Spatial-dependent analysis of biological materials from intact tissue samples

Abstract

Biological research requires the isolation and analysis of materials, such as RNA, DNA, and proteins, from tissue samples.The methods and compositions described herein allow for high-resolution imaging of large intact tissue samples, and then for the isolation of materials in a precise and location-dependent manner.The methods and compositions described herein can be used, for example, for biomarker discovery, cell population identification, pathological analysis, and the generation of expression data in specific regions of interest.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 991,583, filed on March 18, 2020, and U.S. Provisional Application No. 63 / 074,693, filed on September 4, 2020, the contents of each of which are hereby incorporated by reference in their entirety, and for which each priority is claimed.

Background Art

[0002] During most molecular - biology - based methods for tissue analysis, the spatial context of the substances being analyzed is lost. Techniques known in the art for combining sequence analysis with spatial information have limitations, such as, among other constraints, low sequencing depth, sample - volume limitations, analysis of individual tissue sections, or time - consuming reads. Currently, as far as is known, there is no available method that combines the accuracy of isolation and the depth of analysis. The methods and compositions described herein address these and other problems in the art.

Summary of the Invention

[0003] The present technology generally relates to methods and compositions for three - dimensional labeling of cells or other regions of a tissue or tissue sample. The method includes contacting a region of interest (or cell) with an appropriate label and then imaging the cleared tissue or sample that has been labeled by subjecting the region of interest (or cell) to a multiphoton laser. The labeled region can then be isolated from the tissue or sample and subjected to analysis, such as, but not limited to, DNA sequencing, RNA sequencing, proteome analysis, epigenetic analysis, immunohistochemical analysis, chromosome conformation capture, or immunofluorescence analysis. Various methods for isolating regions (or cells) of interest can be used, such as, but not limited to, laser - assisted microdissection, fluorescence - activated cell sorting (FACS), magnetic - activated cell sorting (MACS), or buoyancy - activated cell sorting (BACS). Such analysis can provide information about individual regions of the tissue or sample.

[0004] In one aspect, a method is provided for labeling a region of tissue or tissue sample. The method may include a) providing a three-dimensional tissue or tissue sample; b) making the sample clear; c) contacting the tissue or tissue sample with a photoactivatable label; and d) exposing a region of the tissue or tissue sample to a multiphoton laser to label the region.

[0005] In other aspects, a method is provided for three-dimensional expression profiling of intact tissue or tissue sample. The method may include: a) providing a three-dimensional intact tissue or tissue sample; b) clearing the sample; c) contacting the tissue or tissue sample with a photoactivatable label; d) subjecting a region of the tissue or tissue sample to a multiphoton laser to label the region; e) imaging the labeled region to create an image; f) isolating the labeled region from the tissue or tissue sample; g) determining the DNA, RNA, and / or protein composition of the isolated labeled region; and h) combining the image with the DNA, RNA, and / or protein composition of the isolated labeled region to create a three-dimensional expression profile of the intact tissue or tissue sample.

[0006] In one aspect, a method is provided for isolating a single cell or nucleus in a tissue or tissue sample. The method may include: a) providing a three-dimensional tissue or tissue sample containing the cell or nucleus of interest; b) clearing the sample; c) contacting the tissue or tissue sample with a photoactivatable label; d) subjecting the cell or nucleus to a multiphoton laser to label the cell or nucleus; e) dissociating the labeled cell nucleus from the tissue or tissue sample; and f) isolating the labeled cell or nucleus.

[0007] In other contexts, compositions are provided. Compositions may include a tissue sample, a medium having a refractive index similar to or matching that of the tissue, and a photoactivatable label.

[0008] In one aspect, a cleared tissue sample is provided. The cleared tissue sample may include a photoactivatable label and a medium having a refractive index similar to or matching that of the tissue. [Brief explanation of the drawing]

[0009] [Figure 1] The image shows the mouse brain before (left) and after (right) tissue clearing.

[0010] [Figure 2] This is a series of fluorescence micrographs showing the stability of different fluorescent proteins in cleared tissue (bottom row) or control tissue (top row). In this figure, 293T cells were transfected with plasmids driving the expression of EGFP, mKate2, tdTomato, or Venus, respectively, under the control of the CMV promoter.

[0011] [Figure 3A] This figure shows one embodiment of the use of two-photon microscopy to label target cells or nuclei in cleared tissue using photoreactive labels. Figure 3A shows the labeling of molecules by photoreactive labels and biotin tags having photoreactive groups when exposed to light with wavelengths of 260 nm to 475 nm. Figure 3B shows the advantages of using two-photon microscopy (right) to label target cells or nuclei compared to conventional confocal microscopy (left). Traditional confocal microscopy results in photoactivation of the entire tissue area exposed to both light (red) and the photoactivatable compound (green). In contrast, two-photon microscopy allows for more precise photoactivation and targeting of sites (red star), thereby avoiding nonspecific labeling of non-target tissue. [Figure 3B]This figure shows one embodiment of the use of two-photon microscopy to label target cells or nuclei in cleared tissue using photoreactive labels. Figure 3A shows the labeling of molecules by photoreactive labels and biotin tags having photoreactive groups when exposed to light with wavelengths of 260 nm to 475 nm. Figure 3B shows the advantages of using two-photon microscopy (right) to label target cells or nuclei compared to conventional confocal microscopy (left). Traditional confocal microscopy results in photoactivation of the entire tissue area exposed to both light (red) and the photoactivatable compound (green). In contrast, two-photon microscopy allows for more precise photoactivation and targeting of sites (red star), thereby avoiding nonspecific labeling of non-target tissue.

[0012] [Figure 4] An overview of an analysis using two-photon labeling technology is provided. Tissue with a three-dimensional (3D) cellular structure is cleared, immersed in a photoactivatable dye (e.g., photobiotin), labeled using a two-photon laser (e.g., shown in Figure 3B), and then the labeled nuclei are isolated for analysis, such as single-cell analysis (e.g., SPLiT-seq).

[0013] [Figure 5] The polynucleotide length profiles of untreated samples after reverse transcription, at room temperature (RT) or 4°C (4C) for 3 days, are shown.

[0014] [Figure 6A] This outlines a method for spatially dependent analysis of biological materials from intact tissue samples. Figure 6A shows exemplary tissue clearing (Figure 6A, panel A) and labeling processes (panels B-E), as well as isolation of photolabeled nuclei (panel F). The obtained sample can be analyzed by SPLiT-seq library preparation from the isolated photolabeled nuclei (panels G and H), followed by data analysis (I). In addition (or alternatively), DNA (Figure 6B) or proteins (Figure 6C) can be analyzed from the isolated tissue. [Figure 6B]This outlines a method for spatially dependent analysis of biological materials from intact tissue samples. Figure 6A shows exemplary tissue clearing (Figure 6A, panel A) and labeling processes (panels B-E), as well as isolation of photolabeled nuclei (panel F). The obtained sample can be analyzed by SPLiT-seq library preparation from the isolated photolabeled nuclei (panels G and H), followed by data analysis (I). In addition (or alternatively), DNA (Figure 6B) or proteins (Figure 6C) can be analyzed from the isolated tissue. [Figure 6C] This outlines a method for spatially dependent analysis of biological materials from intact tissue samples. Figure 6A shows exemplary tissue clearing (Figure 6A, panel A) and labeling processes (panels B-E), as well as isolation of photolabeled nuclei (panel F). The obtained sample can be analyzed by SPLiT-seq library preparation from the isolated photolabeled nuclei (panels G and H), followed by data analysis (I). In addition (or alternatively), DNA (Figure 6B) or proteins (Figure 6C) can be analyzed from the isolated tissue.

[0015] [Figure 7A] The RNA quality of formalin-fixed and cleared samples by SPLiT-seq analysis is shown. Figure 7A is a plot showing unique counts per barcode (log2(UMI)) as a function of barcode rank (barcode log10 scale). Figure 7B is a scatter plot showing log2(TPM) for fixed samples on the y axis and log2(TPM) for cleared samples on the x axis (TPM: transcriptions / million). Figure 7C shows an exemplary genome browser window (top) presenting cDNA coverage of a 72kb stretch of mouse (mm10) chromosome 2, with a comparison of fixed and cleared samples (center) and a fixed sample (bottom). Spinal cord was photoactivated with Cy3-PA as shown in Figures 7A-7C. Nuclei were extracted and sorted for label incorporation. SPLiT-seq libraries were prepared and sequenced by HiSeq. [Figure 7B]The RNA quality of formalin-fixed and cleared samples by SPLiT-seq analysis is shown. Figure 7A is a plot showing unique counts per barcode (log2(UMI)) as a function of barcode rank (barcode log10 scale). Figure 7B is a scatter plot showing log2(TPM) for fixed samples on the y axis and log2(TPM) for cleared samples on the x axis (TPM: transcriptions / million). Figure 7C shows an exemplary genome browser window (top) presenting cDNA coverage of a 72kb stretch of mouse (mm10) chromosome 2, with a comparison of fixed and cleared samples (center) and a fixed sample (bottom). Spinal cord was photoactivated with Cy3-PA as shown in Figures 7A-7C. Nuclei were extracted and sorted for label incorporation. SPLiT-seq libraries were prepared and sequenced by HiSeq. [Figure 7C] The RNA quality of formalin-fixed and cleared samples by SPLiT-seq analysis is shown. Figure 7A is a plot showing unique counts per barcode (log2(UMI)) as a function of barcode rank (barcode log10 scale). Figure 7B is a scatter plot showing log2(TPM) for fixed samples on the y axis and log2(TPM) for cleared samples on the x axis (TPM: transcriptions / million). Figure 7C shows an exemplary genome browser window (top) presenting cDNA coverage of a 72kb stretch of mouse (mm10) chromosome 2, with a comparison of fixed and cleared samples (center) and a fixed sample (bottom). Spinal cord was photoactivated with Cy3-PA as shown in Figures 7A-7C. Nuclei were extracted and sorted for label incorporation. SPLiT-seq libraries were prepared and sequenced by HiSeq.

[0016] [Figure 8A]The use of two-photon labeling in spinal cord tissue is demonstrated. Figure 8A is a cross-sectional view of a mouse spinal cord showing different regions of the spinal cord, with the dorsal layer shown as a colored circle. In the enlarged panel, the photoactivated region is highlighted. Figure 8B is a FACS plot of cleared but non-photoactivated nuclei isolated from mouse spinal cord. The Y-axis shows the signal of the nuclear counterstain DAPI, and the X-axis shows the PA dye used in this experiment: Cy3-PA. Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). Figure 8C is a FACS plot of cleared and photoactivated nuclei isolated from mouse spinal cord (shown in Figure 9A). The Y-axis shows the signal of the nuclear counterstain DAPI, and the X-axis shows the PA dye used in this experiment: Cy3-PA. Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). [Figure 8B] The use of two-photon labeling in spinal cord tissue is demonstrated. Figure 8A is a cross-sectional view of a mouse spinal cord showing different regions of the spinal cord, with the dorsal layer shown as a colored circle. In the enlarged panel, the photoactivated region is highlighted. Figure 8B is a FACS plot of cleared but non-photoactivated nuclei isolated from mouse spinal cord. The Y-axis shows the signal of the nuclear counterstain DAPI, and the X-axis shows the PA dye used in this experiment: Cy3-PA. Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). Figure 8C is a FACS plot of cleared and photoactivated nuclei isolated from mouse spinal cord (shown in Figure 9A). The Y-axis shows the signal of the nuclear counterstain DAPI, and the X-axis shows the PA dye used in this experiment: Cy3-PA. Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). [Figure 8C]Shows the use of two-photon labeling of spinal cord tissue. Figure 8A is a cross-sectional view of the mouse spinal cord showing different regions of the spinal cord, with the dorsal layer shown as a colored circle. In the enlarged panel, the region that received photoactivation is highlighted. Figure 8B is a FACS plot of nuclei isolated from the mouse spinal cord that were cleared but not photoactivated. The Y-axis shows the signal of the nuclear counterstain DAPI, and the X-axis shows the PA dye used in this experiment: Cy3-PA. The boxes indicate single nuclei that are negative (left) or positive (right) for incorporated Cy3. Figure 8C is a FACS plot of nuclei isolated from the mouse spinal cord that were cleared and photoactivated (shown in Figure 9A). The Y-axis shows the signal of the nuclear counterstain DAPI, and the X-axis shows the PA dye used in this experiment: Cy3-PA. The boxes indicate single nuclei that are negative (left) or positive (right) for incorporated Cy3.

[0017] [Figure 9A] Shows exemplary compounds for use in the methods of the present invention. Figure 9A shows examples of photoreactive compounds that can be used for photolabeling. Figure 9B shows exemplary modifications to aryl azides induced by ultraviolet (UV) light. Figure 9C shows examples of synthesized photoreactive compounds. [Figure 9B] Shows exemplary compounds for use in the methods of the present invention. Figure 9A shows examples of photoreactive compounds that can be used for photolabeling. Figure 9B shows exemplary modifications to aryl azides induced by ultraviolet (UV) light. Figure 9C shows examples of synthesized photoreactive compounds. [Figure 9C] Shows exemplary compounds for use in the methods of the present invention. Figure 9A shows examples of photoreactive compounds that can be used for photolabeling. Figure 9B shows exemplary modifications to aryl azides induced by ultraviolet (UV) light. Figure 9C shows examples of synthesized photoreactive compounds.

[0018] [Figure 10A]Expression profiling of cleared nuclei derived from spinal cord cells using SPLiT-seq analysis is shown. Figure 10A is a graph showing cell type expression profiles (expression levels of genes associated with the indicated cell type). Figure 10B is a graph generated by UMAP of the data from Figure 10A. [Figure 10B] Expression profiling of cleared nuclei derived from spinal cord cells using SPLiT-seq analysis is shown. Figure 10A is a graph showing cell type expression profiles (expression levels of genes associated with the indicated cell type). Figure 10B is a graph generated by UMAP of the data from Figure 10A.

[0019] [Figure 11] This diagram illustrates an example of how multiplexing can enable multiple labeling events. The tissue can be exposed to a first dye (dye 1), followed by two-photon labeling of the first region of interest (left panel). The same tissue can then be brought into contact with a second dye (dye 2) (optionally after washing), followed by two-photon labeling of the second region of interest (center panel). If desired, the same tissue can be brought into contact with a third dye (dye 3) (optionally after washing), followed by two-photon labeling of the third region of interest (center panel).

[0020] [Figure 12A] Examples of application areas for target compounds for photoactivation across the entire mount are shown. Examples of application areas include tumor heterogeneity, inflammation tumor / immune deserts and proximity to the vascular system (Figure 12A), detection and labeling of low-density cell populations (Figure 12B), generation of expression data, and 3D histology (Figure 12C). [Figure 12B] Examples of application areas for target compounds for photoactivation across the entire mount are shown. Examples of application areas include tumor heterogeneity, inflammation tumor / immune deserts and proximity to the vascular system (Figure 12A), detection and labeling of low-density cell populations (Figure 12B), generation of expression data, and 3D histology (Figure 12C). [Figure 12C]Examples of application areas for target compounds for photoactivation across the entire mount are shown. Examples of application areas include tumor heterogeneity, inflammation tumor / immune deserts and proximity to the vascular system (Figure 12A), detection and labeling of low-density cell populations (Figure 12B), generation of expression data, and 3D histology (Figure 12C). [Modes for carrying out the invention]

[0021] After reading this description, methods for implementing the disclosure in various alternative embodiments and applications will become apparent to those skilled in the art. However, not all embodiments of the invention are described herein. It will be understood that the embodiments presented herein are presented as examples only and are not limiting. Accordingly, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the disclosure as described herein.

[0022] Before disclosing and describing this technology, it should be understood that the aspects described below are not limited to specific compositions, methods for preparing such compositions, or uses thereof, and are therefore naturally subject to change. It should also be understood that the technical terms used herein are intended solely to describe specific aspects and are not intended to limit them.

[0023] Detailed explanations divided into various sections for the convenience of the reader, and disclosures found in any section, may be combined with those in other sections. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of this disclosure. definition

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure pertains. In this specification and the following claims, several terms are referenced that must be defined as having the following meanings.

[0025] The terms used herein are intended solely to describe specific embodiments and are not intended to limit them. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms similarly unless the context explicitly indicates otherwise.

[0026] "Optional" or "optionally" means that the following event or situation may or may not occur, and that the description includes both the cases in which the event or situation occurs and the cases in which it does not.

[0027] The term "approximately" when used before a numerical specification that includes a range, such as temperature, time, quantity, or concentration, indicates an approximate value that may vary by (+) or (-) 10%, 5%, 1%, or any sub-range or sub-value between them. Preferably, when the term "approximately" is used in relation to dosage, it means that the dosage may vary by + / - 10%.

[0028] "Comprising" or "comprises" is intended to mean that a composition and method includes the elements described but does not exclude others. "Consisting essentially of," when used to define a composition and method, shall mean excluding other elements that are essentially important to the combination for the purposes described. Thus, a composition consisting essentially of the elements defined in this invention does not exclude other materials or processes that do not substantially affect the basic and novel features of the claimed technology. "Consisting of" shall mean excluding other components and elements that are more than trace amounts of substantial method processes. Embodiments defined by each of these transitional terms are within the scope of this disclosure. method

[0029] In one aspect, a method is provided for labeling a region of tissue or tissue sample. The method may include (a) providing a three-dimensional tissue or tissue sample; (b) making the sample clear; (c) contacting the tissue or tissue sample with a photoactivatable label; and (d) exposing a region of the tissue or tissue sample to a multiphoton laser to label the region. In some embodiments, the multiphoton laser is a two-photon laser. In some embodiments, the multiphoton laser is a three-photon laser.

[0030] In some embodiments of the methods described herein, the region includes cells, intracellular compartments, aggregates, or secretory aggregates. In some embodiments, the region includes cells. In some embodiments, the region includes intracellular compartments. In some embodiments, the region includes aggregates. In some embodiments, the region includes secretory aggregates. In some embodiments, the intracellular compartment is the nucleus.

[0031] In some embodiments, the method further includes imaging the tissue or tissue sample. In some embodiments, the method further includes imaging the tissue. In some embodiments, the method further includes imaging the tissue sample. In some embodiments, the tissue or tissue sample is fixed tissue or a fixed tissue sample. In some embodiments, the tissue is fixed tissue. In some embodiments, the tissue is a fixed tissue sample. In some embodiments, the tissue sample is fixed tissue. In some embodiments, the tissue sample is a fixed tissue sample.

[0032] In some embodiments of the methods provided herein, the photoactivatable label includes a detectable portion. In some embodiments, the detectable portion includes a luminescent portion. In some embodiments, the luminescent portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion. In some embodiments, the luminescent portion is a fluorescent portion. In some embodiments, the luminescent portion is a chemiluminescent portion. In some embodiments, the luminescent portion is a bioluminescent portion. In some embodiments, the luminescent portion is an electrochemiluminescent portion. In some embodiments, the fluorescent portion includes a fluorophore. In some embodiments, one of the detectable portions includes an antibody or a functional derivative thereof. In some embodiments, the photoactivatable label includes a tag. In some embodiments, the tag is selected from the group including affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags. In some embodiments, the tag is an affinity tag. In some embodiments, the tag is an epitope tag. In some embodiments, the tag is a fluorescent tag. In some embodiments, the tag is an oligonucleotide tag. In some embodiments, the tag is a biotin tag.

[0033] In some embodiments of the methods provided herein, the sample clearing process includes dehydrating the sample and transferring the sample to a medium having a refractive index similar to or consistent with the tissue. In some embodiments, the refractive index is about 1.3 to about 1.6. In some embodiments, the refractive index is about 1.3. In some embodiments, the refractive index is about 1.325. In some embodiments, the refractive index is about 1.35. In some embodiments, the refractive index is about 1.375. In some embodiments, the refractive index is about 1.4. In some embodiments, the refractive index is about 1.425. In some embodiments, the refractive index is about 1.45. In some embodiments, the refractive index is about 1.475. In some embodiments, the refractive index is about 1.5. In some embodiments, the refractive index is about 1.525. In some embodiments, the refractive index is about 1.55. In some embodiments, the refractive index is about 1.575. In some embodiments, the refractive index is about 1.6. In some embodiments, the refractive index is about 1.3, about 1.325, about 1.35, about 1.375, about 1.4, about 1.425, about 1.45, about 1.475, about 1.5, about 1.525, about 1.55, about 1.575, or about 1.6. The refractive index may be any value or subrange within the enumerated range including the endpoints, or any range between any of the listed values.

[0034] In some embodiments, the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, containing or not containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl alcohol containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl alcohol that does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, which does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium comprises a BABB solution.

[0035] As described above in the methods provided herein, in some embodiments, the sample clearing process includes dehydrating the sample. In some embodiments, the sample is dehydrated with a tert-butanol solution. In some embodiments, the tert-butanol solution contains trimethylamine, tetrahydrofuran, ethanol, or methanol. In some embodiments, the tert-butanol solution contains trimethylamine. In some embodiments, the tert-butanol solution contains tetrahydrofuran. In some embodiments, the tert-butanol solution contains ethanol. In some embodiments, the tert-butanol solution contains methanol.

[0036] In some embodiments of the methods provided herein, the photoactivatable label is hydrophobic. In some embodiments of the methods provided herein, the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azido-methylcoumarin group, or a psoralen group. In some embodiments, the photoactivatable label comprises a phenyl azide group. In some embodiments, the photoactivatable label comprises an ortho-hydroxyphenyl azide group. In some embodiments, the photoactivatable label comprises a meta-hydroxyphenyl azide group. In some embodiments, the photoactivatable label comprises a tetrafluorophenyl azide group. In some embodiments, the photoactivatable label comprises an ortho-nitrophenyl azide group. In some embodiments, the photoactivatable label comprises a meta-nitrophenyl azide group. In some embodiments, the photoactivatable label comprises a diazirine group. In some embodiments, the photoactivatable label comprises an azido-methylcoumarin group. In some embodiments, the photoactivatable label comprises a psoralen group. In some embodiments, the photoactivatable label comprises an aryl azide group.

[0037] In some embodiments, the method further includes isolating a labeled region or a portion of a labeled region from a tissue or tissue sample. In some embodiments, the method further includes isolating a labeled region from a tissue. In some embodiments, the method further includes isolating a portion of a labeled region from a tissue. In some embodiments, the method further includes isolating a labeled region from a tissue sample. In some embodiments, the method further includes isolating a portion of a labeled region from a tissue sample. In some embodiments, the labeled region or a portion is isolated by FACS sorting for labeling. In some embodiments, the method further includes analyzing the composition of the isolated labeled region to produce analytical data. In some embodiments, the analysis includes determining the DNA, RNA, and / or protein composition of the isolated labeled region. In some embodiments, the analysis includes determining the DNA, RNA, and protein composition of the isolated labeled region. In some embodiments, the analysis includes determining the DNA, RNA, or protein composition of the isolated labeled region. In some embodiments, the analysis includes determining the DNA composition of the isolated labeled region. In some embodiments, the analysis includes determining the RNA composition of the isolated labeled region. In some embodiments, the analysis includes determining the protein composition of the isolated labeled region. In some embodiments, RNA is analyzed by SPLITseq.

[0038] In some embodiments of the methods described herein, the tissue or sample is imaged to create an image before isolating the labeled region, and the image is further combined with analytical data to create a three-dimensional composition map of the region.

[0039] In one aspect, a method is provided for three-dimensional expression profiling of intact tissue or tissue sample. The method may include: (a) providing a three-dimensional intact tissue or tissue sample; (b) clearing the sample; (c) contacting the tissue or tissue sample with a photoactivatable label; (d) exposing a region of the tissue or tissue sample to a multiphoton laser to label the region; (e) imaging the labeled region to create an image; (f) isolating the labeled region from the tissue or tissue sample; (g) determining the DNA, RNA, and / or protein composition of the isolated labeled region; and (h) combining the image with the DNA, RNA, and / or protein composition of the isolated labeled region to create a three-dimensional expression profile of the intact tissue or tissue sample. In some embodiments, the multiphoton laser is a two-photon laser. In some embodiments, the multiphoton laser is a three-photon laser. In some embodiments, the region is a cell, intracellular compartment, aggregate, or secretory aggregate. In some embodiments, the region is a cell. In some embodiments, the region is an intracellular compartment. In some embodiments, the region is an aggregate. In some embodiments, the region is a secretory aggregate. In some embodiments, the isolated labeled region is a single nucleus. In some embodiments, the RNA composition of the isolated labeled region is determined.

[0040] In one aspect, a method is provided for isolating a single cell or nucleus in a tissue or tissue sample. The method may include (a) providing a three-dimensional tissue or tissue sample; (b) clearing the sample; (c) contacting the tissue or tissue sample with a photoactivatable label; (d) subjecting the cells to a multiphoton laser to label the cells; (e) dissociating the labeled cells from the tissue or tissue sample; and (f) isolating the labeled cells or nucleus. In some embodiments, the multiphoton laser is a two-photon laser. In some embodiments, the multiphoton laser is a three-photon laser.

[0041] In some embodiments, the method further includes imaging the tissue or tissue sample. In some embodiments, the method further includes imaging the tissue. In some embodiments, the method further includes imaging the tissue sample. In some embodiments, the tissue or tissue sample is fixed tissue or a fixed tissue sample. In some embodiments, the tissue is fixed tissue. In some embodiments, the tissue is a fixed tissue sample. In some embodiments, the tissue sample is fixed tissue. In some embodiments, the tissue sample is a fixed tissue sample.

[0042] In some embodiments of the methods provided herein, the photoactivatable label includes a detectable portion. In some embodiments, the detectable portion includes a luminescent portion. In some embodiments, the luminescent portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion. In some embodiments, the luminescent portion is a fluorescent portion. In some embodiments, the luminescent portion is a chemiluminescent portion. In some embodiments, the luminescent portion is a bioluminescent portion. In some embodiments, the luminescent portion is an electrochemiluminescent portion. In some embodiments, the fluorescent portion includes a fluorophore. In some embodiments, the detectable portion includes an antibody or a functional derivative thereof. In some embodiments, the photoactivatable label includes a tag. In some embodiments, the tag is selected from the group including affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags. In some embodiments, the tag is an affinity tag. In some embodiments, the tag is an epitope tag. In some embodiments, the tag is a fluorescent tag. In some embodiments, the tag is an oligonucleotide tag. In some embodiments, the tag is a biotin tag.

[0043] In some embodiments of the methods provided herein, the sample clearing process includes dehydrating the sample and transferring the sample to a medium having a refractive index similar to or consistent with that of the tissue. In some embodiments, the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, containing or not containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl alcohol containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl alcohol that does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, which does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium comprises a BABB solution.

[0044] In some embodiments of the methods provided herein, the sample is dehydrated with a tert-butanol solution. In some embodiments, the tert-butanol solution comprises trimethylamine, tetrahydrofuran, ethanol, or methanol. In some embodiments, the tert-butanol solution comprises trimethylamine. In some embodiments, the tert-butanol solution comprises tetrahydrofuran. In some embodiments, the tert-butanol solution comprises ethanol. In some embodiments, the tert-butanol solution comprises methanol. In some embodiments, the photoactivatable label is hydrophobic.

[0045] In some embodiments of the methods provided herein, the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azido-methylcoumarin group, or a psoralen group. In some embodiments, the photoactivatable label comprises a phenyl azide group. In some embodiments, the photoactivatable label comprises an ortho-hydroxyphenyl azide group. In some embodiments, the photoactivatable label comprises a meta-hydroxyphenyl azide group. In some embodiments, the photoactivatable label comprises a tetrafluorophenyl azide group. In some embodiments, the photoactivatable label comprises an ortho-nitrophenyl azide group. In some embodiments, the photoactivatable label comprises a meta-nitrophenyl azide group. In some embodiments, the photoactivatable label comprises a diazirine group. In some embodiments, the photoactivatable label comprises an azido-methylcoumarin group. In some embodiments, the photoactivatable label comprises a psoralen group. In some embodiments, the photoactivatable label includes an aryl azide group. In some embodiments, the photoactivatable label includes the group shown in Figure 9A.

[0046] In some embodiments, the methods provided herein further include isolating labeled cells or nuclei. In some embodiments, the methods further include isolating labeled cells. In some embodiments, the methods further include isolating labeled nuclei. In some embodiments, labeled cells or nuclei are isolated by fluorescence-activated cell sorting (FACS) for labeling. In some embodiments, labeled cells are isolated by FACS sorting for labeling. In some embodiments, nuclei are isolated by FACS sorting for labeling. In some embodiments, labeled cells or nuclei are isolated by magnetically activated cell sorting (MACS) for labeling. In some embodiments, labeled cells are isolated by MACS sorting for labeling. In some embodiments, labeled nuclei are isolated by MACS sorting for labeling. In some embodiments, labeled cells or nuclei are isolated by buoyancy-activated cell sorting (BACS) for labeling. In some embodiments, labeled cells are isolated by BACS sorting for labeling. In some embodiments, labeled nuclei are isolated by BACS sorting for labeling. In some embodiments, labeled cells or nuclei are isolated by affinity-based column purification (e.g., affinity chromatography) sorting for the label. In some embodiments, labeled cells are isolated by affinity-based column purification sorting for the label. In some embodiments, labeled nuclei are isolated by affinity-based column purification sorting for the label.

[0047] In some embodiments, the methods provided herein further include analyzing isolated labeled cells or nuclei. In some embodiments, the methods further include analyzing isolated labeled cells. In some embodiments, the methods further include analyzing isolated labeled nuclei. In some embodiments, the analysis includes determining the DNA, RNA, and / or protein composition of isolated labeled cells or nuclei. In some embodiments, the analysis includes determining the DNA, RNA, and protein composition of isolated labeled cells or nuclei. In some embodiments, the analysis includes determining the DNA, RNA, or protein composition of isolated labeled cells or nuclei. In some embodiments, the analysis includes determining the DNA composition of isolated labeled cells or nuclei. In some embodiments, the analysis includes determining the RNA composition of isolated labeled cells or nuclei. In some embodiments, the analysis includes determining the protein composition of isolated labeled cells or nuclei. In some embodiments, the analysis includes determining the DNA composition of isolated labeled cells. In some embodiments, the analysis includes determining the RNA composition of isolated labeled cells. In some embodiments, the analysis includes determining the protein composition of isolated labeled cells. In some embodiments, the analysis includes determining the DNA composition of isolated labeled nuclei. In some embodiments, the analysis includes determining the RNA composition of the isolated labeled nuclei. In some embodiments, the analysis includes determining the protein composition of the isolated labeled nuclei. In some embodiments, the RNA is analyzed by single-cell RNA sequencing (scRNAseq). In some embodiments, the RNA is analyzed by SPLITseq.

[0048] In some embodiments of the methods provided herein, the method further includes contacting a tissue or tissue sample with a second photoactivatable label, and subjecting a second cell or nucleus in the tissue or tissue sample to a multiphoton laser, thereby labeling the second cell or nucleus. See, for example, Figure 11. In some embodiments, the multiphoton laser is a two-photon laser. In some embodiments, the multiphoton laser is a three-photon laser.

[0049] In some embodiments of the methods provided herein, the tissue or tissue sample is an entire organ, a tumor, or an animal. In some embodiments, the tissue or tissue sample is an entire organ. In some embodiments, the tissue or tissue sample is a tumor. In some embodiments, the tissue or tissue sample is an animal. In some embodiments, the tissue is an entire organ. In some embodiments, the tissue is a tumor. In some embodiments, the tissue is an animal. In some embodiments, the tissue sample is an entire organ. In some embodiments, the tissue sample is a tumor. In some embodiments, the tissue sample is an animal.

[0050] In some embodiments of the methods provided herein, the wavelength of each photon in a two-photon laser is approximately 500 nm to approximately 1100 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 500 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 505 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 510 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 515 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 520 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 525 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 530 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 535 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 540 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 545 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 550 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 555 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 560 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 565 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 570 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 575 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 580 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 585 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 590 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 595 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 600 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 605 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 610 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 615 nm.In some embodiments, the wavelength of each photon in a two-photon laser is approximately 620 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 625 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 630 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 635 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 640 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 645 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 650 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 655 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 660 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 665 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 670 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 675 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 680 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 685 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 690 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 695 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 700 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 705 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 710 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 715 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 720 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 725 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 730 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 735 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 740 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 745 nm.In some embodiments, the wavelength of each photon in a two-photon laser is approximately 750 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 755 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 760 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 765 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 770 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 775 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 780 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 785 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 790 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 795 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 800 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 805 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 810 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 815 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 820 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 825 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 830 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 835 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 840 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 845 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 850 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 855 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 860 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 865 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 870 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 875 nm.In some embodiments, the wavelength of each photon in a two-photon laser is approximately 880 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 885 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 890 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 895 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 900 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 905 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 910 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 915 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 920 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 925 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 930 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 935 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 940 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 945 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 950 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 955 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 960 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 965 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 970 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 975 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 980 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 985 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 990 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 995 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1000 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1005 nm.In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1010 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1015 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1020 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1025 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1030 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1035 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1040 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1045 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1050 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1055 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1060 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1065 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1070 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1075 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1080 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1085 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1090 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1095 nm. In some embodiments, the wavelength of each photon in a two-photon laser is approximately 1100 nm.In some embodiments, the wavelengths of each photon in the two-photon laser are approximately 905nm, 910nm, 915nm, 920nm, 925nm, 930nm, 935nm, 940nm, 945nm, 950nm, 955nm, 960nm, 965nm, 970nm, 975nm, 980nm, 985nm, 990nm, 995nm, and 1000nm. The wavelengths are approximately 1005 nm, 1010 nm, 1015 nm, 1020 nm, 1025 nm, 1030 nm, 1035 nm, 1040 nm, 1045 nm, 1050 nm, 1055 nm, 1060 nm, 1065 nm, 1070 nm, 1075 nm, 1080 nm, 1085 nm, 1090 nm, 1095 nm, or 1100 nm. The wavelength may be any value or subrange within the stated range including the endpoints, or any range between any of the enumerated values.

[0051] In some embodiments of the methods provided herein, the wavelength of each photon in the three-photon laser is approximately 690 nm to approximately 2500 nm. In some embodiments of the methods provided herein, the wavelength of each photon in the three-photon laser is approximately 690 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 695 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 700 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 705 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 710 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 715 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 720 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 725 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 730 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 735 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 740 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 745 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 750 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 755 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 760 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 765 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 770 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 775 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 780 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 785 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 790 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 795 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 800 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 805 nm.In some embodiments, the wavelength of each photon in a three-photon laser is approximately 810 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 815 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 820 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 825 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 830 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 835 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 840 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 845 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 850 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 855 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 860 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 865 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 870 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 875 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 880 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 885 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 890 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 895 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 905 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 910 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 915 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 920 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 925 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 930 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 935 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 940 nm.In some embodiments, the wavelength of each photon in a three-photon laser is approximately 945 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 950 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 955 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 960 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 965 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 970 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 975 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 980 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 985 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 990 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 995 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1000 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1005 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1010 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1015 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1020 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1025 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1030 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1035 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1040 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1045 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1050 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1055 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1060 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1065 nm.In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1070 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1075 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1080 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1085 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1090 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1095 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1100 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1105 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1110 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1115 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1120 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1125 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1130 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1135 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1140 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1145 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1150 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1155 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1160 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1165 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1170 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1175 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1180 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1185 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 1190 nm.In some embodiments, the wavelength of each photon in a three-photon laser is approximately 1195 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2000 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2005 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2010 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2015 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2020 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2025 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2030 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2035 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2040 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2045 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2050 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2055 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2060 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2065 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2070 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2075 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2080 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2085 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2090 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2100 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2105 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2110 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2115 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 2120 nm.In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2125 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2130 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2135 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2140 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2145 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 21. It is 50 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2155 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2160 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2165 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2170 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2175 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2180 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2185 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2190 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2195 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2200 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2205 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2210 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2215 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2220 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2225 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2230 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2235 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2240 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2245 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2250 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2255 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2260 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2265 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2270 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 2275 nm.In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2280 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2285 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2290 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2295 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2300 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2305 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2310 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2315 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2320 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2325 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2330 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2335 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2340 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2345 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2350 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2350 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2355 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2360 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2365 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2370 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2375 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2380 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2385 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2390 nm. In some embodiments, the wavelength of each photon in the three-photon laser is approximately 2395 nm.In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2400 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2405 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2410 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2415 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2420 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2425 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2430 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2435 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2440 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2445 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2455 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2455 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2460 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2465 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2470 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2475 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2480 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2485 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2490 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2495 nm. In some embodiments, the wavelength of each photon in a three-photon laser is approximately 2500 nm. composition

[0052] In one aspect, a composition is provided. The composition may include a tissue sample, a medium having a refractive index similar to or consistent with the tissue, and a photoactivatable label. In some embodiments, the tissue includes a labeled region. In some embodiments, the region includes cells, intracellular compartments, aggregates, or secretory aggregates. In some embodiments, the region includes cells. In some embodiments, the region includes intracellular compartments. In some embodiments, the region includes aggregates. In some embodiments, the region includes secretory aggregates. In some embodiments, the cells are labeled by a multiphoton method. In some embodiments, the cells are labeled by a two-photon method. In some embodiments, the cells are labeled by a three-photon method. In some embodiments, the photoactivatable label includes a detectable portion. In some embodiments, the detectable portion includes a luminescent portion. In some embodiments, the luminescent portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion. In some embodiments, the luminescent portion is a fluorescent portion. In some embodiments, the luminescent portion is a chemiluminescent portion. In some embodiments, the luminescent portion is a bioluminescent portion. In some embodiments, the luminescent portion is an electrochemiluminescent portion. In some embodiments, the fluorescent portion includes a fluorophore.

[0053] In some embodiments of the compositions provided herein, the photoactivatable label includes a tag. In some embodiments, the tag is selected from the group including affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags. In some embodiments, the tag is an affinity tag. In some embodiments, the tag is an epitope tag. In some embodiments, the tag is a fluorescent tag. In some embodiments, the tag is an oligonucleotide tag. In some embodiments, the tag is a biotin tag. In some embodiments, the detectable portion is an antibody or a functional derivative thereof. In some embodiments, the photoactivatable label is hydrophobic.

[0054] In some embodiments of the compositions provided herein, the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azido-methylcoumarin group, or a psoralen group. In some embodiments, the photoactivatable label comprises a phenyl azide group. In some embodiments, the photoactivatable label comprises an ortho-hydroxyphenyl azide group. In some embodiments, the photoactivatable label comprises a meta-hydroxyphenyl azide group. In some embodiments, the photoactivatable label comprises a tetrafluorophenyl azide group. In some embodiments, the photoactivatable label comprises an ortho-nitrophenyl azide group. In some embodiments, the photoactivatable label comprises a meta-nitrophenyl azide group. In some embodiments, the photoactivatable label comprises a diazirine group. In some embodiments, the photoactivatable label comprises an azido-methylcoumarin group. In some embodiments, the photoactivatable label comprises a psoralen group.

[0055] In some embodiments of the compositions provided herein, the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, which may or may not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium comprises a solution of benzyl alcohol, which comprises one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium comprises a solution of benzyl alcohol, which does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, which comprises one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, which does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium comprises a BABB solution. In some embodiments, one or more of the described components can be explicitly excluded.

[0056] In one aspect, a cleared tissue sample is provided. The cleared tissue sample may include a photoactivatable label and a medium having a refractive index similar to or substantially consistent with the tissue. In some embodiments, for compositions provided herein, the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, containing or not containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl alcohol containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl alcohol that does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol. In some embodiments, the medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, which does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium comprises a BABB solution. In some embodiments, one or more of the enumerated components can be explicitly excluded.

[0057] In some embodiments, the cleared tissue provided herein includes labeled cells. In some embodiments, the cells are labeled by multiphoton technology. In some embodiments, the photoactivatable label includes a detectable portion. In some embodiments, the detectable portion includes a luminescent portion. In some embodiments, the luminescent portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion. In some embodiments, the luminescent portion is a fluorescent portion. In some embodiments, the luminescent portion is a chemiluminescent portion. In some embodiments, the luminescent portion is a bioluminescent portion. In some embodiments, the luminescent portion is an electrochemiluminescent portion. In some embodiments, the fluorescent portion includes a fluorophore. In some embodiments, the photoactivatable label includes a tag. In some embodiments, the tag is selected from the group including affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags. In some embodiments, the tag is an affinity tag. In some embodiments, the tag is an epitope tag. In some embodiments, the tag is a fluorescent tag. In some embodiments, the tag is an oligonucleotide tag. In some embodiments, the tag is a biotin tag. In some embodiments, the detectable portion is an antibody or a functional derivative thereof. In some embodiments, the photoactivatable label is hydrophobic. In some embodiments, one or more of the listed labels can be explicitly excluded.

[0058] In some embodiments of the cleared tissues provided herein, the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azido-methylcoumarin group, or a psoralen group. In some embodiments, the photoactivatable label comprises a phenyl azide group. In some embodiments, the photoactivatable label comprises an ortho-hydroxyphenyl azide group. In some embodiments, the photoactivatable label comprises a meta-hydroxyphenyl azide group. In some embodiments, the photoactivatable label comprises a tetrafluorophenyl azide group. In some embodiments, the photoactivatable label comprises an ortho-nitrophenyl azide group. In some embodiments, the photoactivatable label comprises a meta-nitrophenyl azide group. In some embodiments, the photoactivatable label comprises a diazirine group. In some embodiments, the photoactivatable label comprises an azido-methylcoumarin group. In some embodiments, the photoactivatable label comprises a psoralen group. In some embodiments, one or more of the enumerated labels can be explicitly excluded. Purpose

[0059] The methods and compositions described herein can be used in any application where three-dimensional tissue analysis is desired. The following uses are illustrative and not intended to limit you.

[0060] In some embodiments, heterogeneity in a tumor or tumor sample (e.g., biopsy) can be detected using the compositions and methods described herein. A patient-derived tumor sample may be cleared as described herein. The tumor sample may then be brought into contact with a photoactivatable label, and one or more regions of interest may be subjected to a multiphoton laser (e.g., a two-photon laser). The regions of interest may include various regions within the tumor, as well as tumor-adjacent "normal" cells. The labeled regions can be isolated and analyzed by one or more analytical methods, such as DNA, RNA, and / or protein analysis. The tumor sample may be imaged in any or more steps before isolation or analysis, for example, before or after clearing, before or after contact with the photoactivatable label, etc. DNA, RNA, and / or protein profiles determined from the analysis can be combined with the images to provide a detailed map of the tumor. Analysis of tumor DNA, RNA, and / or proteins in various regions may enable the construction of a three-dimensional evolutionary model of tumor heterogeneity.

[0061] In some embodiments, the compositions and methods described herein can be used to identify immune cells (or their absence) in a sample, such as a tumor sample. For example, one or more regions of a sample can be labeled, imaged, and isolated as described herein. The isolated regions can be analyzed, for example, for RNA and / or proteins expressed (preferentially expressed) by one or more immune cells of interest.

[0062] In some embodiments, the compositions and methods described herein can be used to identify the location of blood vessels in a sample, such as a tumor sample, and / or analyze DNA, RNA, or protein expression based on proximity to blood vessels. For example, one or more regions of a sample can be labeled, imaged, and isolated based on proximity to blood vessels, as described herein. The isolated regions can be analyzed, for example, for RNA and / or proteins expressed (preferentially expressed) by one or more cells of interest. Similarly, regions of metastasis or suspected metastasis can be analyzed using these methods.

[0063] In some embodiments, the compositions and methods described herein can be used to locate low-density populations of cells. Some cell types are found in small numbers in certain tissues and / or are dispersed in distinct regions within the tissue. The compositions and methods described herein may be useful in identifying / locating those cells or regions within the tissue.

[0064] In some embodiments, the compositions and methods described herein can be used to profile specific cell types in a sample. For example, but not limited to, the spinal cord consists of multiple layers / regions of dorsal root ganglia, each layer or region containing different cell types. The compositions and methods described herein may be useful in identifying differences (and / or similarities) between cells in various layers. Thus, any tissue composed of multiple cell types can be analyzed.

[0065] In some embodiments, the compositions and methods described herein can be used to generate data about a region of interest in a tissue or sample, such as expression (e.g., RNA or protein) or mutation (e.g., DNA) data, after 3D histological analysis of the tissue or sample.

[0066] In some embodiments, the compositions and methods described herein can be used to label and extract precise regions of interest within a tissue or sample. For example, the region of interest can be labeled within a resolution of 1.6 × 1.6 × 3 μm (using a 20x lens).

[0067] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes taking them into account should be proposed to those skilled in the art and should be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein in their entirety by reference for all purposes. [Examples]

[0068] Those skilled in the art will understand that the descriptions of the preparation and use of particles provided herein are for illustrative purposes only and that this disclosure is not limited by such examples. Example 1. Dehydration and clearing of tissue

[0069] Dehydration and tissue clearing

[0070] To collect organs free of residual blood, mice were perfused with warm PBS / heparin (5 U / ml; 50-100 ml / mouse at steady pressure (gravity flow or perfusion system; 1-1.5 mH2O (Leica))), followed by perfusion with 2% PFA (Electron Microscopy Sciences, diluted in PBS). Depending on sample permeability, the removed organs were post-fixed overnight in PFA at 4°C. Samples were then sterilized in 30% and 50% tert-butanol (pH=9.5, RT), followed sequentially in 70%, 80%, 96%, and 100% tert-butanol (pH=9.5, 30°C) for X hours each (where X is the time determined by Fick's diffusion law in a mouse brain incubated for 24 hours, serving as a reference; T=(1 / [2D])r 2The sample was incubated (D = diffusion coefficient (estimated from mouse brain dimensions and incubation time = 0.00015349), r = distance closest to the center of the sample) and finally cleared in BABB (benzyl alcohol:benzyl benzoate in a volume ratio of 1:2, pH = 9.5, 30°C). After the final clearing step, the organ can be stored in BABB solution at 4°C for at least one year. An example of a cleared mouse brain after this protocol, with approximately 24 hours of incubation per step, can be seen in Figure 1.

[0071] Stability experiment

[0072] To demonstrate that the above protocol did not alter cell stability and integrity, HEK293 cells were transfected with plasmids encoding EGFP, mKate2, tdTomato, or Venus, respectively. Cells were seeded onto Millipore EZ slides, fixed with 4% PFA, and then processed according to the above protocol with an incubation time of 15 minutes per step. For mounting, BABB was used as the mounting medium, and the cells were covered using glass coverslips. Cells were then imaged using a Leica SP8 upright laser scanning microscope with wavelengths matching the excitation / emission spectra of the fluorescent proteins. Control slides were not subjected to the clearing protocol and were mounted using VectaShield mounting medium. The results of these stability experiments are shown in Figure 2. Example 2. Two-photon labeling of tissue

[0073] For spatially defined activation of photoreactive compounds deep within tissue, samples were incubated with appropriate compounds dissolved in BABB (12.5 μg / ml) at room temperature (RT) for the time described by Fick's law of diffusion, or overnight at 4°C. Successful conjugation of compounds to sites of interest was achieved by using a 20x lens (Leica HCX APO L 20x / 0.95 IMM) and a 700 nm lens at a laser output of 75 mW with a resolution of 512 x 512 pixels and a pixel residence time of 10 μs. Based on the composition of photobiotin (biotin-linker-phenylazide; Sigma A1935-1MG), photoactivatable Pacific Blue, Cy3, Cy5, and Alexa 488 readily soluble in pH-adjusted BABB at 1 mg / ml (stock concentration) were synthesized. Immediately after photolabeling of the sample, the sample was washed with BABB (pH=9.5, 30°C), then with 100% tert-butanol (pH=9.5, 30°C) for a time determined by Fick's law (by transferring the sample to each solution), followed by a final washing step with 100% DMSO (pH=9.5, 30°C), and then stored in 100% DMSO at 4°C until further processing. Example 3. Isolation and Purification of Photolabeled Nuclei

[0074] All instruments were treated with RNAse. Photolabeled tissues stored in DMSO were rehydrated by incubating in PBS supplemented with an RNAse inhibitor (Takara) on a shaker at 20°C for 30 minutes. The tissue was shredded into bits less than 1 mm and transferred to lysis buffer (975 μl of HBSS containing divalent nucleotides, 10 μl of proteinase K, 10 μl of RNAse-free DNAse, and 0.2 U / ml of RNAse inhibitor). After incubation at 30°C for 15 minutes, the tissue bits were transferred to a Dounce homogenizer, and 1 ml of HBSS+ solution (HBSS, 3% BSA (fatty acid-free), and 0.2 U / ml RNAse inhibitor) was added, followed by homogenization with 5-10 strokes (avoiding air bubbles). The homogenate was filtered through a pre-moistened 70 μm filter and rotated at 300 rcf for 3-5 minutes. The supernatant was discarded, the pellet was resuspended in HBSS+, the nuclei were broken up by grinding with a 200 μl tip, pelletized at 300 rcf, resuspended in 1 ml of HBSS+, mixed with an equal volume of cold 25% Optiprep (Sigma-Aldrich), and then placed on ice. On ice, a gradient was prepared in a 15 ml tube by adding 2 ml of 40% Optiprep to the bottom and pipetting 2 ml of 25% Optiprep to the top, carefully layering the nucleus mixture on top. The tube was then rotated in a pre-cooled oscillating bucket rotor at 2500 rcf for 20 minutes. The nuclei located at the interface of the 25% and 40% Optiprep solutions (located beneath a fluffy, opaque layer of fragments, which may appear transparent with a brownish hue) were collected, resuspended in 3 volumes of HBSS+, and spun at 250 rcf for 10 minutes. Next, the pellet was washed with HBSS+, resuspended in HBSS+, and stained for biotin incorporation with appropriate nuclear labeling or streptavidin-conjugated dye for FACS (PI or DAPI). After incubation at RT for 5 minutes, the samples were spun down and resuspended in HBSS+. For selection, single nuclei (using nuclear labeling) and unlabeled controls were prepared, treated identically to the samples except for exposure to the light used for photoactivation and gating of photolabeled nuclei. Example 4. Image acquisition and analysis

[0075] The cleared samples were mounted on insect pins (Austerlitz) fixed to an inert silicone rubber surface (Momentive, RTV615), completely covered with BABB, and imaged using a white light laser and a Leica SP8 microscope equipped with a Leica BABB immersion lens HCX PL FLUOTAR 5x / 0.15IMM lens for low resolution and an HCX APO L 20x / 0.95IMM lens for high resolution. The acquired Leica image containers were converted to Imaris containers (Imaris File Converter 9.1.2, Bitplane) and transferred to a power workstation (Dual Xeon E5-2687W v4, 1TB memory, GeForce Titan (Pascal)) for image analysis using Imaris 9.3.1 (Bitplane). Where necessary, signal intensity was compensated using non-signal channels, and image data was deconvolved using the Matlab (MathWorks) adaptthresh function or Huygens (Scientific Volume Imaging BV). Example 5. SPLiT-seq of cleared nuclei from spinal cord

[0076] Cleared nuclei were isolated from Cy3-PA photolabeled mouse spinal cord, sorted for dye uptake, and libraries were prepared according to the SPLiT seq method. RNA sequencing was performed using a MiSeq instrument. Figure 7A shows the plotted number of unique barcodes relative to the total number of barcodes detected. The two sublibraries were then mixed in a 2:1 ratio (fixed:cleared) (nuclei isolated from fixed but uncleared tissue and nuclei isolated from fixed and cleared tissue). As shown in Figure 7B, TPM counts were plotted for both libraries, showing a linear correlation and indicating that the clearing process does not alter the sequencing results. Figure 7C shows random regions from mouse chromosome 2 with reads generated from fixed but uncleared, as well as fixed and cleared, mouse spinal cord. Example 6. 3D analysis of DNA / RNA / protein composition in mouse lungs

[0077] Appropriately prepared mouse lung tissue is subjected to a clearing protocol, imaged, and photoactivatable compounds are added in situ (without moving the tissue). Meanwhile, regions of interest are identified using previously generated, programmed, and photoactivated data. The tissue is then unmounted, washed, and rehydrated. The tissue can then be processed by extracting the nuclei using a Dounce homogenizer or by targeting and isolating the nuclei, and then staining them with the relevant compound (e.g., streptavidin if photobiotin is used as the photoactivatable compound). Cleared nuclei are isolated from the photolabeled lungs, stained with streptavidin-conjugated dye, and sorted for dye uptake. The library is prepared according to the SPLiT seq method, and the RNA is sequenced using an appropriate method. Alternatively, the RNA can be analyzed by any method including RT-PCR, electrophoresis, etc.

[0078] In the case of DNA sequencing, the process is similar; after nuclear isolation and sorting, the DNA can be sequenced using an appropriate method. The DNA can also be analyzed by, for example, PCR, electrophoresis, or any other suitable method.

[0079] For protein analysis, the composition of proteins incorporating the PA compound can be determined using mass spectrometry readings (e.g., liquid chromatography / mass spectrometry) or other suitable protein analysis methods. Other protein analysis methods may include electrophoresis, protein blotting, Edman degradation, or other protein sequencing. Example 7. 3D analysis of DNA / RNA / protein composition in the spinal cord.

[0080] Figure 8A is a cross-sectional view of a mouse spinal cord, showing different regions of the spinal cord, with the dorsal layer indicated as a colored circle. In the magnified panel, the photoactivated region is highlighted. It should be noted that in this embodiment, the PA region is not further refined—this is possible because the resolution correlates with the optical resolution of the lens used. For example, a 20x lens provides a resolution of 0.89 μm × 0.89 μm × 1.6 μm.

[0081] Figure 8B is a FACS plot of cleared but non-photoactivated nuclei isolated from mouse spinal cord using the procedure described herein. The Y-axis shows the signal for the nuclear counterstain DAPI, and the X-axis shows the PA dye (Cy3-PA). Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right).

[0082] Figure 8C shows a FACS plot of cleared and photoactivated nuclei isolated from mouse spinal cord (shown in Figure 9A). The Y-axis shows the signal of the nuclear counterstain DAPI, and the X-axis shows the PA dye (Cy3-PA). Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). Example 8. SPLiT-seq of cleared nuclei derived from spinal cord.

[0083] To demonstrate that cleared tissue can be analyzed by SPLiT-seq, mouse spinal cord was cleared as described in Example 1. Nuclei were isolated from the cleared tissue and subjected to SPLiT-seq analysis. As shown in Figures 10A and 10B, SPLiT-seq expression profiling showed the expression of markers for expected cell types present in the cleared tissue, including neurons, oligodendrocytes, and astrocytes. RNA expression profiles were determined based on RNA expression databases. Figure 10A shows nFeature plots (left) and n-count plots (right) of RNA expression levels. Figure 10B shows a UMAP plot of RNA expression levels. References 1. Olson,E.,Levene,M.and Torres,R.(2016).Multiphoton microscopy with clearing for three dimensional histology of kidney biopsies.Biomedical Optics Express,7(8),p.3089. 2. Rodriques,S.,Stickels,R.,Goeva,A.,Martin,C.,Murray,E.,Vanderburg,C.,Welch,J.,Chen,L.,Chen,F.and Macosko,E.(2019).Slide-seq:A scalable technology for measuring genome-wide expression at high spatial resolution.Science,363(6434),pp.1463-1467. 3. Schwarz,M.K.,Scherbarth,A.,Sprengel,R.,Engelhardt,J.,Theer,P.,&Giese,G.(2015).Fluorescent-protein stabilization and high-resolution imaging of cleared,intact mouse brains.PLoS ONE,10,1-26. 4. Wang,X.,Allen,W.,Wright,M.,Sylwestrak,E.,Samusik,N.,Vesuna,S.,Evans,K.,Liu,C.,Ramakrishnan,C.,Liu,J.,Nolan,G.,Bava,F.and Deisseroth,K.(2018).Three-dimensional intact-tissue sequencing of single-cell transcriptional states.Science,361(6400),p.eaat5691. The present invention includes the following embodiments. [1] A method for labeling a region of tissue or tissue sample, a) To provide a three-dimensional tissue or tissue sample; b) To make the sample transparent; c) Contacting the tissue or tissue sample with a photoactivatable label; and d) A method comprising subjecting the tissue or region of a tissue sample to a multiphoton laser and thereby labeling the region. [2] The method according to [1], wherein the multiphoton laser is a two-photon laser. [3] The method according to [1], wherein the multiphoton laser is a 3-photon laser. [4] The method according to any one of [1] to [3], wherein the region comprises cells, intracellular compartments, aggregates, or secretory aggregates. [5] The method according to [4], wherein the intracellular compartment is the nucleus. [6] The method according to any one of [1] to [5], further comprising imaging the tissue or tissue sample. [7] The method according to any one of [1] to [6], wherein the tissue or tissue sample is fixed tissue or fixed tissue sample. [8] The method according to any one of [1] to [7], wherein the photoactivatable label includes a detectable portion. [9] The method according to [8], wherein the detectable portion includes a light-emitting portion.

[10] The method according to [9], wherein the light-emitting portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion.

[11] The method according to

[10] , wherein the fluorescent portion comprises a fluorophore.

[12] The method according to [8], wherein one of the detectable portions comprises an antibody or a functional derivative thereof.

[13] The method according to any one of [1] to [7], wherein the photoactivatable label includes a tag.

[14] The method according to

[13] , wherein the tag is selected from the group including affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags.

[15] The method according to any one of [1] to

[14] , wherein the sample clearing process comprises dehydrating the sample and transferring the sample to a medium having a refractive index similar to or consistent with the tissue.

[16] The method according to

[15] , wherein the refractive index is approximately 1.3 to approximately 1.6.

[17] The method according to

[15] , wherein the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, which comprises or does not comprise one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol.

[18] The method according to

[17] , wherein the medium comprises a BABB solution.

[19] The method according to any one of [1] to

[17] , wherein the sample is dehydrated with a tert-butanol solution.

[20] The method according to

[19] , wherein the tert-butanol solution comprises trimethylamine, tetrahydrofuran, ethanol, or methanol.

[21] The method according to any one of [1] to

[19] , wherein the photoactivatable label is hydrophobic.

[22] The method according to any one of [1] to

[21] , wherein the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azide-methylcoumarin group, or a psoralen group.

[23] The method according to

[22] wherein the photoactivatable label comprises an aryl azide group.

[24] The method according to any one of [1] to

[23] , further comprising isolating the labeled region or a portion of the labeled region from the tissue or tissue sample.

[25] The method according to

[24] , wherein the labeled region or a portion thereof is isolated by FACS sorting for the label.

[26] The method according to

[24] or

[25] , further comprising analyzing the composition of the isolated labeled region to obtain analytical data.

[27] The method according to

[26] , comprising determining the DNA, RNA, and / or protein composition of the isolated labeled region.

[28] The method according to

[27] , wherein the RNA is analyzed by sequencing.

[29] The method according to

[27] , wherein the RNA is analyzed by SPLITseq.

[30] The method according to

[27] , wherein the protein is analyzed by mass spectrometry.

[31] The method according to

[27] , wherein the DNA is analyzed by DNA sequencing.

[32] A method for imaging the tissue or tissue sample before isolating the labeled region to create an image, further comprising combining the image with the analysis data to create a three-dimensional composition map of the region, according to any one of

[26] to

[31] .

[33] A method for three-dimensional expression profiling of intact tissue or tissue sample, a) To provide a three-dimensional intact tissue or tissue sample; b) To make the sample transparent; c) Contacting the tissue or tissue sample with a photoactivatable label; d) Exposing the tissue or region of the tissue sample to a multiphoton laser to label the region; e) Creating an image by imaging the marked area; f) Isolating the labeled region from the tissue or tissue sample; g) Determining the DNA, RNA, and / or protein composition of the isolated labeled region; h) A method comprising combining the image with the DNA, RNA, and / or protein composition of the isolated labeled region to create a three-dimensional expression profile of the intact tissue or tissue sample.

[34] The method according to

[33] , wherein the multiphoton laser is a two-photon laser.

[35] The method according to

[33] , wherein the multiphoton laser is a 3-photon laser.

[36] The method according to any one of claims 33 to 35, wherein the region is a cell, an intracellular compartment, an aggregate, or a secretory aggregate.

[37] The method according to any one of 33 to 36, wherein the isolated labeled region is a single nucleus.

[38] The RNA composition of the isolated labeled region is determined by the method described in any one of [33-37].

[39] The RNA composition is determined by sequencing [the method described in 38].

[40] The RNA composition is determined by SPLITseq, as described in

[38] .

[41] The protein composition of the isolated labeled region is determined by the method described in any one of [33-40].

[42] The protein composition is determined by mass spectrometry [the method described in 41.

[43] The DNA composition of the isolated labeled region is determined by the method described in any one of [33-40].

[44] The DNA composition is determined by DNA sequencing [the method described in 43].

[45] A method for isolating a single cell or nucleus in tissue or tissue sample, a) To provide a three-dimensional tissue or tissue sample; b) To make the sample transparent; c) Contacting the tissue or tissue sample with a photoactivatable label; d) Labeling the cells by exposing them to a multiphoton laser; e) Dissociating the labeled cells from the tissue or tissue sample; and f) A method comprising isolating the labeled cells or nuclei.

[46] The method according to

[45] , wherein the multiphoton laser is a two-photon laser.

[47] The method according to

[45] , wherein the multiphoton laser is a 3-photon laser.

[48] The method according to any one of

[45] to

[47] , further comprising imaging the tissue or tissue sample.

[49] The method according to any one of [33 to

[48] , wherein the tissue or tissue sample is fixed tissue or fixed tissue sample.

[50] The method according to any one of

[33] to

[49] , wherein the photoactivatable label includes a detectable portion.

[51] The method according to

[50] , wherein the detectable portion includes a light-emitting portion.

[52] The method according to

[51] , wherein the light-emitting portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion.

[53] The method according to

[52] , wherein the fluorescent portion comprises a fluorophore.

[54] The method according to

[50] , wherein the detectable portion comprises an antibody or a functional derivative thereof.

[55] The method according to any one of [33 to 49], wherein the photoactivatable label includes a tag.

[56] The method according to

[55] , wherein the tag is selected from the group including affinity tags, epitope tags, fluorescent tags, or biotin tags.

[57] The method according to any one of

[33] to

[56] , wherein the sample clearing process comprises dehydrating the sample and transferring the sample to a medium having a refractive index similar to or consistent with the tissue.

[58] The method according to

[57] , wherein the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, which may or may not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol.

[59] The method according to

[58] , wherein the medium comprises a BABB solution.

[60] The method according to any one of

[33] to

[59] , wherein the sample is dehydrated with a tert-butanol solution.

[61] The method according to

[60] , wherein the tert-butanol solution comprises trimethylamine, tetrahydrofuran, ethanol, or methanol.

[62] The method according to any one of

[33] to

[61] , wherein the photoactivatable label is hydrophobic.

[63] The method according to any one of

[33] to

[62] , wherein the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azide-methylcoumarin group, or a psoralen group.

[64] The method according to

[63] , wherein the photoactivatable label comprises an aryl azide group.

[65] The method according to any one of

[45] to

[64] , further comprising isolating the labeled cells or nuclei.

[66] The method according to any one of

[33] -

[44] or

[65] , wherein the labeled cells or nuclei are isolated by FACS sorting for the label.

[67] The method according to

[65] or

[66] , further comprising analyzing the isolated labeled cells or nuclei.

[68] The method according to

[67] , comprising determining the DNA, RNA, and / or protein composition of the isolated labeled cells or nuclei.

[69] The method according to

[38] or

[68] , wherein the RNA is analyzed by single-cell RNA sequencing (scRNAseq).

[70] The method according to

[68] , wherein the RNA composition is determined by sequencing.

[71] The method according to

[68] , wherein the protein composition of the isolated labeled region is determined.

[72] The method according to

[71] , wherein the protein composition is determined by mass spectrometry.

[73] The method according to

[68] , wherein the DNA composition of the isolated labeled region is determined.

[74] The method according to

[73] , wherein the DNA composition is determined by DNA sequencing.

[75] The method described in

[68] , wherein the RNA is analyzed by SPLITseq.

[76] The method according to any one of [1] to

[75] , further comprising contacting the tissue or tissue sample with a second photoactivatable label, and subjecting the second cells or nuclei in the tissue or tissue sample to a multiphoton laser, thereby labeling the second cells or nuclei.

[77] The method according to

[76] , wherein the multiphoton laser is a two-photon laser.

[78] The method according to

[76] , wherein the multiphoton laser is a 3-photon laser.

[79] The method according to any one of [1] to

[78] , wherein the tissue or tissue sample is an entire organ, a tumor, or an animal.

[80] The method according to any one of [1] to

[79] , wherein the wavelength of each photon in the two-photon laser is between approximately 175 nm and approximately 350 nm.

[81] The method according to any one of [1] to

[80] , wherein the tissue or tissue sample is derived from a tumor.

[82] A composition comprising a tissue sample, a medium having a refractive index similar to or matching that of the tissue, and a photoactivatable label.

[83] The composition according to

[82] , wherein the aforementioned structure includes a labeled region.

[84] The composition according to

[83] , wherein the region comprises cells, intracellular compartments, aggregates, or secretory aggregates.

[85] The composition according to

[83] or

[84] , wherein the cells are labeled by multiphoton technology.

[86] The composition according to

[85] , wherein the cells are labeled by a two-photon method.

[87] The composition according to

[85] , wherein the cells are labeled by a three-photon method.

[88] The composition according to any one of

[82] to

[87] , wherein the photoactivatable label comprises a detectable portion.

[89] The composition according to

[88] , wherein the detectable portion includes a light-emitting portion.

[90] The composition according to

[89] , wherein the light-emitting portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion.

[91] The composition according to

[90] , wherein the fluorescent portion comprises a fluorophore.

[92] The composition according to any one of

[82] to

[87] , wherein the photoactivatable label comprises a tag.

[93] The composition according to

[92] , wherein the tag is selected from the group including affinity tags, epitope tags, fluorescent tags, or biotin tags.

[94] The composition according to

[88] , wherein the detectable portion is an antibody or a functional derivative thereof.

[95] The composition according to any one of

[82] to

[94] , wherein the photoactivatable label is hydrophobic.

[96] The composition according to any one of

[82] to

[95] , wherein the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azide-methylcoumarin group, or a psoralen group.

[97] The composition according to any one of

[82] to

[96] , wherein the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, which may or may not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol.

[98] The composition according to

[97] , comprising the aforementioned medium a BABB solution.

[99] A cleared tissue sample comprising a photoactivatable label and a medium having a refractive index substantially matching the refractive index of the tissue.

[0100] The cleared tissue according to

[99] , wherein the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, which may or may not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or quadrol.

[0101] The cleared tissue according to

[0100] , wherein the medium contains a BABB solution.

[0102] Cleared tissue as described in any one of items

[99] to

[0101] , including labeled cells.

[0103] The cleared tissue described in

[0102] , wherein the cells are labeled by multiphoton technology.

[0104] A transparentized composition according to any one of

[99] to

[0103] , wherein the photoactivatable label comprises a detectable portion.

[0105] The transparent tissue according to

[0104] , wherein the detectable portion includes a light-emitting portion.

[0106] The cleared tissue according to

[0105] , wherein the light-emitting portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion.

[0107] The cleared tissue according to

[0106] , wherein the fluorescent portion contains a fluorophore.

[0108] The transparent tissue according to any one of

[99] to

[0107] , wherein the photoactivatable label includes a tag.

[0109] The cleared tissue according to

[0108] , wherein the tag is selected from the group including affinity tags, epitope tags, fluorescent tags, or biotin tags.

[0110] The cleared tissue according to

[0104] , wherein the detectable portion is an antibody or a functional derivative thereof.

[0111] The cleared tissue according to any one of

[99] to

[0110] , wherein the photoactivatable label is hydrophobic.

[0112] The cleared tissue according to any one of

[99] to

[0111] , wherein the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrahydrophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azide-methylcoumarin group, or a psoralen group.

[0113] The composition or cleared tissue according to any one of

[82] to

[0112] , wherein the tissue or tissue sample is derived from a tumor.

Claims

1. A method for labeling a region of tissue or tissue sample, (a) To make a three-dimensional tissue or tissue sample transparent, provided that the three-dimensional tissue or tissue sample is human tissue or a human tissue sample, it is not in a state where it is inside or on the surface of a human body; (b) Contacting the tissue or tissue sample with a photoactivatable label; (c) Exposing the tissue or region of the tissue sample to a multiphoton laser and thereby labeling the region; (d) Isolating from the tissue or tissue sample a region labeled by step (c) or a portion of a region labeled by step (c); and (e) Analyzing the composition of the region isolated by step (d) to produce analytical data, wherein the analysis includes determining the DNA, RNA, and / or protein composition of the region isolated by step (d); Methods that include...

2. The method according to claim 1, wherein the region includes cells, intracellular compartments, aggregates, or secretory aggregates.

3. The method according to claim 1 or 2, further comprising imaging the tissue or tissue sample.

4. The method according to any one of claims 1 to 3, wherein the photoactivatable label includes a detectable portion or a tag.

5. The method according to any one of claims 1 to 4, wherein the step (a) of making the three-dimensional tissue or tissue sample clear comprises dehydrating the sample and transferring the sample to a medium that is similar to, identical to, or has a refractive index of 1.3 to 1.6 of the tissue.

6. The method according to any one of claims 1 to 5, wherein the photoactivatable label is hydrophobic.

7. The method according to any one of claims 1 to 6, wherein the RNA composition is analyzed by sequencing or SPLITseq, the protein composition is analyzed by mass spectrometry, and / or the DNA composition is analyzed by DNA sequencing.

8. A method according to any one of claims 1 to 7, wherein the tissue or tissue sample is imaged before step (c) to create an image, further comprising combining the image with the analysis data to create a three-dimensional composition map of the region.

9. A method for three-dimensional expression profiling of intact tissue or tissue sample, (a) To make a three-dimensional intact tissue or tissue sample transparent, provided that the three-dimensional intact tissue or tissue sample is not in a state within or on the surface of a human body if it is human tissue or a human tissue sample; (b) Contacting the tissue or tissue sample with a photoactivatable label; (c) Creating an image by imaging the tissue or tissue sample; (d) Exposing the tissue or region of the tissue sample to a multiphoton laser and thereby labeling the region; (e) Isolating from the tissue or tissue sample a region labeled by step (d) or a portion of a region labeled by step (d); (f) Determining the composition of the region isolated by step (e); (g) A method comprising combining the image with the composition of the region isolated by step (e) to create a three-dimensional expression profile of the intact tissue or tissue sample.

10. The method according to claim 9, wherein the region is a cell, an intracellular compartment, an aggregate, or a secretory aggregate.

11. The method according to claim 9 or 10, wherein the RNA composition, protein composition, and / or DNA composition of the isolated region are determined, wherein the RNA composition is determined by sequencing or SPLITseq, the protein composition is determined by mass spectrometry, and / or the DNA composition is determined by DNA sequencing.

12. The method according to any one of claims 9 to 11, wherein the photoactivatable label includes a detectable portion or a tag.

13. The method according to any one of claims 9 to 12, wherein the step (a) of making the three-dimensional intact tissue or tissue sample clear comprises dehydrating the sample and transferring the sample to a medium similar to, matching, or having a refractive index of 1.3 to 1.6 of the tissue.

14. The method according to any one of claims 9 to 13, wherein the photoactivatable label is hydrophobic.

15. The method according to any one of claims 9 to 14, further comprising imaging the tissue or tissue sample a second time after labeling in step (d) to create a second image.

16. A method for isolating a single cell or nucleus in tissue or tissue sample, (a) To make a three-dimensional tissue or tissue sample transparent, provided that the three-dimensional tissue or tissue sample is human tissue or a human tissue sample, it is not in a state where it is inside or on the surface of a human body; (b) Contacting the tissue or tissue sample with a photoactivatable label; (c) Exposing cells or nuclei to a multiphoton laser to label the cells or nuclei; (d) Dissociating the cells or nuclei labeled by step (c) from the tissue or tissue sample; and (e) A method comprising isolating cells or nuclei labeled by step (c).

17. The method according to claim 16, further comprising imaging the tissue or tissue sample.

18. The method according to claim 16 or 17, wherein the photoactivatable label includes a detectable portion or tag.

19. The method according to any one of claims 16 to 18, wherein the step (a) of making the three-dimensional tissue or tissue sample clear comprises dehydrating the sample and transferring the sample to a medium that is similar to, identical to, or has a refractive index of 1.3 to 1.6 of the tissue.

20. The method according to any one of claims 16 to 19, wherein the photoactivatable label is hydrophobic.

21. Further comprising analyzing the cells or nuclei labeled by step (c), Here, the analysis includes determining the DNA composition, RNA composition and / or protein composition of the cells or nuclei isolated by step (e), The method according to claim 16, wherein the RNA composition is determined by sequencing, the protein composition is determined by mass spectrometry, and / or the DNA composition is determined by DNA sequencing.

22. The method according to any one of claims 1 to 21, further comprising contacting the tissue or tissue sample with a second photoactivatable label, and subjecting the second cells or nuclei in the tissue or tissue sample to a multiphoton laser to label the second cells or nuclei.

23. The method according to any one of claims 1 to 22, wherein the multiphoton laser is a two-photon laser or a three-photon laser.

24. The aforementioned photoactivatable label is (a) Detectable portion; (b) Light-emitting part; (c) Fluorescent portion; (d) Chemiluminescent portion; (e) Bioluminescent part; (f) Electrochemiluminescence portion; (g) Fluorophores; (h) Antibodies or functional derivatives thereof; (i) Tags; (j) Affinity tags; (k) Epitope tag; (l) Fluorescent tag; (m) oligonucleotide tag; or (n) Biotin Tag The method according to any one of claims 1 to 23, comprising one or more selected from.

25. The method according to claim 5, 13, or 19, wherein the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB), or a derivative thereof, which comprises or does not comprise one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol.

26. The method according to claim 5, 13, or 19, wherein the tissue or tissue sample is dehydrated with a tert-butanol solution or a tert-butanol solution containing trimethylamine, tetrahydrofuran, ethanol, or methanol.

27. The method according to any one of claims 1 to 26, wherein the photoactivatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azido-methylcoumarin group, or a psoralen group.

28. The method according to any one of claims 1 to 27, wherein the labeled region or a portion thereof is isolated by FACS sorting for photoactivatable labeling.

29. The method according to any one of claims 1 to 28, wherein the tissue or tissue sample is an entire organ, a tumor or an animal, or derived from a tumor.

30. The method according to claim 23, wherein the wavelength of each photon in the two-photon laser is approximately 175 nm to approximately 350 nm.

Images