT-CreER x TdTomatofl / flmouse (white) injected retro-orbitally with Lectin-Dylight649 to label vasculature (fire) acquired by light sheet microscopy. (B) Extended display near lateral ventricle from A displaying blood vessels in the core of the brain and preservation of fine neuron dendrites in the cortex. (C) 3D whole-mount projection of a brain from a 16-week old mouse expressing CD1 Ic-eYFP which was decolorized, delipi dated, and incubated in ADAPT:RI followed by imaging with light sheet microscopy. (D) 500-micron coronal maximum intensity projection from whole brain of CD1 Ic-eYFP where white arrow points to a CD1 Ic-positive neuron. (E) A 50-micron x-y maximum intensity projection of the brain borders from a light sheet image of the whole skull from a LyvelCreER x TdTomatofl / fl(red) mouse injected i.v. with Lectin-Dylight649 and CD31-AF647 to label blood vessels (white). The layers of the brain borders are annotated with the following abbreviations: muscle [MS], skull [SK], dura mater [D], leptomeninges [LM], brain parenchyma [B], (F) Dorsal view of the light sheet imaging volume acquired from the whole skull in E where arrowheads point to dural lymphatics and full arrows point to meningeal macrophages. (G) Extended display of Lyvel positive skull channels where the asterisk denotes consecutive skull channels bridging the skull bone marrow and meninges. (H) A graphical depiction of the relationship between the depth of light penetration in light sheet images of intact mouse skulls and the corresponding tissue preparation times reported for the different clearing protocols in the literature including the 8-day time frame determined here for ADAPT-3D.

[0017] FIG. 4 shows the effect of ADAPT-3D on finicky antigens and compatibility with deep immunolabeling. (A) Maximum intensity projections of tight junctions (arachnoid barrier:occludin in red and claudin-11 in green, endothelial-cell specific: claudin-5 in grey) found in leptomeninges from mouse imaged by confocal microscopy. B) Extended display showing en face and z-side projections of mouse ileum that was immunolabeled with alpha smooth muscle actin (yellow), lymphatic vasculature (LYVE-1, magenta), myeloid cells (S100A9, cyan), and nuclei (DAPI, grey) followed by imaging with confocal microscopy. C) Extended display showing en face and z-side projections of ileum from a 16-week-old mouse that expresses TnUARE?a model of ileitis. D) Extended display showing en face, z-side, and 3D projections of fixed human ileum applied with decolorization, delipidation, immunolabeling with CD 163 (green), IB Al (red), and nuclei (DAPI, grey) followed by refractive index matching.

[0018] FIG. 5 shows decolorization and ADAPT-3D tissue processing from sections to whole organs. (A) Full thickness cross sections of piglet colon untreated (top) or incubated in ADAPT:RI for 10 minutes (bottom) without decolorization or delipidation. (B) Effect of decolorization solution on full-thickness pieces of piglet colon using SHANEL (top) or ADAPT- 3D (bottom) captured at 40 and 80 minutes using a stereomicroscope. (C) Two unprocessed LysM-TdTomato sections immersed in ADAPT:RI (top) and the exact same sections reimmersed in ADAPT:RI after an overnight incubation in ADAPT:DC (bottom). (D) A 1mm LysM- TdTomato section at every ADAPT-3D tissue processing step including 6 hours of decolorization, 2 hours washing, 3 hours of delipidation, 2 hours washing, and 4 hours full strength RI matching. (E) The whole mouse spleen and left lung lobe cleared with the complete ADAPT-3D method (left and middle) including 48 hours of decolorization and 36 hours of delipidation, and the medial liver lobe from a ProxlERCre x TdTomato fl / fl mouse cleared with ADAPT-3D (right) that was decolorized for 72 hours, delipidated for 36 hours, and RI matched overnight.

[0019] FIG. 6 shows brain section tissue size and integrity maintained after clearing with ADAPT- 3D in comparison with CUBIC. (A) Stereoscope images of 1 mm fixed brain section from a LysM- TdTomato mouse before treatment (top), immediately after 24hrs in CUBIC-L at 37°C (middle), and after washing out of CUBIC-L buffer (bottom). (B) Matched brain section from the contralateral hemisphere to that in A before treatment (top), captured immediately after 24 hours in ADAPDPDL (middle), and after washing out of ADAPUPDL (bottom). (C) Stereoscope images of 1 mm fixed brain section from a LysM-TdTomato mouse before treatment (top), immediately after 6 hours in 50% CUBIC-L diluted in water at room temperature (middle), andafter washing into PBS (bottom). (D) Matched brain section from the contralateral hemisphere to that in C before treatment (top), captured immediately after 6 hours in ADAPT:DC (middle), and after washing out of ADAPT:DC (bottom). (E) The leptomeninges on the cortical edge of a section processed with CUBIC (top) vs a section processed with ADAPT-3D (bottom) where delipidation was performed for 3 hours for both methods or (F) for 24 hours.DETAILED DESCRIPTION

[0020] The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein.

[0021] Light sheet microscopy and preparative clearing methods that improve light penetration in 3D tissues have revolutionized imaging in biomedical research. While most clearing methods focus on removing molecules that scatter light, the methods generally involve immersing tissues in solutions that minimize refraction of light to enhance detection of fluorescent signal deeper into tissues. Here, a new tissue preparative method was developed called ADAPT-3D with broad applicability across species and tissue types. This method enables efficient antibody staining and detection of endogenous fluorophores and offers advantages in terms of speed at which tissue staining and clearing is achieved. In about 4 days from tissue harvest to imaging, human intestinal tissue could be fixed, decolored and delipidated to remove light-interfering substances and stained with antibodies for imaging. In the intact mouse skull and brain, involving an 8-day protocol from tissue harvest to completion of imaging, the aqueous and non-shrinking ADAPT-3D method allowed the specialized channels between skull and underlying tissue to be detected without meningeal tearing. Overall, ADAPT-3D provides a highly versatile preparative method for 3D fixed tissue imaging with superior time savings, sensitivity and preservation of tissue morphology compared with previously described methods.

[0022] The present disclosure relates to methods and compositions for rendering biological samples optically transparent while preserving their fluorescent properties. More specifically, the disclosure provides treatment solutions and processing techniques for optically clearing and three-dimensionally imaging intact tissues or whole organs while maintaining tissue morphology.

[0023] Tissue clearing and three-dimensional imaging methods have become valuable tools for studying complex biological structures. These approaches allow researchers to visualize large tissue volumes with high resolution, providing insights into cellular organization and interactions that may not be apparent from traditional two-dimensional histological sections. However, existing tissue clearing techniques often have limitations such as long processing times, loss of fluorescent signals, or tissue distortion.

[0024] The methods and compositions described herein, referred to as ADAPT-3D (Accelerated Deep Adaptable Processing of Tissue for 3-Dimensional Imaging), address many of the limitations of prior approaches. ADAPT-3D may provide rapid tissue clearing, preservation of endogenous and exogenous fluorescent labels, and minimal impact on tissue morphology. The disclosed treatment solutions and processing steps may be compatible with various microscopy techniques, enabling flexible experimental designs.

[0025] In some cases, the ADAPT-3D approach may involve treating biological samples with a series of specialized solutions. These may include fixatives, decolorization buffers, delipidation buffers, refractive index matching solutions, and decalcification solutions. Each component may be optimized to maintain sample integrity while enhancing optical transparency.

[0026] The refractive index matching solution may play a key role in rendering samples transparent. This solution may contain a combination of contrast agents, carbohydrates, nitrogencontaining compounds, and antioxidants carefully formulated to match the refractive index of biological tissues. By minimizing light scattering within the sample, this solution may allow for deep tissue imaging.

[0027] In addition to tissue processing, the ADAPT-3D method may incorporate advanced image acquisition and processing techniques. These may include the use of various microscopy modalities and computational approaches such as deconvolution to enhance image quality and resolution.

[0028] The ADAPT-3D approach may be applicable to a wide range of biological samples, from small tissue sections to intact organs. By preserving both tissue structure and molecular signals, this method may enable new insights into complex biological systems across multiple scales. The ADAPT-3D method may be applicable to various types of biological samples, including intact tissues and whole organs. By combining optimized tissue processing steps with advanced imaging and computational techniques, this method may enable detailed three-dimensional visualization of complex biological structures while preserving sample integrity and fluorescent signals.

[0029] The treatment solution for rendering biological samples optically transparent may comprise a combination of components carefully formulated to maintain sample integrity while enhancing optical clarity. In some cases, the treatment solution may include a contrast agent, a carbohydrate, a nitrogen-containing compound, and an antioxidant.

[0030] The contrast agent in the treatment solution may comprise an iodinated compound. In some cases, the iodinated compound may be iohexol, iodixanol, or a combination thereof. These X-ray contrast reagents may contribute to matching the refractive index of the biological sample. The concentration of the iodinated compound in the treatment solution may vary depending on the specific application and sample characteristics.

[0031] The iodixanol solution is typically a 60 wt.% solution of iodixanol sold as OptiPrep™ by StemCell Technologies.

[0032] The iohexol can be present at a concentration from about 20 w / v% to about 30 w / v%, from about 21 w / v% to about 30 w / v%, from about 22 w / v% to about 30 w / v%, from about 23 w / v% to about 30 w / v%, from about 24 w / v% to about 30 w / v%, from about 20 w / v% to about 29 w / v%, from about 20 w / v% to about 28 w / v%, from about 20 w / v% to about 27 w / v%, from about 20 w / v% to about 26 w / v%, from about 21 w / v% to about 29 w / v%, from about 22 w / v% to about 28 w / v%, from about 23 w / v% to about 27 w / v%, or from about 24 w / v% to about 26 w / v%, or about 25.3 w / v%, based on the weight of iohexol in the total volume of the treatment solution.

[0033] The carbohydrate component of the treatment solution may comprise a sugar. In some cases, the sugar may be sucrose. Other sugars that may be used include fructose, glucose, galactose, maltose, lactose, or combinations thereof.

[0034] The carbohydrate or sugar may be present at a specific concentration range within the treatment solution, which may be optimized for particular tissue types or imaging requirements. For example, the sugar can be present at a concentration from about 25 w / v% to about 35 w / v%, from about 26 w / v% to about 35 w / v%, from about 27 w / v% to about 35 w / v%, from about 28 w / v% to about 35 w / v%, from about 29 w / v% to about 35 w / v%, from about 25 w / v% to about 34 w / v%, from about 25 w / v% to about 33 w / v%, from about 25 w / v% to about 32 w / v%, from about 25 w / v% to about 31 w / v%, from about 26 w / v% to about 34 w / v%, fromabout 27 w / v% to about 33 w / v%, from about 28 w / v% to about 32 w / v%, from about 29 w / v% to about 31 w / v%, or about 30 w / v%, based on the weight of sugar in the total volume of the treatment solution.

[0035] The nitrogen-containing compound in the treatment solution may comprise urea. In some cases, other nitrogen-containing compounds such as biuret or alkyl ureas may be used, either alone or in combination with urea.

[0036] The concentration of the nitrogen-containing compound may be adjusted within a specific range to achieve desired clearing effects while preserving sample integrity. For example, the concentration can be from about 20 w / v% to about 30 w / v%, from about 21 w / v% to about 30 w / v%, from about 22 w / v% to about 30 w / v%, from about 23 w / v% to about 30 w / v%, from about 24 w / v% to about 30 w / v%, from about 20 w / v% to about 29 w / v%, from about 20 w / v% to about 28 w / v%, from about 20 w / v% to about 27 w / v%, from about 20 w / v% to about 26 w / v%, from about 21 w / v% to about 29 w / v%, from about 22 w / v% to about 28 w / v%, from about 23 w / v% to about 27 w / v%, or from about 24 w / v% to about 26 w / v%, or about 25 w / v%, based on the weight of nitrogen-containing compound in the total volume of the treatment solution.

[0037] An antioxidant may be included in the treatment solution to help preserve fluorescent properties of the biological sample. In some cases, the antioxidant may comprise n-propyl gallate.

[0038] The antioxidant may be present at a specific concentration range within the treatment solution. For example, the antioxidant can be present at a concentration from about 0.1 w / v% to about 1 w / v%, from about 0.2 w / v% to about 1 w / v%, from about 0.3 w / v% to about 1 w / v%, from about 0.4 w / v% to about 1 w / v%, from about 0.1 w / v% to about 0.9 w / v%, from about 0.1 w / v% to about 0.8 w / v%, from about 0.1 w / v% to about 0.7 w / v%, from about 0.1 w / v% to about 0.6 w / v%, from about 0.2 w / v% to about 0.9 w / v%, from about 0.3 w / v% to about 0.8 w / v%, from about 0.4 w / v% to about 0.7 w / v%, from about 0.4 w / v% to about 0.6 w / v%, or from about 0.5 w / v%, based on the weight of antioxidant in the total volume of the treatment solution.

[0039] In some cases, the treatment solution may further comprise a reaction inhibitor of a color-developing reaction. This reaction inhibitor may be specifically targeted at inhibiting Maillard-reactive browning reactions. In some cases, the reaction inhibitor may comprise 1- thioglycerol.

[0040] The concentration of the reaction inhibitor may be adjusted within a specific range to effectively prevent unwanted color development during the clearing process. For example, the reaction inhibitor can be present at a concentration from about 0.2 v / v% to about 2 v / v%, from about 0.5 v / v% to about 1.5 v / v%, from about 0.6 v / v% to about 1.5 v / v%, from about 0.7 v / v% to about 1.5 v / v%, from about 0.8 v / v% to about 1.5 v / v%, from about 0.9 v / v% to about 1.5 v / v%, from about 0.5 v / v% to about 1.4 v / v%, from about 0.5 v / v% to about 1.3 v / v%, from about 0.5 v / v% to about 1.2 v / v%, from about 0.5 v / v% to about 1.1 v / v%, from about 0.6 v / v% to about 1.4 v / v%, from about 0.7 v / v% to about 1.3 v / v%, from about 0.8 v / v% to about 1.2 v / v%, from about 0.9 v / v% to about 1.1 v / v%, or about 1 v / v%, based on the volume of reaction inhibitor in the total volume of the treatment solution.

[0041] The combination of these components may result in a treatment solution with a refractive index from about 1.45 to about 1.55. This refractive index range may be particularly effective for rendering biological samples optically transparent while preserving their fluorescent properties.

[0042] The composition of the treatment solution, comprising an X-ray contrast reagent, a sugar, an amine, an inhibitor of Maillard-reactive browning, and an antioxidant, may be optimized to achieve rapid and effective optical clearing of biological samples. The specific concentrations and combinations of these components may be adjusted based on factors such as sample type, size, and desired imaging depth.

[0043] The ADAPT-3D method may involve several steps for processing biological samples to achieve optical transparency and enable three-dimensional imaging. In some cases, the method may include treating the biological sample with a treatment solution, acquiring an image of the optically transparent biological sample, and processing the acquired image to generate a three- dimensional visualization of the biological sample.

[0044] The treatment process may begin with fixing the biological sample. In some cases, the fixative may be a solution comprising paraformaldehyde and a sugar, such as sucrose, at a pH ranging from about 7 to about 9. This fixative composition may help preserve endogenous fluorescent reporter proteins and prevent masking of tissue antigens.

[0045] The ADAPT-3D method may utilize a specialized fixative solution to prepare biological samples for optical clearing and imaging. In some cases, the fixative solution may comprise paraformaldehyde and a sugar. The paraformaldehyde may serve as a crosslinkingagent to stabilize tissue structures, while the sugar may help preserve sample morphology and fluorescent signals.

[0046] The fixative solution may have a pH ranging from about 7 to about 9. This pH range may be optimized to maintain sample integrity while allowing for effective fixation. In some cases, the pH may be adjusted to around 9.0, which may provide improved preservation of certain fluorescent proteins and tissue antigens.

[0047] A key feature of the fixative solution may be its ability to preserve endogenous fluorescent reporter proteins within the biological sample. Many fluorescent proteins, such as green fluorescent protein (GFP) and its variants, may be sensitive to fixation conditions. The combination of paraformaldehyde, sugar, and controlled pH in the ADAPT-3D fixative solution may help maintain the fluorescence of these proteins throughout the clearing and imaging process.

[0048] In addition to preserving fluorescent signals, the fixative solution may be formulated to prevent masking of tissue antigens. Antigen masking can occur when fixation alters protein structures in a way that interferes with antibody binding. The ADAPT-3D fixative solution may minimize this issue, potentially allowing for more effective immunostaining of cleared samples.

[0049] The sugar component of the fixative solution may be sucrose. In some cases, the concentration of sucrose may range from about 10 w / v% to about 30 w / v%. The inclusion of sucrose in the fixative may serve multiple purposes, including osmotic balance maintenance and protein stabilization.

[0050] The paraformaldehyde concentration in the fixative solution may typically be around 4% (w / v), although this concentration may be adjusted based on specific sample requirements. In some cases, lower concentrations of paraformaldehyde may be used to further reduce the risk of antigen masking or fluorescent protein quenching.

[0051] The fixative solution may be applied to biological samples through various methods, such as perfusion for whole organs or immersion for smaller tissue samples. The duration of fixation may vary depending on sample size and type, but may generally range from a few hours to overnight at a controlled temperature, often around 4°C.

[0052] By carefully balancing the components and conditions of the fixative solution, the ADAPT-3D method may achieve effective tissue stabilization while maintaining compatibility with subsequent clearing and imaging steps. This approach may allow for high-quality three-dimensional visualization of complex biological structures with preserved fluorescent signals and accessible antigenic sites.

[0053] Following fixation, the biological sample may be treated with a decolorization solution. The decolorization solution may comprise a detergent that forms small micelles or salt- free amines, one or more N-alkyldiethanolamines, and a humectant. In some cases, the detergent may be (3 -cholamidopropyl)dimethylammonio)-l -propanesulfonate (CHAPS). The N- alkyldiethanolamines may include N-methyldiethanolamine and N-butyldiethanolamine. The humectant may be 1,2-hexanediol.

[0054] The ADAPT-3D method may involve treating biological samples with a decolorization solution to remove pigments and enhance tissue transparency. The decolorization solution may comprise a combination of components designed to effectively remove colorcausing molecules while preserving sample integrity.

[0055] In some cases, the decolorization solution may include a detergent that forms small micelles or salt-free amines. One such detergent may be (3-cholamidopropyl)dimethylammonio)- 1 -propanesulfonate (CHAPS). CHAPS may be effective at solubilizing and removing pigments from tissues without causing significant damage to cellular structures.

[0056] The decolorization solution may also contain one or more N-alkyldiethanolamines. In some cases, these may include N-methyldiethanolamine and N-butyldiethanolamine. These compounds may help facilitate the penetration of the decolorization solution into tissues and assist in the removal of pigments.

[0057] A humectant may be included in the decolorization solution to help maintain tissue hydration during the decolorization process. In some cases, the humectant may be 1,2- hexanediol. This compound may help prevent excessive dehydration of the sample, which could otherwise lead to tissue shrinkage or distortion.

[0058] The combination of a detergent (such as CHAPS), N-alkyldiethanolamines (such as N-methyldiethanolamine and N-butyldiethanolamine), and a humectant (such as 1,2-hexanediol) in the decolorization solution may provide effective pigment removal while minimizing potential damage to the biological sample. This formulation may allow for improved tissue transparency without compromising structural integrity or molecular signals within the sample.

[0059] The decolorization step may be particularly important for samples containing high levels of endogenous pigments, such as hemoglobin in blood-rich tissues or melanin in skinsamples. By removing these light-absorbing molecules, the decolorization solution may significantly enhance the optical clarity of the biological sample, facilitating subsequent imaging steps.

[0060] The duration and conditions of the decolorization treatment may vary depending on the specific sample type and size. In some cases, multiple applications or extended incubation times may be necessary to achieve optimal pigment removal and tissue transparency.

[0061] After decolorization, the biological sample may be subjected to a partial delipidation step. The partial delipidation solution may comprise tetrahydrofuran and a humectant such as 1,2-hexanediol. This step may help remove lipids from the sample while minimizing tissue shrinkage and preserving morphology.

[0062] The ADAPT-3D method may involve treating biological samples with a partial delipidation solution to remove lipids while preserving tissue morphology. In some cases, the partial delipidation solution may comprise tetrahydrofuran and a humectant.

[0063] Tetrahydrofuran may serve as an effective solvent for lipid extraction from biological tissues. The use of tetrahydrofuran in the partial delipidation solution may allow for efficient removal of lipids without causing excessive damage to other cellular structures.

[0064] The inclusion of a humectant in the partial delipidation solution may help maintain tissue hydration during the delipidation process. In some cases, the humectant may be 1,2- hexanediol. This compound may help prevent excessive dehydration of the sample, which could otherwise lead to tissue shrinkage or distortion.

[0065] The combination of tetrahydrofuran and a humectant in the partial delipidation solution may provide a balance between effective lipid removal and tissue preservation. This formulation may allow for improved tissue transparency without compromising structural integrity or causing significant shrinkage of the biological sample.

[0066] The partial delipidation step may be particularly important for samples containing high levels of lipids, such as adipose tissue or myelin-rich nervous tissue. By removing these light-scattering molecules, the partial delipidation solution may significantly enhance the optical clarity of the biological sample, facilitating subsequent imaging steps.

[0067] In some cases, the concentration of tetrahydrofuran in the partial delipidation solution may be adjusted based on the specific sample type and desired degree of delipidation.Higher concentrations may provide more thorough lipid removal but may also increase the risk of tissue distortion.

[0068] The duration and conditions of the partial delipidation treatment may vary depending on the specific sample type and size. In some cases, multiple applications or extended incubation times may be necessary to achieve optimal lipid removal and tissue transparency while maintaining sample morphology.

[0069] By carefully balancing the components and application of the partial delipidation solution, the ADAPT-3D method may achieve effective lipid removal while preserving tissue structure. This approach may allow for high-quality three-dimensional visualization of complex biological structures with minimal distortion or shrinkage.

[0070] In some cases, particularly when processing samples containing bone or calcified tissues, a decalcification step may be performed. The decalcification solution may comprise a chelator such as ethylenediaminetetraacetic acid (EDTA), one or more N-alkyldiethanolamines (e.g., N-methyldi ethanolamine and N-butyl di ethanol amine), and imidazole.

[0071] The decalcification solution used in the ADAPT-3D method may comprise a combination of components designed to effectively remove calcium from tissues while preserving sample integrity. In some cases, the decalcification solution may include a chelator, one or more N-alkyldiethanolamines, and imidazole.

[0072] The chelator in the decalcification solution may be ethylenediaminetetraacetic acid (EDTA). EDTA may bind to calcium ions in the tissue, facilitating their removal and softening calcified structures. The use of EDTA as a chelator may allow for gentler decalcification compared to strong acid-based methods, potentially helping to preserve tissue morphology and molecular targets.

[0073] The N-alkyldiethanolamines in the decalcification solution may include compounds such as N-methyldiethanolamine and N-butyldiethanolamine. These components may help enhance the penetration of the decalcification solution into tissues and may assist in maintaining pH balance during the decalcification process.

[0074] Imidazole may be included in the decalcification solution to further modulate pH and potentially enhance the efficiency of calcium removal. The combination of imidazole with the chelator and N-alkyldiethanolamines may create a balanced environment for effective decalcification while minimizing damage to tissue structures.

[0075] The decalcification step may be integrated into the overall ADAPT-3D workflow, typically following initial fixation and preceding the decolorization and delipidation steps. By removing calcium from bone or calcified tissues, the decalcification process may enhance the optical clarity of these samples and improve the penetration of subsequent clearing solutions.

[0076] The duration of the decalcification treatment may vary depending on the size and calcium content of the sample. In some cases, the process may be monitored by periodically testing the flexibility of the tissue or through radiographic assessment to determine when sufficient decalcification has been achieved.

[0077] Following decalcification, samples may proceed through the subsequent steps of the ADAPT-3D method, including decolorization, delipidation, and refractive index matching. The removal of calcium during the decalcification step may facilitate more uniform clearing and staining of the entire sample, including regions that were previously calcified.

[0078] The integration of the decalcification step with other components of the ADAPT-3D method may allow for comprehensive processing of complex biological samples containing both soft tissues and calcified structures. This approach may enable three-dimensional visualization of cellular and tissue organizations in contexts such as bone-soft tissue interfaces or calcified blood vessels within organs.

[0079] The final step in the treatment process may involve applying the refractive index matching (RIM) or treatment solution to the biological sample. The RIM solution may contain a combination of components including an X-ray contrast reagent (e.g., iodixanol and iohexol), a sugar (e.g., sucrose), a nitrogen-containing compound or an amine (e.g., urea), an inhibitor of Maillard-reactive browning (e.g., 1 -thioglycerol), and an antioxidant (e.g., n-propyl gallate). This solution may render the biological sample optically transparent while preserving its fluorescent properties.

[0080] Once the biological sample has been treated and rendered optically transparent, image acquisition may be performed using various microscopy techniques. In some cases, the images may be acquired using epifluorescent microscopy, stereomicroscopy, light sheet microscopy, or confocal microscopy. The choice of microscopy technique may depend on factors such as the size of the sample, desired resolution, and specific experimental requirements.

[0081] The acquired images may then be processed to generate a three-dimensional visualization of the biological sample. In some cases, the image processing may includedeconvolution techniques. Deconvolution may help improve image quality by reducing out-of- focus light and enhancing resolution, particularly for images acquired using widefield microscopy techniques.

[0082] The ADAPT-3D method may utilize a specialized refractive index matching (RIM) solution to render biological samples optically transparent while preserving fluorescent properties. The RIM solution may comprise a combination of components carefully formulated to match the refractive index of biological tissues, thereby minimizing light scattering and enabling deep tissue imaging.

[0083] In some cases, the RIM solution may contain X-ray contrast reagents as key components. These may include iodixanol and iohexol. The concentration of iohexol in the RIM solution may range from about 25.33% w / v to about 30% w / v. These contrast reagents may contribute significantly to achieving the desired refractive index for tissue clearing.

[0084] The RIM solution may also include a sugar component, which may be D-sucrose.The concentration of sucrose may range from about 10% w / v to about 30% w / v. The inclusion of sugar in the RIM solution may help maintain osmotic balance and stabilize tissue structures during the clearing process.

[0085] An amine compound may be incorporated into the RIM solution. In some cases, this may be urea at a concentration of about 25% w / v. Urea may assist in denaturing and solubilizing proteins, further enhancing tissue transparency.

[0086] To prevent unwanted color development during the clearing process, the RIM solution may contain an inhibitor of Maillard-reactive browning. This inhibitor may be 1- thioglycerol, with a concentration ranging from about 0.5% w / v to about 1% w / v.

[0087] An antioxidant may be included in the RIM solution to help preserve fluorescent properties of the biological sample. In some cases, the antioxidant may be n-propyl gallate, with a concentration ranging from about 0.01% w / v to about 0.5% w / v.

[0088] The combination of these components in the RIM solution may result in a refractive index from about 1.45 to about 1.55. This refractive index range may be particularly effective for rendering biological samples optically transparent while minimizing distortion of tissue morphology.

[0089] The RIM solution may be applied to biological samples as the final step in the ADAPT-3D clearing process. The duration of treatment with the RIM solution may vary depending on sample size and type, but may typically range from a few hours to overnight.

[0090] By carefully matching the refractive index of the clearing solution to that of the tissue, the RIM solution may allow light to pass through the sample with minimal scattering. This may enable deep tissue imaging and high-resolution three-dimensional visualization of complex biological structures.

[0091] The preservation of fluorescent signals may be a key feature of the ADAPT-3D RIM solution. Unlike some organic solvent-based clearing methods that may quench fluorescence, the aqueous nature of the RIM solution and the inclusion of antioxidants may help maintain the intensity of both endogenous fluorescent proteins and exogenous fluorescent labels.

[0092] In some cases, the RIM solution may be compatible with various microscopy techniques, including widefield epifluorescence, confocal, and light-sheet microscopy. This versatility may allow researchers to choose the most appropriate imaging modality for their specific experimental requirements.

[0093] The composition of the RIM solution may be adjusted based on specific sample characteristics or imaging needs. For example, the concentrations of individual components may be fine-tuned to optimize clearing efficiency for different tissue types or to achieve a particular refractive index.

[0094] The ADAPT-3D method may incorporate a rapid immunolabeling technique using a nonionic detergent solution to enable efficient antibody penetration and staining of tissues. This approach may allow for adaptable and accelerated immunolabeling of biological samples.

[0095] The ADAPT-3D block and stain solution or buffer can contain glycine, one or more non-ionic surfactants, donkey serum, alpaca serum, bovine serum albumin (BSA), peroxide (H2O2), a solvent (e.g., dimethyl sulfide or dimethyl sulfoxide), and tris-buffered saline (TBS).

[0096] In some cases, the nonionic detergent solution used for rapid immunolabeling may comprise a combination of Tween-20 and Triton X-100 in dimethyl sulfoxide (DMSO). The concentration of Tween-20 in the solution may be approximately 0.167% (v / v), while the concentration of Triton X-100 may be approximately 0.33% (v / v).

[0097] The use of nonionic detergents in the immunolabeling solution may help permeabilize cell membranes, facilitating the entry of antibodies into cells and tissues. DMSOmay serve as a carrier solvent, potentially enhancing the penetration of antibodies into the sample.

[0098] The rapid immunolabeling technique may be applied to biological samples that have undergone prior clearing steps, such as decolorization and delipidation. In some cases, the cleared samples may be incubated in the nonionic detergent solution containing the desired antibodies.

[0099] The duration of the immunolabeling process using this rapid technique may vary depending on factors such as sample size and antibody characteristics. However, the use of the nonionic detergent solution may significantly reduce the time required for antibody penetration compared to traditional immunolabeling methods.

[0100] In some cases, the rapid immunolabeling technique may be compatible with a wide range of antibodies, including both primary and secondary antibodies. The method may allow for multiplexed staining, enabling the visualization of multiple targets within the same sample.

[0101] Following the immunolabeling step, samples may be washed to remove excess antibodies and then treated with the refractive index matching solution to restore optical transparency. The labeled samples may then be imaged using various microscopy techniques to visualize the distribution of target molecules within the three-dimensional tissue structure.

[0102] The adaptability of this rapid immunolabeling technique may allow researchers to optimize staining protocols for specific tissue types or antibodies. Factors such as detergent concentrations, incubation times, and antibody dilutions may be adjusted to achieve optimal staining results while maintaining sample integrity.

[0103] By incorporating this rapid immunolabeling technique, the ADAPT-3D method may enable efficient visualization of specific molecular targets within cleared and optically transparent biological samples. This approach may facilitate the study of complex cellular interactions and protein distributions in three-dimensional tissue contexts.

[0104] The ADAPT-3D method may be compatible with various microscopy techniques for image acquisition of optically cleared biological samples. In some cases, epifluorescence microscopy may be used to acquire images rapidly over large sample areas. Confocal microscopy may provide improved optical sectioning capabilities for three-dimensional imaging. Light sheet microscopy may enable fast acquisition of large tissue volumes with reducedphotobleaching. Stereomicroscopy may be suitable for imaging larger samples at lower magnifications.

[0105] The image acquisition process may involve capturing multiple z-stacks through the depth of the cleared sample. In some cases, tiling approaches may be used to image large sample areas by acquiring and stitching together multiple adjacent fields of view. The specific acquisition parameters such as exposure time, laser power, and z-step size may be optimized based on the sample characteristics and microscopy technique used.

[0106] Following image acquisition, deconvolution techniques may be applied to enhance image quality and resolution. In some cases, deconvolution may involve computational processing to remove out-of-focus light and restore the original object based on the point spread function of the optical system. Iterative deconvolution algorithms may be used to progressively improve image clarity through multiple processing cycles.

[0107] The deconvolution process may utilize depth-variant point spread functions to account for changes in the optical properties of the sample at different imaging depths. This approach may help compensate for spherical aberrations and other depth-dependent optical effects in thick cleared samples.

[0108] In some cases, the deconvolution software may allow for adjustment of parameters such as the number of iterations, regularization strength, and noise suppression to optimize the balance between resolution enhancement and artifact introduction. The specific deconvolution settings may be tailored to the characteristics of the acquired images and the desired visualization outcomes.

[0109] The combination of optimized image acquisition and deconvolution processing in the ADAPT-3D method may enable high-resolution three-dimensional visualization of optically cleared biological samples across various scales and tissue types. This approach may facilitate detailed analysis of cellular and tissue structures in their native three-dimensional context.

[0110] The ADAPT-3D method may be applied to a wide range of biological samples and experimental contexts, demonstrating versatility across tissue types and species. In some cases, the method may be used for rapid optical clearing and imaging of human donor kidney tissue in a clinical diagnostic setting.

[0111] When applied to human kidney samples, the ADAPT-3D method may allow for visualization of intact glomeruli and tubules within the three-dimensional tissue structure. Thiscapability may be particularly valuable in the context of transplant evaluation, where rapid assessment of kidney tissue quality and structure may be critical for decision-making.

[0112] In some cases, the ADAPT-3D protocol may be optimized to achieve optical transparency of human kidney tissue samples measuring approximately 1 cm x 1 cm x 0.5 mm in size. The method may allow for clearing and imaging of such samples within a timeframe compatible with clinical evaluation processes for organ transplantation.

[0113] The ability to visualize intact glomeruli and tubules in three dimensions may provide more comprehensive information about kidney tissue structure and function compared to traditional two-dimensional histological sections. In some cases, this approach may allow clinicians to assess the spatial relationships between different kidney structures and identify any abnormalities or pathological changes that may impact transplant suitability.

[0114] The ADAPT-3D method may also be applied to various other tissue types and species in research settings. For example, the method may be used to study brain tissue from rodent models, allowing for visualization of neuronal networks and vascular structures in their native three-dimensional context. In some cases, the method may be adapted for use with larger tissue samples from species such as pigs or non-human primates.

[0115] The versatility of the ADAPT-3D method may extend to different organ systems beyond the kidney and brain. For instance, the method may be applied to study cardiac tissue, liver samples, or complex structures such as the gastrointestinal tract. In each case, the protocol may be optimized to address the specific challenges posed by different tissue compositions and structures.

[0116] In research applications, the ADAPT-3D method may facilitate studies of tissue development, disease progression, and therapeutic responses. The ability to visualize large tissue volumes with high resolution may enable researchers to track cellular interactions, analyze vascular networks, or examine the distribution of specific molecular markers throughout intact organs or tissue samples.

[0117] The efficiency of the ADAPT-3D method may make it suitable for high-throughput screening applications in both research and clinical settings. In some cases, the rapid processing times and compatibility with various imaging modalities may allow for analysis of multiple samples or experimental conditions within a relatively short timeframe.

[0118] Furthermore, the ADAPT-3D method may be combined with other advanced imaging and analysis techniques to enhance its utility. For example, the method may be used in conjunction with multiphoton microscopy, super-resolution imaging, or automated image analysis algorithms to extract quantitative data from cleared and imaged tissue samples.

[0119] In summary, the ADAPT-3D method may offer a versatile and efficient approach for optical clearing and three-dimensional imaging of biological samples across various applications. From rapid evaluation of human donor tissues in clinical settings to detailed analysis of complex organ structures in research contexts, the method may provide valuable insights into tissue architecture and function across multiple scales and species.

[0120] The present disclosure is based, at least in part, on the discovery of methods and compositions to optically clear intact tissue and whole organs for fluorescent three-dimensional imaging.

[0121] Disclosed herein are aqueous-based clearing methods using a solution for tissue preparation and refractive index matching (RIM) called ADAPT-3D, accelerated deep adaptable processing of tissue for 3-dimensional imaging.

[0122] The present teachings include methods to optically clear and three-dimensionally image an intact tissue or whole organ while preserving tissue morphology, the method comprising (i) a fixative, (ii) a decolorization buffer, (iii) a delipidation buffer, (iv) a refractive index matching (RIM) solution, (v) acquiring an image(s) using microscopy, and (vi) deconvolution of the acquired image(s).

[0123] In one aspect, the method further comprises an adaptable rapid immunolabeling of the intact tissue or whole organ using a nonionic detergent solution. In another aspect, the fixative preserves an endogenous fluorescent reporter protein and prevents the masking of a tissue antigen. In another aspect, the fixative is comprised of 4% (w / v) paraformaldehyde (PF A), at least 10% (w / v) sucrose, and has a pH of 9.0, and optionally includes triethanolamine. In another aspect, the decolorization buffer is comprised of 10% ((3- cholamidopropyl)dimethylammonio)- 1 -propanesulfonate (CHAPS), 25% N- methyldiethanolamine, 5% N-butyldiethanolamine, and 10% 1,2-hexanediol. In another aspect, the delipidation buffer prevents tissue shrinkage and preserves tissue morphology. In yet another aspect, the delipidation buffer is comprised of 50% tetrahydrofuran and 50% 1,2-hexanediol. In another aspect, the RIM solution comprises iodixanol, iohexol (26% (w / v), 30% (w / v) sucrose,and 25% (w / v) Urea. In another aspect, the nonionic detergent solution is comprised of 0.167% (v / v) Tween-20 and 0.33% (v / v) Triton X-100 in dimethyl sulfoxide (DMSO). In another aspect, the image(s) can be acquired using multiple modes of microscopy, including epifluorescent microscopy, stereomicroscopy, light sheet microscopy, and confocal microscopy. In another aspect, tissue optical transparency may be achieved after consecutive use of the decolorization buffer for 80 minutes, the delipidation buffer for 120 minutes, and the RIM solution for 60 minutes. In another aspect, the tissue may be 1 cm x 1 cm x 0.5 mm.

[0124] The present teachings also include a composition of a refractive index matching (RIM) solution for use in imaging optically cleared three-dimensional images, the composition comprising an X-ray contrast reagent, a sugar, an amine, and an antioxidant. In one aspect, the X-ray contrast reagent may be iodixanol, iohexol, or any combination thereof. In one aspect, the sugar may be sucrose at 30% (w / v). In one aspect, the amine may be urea at 25% (w / v). In one aspect, the antioxidant may be n-propyl gallate at 0.5% (w / v).

[0125] As shown herein, the disclosed protocol achieves very rapid, even, and thorough tissue clearing and RIM in aqueous solutions that minimize tissue distortion, and the method is compatible with endogenous fluorophores, antibody labeling or tissue dyes. A key advantage is the speed at which the method can be applied. With ADAPT-3D, staining and clearing is sufficiently thorough and rapid that applications requiring timely decisions in a clinically relevant scenario are possible. With improvements in methods for tissue transparency, various modalities of light microscopy may be used.

[0126] The present teachings include methods to clear, stain, acquire and reconstruct images on human donor wedge biopsies in a mock work-up for evaluation for kidney transplant suitability. In less than 2h, high-quality, valuable data of 1 mm3 kidney was generated. Such rapid turnaround was achieved after ADAPT-3D tissue preparation by acquiring images on a customized but affordable epifluorescence microscope followed by 3D deconvolution, allowing imaging acquisition in a fraction of the usual time needed. This approach is amenable to multiple research and clinical applications, boasting substantial time saving and equipment that is affordable both in setup and maintenance. Thus, a combined set of innovations from tissue processing to image acquisition and analysis generate a start-to-finish platform to fill a niche in laboratories wherein 3D imaging in time- and money-saving settings is desired or is a necessity. Taken together, ADAPT-3D represents an adaptation in the field of fluorescence 3D imagingthat is amenable to range of imaging builds from light sheet or tile-scanning confocal microscopy to more rapid and affordable epifluorescence imaging.

[0127] With advances to the capabilities of light microscopy, several methods to optically clear tissues have emerged, but most methods have been optimized for the murine brain that comprises high water and lipid content, low density, and stiffness. Other tissues from the mouse or other species, however, comprise less water and lipid content than the brain; tissues that are dense in erythrocytes require further depigmentation. Nevertheless, previous methods are suited best for simply endogenous fluorescent reporters, immunolabeling while others perform poorly with heme-rich tissues. Although each method has its advantages, some of the approaches require further setups with active perfusion, which not only require more reagents but also another technical skill. Therein arose a need for an approach (ADAPT-3D) that can be adapted with a passive, yet practical approach while retaining the familiar immunohistochemical workflow of fixation, depigmentation, and labeling.

[0128] ADAPT-3D features several advancements that harness different approaches to address challenges in 3D tissue clearing and imaging: (1) achieves scalable optical transparency for intact tissues up to whole organs of the mouse while preserving morphology within 5 days using non-specialized equipment for most common laboratories; (2) preserves endogenous fluorescent reporter intensities while simultaneously being compatible with several fluorescent dyes including charged dyes for nuclear labeling; (3) enables deep accelerable labeling and relabeling with multiplexed, in-house conjugated antibodies; and (4) adaptability for different modes of microscopy including the stereoscope. Thereby, ADAPT-3D serves the underlying premise of a modular, passive clearing approach that can be adapted depending on the need while utilizing few toxic chemicals for most laboratories to use in a practical way. The flexibility achieved within these approaches allowed for both time and sample-efficient 3D imaging. Also, the process allowed for new biological observations in several different tissues and contexts, including comparisons across species of intestines from human patients and the translationally relevant piglet.Microscopy and Optical clearing

[0129] The absorption and scattering of light in biological tissues has hampered optical imaging's depth and resolution. High-resolution techniques such as optical coherence tomography (OCT) limit imaging to the tissue's surface layer. OCT is reliable down to a fewmillimeters with a resolution of a few microns, while photoacoustic tomography (PAT), with a resolution of a few hundred microns, observes a depth of a few centimeters of the tissue, or diffuse optical tomography (DOT), which has a greater imaging depth (several centimeters), has a much lower resolution (a few millimeters). Conventional optical clearing techniques aim to decrease scattering and improve image's depth and resolution. Agent-based optical clearing techniques minimize light scattering in various ways. Examples of how optical clearing agents (OCAs) works include dissociating collagen fibers, matching the refractive index of tissue components and interstitial fluid, and tissue dehydration.

[0130] The different refractive indexes (RI) of the major components of biological tissue, i.e. water, lipids and proteins result in light scattering when light passes through the tissue. Tissue clearing modifies the optical properties of usually opaque samples to render them transparent while keeping their structure and fluorescent labels intact. After clearing, light can travel many millimeters through a specimen unrestricted from absorption and scattering, ideal for high-resolution microscopic imaging deep within the specimen.

[0131] Various 3D tissue clearing techniques are available for section-less 3D deep tissue imaging. These methods can be classified into organic solvent-based clearing methods or aqueous-based clearing methods. Organic solvent-based methods including Benzoic Acid Benzyl Benzoate, dibenzyl ether, 3D imaging of solvent-cleared organs, ultimate DISCO use organic reagents of high refractive index (RI), to achieve rapid and high transparency but, there are serious disadvantages in that the tissues are significantly shrunk and most of the fluorescence signals of the proteins disappear.

[0132] Aqueous-based methods generally show good fluorescence preservation and maintain tissue size within a reasonable range. They utilize (1) simple immersion in a solution containing high RI materials such as -2,2 '-thiodi ethanol , (2) hyperhydration of the sample by urea and lipid removal (3) hydrogel embedding followed by active or passive removal of lipid. Immersion in high RI solutions such as fructose-based SeeDeepBrain (SeeDB) is effective for passive clearing of relatively thin samples, but due to high viscosity, its application to thick samples is limited. To avoid these issues, low viscosity alternative-based methods such as diatrizoic acid (FocusClear), or iohexol (RIMS). However, they also suffer from long clearing time or insufficient clearing efficiency due to poor tissue penetration. For better clearingperformance, urea-based hyperhydration followed by detergent-mediated lipid removal has been adopted in Scale, ScaleS, and CUBIC.

[0133] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0134] In some cases, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain cases of the present disclosure are to be understood as being modified in some instances by the term "about." In some cases, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some cases, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular case. In some cases, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some cases of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some cases of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0135] In some cases, the terms "a" and "an" and "the" and similar references used in the context of describing a particular case (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some cases, the term "or" as used herein, including the claims, is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0136] The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that "comprises," "has" or "includes" one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0137] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain cases herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0138] Groupings of alternative elements or cases of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0139] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0140] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent cases are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0141] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific cases that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 - Rapid-acting formulations that decolor, delipidate and match refractive index of tissue while preserving tissue morphology

[0142] Recent literature to develop aqueous, fast-acting clearing, or RIM (refractive index matching), solutions while minimizing toxic chemicals focused on combinations of x-ray contrast solution with urea21-22. Fast action was reported to be 2-3 days for the RIM step alone, not including additional time for staining21, 22. A solution of 7M urea in 80% iohexol in a method referred to as EZ Clear was especially appealing22. However, the preparation of the EZ Clear mixture for RIM is lengthy and requires heating. Furthermore, the solution is subject to crystallization, including after being applied to tissue, due to urea being near its saturation point especially as imaging time lengthens. We aimed to find an improved solution while working a similar theme of chemicals. Combinations of components were tested to achieve optical clarity in tissues within hours, preservation of signal to noise, retention of endogenous fluorescent tracers, low toxicity, minimal browning from Maillard reactions, a refractive index as high as possible, maintenance of tissue morphology, and ease of preparation and use. We determined that iodixanol could be used as a base solution, allowing us to start at a refractive index of 1.43 and to proceed without the need for heating when other chemicals were added. To counteract tissue swelling that occurred with iodixanol used alone, we paid close attention to the inclusion of sugars that would partially dehydrate tissue and thereby offset the expansion effect of iodixanol. Although we aimed to reduce the high, crystallization-prone urea concentration associated with EZ Clear, we observed that inclusion of some urea accelerated the speed at which the solution permeated the tissue to achieve RIM. Ultimately, a particular combination of sucrose (30%wt / vol), urea (25% wt / vol), iohexol (-26% wt / vol, alternatively known as Histodenz) dissolved into commercially prepared iodixanol (alternatively known as OptiPrep) satisfied all desired characteristics outlined above and resulted in refractive index of 1.50-1.51. We named this formulated RIM solution ADAPT:RI (Table). For a complete prepatory workflow for passive immersion of samples, we formulated four additional solutions: (i) a fixative that preserves the fluorescence of endogenous fluorophores (ADAPT :Fix), (ii) a decolorization buffer to remove pigments (ADAPT:DC), (iii) a gentle delipidation buffer for partial removal of lipids (ADAPT:PDL), (iv) and a decalcification buffer for bones (ADAPT:Decal, Table).

[0143] As an initial step to prepare samples for processing across diverse applications, we optimized fixation conditions to retain intensity of sensitive endogenous fluorescent reporters while limiting masking of antigens that would be detected by immunolabeling. Based on prior studies using recombinant fluorescent reporter proteins (FP), we investigated how altering pH, temperature, and adding sugars might augment the intensity of the FPs24'26. Systematic surveys of different fixation conditions using 4% w / v paraformaldehyde (PF A) as the base fixative were performed by measuring the fluorescent intensity of lymphoid follicles containing CD1 lc-eYFP+cells within Peyer’s Patches of mice (Fig. 1A, bottom). The lowest average CD1 Ic-eYFP signal intensity was observed after fixing tissue with the most widely used formulation of 4% PFA at neutral pH 7.6 (Fig. 1A). As is well known, the addition of the known protein stabilizing agent sucrose to a concentration of at least 10% (w / v) further improved retention of fluorescence intensity (Fig. 1A). Adjustment to pH 9.0 alone provided a significant retention of signal intensity. Addition of at least 10% (w / v) sucrose to 4% (w / v) PFA at pH 9.0 was as efficient for preserving fluorescence intensity of eYFP as 30% sucrose at pH 7.6 (Fig. 1 A). For some tissues, however, fixation with 30% sucrose was sufficiently dehydrating as to cause tissue compression.

[0144] To test the utility of the ADAPT :RI solution for RIM, we evaluated whether use of the solution alone could render full-thickness samples of human, piglet, and mouse colon transparent. In thicker human and piglet colon tissues (1.5-2.0 mm), increased light scattering from pigments and lipids further limits translucency. (Fig. IB, Fig. 5A). ADAPT-3D:RI renders mouse colon partially transparent while the presence of endogenous pigments prevents full transparency (Fig. 1C, right). After removal of pigments using the previously published SHANEL decolorization solution containing CHAPS and N-methyldiethanolamine23, AD APT-3D:RI rendered mouse tissue visibly transparent to the naked eye within 10 minutes at room temperature (Fig. 1C, left).

[0145] To generate a faster-acting decolorant, we adapted the previously the SHANEL decoloring solution23by adding limited amounts of N-butyldiethanolamine to increase tissue permeability while preserving morphology and 1,2 hexanediol, an inert emulsifying and humidifying agent (Table). To develop the solutions of ADAPT-3D, we consulted in silica resources including those from previous chemical screening used to generate the CUBIC-L protocol18, Reaxys, Open Reaction Database, and WolframAlpha for potential unwanted reactivity between chemicals and found none. Consistent with this conclusion, we noted that the preparation of this solution did not generate a notable exothermic or endothermic reaction. Reagents like hexanediol are used in skin and hair care products and thus we deemed them to have a favorable safety profde. In a comparison of decolorization alone (without RIM solution), ADAPT-3D:DC achieves greater transparency of piglet colon relative to the previously formulated SHANEL decolorization23over the same 80-minute timeframe (Fig. 5B).

[0146] To promote partial removal of light-interfering lipids, we aimed to develop a protocol that, while removing tissue lipids, would not lead to significant shrinkage that is caused by some solutions, such as graded exposure to tetrahydrofuran21that has been long recognized for its delipidation properties.9, 27, 28Although shrinkage can be minimized with a multistep gradient of tetrahydrofuran and reversed by rehydration21, any initial tissue shrinkage can cause irreversible structural tears in tissue. We predicted that combining tetrahydrofuran with a known delipi dating agent 1,2-hexanediol29that also has hydrating properties would create an effective single step delipidation while preserving tissue size. Following an initial 80-minute decolorization step, treatment of mouse and pig colons with a balanced mixture of tetrahydrofuran and 1,2-hexanediol (Table) for 2 hours enabled effective ADAPT:RI of the mouse colon within 30 minutes (Fig. ID, top row) while the thicker piglet colon took 1 hour to achieve visible transparency in this 3-hour trial (Fig. ID, bottom row).

[0147] Although lipids are present in all tissues, including the intestine, we next evaluated the efficacy of ADAPT :PDL for preparing lipid-rich white matter regions of the mouse brain for RIM. In a 1 mm thick brain section from a LysM-Cre;tdTomato mouse (Fig. IE), ADAPT:DC alone without ADAPT:PDL was insufficient to achieve complete optical transparency after RIM particularly in the lipid-rich white matter (Fig. IF). However, a short 3-hour incubation inADAPT:PDL was sufficient to enable complete visible transparency by ADAPT:RI (Fig. 1G). Decolorization alone did improve transparency after RI matching possibly due to the moderate delipidating effects of CHAPS, N-butyldiethanolamine, and 1,2-hexanediol in ADAPT:DC in addition to the removal of pigments (Fig. 5C). To assess how differences in tissue transparency by eye affected 3D fluorescence imaging, we acquired full-thickness z-stacks of brain sections from LyzM-Cre;tdTomato mice, with nuclear labeling in the cortex and brainstem (Fig. 1H). Without delipidation, the brainstem remained visibly opaque, and labeling with anti-Histone- ATTO488 was limited to a depth of -150 pm (Fig. II, left). A 3-hour step with ADAPT:PDL enabled clear nuclear detection throughout the full thickness of the section (Fig. II, right). While the cortex appeared more transparent by eye, fluorescence imaging revealedreduced nuclear signal detection at depth, with near-complete loss beneath the lipid-rich corpus callosum (Fig. 1 J, left). Delipidation restored uniform nuclear detection across the entire cortex, including the corpus callosum (Fig. 1 J, right).

[0148] Having established ADAPT:PDL as a key component of the broader ADAPT-3D workflow in brain sections, we next evaluated its clearing speed and ability to preserve tissue size in whole mouse brains relative to the widely used organic-based iDISCO method. The iDISCO+ protocol, involving five steps with methanol dehydration and incubation with dichloromethane, cleared fixed brains in 4 days but caused -40% shrinkage (Fig. IK), consistent with previous reports of tissue shrinkage and detachment from the dural membrane in whole skull clearing.27In contrast, Two days of the single-step ADAPT-3D delipidation following 1 overnight incubation in decolorization, facilitated successful ADAPT-3D RIM of the fixed brain in 4 hours without causing shrinkage (Fig. IL). By combining tetrahydrofuran’s delipidation power with the hydrating and delipidating properties of 1,2-hexanediol, the ADAPT-3D buffer matches iDISCO’ s speed while eliminating shrinkage and reducing the number of handling steps.

[0149] We applied our newly formulated ADAPT-3D four step protocol of fixation, decolorization, delipidation, and RIM (Fig. 5D) to make a variety of tissues transparent including, but not limited to, heme-rich spleen (Fig. 5E), lung (middle), and a liver from a mouse with endogenously fluorescent hepatocytes and lymphatic vessels (i.e., ProxlCreERx TdTomatofl / flreporter mice) such that the transparent tissue retained red intensity from the tdTomato reporter (Fig. 5E, right panel). These solutions collectively support rapid tissueprocessing for 3D imaging while avoiding significant changes in tissue morphology at all stages and reduce the time of decolorization, delipidation, and RIM, such that even relatively thick tissues, like the 1.5 mm pig colon wall, could be effectively prepared for imaging in under 5 h. Overall, ADAPT-3D is simple to prepare, store, and use at room temperature, which are features that highlight strong potential for broad adaptability to fluorescence imaging.Example 2 - ADAPT-3D maintains tissue integrity and fluorescent signal with partial delipidation in comparison to CUBIC

[0150] Since ADAPT-3D effectively rendered whole organs transparent while maintaining overall size, we next evaluated how decolorization and delipidation affect not only the transparency but also tissue integrity across both macroscopic and microscopic scales. To benchmark its performance, ADAPT:PDL, containing a chemical identified from the CUBIC chemical screen, was compared to commercially available aqueous CUBIC intended for preserving fluorescent reporter intensity. After 12 hours of delipidation in CUBIC -L, a 1 mm thick brain section (Fig. 2A, top) drastically swelled (Fig. 2A, middle), and upon washing with PBS, the cortex and hippocampus detached from the midbrain along the 3rdventricle (Fig. 2A, bottom). Conversely, the balanced ADAPT:PDL preserved relative size of the brain section over the same 12-hour time period of (Fig. 2B, top-middle) and maintained structural integrity without tearing after washing (Fig. 2B, bottom). A similar detachment between the midbrain and cerebral cortex was observed in an adjacent section after 24 hours of CUBIC-L treatment (Fig. 6A), while ADAPT:PDL still maintained tissue size at 24 hours (Fig. 6B). Even the recommended CUBIC-L pretreatment at 50% dilution prepared in water before delipidation caused swelling and dislocation of the cerebellum on its own (Fig. 6C), while the analogous ADAPT DC step preserved tissue morphology (Fig. 6D).

[0151] Both CUBIC-R+(M) and ADAPT-3D achieved refractive index matching of 1 mm sections to resolve at least 3.56 line pairs per mm; however, CUBIC-treated tissue was visibly enlarged (Fig. 2C). CUBIC is known to swell, a feature that can be advantageous in certain scenarios for increasing imaging resolution of the same tissue. Confocal imaging of the first infocus plane revealed that brain sections in CUBIC-R+(M) were double the size of ADAPT-3D sections (Fig 2D, E). The fluorescent intensity of nuclei labeled with an ATTO-488 conjugated nanobody against histones was ten times brighter in the ADAPT-3D processed section comparedto the CUBIC section when acquired with matched acquisition settings (Fig. 2D, F). These matched brain sections from contralateral hemispheres of the same brain were fixed with ADAPT-3D fixative to preserve endogenous fluorophores, yet the fluorescence intensity of endogenous tdTomato signal was nearly three times brighter in the ADAPT-3D processed sections (Fig. 2G). These differences are particularly surprising considering that N- methylnicotinamide in CUBIC-R+(M) was selected for its superior preservation of fluorescence relative to nicotinamide found in the basic CUBIC-R+(N) solution. After digitally increasing the brightness of the CUBIC sample image, we observed that the ventricular space was distorted and lacked an intact choroid plexus (Fig. 2H) whereas ADAPT-3D maintained both ventricular architecture without swelling or collapse and an intact choroid plexus (Fig. 21). Only ADAPT- 3D tissue clearing enabled tracing of the choroid plexus in 3D from the lateral into the 3rdventricle in a volumetric image acquired on the confocal while CUBIC clearing failed to preserve the ventricular space and tore the choroid plexus. Swelling during CUBIC delipidation also caused the thin leptomeningeal membrane lining the surface of the brain to break into fragments (Fig. 2J) while ADAPT-3D tissue processing preserved the continuous leptomeningeal layer (Fig. 2K). The preservation of tissue size by ADAPT-3D at all tissue processing steps and refractive index matching maintains macroscale features for 3D visualization.

[0152] To minimize disruption of microscale features such as cellular membranes and intracellular compartments caused by excessive lipid removal, ADAPT :PDL was designed to partially delipidate tissues.

[0153] Notably, after 3 hours of delipidation, ADAPT :PDL-treated sections retained the characteristic white appearance of lipid-rich fiber tracts following washing in PBS (Fig. 2L, bottom), whereas sections delipidated with CUBIC-L at 37°C degrees began to lose this contrast (Fig. 2L, top). By 24 hours, CUBIC-L treated sections exhibited partial transparency in the cortex (Fig. 6A, bottom) consistent with extensive lipid removal, while ADAPT-3D processed sections remained relatively opaque and preserved white matter tracts (Fig. 6B, bottom). These observations support that ADAPT-3D only partially delipidates brain tissue in contrast to CUBIC, which more aggressively removes lipids from myelinated and non-myelinated regions. Despite this gentler approach, ADAPTRI effectively RI matched tissues even after only a partial removal of lipid for 3 hours (Fig. 1G).

[0154] To assess the effects of ADAPT-3D delipidation at a cellular level, we incubated fixed peritoneal cells in ADAPT:PDL for 15 minutes and stained for lipids and intracellular compartments. Peritoneal cells from LysM-Cre;AbcalxAbcglfl / flmice, contain lipid droplets that stain positive for the neutral lipid dye LipidSpot (Fig. 2M, left), but after 15 minutes of incubation in ADAPT:PDL the macrophages no longer stain positive for lipid spot (Fig. 2M, right). However, the cell membrane still stains positive with the lipophilic dye DilCis(3) that inserts into the lipid bilayer after 15 minutes of ADAPT:PDL treatment (Fig. 2N). Endosomes labeled with anti-EEAl apear as punctated compartments throughout the cell (Fig. 20, left), and are still retained following ADAPT:PDL treatment, (Fig. 20, right). Therefore, partial removal of lipid by ADATP-3D also preserves cellular membranes and subcellular structures while removing certain light scattering lipids like those found in lipid droplets.

[0155] In conclusion, ADAPT-3D effectively clears brain tissue for 3D imaging with a partial delipidating approach while preserving anatomical features and the intensity of fluorescent staining in comparison to CUBIC. ADAPT-3D and CUBIC both successfully lead to optically transparent tissues. However, only ADAPT-3D was able to maintain tissue size throughout all steps and avoid damage to the ventricular and leptomeningeal compartments. While CUBIC’s optimized RIM solution is sufficient for visualization of endogenous and antibody-conjugated fluorophores under high laser power, ADAPDRI preserves fluorescence intensity up to tenfold higher under identical imaging conditions. At a cellular level, ADAPT :PDL effectively removes neutral lipids while preserving membrane lipids and intracellular compartments. Consistently, sections processed with ADAPT-3D still have clearly distinguishable white matter and remain opaque after delipidation unlike samples delipidated according to the CUBIC protocol.Example 3 - Light-sheeting imaging with ADAPT-3D including at the intact skull-meningeal interface

[0156] ADAPT-3D effectively clears whole brains and facilitated full-thickness confocal imaging in 1 mm sections. Thus we aimed to test its capacity to be coupled with light sheet imaging of tissues with endogenous fluorophore reporter proteins. We applied the ADAPT-3D protocol to prepare the brain of a ChAT-Cre;TdTomatofl / flmouse where cholinergic neurons are labeled with TdTomato30in a total of 4 days including 4 hours of RIM. The vasculature in thismouse, which was labeled with an intravenous injection of Dylight649-labeled Lycopersicon Esculentum lectin, was readily visualized into the core of the brain and highlighted the choroid plexus in the lateral ventricle (Fig. 3A, 3B). The bright cell bodies of cholinergic neurons were observed throughout the cortex, as were the dimer axons that extended into fine dendrites observed in the first cortical layer (Fig. 3B). We also processed the brain from a CD1 Ic-eYFP transgenic mouse31to test the ability of ADAPT-3D to preserve the more sensitive endogenous fluorophore eYFP32-33durinng light sheet imaging of the whole brain (Fig. 3C). In some CD1 lc-EYFP+transgenic mice31, we unexpectedly identified eYFP+cells with the morphology of dentate gyrus granule cells (Fig. 3D). This finding likely points to an off-target expression pattern in a mouse strain designed to study antigen-presenting dendritic cells of the immune system, but nonetheless nicely highlights the depth and preservation of eYFP that ADAPT-3D offers in whole mouse brain light-sheet imaging. Thus, in 5 days from tissue collection to imaging, ADAPT-3D methodology cleared the whole mouse brain for light-sheet microscopy with strong retention of the fluorescent intensities of endogenous reporter proteins eYFP and tdTomato.

[0157] A challenge for 3D imaging is the examination of bone with adjacent soft tissues such as in the skull-brain interface where reported vascularized skull channels exist through which immune cells can migrate into the dura mater34’36. Previous studies highlight this challenge with use of popular methods like iDISCO, where the organic nature of the iDISCO solutions cause dehydration and consequent shrinking of soft tissue that tear the fragile space leptomeninges between the brain and skull, while simultaneously extinguishing endogenous fluorophores.37As a result, visualization of the leptomeningeal space including its skull channels has been limited.

[0158] We sought to leverage the ability of ADAPT-3D to preserve both tissue size and endogenous fluorophores to visualize intact brain borders in a cleared whole mouse skull without the need for time intensive antibody labeling. We decalcified the fixed intact brain and skull using EDTA with N-butyldiethanolamine, imidazole, and 1,2-hexanediol at pH 9.0 (Table) over a period of 3 days at room temperature while preserving endogenous fluorophores. Light sheet imaging of a whole skull from a LYVElCreERx TdTomato11 flmice injected i.v. with Lectin- Dylight649 cleared with ADAPT-3D captured the intact layers at the brain border without shrinkage (Fig 3E). Looking into the brain through the intact skull in a 3D we observedmeningeal macrophages (Fig. 2F, arrows) and dural lymphatic vessels (Fig. 2F, arrowheads). In fact, there were distinctive LYVEU channels bridging the skull and the leptomeningeal space, some of which were Lectin+and some that were not (Fig. 2G, asterisks). The time from tissue acquisition and the initiation of processing to the time of light-sheet imaging with an intact skull was 8 days (1 day fixation, 2 days of decalcification, 2 days decolorization, 1 day delipidation, 2 days RIM, such that light-sheet imaging was set up on the 8thday). ADAPT-3D compared favorably with depth of clearing of brains or brain slices with an intact skull, as it was significantly faster from start to finish than all reported methods investigating light-sheet skullbrain imaging including iDISCO,37SHANEL,5or HYBRID.5A comparison of total time frame used in ADAPT-3D relative to these other published processes is charted in Fig. 21. Among other aqueous-based methods, CUBIC and its variants rendered marmoset brain hemispheres transparent in 29 days without attached skull.38Table. ADAPT-3D Refractive lindex Matching Solution. (ADAPT:RI)Table. ADAPT-3D Decolorization Solution (ADAPT:DC).Table. ADAPT-3D Partial Delipidation Solution (ADAPT:PDL)Table. ADAPT-3D Decalcification Solution (ADAPT:Decal)Table. ADAPT-3D Block and Stain Buffer (ADAPT:BS)Table. ADAPT-3D Fixative (ADAPT.Fix)Example 4 - Preserving antigens with deep immunolabeling

[0159] Having observed that fluorescent reporter proteins are preserved with strong intensity, we next asked how immunolabeling was affected in the ADAPT-3D protocol. In particular, some antigens such as tight junctions are commonly masked with traditional fixation using 4% PFA at neutral pH, which have led some to seek alternative fixatives including those using methanol39’40. Our modified fixative (Fig. 1A) combined with ADAPT-3D clearing enabled detection of claudin-11 and occludin tight junctional proteins along the arachnoid barrier and claudin-5- and occludin-expressing endothelial cells in the leptomeninges (Fig. 4A). Use of the modified methanol fixative in a side-by-side comparison revealed our approach was superior.

[0160] As a further illustration of the utility of ADAPT-3D with immunolabeling protocols, we imaged full thickness preparations of the mouse ileum, staining for smooth muscle actin (SMA) and S100A9 in ileum of wildtype littermates (Fig. 4B) and TNFAAREmice (Fig. 4C) that develop transmural ileitis41. Widened, edematous villi and infiltrated neutrophils associated with ileitis in TNFAAREmice were evident (Fig. 3B, 3C). ADAPT-3D was also used to visualize macrophage subpopulations differentially expressing CD 163 and IB Al in villi of the human intestine (Fig. 4D). Villus height was readily measured, here averaging 344.6 ± 38.4 pm mean ± S.E.M.). Videos can depict 3D reconstruction of ileum across species. IBAU macrophages could be easily visualized throughout not only the villi but also concentrated populations particularly along the submucosal regions along the lower depths of the villi.

[0161] In summary, we show that ADAPT-3D is a superior method for processing tissue, decalcifying bone and achieving RIM that holds numerous advantages for 3D tissue imaging. It is remarkably fast-acting compared with the reported tissue processing times of other aqueous methods. The use of compounds in a mixture and ratio that readily penetrates tissue likely underlies its fast action and ease of use. As we illustrate, ADAPT-3D is also designed to prevent morphological changes that result from dehydration, and is readily applicable toward optimalpreservation of fluorescent reporters without the need to add compounds to “boost” these reporters. It also readily accommodates antibody staining. Finally, it holds advantage in the area of low toxicity. Recently, di chloromethane, a component in iDISCO and other related methods, was regulated by the environmental protection agency such that laboratory exposure will require monitoring (40 Code of Federal Regulations Part 751). ADAPT-3D does not use this regulated chemical, adding another attractive characteristic that should favor its adaptability for imaging applications in light microscopy. We suggest that this protocol will be of strong interest to pathologists and scientists interested in 3D fluorescence imaging.Example 5 - Rapid optical transparency of human donor kidney in a clinical diagnostic context

[0162] ADAPT-3D, accelerated deep adaptable processing of tissue for 3-dimensional imaging, can achieve rapid and thorough tissue clearing and RIM in an aqueous solution that minimizes tissue distortion and is compatible with endogenous fluorophores, antibody labeling, and tissue dyes.

[0163] In a clinically relevant scenario, donor organs are evaluated for transplant viability within a finite, defined time. However, this has traditionally used frozen sections of 5 microns where artifacts lower congruence of agreement between pathologist interpretation, and ultimately leads to a higher rate of discarding tissues. We tested our rapid, aqueous approach for a fluorescent Periodic Acid Schiff stain along with a lipophilic dye. Human kidney tissue measuring approximately 1 cm x 1 cm x 0.5 mm (x, y, z), equivalent to 150 individual frozen section slides, can be visualized with a fluorescent analog of the Periodic Acid-Schiff stain in under 180 minutes, a timeframe that can be further decreased for more realistic evaluation periods. It highlights glomeruli in continuity with tubules in intact wedge biopsies. It also reveals thickened and atrophic tubules surrounding specific glomeruli, as well as neighboring glomeruli with varying basement membrane thicknesses. Thereby, ADAPT-3D rapidly enabled continuous visualization of histologically normal glomeruli yet pathological tubules in a clinically applicable setting.Example 5 - Methods

[0164] Mouse tissue. All mice were bred and housed in specific pathogen-free facilities at Washington University School of Medicine under standard housing conditions (12-hour light anddark cycles with feeding ad libitum). The Institutional Animal Care and Use Committee (IACUC) approved all experiments and procedures (protocol 22-0433). Apart from CD1 Ic-eYFP and CD1 Ic-eYFP x ProxlCreER-tdTomatofl / flmice, all other mice were on a C57BL / 6 background. CD1 Ic-EYFP mice31were a gift from Michel Nussenzweig (Rockefeller University). ProxlCreERmice43were from Jackson Laboratory (Jax# 022075) and crossed to tdTomatofl / flmice44from Jackson Laboratory (Jax #007909). ChAT-TdTomatofl / flreporter mice (Jax #028861), originating from a cross between were a gift from the Rodney Newberry laboratory at Washington University. For tdTomato expression in inducible Cre mice, 12-week- old CD 1 Ic-eYFP x ProxlCreER-tdTomatofl / flmice were treated with 2 mg tamoxifen (Sigma Aldrich, T5648), given by gavage and dissolved in sterile com oil to 20 mg / mL for 3 total doses within a 7-day period. TNFAARE / +mice41were obtained in 2016 through the Cleveland Digestive Disease Research Core Center (NIH P30 DK097948) and continuously bred at Washington University. These mice were kept and bred as heterozygotes and always cohoused with wild-type littermates. LYVECreERtdTomatofl / flmice were generated by the Kipnis laboratory as described45, and administered 2 mg tamoxifen by oral gavage 3 times over 1 week before euthanasia to process samples for imaging.

[0165] General ADAPT-3D preparation and immunostaining. The following is a generalizable workflow for the processing of samples. However, the specific durations and volumes varied depending on the tissue type and size. In general, following fixation in modified ADAPT-3D fixative at 4°C ranging from 4 hours to overnight, all steps are performed at room temperature. Samples were rinsed twice in IX phospho-buffered saline (PBS) containing 10 U / mL heparin with at least 5 times of excess volume of the tissue. If bones are included, samples were immersed in excess volume of Decalcification Buffer (Table) at room temperature with daily change until soft to the touch. Hours after incubation with Decolorization / Delipidation Buffer (Table), samples become visibly partially transparent while incubation is generally performed for 12 hours every 1 mm of tissue; they were then washed in IX PBS containing 10 U / mL heparin where visible transparency appears to reverse, which generally took about 1 hour at room temperature. Samples were incubated in Partial Delipidation Buffer (Table) until some transparency (especially apparent for the brain) was observed followed by washing in 0.2X PBS for at least 1 hour at room temperature until exchanged for IX PBS containing 10 U / mL heparin. Finally, if just visualizing fluorescent reporter proteins, samples are immersed in RefractiveIndex Matching Solution until transparent. If immunolabeling was planned, samples were incubated in ADAPT-3D blocking buffer (containing 0.1 mM Glycine, 0.167% Tween-20, 0.33% Triton X-100, 1% Donkey Serum, 1% Alpaca Serum, 1% BSA, 0.05% Hydrogen peroxide, 5% v / v DMSO) with antibodies (see protocol below) rinsed with IX PBS containing 10 U / mL heparin and 0.2% Tween-20, and then refractive index matching solution (Table) until transparent. For most tissues, they were acclimatized in 0.5X ADAPT-3D Refractive Index Matching solution diluted in IX PBS for 30 minutes to 1 hour at room temperature before exchanging into IX ADAPT-3D Refractive Index Matching solution until transparent.

[0166] Comparison of ADAPT-3D to CUBIC. Whole brains from LysMcre-tdTomato mice were fixed in 4% PFA (PH 9 10% (v / v) sucrose) for 24 hours, washed in PBS-H, and then prepared into 1 mm sections using a vibratome. Matched sections from contralateral brain hemispheres of the same mouse were then cleared in a side-by-side comparison of ADAPT-3D or commercial CUBIC reagents (TCI Chemicals) while varying the time of delipidation. ADAPT-3D samples were incubated in Decolorization buffer with 0.2x PBS for 6 hours while CUBIC samples were pretreated for 6 hours in 50% CUBIC-L diluted in water at room temperature. ADAPT-3D samples were washed with PBS for 3x30 minutes shaking, while CUBIC samples were switched directly into 100% CUBIC-L. Separate sections were delipidated for 0, 3, 12, or 24 hours in ADAPT-3D Delipidation buffer at room temperature or CUBIC-L at 37°C shaking. ADAPT-3D samples were rinsed for 2 hours in PBS with the first 30-minute wash in 0. lx PBS while CUBIC samples were washed for a total of 2 hours with three changeouts. All sections were labeled with anti-H2A-H2B (Hi stone-Lab el Atto488, Chromotek, tba488, 1 :200) either in ADAPT-3D blocking buffer or in PBS with 0.5% Triton X-100 and 0.01% NaNs for CUBIC samples for 20 hours. Samples were washed overnight at room temperature in PBS-H with 0.2% (v / v) Tween-20 for ADAPT-3D treated samples or in PBS for CUBIC samples. CUBIC antibody labeling and washing was performed in accordance with basic CUBIC immunolabeling of 1.5mm brain sections. Samples were refractive indexed matched first for 1 hour in 50% ADAPT-3D diluted in PBS or 50% CUBIC -R+(M) diluted in milliQ water and then incubated for 4 hours in their respective undiluted refractive index matching solutions before imaging. Fluorescent images were captured with a lOx lens (0.3 NA, Leica) in the first in-focus plane and then full thickness Z-stacks were acquired from the brain stem and cortex.

[0167] Mouse tissue immunostaining and imaging. For tight junctional protein staining, following euthanasia, C57BL / 6J mice were perfused by transcardiac perfusion with PBS containing 10 U / mL heparin then by modified ADAPT-3D fixative. For preparation of mouse intestines, samples were fixed overnight in modified ADAPT-3D fixative, rinsed twice with IX PBS containing 10 U / mL Heparin for 30 minutes each, incubated in Decolorization / Delipidation Buffer for at least 60 minutes, incubated overnight with antibodies against alpha smooth muscle actin-Cy3 (clone 1A4, 1:200, Sigma-Aldrich, C6198), Lyvel (Abeam, abl4917, 1 :300), S100A9 (Bio-techne R&D, AF2065, 1 :400) in ADAPT-3D blocking buffer. Samples were rinsed in IX PBS containing 10 U / mL and 0.2% (v / v) Tween-20 for 30 minutes at room temperature. They were then mounted in 0.5X ADAPT-3D Refractive Index Matching solution in IX PBS for 30 minutes at room temperature before incubating in IX ADAPT-3D Refractive Index Matching solution until transparent. For comparisons of preserving tight junctions in leptomeninges, samples were fixed with either a 4-h incubation with 100% methanol (wt / vol) or left in ADAPT- 3D fixative. The following pre-treatments of ADAPT-3D methodology as described above were performed including decolorization and delipidation. For immunolabeling, dorsal cortices were roughly dissected with a razor blade (0.5-1 mm thick) and stained with antibodies against Occludin (clone OC-3F10, Invitrogen, Cat. No., 331594, 1:200), Claudin-5 (clone 4C3C2, Invitrogen, Cat. No., 352588, 1: 100), Claudin-11 (Invitrogen, Cat. No., 36-4500, 1 : 100) in ADAPT-3D blocking buffer. Samples were rinsed in IX PBS containing 10 U / mL and 0.2% (v / v) Tween-20 at room temperature. Finally, cortices were mounted in 0.8 mm CoverWellTM imaging chambers containing refractive index matching solution.

[0168] Piglet tissue. Piglets were procured at 7 days of age and studied from an approved class A vendor (Oak Hill) then housed for 14 days before euthanasia. During this period, they were fed with Nutra-Start Liqui-Wean formula (Milk Specialties Global, Eden Prairie, MN) at Saint Louis University in accordance with approved IACUC protocol [2346, United States Department of Agriculture (USDA) registration is 43-R-011 to AKJ. Euthanasia was performed through an overdose injection of sodium pentobarbital. Tissue was then immediately collected and transferred to fixative in large buckets to allow for nearly 5 times the volume of tissue. Following fixation, tissues were washed with IX PBS containing 0.3M glycine for 1 hour at room temperature, for a total of 3 changes. They were then stored at 4°C until proceeding with the protocol.

[0169] Human tissue preparation and immunostaining. Human intestinal tissue samples were collected according to approved procedures in IRB protocol #201111038 to GJR, Washington University. No tissues were acquired from prisoners. For some intestinal samples, tissue was perfused with fixative directly into the ileocolic artery of the mesentery followed by immersion in fixative overnight at 4° C. 1 cm3samples were rinsed in IX PBS containing 10 U / mL heparin for 2 hours, incubated in decolorization / delipidation buffer for 2 days, partial delipidation buffer for 1 day, followed by blocking buffer containing antibodies against CD 163 (EDHu-1, Bio-Rad, 1 :100), IBA1 (Fujifilm Wako, 019-19741, 1 : 100), and DAPI (Sigma- Aldrich, D9542, 1 :200) for 2 days. Samples were rinsed in IX PBS containing 10 U / mL Heparin and 0.2% Tween-20 for 2 hours and then incubated in refractive index matching solution until imaging.

[0170] Peritoneal cell isolation and imaging. Peritoneal cells were collected by lavaging the peritoneal cavity with 5 mL of PBS supplemented with 5 mM EDTA. Cells were then centrifuged at 300xg for 5 minutes and resuspended in DMEM-F12 media containing (10% w / v FBS, lx sodium pyruvate (), IX MEM Non-Essential Amino Acids Solution (100X, 11140050), 100 U / mL penicillin-streptomycin. Following resuspensions, cells were plated on 8-well chambered slides (ibidi, 80841) that were manually coated with poly-L-lysine. After 2 hours of incubation, cells were fixed with 4% paraformaldehyde for 15 minutes on ice, permeabilized with ADAPT:BS, and incubated with a primary antibody against EEA1 (Cell Signaling Technology, C45B10) at 1 :200 overnight at 4°C. On the following day, cells were washed with PBS containing 0.2% (v / v) Tween-20 for 5 minutes then incubated with Alexa Fluor® 647 AffmiPure-VHH® Fragment Alpaca Anti -Rabbit IgG (Jackson Laboratories, 611-604-215) at 1 :400 dilution for 1 hour at room temperature. Following 2 washes with PBS containing 0.2% (v / v) Tween-20, cells were incubated with CellBrite Orange (1 :200) for 30 minutes diluted in IX PBS. After washing with PBS containing Tween-20, slides were mounted with FluoroBrite containing DAPI. Confocal microscopy was performed with an inverted Leica SP8 microscope that is equipped with 7 lasers with full spectral hybrid detectors (40X Objective lens, NA1.3, oil immersion) and set to pseudo-zoom by a factor of 7.5X.

[0171] Stereomicroscopy and confocal microscopy and tissue processing.Stereomicroscopy was performed using a Leica M205FA stereoscope equipped with a K8 digital color camera (2048 x 2048 pixels) at 12-bit for the acquisition of CD1 Ic-eYFP-positive signal.Widefield microscopy was performed using a Zeiss Z1 examiner with an Objective LD SC Plan- Apochromat 20x / 1.0 Corr M32 85mm equipped with a Colibri 7 light source. Confocal microscopy was performed with an inverted Leica SP8 microscope that is equipped with 7 lasers with full spectral hybrid detectors (20X Objective lens, NA0.75, oil immersions, or 25X Objective lens, NA0.95, water immersion). Alternatively, high-magnification images were acquired with the Stellaris TCS SP8 confocal microscope (Leica) using either a lOx objective (NA 0.4, Leica) with 2-2.5X digital zoom.

[0172] For comparison of visual transparency in organs like brain, spleen, and lung, stereomicroscopy was used. Following euthanasia with CO2, 12-week-old C57BL / 6J mice were perfused by transcardiac administration with IX PBS containing 10 U / mL heparin (PBS-H) followed by either 4% PFA (pH 9.0) for ADAPT-3D or 4% PFA (pH 7.6) for iDISCCF brains and post-fixed overnight at 4°C. For ADAPT-3D processing of the lung and spleen, following fixation, they were washed in PBS-H for 30 minutes at room temperature and then incubated in ADAPT-3D Decolorization buffer for 48 hours. After washing in PBS-H for 30 minutes at room temperature, they were then incubated in ADAPT-3D delipidation buffer for 24 hours, washed in PBS-H again for 30 minutes at room temperature, and then incubated in RIM solution for 4 hours.

[0173] For clearing using iDISCO1methodology, brains were processed according to the protocol available at http: / / idisco.info with the only exception that the refractive index matching solution was ethyl cinnamate. Briefly, after overnight fixation, brains were dehydrated in graded methanol solutions starting at 20% v / v in MilliQ water for 1 hour at room temperature, progressing to 100% methanol, followed by 1 hour in chilled 100% methanol. The brains were delipidated with a 2: 1 dichloromethane:methanol solution at room temperature and washed in 100% methanol two times and chilled at 4°C. Then, they were incubated overnight in chilled fresh 5% hydrogen peroxide in methanol at 4°C, rehydrated in a decreasing methanol series at room temperature until they were twice washed in IX PBS containing 0.2% Triton X-100 for 1 hour at room temperature. Brains were again dehydrated in a progressive series of methanol into a 2: 1 dichloromethane:methanol solution when they were incubated for 3 hours at room temperature, twice washed in 100% dichloromethane for 15 minutes, then incubated in ethyl cinnamate for 4 hours. Following iDISCO+or ADAPT-3D processing, samples were imagedwith Leica M205 stereoscope that is mounted with DFC7000T camera. Areas were quantified by outlining brains at fixation or after refractive index matching.

[0174] Light-sheet microscopy and processing. Lyvel-CreER mice were injected retro- orbitally with a mixture of 40 micrograms of Lectin-Dylight649. After 5 minutes, mice were euthanized. Intact skull with brains were fixed overnight at 4°C, twice washed in IX PBS containing 10 U / mL heparin (PBS-H) for 30 minutes each at room temperature, incubated in ADAPT-3D decalcification buffer with daily change for 3 days, washed in PBS-H once for 30 minutes at room temperature, and incubated in ADAPT-3D Decolorization buffer for approximately 48 hours at room temperature. They were washed in PBS-H once for 30 minutes at room temperature followed by incubation in ADAPT-3D delipidation buffer for approximately 36 hours, washed in PBS-H twice for 30 minutes at room temperature. Finally, they were incubated in 0.5X RIM in PBS to acclimatize the tissue for 1 hour at room temperature and then IX RIM overnight.

[0175] For intact brains from CD1 Ic-eYFP and ChAT-tdTomato reporter mice, following retro-orbital injection, the steps above were performed with the exception of decalcification. Following incubation in ADAPT-3D refractive index solution, samples were then placed in immersion oil (Cargille Labs, NA 1.52). Images were acquired on Miltenyi Ultra Microscope Blaze with a 4X / NA0.35 using 633 nm at 13% power with 40 millisecond exposure, 568 nm at 11% power with 20 millisecond exposure. Following acquisition, ome-tiff file formats were used to stitch in Stitchy™ (Translucence Biosystems) with default settings and exported as *.ims.

[0176] Image visualization and analysis. 3D visualization was visualized on Imaris software (Bitplane Inc.) on vlO.1.1 software. The area and fluorescence intensity of CUBIC and ADAPT-3D processed thick sections were measured using the mean gray intensity measurement function of image! software (vl.54p) after tracing the edge of the section for each replicate.

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[0178] A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.

Claims

CLAIMS1. A treatment solution for rendering a biological sample optically transparent, the treatment solution comprising: a contrast agent; a carbohydrate; a nitrogen-containing compound; and an antioxidant; wherein the treatment solution renders the biological sample optically transparent while preserving fluorescent properties of the biological sample.

2. The treatment solution of claim 1, wherein the contrast agent comprises an iodinated compound.

3. The treatment solution of claim 2, wherein the iodinated compound comprises iohexol, iodixanol, or a combination thereof.

4. The treatment solution of claim 2, wherein the iodinated compound comprises iohexol, iodixanol, or a combination thereof.

5. The treatment solution of claim 1 or 2, wherein the iohexol is present at a concentration from about 20 w / v% to about 30 w / v% based on the total weight and volume of the treatment solution.

6. The treatment solution of any one of claims 1 to 5, wherein the carbohydrate comprises a sugar.

7. The treatment solution of claim 6, wherein the sugar comprises sucrose, fructose, glucose, galactose, maltose, lactose, or a combination thereof.

8. The treatment solution of claim 6, wherein the sugar comprises sucrose.

9. The treatment solution of any one of claims 1 to 8, wherein the carbohydrate or sugar is present at a concentration of from about 25 w / v% to about 35 w / v% based on the total weight and volume of the treatment solution.

10. The treatment solution of any one of claims 1 to 9, wherein the nitrogen-containing compound comprises urea, biuret, an alkyl urea, or a combination thereof.

11. The treatment solution of claim 10, wherein the nitrogen-containing compound comprises urea.

12. The treatment solution of any one of claims 1 to 11, wherein the nitrogen-containing compound is present at a concentration of from about 20 w / v% to about 30 w / v%.

13. The treatment solution of any one of claims 1 to 12, wherein the antioxidant comprises n- propyl gallate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), or a combination thereof.

14. The treatment solution of claim 13, wherein the antioxidant comprises n-propyl gallate.

15. The treatment solution of any one of claims 1 to 14, wherein the antioxidant is present at a concentration of from about 0.1 w / v% to about 1 w / v% based on the total weight and volume of the treatment solution.

16. The treatment solution of any one of claims 1 to 15, further comprising a reaction inhibitor of a color-developing reaction.

17. The treatment solution of claim 16, wherein the reaction inhibitor of a color-developing reaction is a reaction inhibitor of a Maillard-reactive browning reaction.

18. The treatment solution of claim 16 or 17, wherein the reaction inhibitor comprises 1- thioglycerol.

19. The treatment solution of any one of claims 16 to 18, wherein the reaction inhibitor is present at a concentration of from about 0.5 v / v% to about 1.5 v / v% based on the total weight and volume of the treatment solution.

20. The treatment solution of any one of claims 1 to 19, wherein the treatment solution has a refractive index from about 1.45 to about 1.55.

21. The treatment solution of claim 20, wherein the treatment solution has a refractive index from about 1.50 to about 1.51.

22. A method for processing a biological sample, the method comprising: treating the biological sample with a treatment solution of any one of claim 1 to 21; acquiring an image of the optically transparent biological sample; and processing the acquired image to generate a three-dimensional visualization of the biological sample.

23. The method of claim 22, further comprising fixing the biological sample by treating the biological sample with a fixative solution comprising paraformaldehyde and a sugar at a pH of from about 7 to about 9.

24. The method of claim 23, wherein the sugar comprises sucrose.

25. The method of any one of claims 22 to 25, further comprising treating the biological sample with a decolorization solution.

26. The method of claim 25, wherein the decolorization solution comprises a detergent that forms small micelles or salt-free amines, one or more N-alkyldiethanolamines, and a humectant.

27. The method of claim 26, wherein the detergent that forms small micelles or salt-free amines comprises (3-cholamidopropyl)dimethylammonio)-l-propanesulfonate (CHAPS).

28. The method of claim 26 or 27, wherein the one or more N-alkyldiethanolamines comprises N-methyldiethanolamine, N-butyldiethanolamine, N-propyldiethanolamine, N- ethyldiethanolamine, or a combination thereof.

29. The method of any one of claims 26 to 28, wherein the humectant comprises 1,2- hexanediol.

30. The method of any one of claims 22 to 29, further comprising treating the biological sample with a partial delipidation solution.

31. The method of claim 30, wherein the partial delipidation solution comprises tetrahydrofuran and a humectant.

32. The method of claim 31, wherein the humectant comprises 1,2-hexanediol.

33. The method of any one of claims 22 to 32, further comprising treating the biological sample with a decalcification solution.

34. The method of claim 33, wherein the decalcification solution comprises a chelator, one or more N-alkyldiethanolamine, and imidazole.

35. The method of claim 34, wherein the chelator comprises ethylenediaminetetraacetic acid (EDTA)36. The method of claim 34 or 35, wherein the one or more N-alkyldiethanolamine comprises N-methyldiethanolamine, N-butyldiethanolamine, or a combination thereof.

37. The method of any one of claims 22 to 36, wherein the image is acquired using microscopy.

38. The method of any one of claims 22 to 37, wherein the biological sample comprises tissue.

39. The method of any one of claims 22 to 38, wherein the processing comprises deconvolution.

40. A method to optically clear and three-dimensionally image an intact tissue or whole organ while preserving tissue morphology, the method comprising: treating the intact tissue or whole organ with at least one of a fixative, a decolorization buffer, a delipidation buffer, a refractive index matching (RIM) solution, and a decalcification solution; acquiring image(s) of the treated intact tissue or whole organ using microscopy; and deconvolution of the acquired image(s).

41. The method of claim 40, the method further comprising adaptable rapid immunolabeling using a nonionic detergent solution.

42. The method of claim 40, wherein the fixative preserves an endogenous fluorescent reporter protein and prevents the masking of a tissue antigen.

43. The method of claim 42, wherein the fixative is comprised of 4% (w / v) paraformaldehyde (PF A), at least 10% (w / v) sucrose, and has a pH of 9.0.

44. The method of claim 40, wherein the decolorization buffer is comprised of up to about 20% ((3-cholamidopropyl)dimethylammonio)- 1 -propanesulfonate (CHAPS), about 25% N- methyldiethanolamine, about 5% N-butyldiethanolamine, and about 10% 1,2-hexanediol.

45. The method of claim 40, wherein the delipidation buffer prevents tissue shrinkage and preserves tissue morphology.

46. The method of claim 45, wherein the delipidation buffer is comprised of about 50% tetrahydrofuran and about 10% 1,2-hexanediol.

47. The method of claim 40, wherein the RIM solution comprises iodixanol, from about 25.33% w / v to about 30% w / v iohexol, from about 10% w / v to about 30% w / v sucrose, 25% w / v Urea, from about 0.5% v / v to about 1% v / v 1 -thioglycerol, and from about 0.01% w / v to about 0.5% w / v n-propyl gallate.

48. The method of claim 41, wherein the nonionic detergent solution is comprised of 0.167% (v / v) Tween-20 and 0.33% (v / v) Triton X-100 in dimethyl sulfoxide (DMSO).

49. The method of claim 40, wherein the image(s) can be acquired using multiple modes of microscopy, including epifluorescent microscopy, stereomicroscopy, light sheet microscopy, and confocal microscopy.

50. The method of claim 40, wherein tissue optical transparency may be achieved after consecutive use of the decolorization buffer for 80 minutes, the delipidation buffer for 120 minutes, and the RIM solution for 60 minutes.

51. The method of claim 40, wherein the tissue may be 1 cm x 1 cm x 0.5 mm in size.

52. A composition of a refractive index matching (RIM) solution for use in imaging optically cleared three-dimensional images, the composition comprising an X-ray contrast reagent, a sugar, an amine, an inhibitor of Maillard-reactive browning, and an antioxidant.

53. The composition of claim 52, wherein the X-ray contrast reagents may be iodixanol, iohexol, and any combination thereof.

54. The composition of claim 53, wherein iohexol may be at a concentration ranging from about 25.33% w / v to about 30% w / v.

55. The composition of claim 52, wherein the sugar may be D-sucrose ranging from about 10% w / v to about 30% w / v.

56. The composition of claim 52, wherein the amine may be urea at about 25% (w / v).

57. The composition of claim 52, wherein the inhibitor of Maillard-reactive browning may be 1 -thioglycerol ranging from about 0.5% w / v to about 1% w / v.

58. The composition of claim 52, wherein the antioxidant may be n-propyl gallate ranging from about 0.01% w / v to about 0.5% w / v.

Images