Spatially dependent analysis of biological material 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 March 18, 2020, and U.S. Provisional Application No. 63 / 074,693, filed September 4, 2020, the contents of each of which are incorporated herein by reference in their entirety and to which priority is claimed from each. [Background technology]

[0002] In most of the molecular biology-based methods for tissue analysis, the spatial context of the analyzed material is lost.The techniques known in the art that combine sequence analysis with spatial information have limitations, such as low sequencing depth, limited sample volume, analysis of individual tissue sections, or time-consuming reading, among other constraints.To the best of our knowledge, there is currently no available method that combines isolation accuracy and analysis depth.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 tissues or tissue samples. The method involves imaging cleared tissues or samples that have been labeled by contacting the region (or cells) of interest with an appropriate label and then subjecting the region (or cells) to a multiphoton laser. The labeled region can then be isolated from the tissue or sample and subjected to analysis, including, but not limited to, DNA sequencing, RNA sequencing, proteomic analysis, epigenetic analysis, immunohistochemistry, chromosome conformation capture, or immunofluorescence analysis. Various methods for isolating the labeled region (or cells) of interest can be used, including, but not limited to, laser-assisted microdissection, fluorescence-assisted cell sorting (FACS), magnetically 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 for labeling a region of a tissue or tissue sample is provided. The method can 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; and d) subjecting the region of the tissue or tissue sample to a multiphoton laser, thereby labeling the region.

[0005] In another aspect, a method for three-dimensional expression profiling of an intact tissue or tissue sample is provided. 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, thereby labeling the region; e) imaging the labeled region to generate 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; h) combining the image with the DNA, RNA, and / or protein composition of the isolated labeled region to generate a three-dimensional expression profile of the intact tissue or tissue sample.

[0006] In one aspect, a method for isolating a single cell or nucleus in a tissue or tissue sample is provided.The method can include: a) providing a three-dimensional tissue or tissue sample containing a 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, thereby labeling 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 another aspect, a composition is provided. The composition can include a tissue sample, a medium having a refractive index similar to or matching 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 matched to the tissue. [Brief explanation of the drawings]

[0009] [Figure 1] A mouse brain is shown before (left) and after (right) tissue clearing.

[0010] [Figure 2] A series of fluorescence micrographs showing the stability of different fluorescent proteins in cleared (bottom row) or control (top row) tissues. 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] Figure 3A illustrates one embodiment of the use of two-photon microscopy to label cells or nuclei of interest in cleared tissue using photoreactive labels. Figure 3A shows the labeling of molecules with a photoreactive label bearing a photoreactive group and a biotin tag upon exposure to light with wavelengths between 260 nm and 475 nm. Figure 3B illustrates the advantages of using two-photon microscopy (right) to label cells or nuclei of interest compared to traditional confocal microscopy (left). Traditional confocal microscopy results in photoactivation of the entire area of ​​tissue exposed to both light (red) and the photoactivatable compound (green). In contrast, two-photon microscopy allows for more precise photoactivation and targeting of the target site (red star), thereby avoiding nonspecific labeling of non-target tissue. [Figure 3B]Figure 3A illustrates one embodiment of the use of two-photon microscopy to label cells or nuclei of interest in cleared tissue using photoreactive labels. Figure 3A shows the labeling of molecules with a photoreactive label bearing a photoreactive group and a biotin tag upon exposure to light with wavelengths between 260 nm and 475 nm. Figure 3B illustrates the advantages of using two-photon microscopy (right) to label cells or nuclei of interest compared to traditional confocal microscopy (left). Traditional confocal microscopy results in photoactivation of the entire area of ​​tissue exposed to both light (red) and the photoactivatable compound (green). In contrast, two-photon microscopy allows for more precise photoactivation and targeting of the target site (red star), thereby avoiding nonspecific labeling of non-target tissue.

[0012] [Figure 4] An example of an analysis using two-photon labeling technology is outlined below. Tissues with three-dimensional (3D) cellular structures are cleared, immersed in a photoactivatable dye (e.g., photobiotin), and labeled using a two-photon laser (e.g., as shown in Figure 3B), followed by isolation of labeled nuclei for subsequent analysis, e.g., single-cell analysis (e.g., SPLiT-seq).

[0013] [Figure 5] Polynucleotide length profiles of treated samples without EDC treatment after 3 days at room temperature (RT) or 4°C (4C) after reverse transcription are shown.

[0014] [Figure 6A] An overview of a method for spatially dependent analysis of biological material from intact tissue samples is shown. Figure 6A shows an exemplary tissue clearing (Figure 6A, panel A) and labeling process (panels B-E), as well as the isolation of photolabeled nuclei (panel F). The resulting sample can be analyzed by SPLiT-seq library preparation from isolated photolabeled nuclei (panels G and H), followed by subsequent data analysis (I). Additionally (or alternatively), DNA (Figure 6B) or protein (Figure 6C) from the isolated tissue can be analyzed. [Figure 6B]An overview of a method for spatially dependent analysis of biological material from intact tissue samples is shown. Figure 6A shows an exemplary tissue clearing (Figure 6A, panel A) and labeling process (panels B-E), as well as the isolation of photolabeled nuclei (panel F). The resulting sample can be analyzed by SPLiT-seq library preparation from isolated photolabeled nuclei (panels G and H), followed by subsequent data analysis (I). Additionally (or alternatively), DNA (Figure 6B) or protein (Figure 6C) from the isolated tissue can be analyzed. [Figure 6C] An overview of a method for spatially dependent analysis of biological material from intact tissue samples is shown. Figure 6A shows an exemplary tissue clearing (Figure 6A, panel A) and labeling process (panels B-E), as well as the isolation of photolabeled nuclei (panel F). The resulting sample can be analyzed by SPLiT-seq library preparation from isolated photolabeled nuclei (panels G and H), followed by subsequent data analysis (I). Additionally (or alternatively), DNA (Figure 6B) or protein (Figure 6C) from the isolated tissue can be analyzed.

[0015] [Figure 7A] SPLiT-seq analysis demonstrates RNA quality for formalin-fixed and cleared samples. 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: transcripts per million). Figure 7C shows an exemplary genome browser window (top) displaying cDNA coverage of a 72 kb stretch of mouse (mm10) chromosome 2, along with a comparison of a fixed and cleared sample (middle) and a fixed sample (bottom). Spinal cords were photoactivated with Cy3-PA as shown in Figures 7A-7C. Nuclei were extracted and sorted for label incorporation. SPLiT-seq libraries were generated and sequenced by HiSeq. [Figure 7B]SPLiT-seq analysis demonstrates RNA quality for formalin-fixed and cleared samples. 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: transcripts per million). Figure 7C shows an exemplary genome browser window (top) displaying cDNA coverage of a 72 kb stretch of mouse (mm10) chromosome 2, along with a comparison of a fixed and cleared sample (middle) and a fixed sample (bottom). Spinal cords were photoactivated with Cy3-PA as shown in Figures 7A-7C. Nuclei were extracted and sorted for label incorporation. SPLiT-seq libraries were generated and sequenced by HiSeq. [Figure 7C] SPLiT-seq analysis demonstrates RNA quality for formalin-fixed and cleared samples. 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: transcripts per million). Figure 7C shows an exemplary genome browser window (top) displaying cDNA coverage of a 72 kb stretch of mouse (mm10) chromosome 2, along with a comparison of a fixed and cleared sample (middle) and a fixed sample (bottom). Spinal cords were photoactivated with Cy3-PA as shown in Figures 7A-7C. Nuclei were extracted and sorted for label incorporation. SPLiT-seq libraries were generated and sequenced by HiSeq.

[0016] [Figure 8A]We demonstrate the use of two-photon labeling of spinal cord tissue. Figure 8A is a cross-section of a mouse spinal cord showing different regions of the spinal cord, with the dorsal layers shown as colored circles. The magnified panel highlights the area that underwent photoactivation. Figure 8B is a FACS plot of cleared but non-photoactivated nuclei isolated from a mouse spinal cord. The Y-axis shows the signal of the nuclear counterstain, DAPI, and the X-axis shows the PA dye, Cy3-PA, used in this experiment. 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 a 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, used in this experiment. Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). [Figure 8B] We demonstrate the use of two-photon labeling of spinal cord tissue. Figure 8A is a cross-section of a mouse spinal cord showing different regions of the spinal cord, with the dorsal layers shown as colored circles. The magnified panel highlights the area that underwent photoactivation. Figure 8B is a FACS plot of cleared but non-photoactivated nuclei isolated from a mouse spinal cord. The Y-axis shows the signal of the nuclear counterstain, DAPI, and the X-axis shows the PA dye, Cy3-PA, used in this experiment. 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 a 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, used in this experiment. Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). [Figure 8C]We demonstrate the use of two-photon labeling of spinal cord tissue. Figure 8A is a cross-section of a mouse spinal cord showing different regions of the spinal cord, with the dorsal layers shown as colored circles. The magnified panel highlights the area that underwent photoactivation. Figure 8B is a FACS plot of cleared but non-photoactivated nuclei isolated from a mouse spinal cord. The Y-axis shows the signal of the nuclear counterstain, DAPI, and the X-axis shows the PA dye, Cy3-PA, used in this experiment. 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 a 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, used in this experiment. Boxes indicate single nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right).

[0017] [Figure 9A]

[0033] Figures 9A and 9B show exemplary compounds for use in the methods of the present invention. Figure 9A shows an example of a photoreactive compound that can be used for photolabeling. Figure 9B shows exemplary modifications to aryl azides induced by ultraviolet (UV) light. Figure 9C shows an example of a synthesized photoreactive compound. [Figure 9B]

[0033] Figures 9A and 9B show exemplary compounds for use in the methods of the present invention. Figure 9A shows an example of a photoreactive compound that can be used for photolabeling. Figure 9B shows exemplary modifications to aryl azides induced by ultraviolet (UV) light. Figure 9C shows an example of a synthesized photoreactive compound. [Figure 9C]

[0033] Figures 9A and 9B show exemplary compounds for use in the methods of the present invention. Figure 9A shows an example of a photoreactive compound that can be used for photolabeling. Figure 9B shows exemplary modifications to aryl azides induced by ultraviolet (UV) light. Figure 9C shows an example of a synthesized photoreactive compound.

[0018] [Figure 10A]Figure 10 shows expression profiling of cleared nuclei from spinal cord using SPLiT-seq analysis. Figure 10A shows a graph depicting the cell type expression profile (expression levels of genes associated with the indicated cell type). Figure 10B shows a graph generated by UMAP of the data in Figure 10A. [Figure 10B] Figure 10 shows expression profiling of cleared nuclei from spinal cord using SPLiT-seq analysis. Figure 10A shows a graph depicting the cell type expression profile (expression levels of genes associated with the indicated cell type). Figure 10B shows a graph generated by UMAP of the data in Figure 10A.

[0019] [Figure 11] 1 is an illustration of an example of how multiplexing can enable multiple labeling events. Tissue can be exposed to a first dye (dye 1), followed by two-photon labeling of a first region of interest (left panel). The same tissue can then be contacted (optionally after washing) with a second dye (dye 2), followed by two-photon labeling of a second region of interest (center panel). Optionally, the same tissue can be contacted (optionally after washing) with a third dye (dye 3), followed by two-photon labeling of a third region of interest (center panel).

[0020] [Figure 12A] Examples of application areas for targeting compounds for whole mount photoactivation are shown, including tumor heterogeneity, inflammatory tumor / immune deserts, and proximity to vasculature (Figure 12A), discovery and labeling of sparse cell populations (Figure 12B), generation of expression data, and 3D histology (Figure 12C). [Figure 12B] Examples of application areas for targeting compounds for whole mount photoactivation are shown, including tumor heterogeneity, inflammatory tumor / immune deserts, and proximity to vasculature (Figure 12A), discovery and labeling of sparse cell populations (Figure 12B), generation of expression data, and 3D histology (Figure 12C). [Figure 12C]Examples of application areas for targeting compounds for whole mount photoactivation are shown, including tumor heterogeneity, inflammatory tumor / immune deserts, and proximity to vasculature (Figure 12A), discovery and labeling of sparse cell populations (Figure 12B), generation of expression data, and 3D histology (Figure 12C). DETAILED DESCRIPTION OF THE INVENTION

[0021] After reading this description, it will be apparent to one skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, not all of the various embodiments of the present invention are described herein. It will be understood that the embodiments presented herein are presented by way of example only and not by way of limitation. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present disclosure as described herein.

[0022] Before the present technology is disclosed and described, it is to be understood that the aspects described below are not limited to particular compositions, methods of preparing such compositions, or uses thereof, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0023] The detailed description is divided into various sections solely for the convenience of the reader, and disclosure found in any section may be combined with that of other sections. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the disclosure. definition

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and in the claims that follow, reference will be made to several terms that shall be defined to have the following meanings.

[0025] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0026] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0027] The term "about," when used before a numerical designation including a range, e.g., temperature, time, amount, concentration, etc., indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, when used in reference to a dose, the term "about" means that the dose may vary by + / - 10%.

[0028] "Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the described purpose. Thus, a composition consisting essentially of the elements defined in the present invention does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed technology. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. method

[0029] In one aspect, a method for marking a region of tissue or tissue sample is provided.The method can 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; and (d) subjecting the region of tissue or tissue sample to a multiphoton laser, thereby marking 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] For the methods described herein, in some embodiments, the region comprises a cell, a subcellular compartment, an aggregate, or a secreted aggregate. In some embodiments, the region comprises a cell. In some embodiments, the region comprises a subcellular compartment. In some embodiments, the region comprises an aggregate. In some embodiments, the region comprises a secreted aggregate. In some embodiments, the subcellular compartment is a nucleus.

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

[0032] For the methods provided herein, in some embodiments, the photoactivatable label comprises a detectable moiety. In some embodiments, the detectable moiety comprises a luminescent moiety. In some embodiments, the luminescent moiety is a fluorescent moiety, a chemiluminescent moiety, a bioluminescent moiety, or an electrochemiluminescent moiety. In some embodiments, the luminescent moiety is a fluorescent moiety. In some embodiments, the luminescent moiety is a chemiluminescent moiety. In some embodiments, the luminescent moiety is a bioluminescent moiety. In some embodiments, the luminescent moiety is an electrochemiluminescent moiety. In some embodiments, the fluorescent moiety comprises a fluorophore. In some embodiments, one of the detectable moieties comprises an antibody or a functional derivative thereof. In some embodiments, the photoactivatable label comprises a tag. In some embodiments, the tag is selected from the group comprising an affinity tag, an epitope tag, a fluorescent tag, an oligonucleotide tag, or a biotin tag. 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 matching that of 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 can be any value or subrange within the recited range, including the endpoints, or any range between any of the recited values.

[0034] In some embodiments, the vehicle comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, with or without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the vehicle comprises a solution of benzyl alcohol with one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the vehicle comprises a solution of benzyl alcohol without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the vehicle comprises a solution of benzyl benzoate (BABB) or a derivative thereof, with 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, free of 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 for 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 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.

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

[0038] In some embodiments of the methods described herein, the tissue or sample is imaged to generate an image prior to isolating the labeled region, and further includes combining the image with analytical data to generate a three-dimensional compositional map of the region.

[0039] In one aspect, a method for three-dimensional expression profiling of an intact tissue or tissue sample is provided. 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, thereby labeling the region; (e) imaging the labeled region to generate 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 generate 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, a subcellular compartment, an aggregate, or a secretory aggregate. In some embodiments, the region is a cell. In some embodiments, the region is a subcellular compartment. In some embodiments, the region is an aggregate. In some embodiments, the region is a secreted 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 single cells or nuclei in tissue or tissue sample.The method can 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 cells to a multiphoton laser, thereby labeling the cells; (e) dissociating the labeled cells from the tissue or tissue sample; and (f) isolating the labeled cells or nuclei.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 comprises imaging the tissue or tissue sample. In some embodiments, the method further comprises imaging the tissue. In some embodiments, the method further comprises imaging the tissue sample. In some embodiments, the tissue or tissue sample is a fixed tissue or fixed tissue sample. In some embodiments, the tissue is a fixed tissue. In some embodiments, the tissue is a fixed tissue sample. In some embodiments, the tissue sample is a fixed tissue. In some embodiments, the tissue sample is a fixed tissue sample.

[0042] For the methods provided herein, in some embodiments, the photoactivatable label comprises a detectable moiety. In some embodiments, the detectable moiety comprises a luminescent moiety. In some embodiments, the luminescent moiety is a fluorescent moiety, a chemiluminescent moiety, a bioluminescent moiety, or an electrochemiluminescent moiety. In some embodiments, the luminescent moiety is a fluorescent moiety. In some embodiments, the luminescent moiety is a chemiluminescent moiety. In some embodiments, the luminescent moiety is a bioluminescent moiety. In some embodiments, the luminescent moiety is an electrochemiluminescent moiety. In some embodiments, the fluorescent moiety comprises a fluorophore. In some embodiments, the detectable moiety comprises an antibody or a functional derivative thereof. In some embodiments, the photoactivatable label comprises a tag. In some embodiments, the tag is selected from the group comprising an affinity tag, an epitope tag, a fluorescent tag, an oligonucleotide tag, or a biotin tag. 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 with a refractive index similar to or matching that of the tissue. In some embodiments, the medium includes a solution of benzyl alcohol, benzyl benzoate (BABB), or a derivative thereof, with or without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium includes a solution of benzyl alcohol with one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium includes a solution of benzyl alcohol without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium includes a solution of benzyl alcohol with one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the medium includes a solution of benzyl benzoate (BABB) or a derivative thereof with 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, free of 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] For the methods provided herein, in some embodiments, 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, hi some embodiments, the photoactivatable label comprises the group shown in Figure 9A.

[0046] In some embodiments, the methods provided herein further comprise isolating labeled cells or nuclei. In some embodiments, the methods further comprise isolating labeled cells. In some embodiments, the methods further comprise isolating labeled nuclei. In some embodiments, the labeled cells or nuclei are isolated by fluorescence-activated cell sorting (FACS) sorting for the label. In some embodiments, the labeled cells are isolated by FACS sorting for the label. In some embodiments, the nuclei are isolated by FACS sorting for the label. In some embodiments, the labeled cells or nuclei are isolated by magnetic-activated cell sorting (MACS) sorting for the label. In some embodiments, the labeled cells are isolated by MACS sorting for the label. In some embodiments, the labeled nuclei are isolated by MACS sorting for the label. In some embodiments, the labeled cells or nuclei are isolated by buoyancy-activated cell sorting (BACS) sorting for the label. In some embodiments, the labeled cells are isolated by BACS sorting for the label. In some embodiments, the labeled nuclei are isolated by BACS sorting for the label. 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 comprise analyzing the isolated labeled cells or nuclei. In some embodiments, the methods further comprise analyzing the isolated labeled cells. In some embodiments, the methods further comprise analyzing the isolated labeled nuclei. In some embodiments, the analysis comprises determining the DNA, RNA, and / or protein composition of the isolated labeled cells or nuclei. In some embodiments, the analysis comprises determining the DNA, RNA, and protein composition of the isolated labeled cells or nuclei. In some embodiments, the analysis comprises determining the DNA, RNA, or protein composition of the isolated labeled cells or nuclei. In some embodiments, the analysis comprises determining the DNA composition of the isolated labeled cells or nuclei. In some embodiments, the analysis comprises determining the RNA composition of the isolated labeled cells or nuclei. In some embodiments, the analysis comprises determining the protein composition of the isolated labeled cells or nuclei. In some embodiments, the analysis comprises determining the DNA composition of the isolated labeled cells. In some embodiments, the analysis comprises determining the RNA composition of the isolated labeled cells. In some embodiments, the analysis comprises determining the protein composition of the isolated labeled cells. In some embodiments, the analysis comprises determining the DNA composition of the isolated labeled cells. In some embodiments, the analysis comprises determining the RNA composition of the isolated labeled nuclei. In some embodiments, the analysis comprises 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 comprises contacting the 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, FIG. 11. In some embodiments, the multiphoton laser is a two-photon laser. In some embodiments, the multiphoton laser is a three-photon laser.

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

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

[0051] For the methods provided herein, in some embodiments, the wavelength of each photon in the three-photon laser is about 690 nm to about 2500 nm. For the methods provided herein, in some embodiments, the wavelength of each photon in the three-photon laser is about 690 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 695 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 700 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 705 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 710 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 715 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 720 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 725 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 730 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 735 nm. In some embodiments, the wavelength of each photon in the three-photon laser is about 740 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 745 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 750 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 755 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 760 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 765 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 770 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 775 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 780 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 785 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 790 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 795 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 800 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 805 nm.In some embodiments, the wavelength of each photon of the three-photon laser is about 810 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 815 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 820 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 825 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 830 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 835 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 840 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 845 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 850 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 855 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 860 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 865 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 870 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 875 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 880 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 885 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 890 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 895 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 905 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 910 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 915 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 920 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 925 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 930 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 935 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 940 nm.In some embodiments, the wavelength of each photon of the three-photon laser is about 945 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 950 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 955 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 960 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 965 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 970 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 975 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 980 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 985 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 990 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 995 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1000 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1005 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1010 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1015 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1020 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1025 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1030 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1035 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1040 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1045 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1050 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1055 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1060 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1065 nm.In some embodiments, the wavelength of each photon of the three-photon laser is about 1070 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1075 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1080 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1085 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1090 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1095 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1100 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1105 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1110 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1115 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1120 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1125 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1130 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1135 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1140 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1145 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1150 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1155 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1160 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1165 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1170 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1175 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1180 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1185 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 1190 nm.In some embodiments, the wavelength of each photon of the three-photon laser is about 1195 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2000 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2005 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2010 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2015 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2020 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2025 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2030 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2035 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2040 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2045 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2050 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2055 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2060 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2065 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2070 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2075 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2080 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2085 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2090 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2100 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2105 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2110 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2115 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2120 nm.In some embodiments, the wavelength of each photon of the three-photon laser is about 2125 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2130 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2135 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2140 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2145 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 21. 50 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2155 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2160 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2165 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2170 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2175 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2180 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2185 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2190 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2195 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2200 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2205 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2210 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2215 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2220 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2225 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2230 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2235 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2240 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2245 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2250 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2255 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2260 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2265 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2270 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2275 nm.In some embodiments, the wavelength of each photon of the three-photon laser is about 2280 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2285 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2290 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2295 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2300 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2305 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2310 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2315 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2320 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2325 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2330 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2335 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2340 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2345 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2350 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2350 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2355 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2360 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2365 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2370 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2375 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2380 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2385 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2390 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2395 nm.In some embodiments, the wavelength of each photon of the three-photon laser is about 2400 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2405 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2410 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2415 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2420 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2425 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2430 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2435 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2440 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2445 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2450 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2455 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2460 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2465 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2470 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2475 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2480 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2485 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2490 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 2495 nm. In some embodiments, the wavelength of each photon of the three-photon laser is about 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 matching that of the tissue, and a photoactivatable label. In some embodiments, the tissue includes a labeled region. In some embodiments, the region includes a cell, an intracellular compartment, an aggregate, or a secreted aggregate. In some embodiments, the region includes a cell. In some embodiments, the region includes an intracellular compartment. In some embodiments, the region includes an aggregate. In some embodiments, the region includes a secreted aggregate. In some embodiments, the cell is labeled by a multiphoton method. In some embodiments, the cell is labeled by a two-photon method. In some embodiments, the cell is labeled by a three-photon method. In some embodiments, the photoactivatable label includes a detectable moiety. In some embodiments, the detectable moiety includes a light-emitting moiety. In some embodiments, the light-emitting moiety is a fluorescent moiety, a chemiluminescent moiety, a bioluminescent moiety, or an electrochemiluminescent moiety. In some embodiments, the light-emitting moiety is a fluorescent moiety. In some embodiments, the light-emitting moiety is a chemiluminescent moiety. In some embodiments, the light-emitting moiety is a bioluminescent moiety. In some embodiments, the light-emitting moiety is an electrochemiluminescent moiety. In some embodiments, the fluorescent moiety includes a fluorophore.

[0053] For the compositions provided herein, in some embodiments, the photoactivatable label comprises a tag. In some embodiments, the tag is selected from the group comprising an affinity tag, an epitope tag, a fluorescent tag, an oligonucleotide tag, or a biotin tag. 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 moiety is an antibody or a functional derivative thereof. In some embodiments, the photoactivatable label is hydrophobic.

[0054] For compositions provided herein, in some embodiments, 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 vehicle comprises a solution of benzyl alcohol, benzyl benzoate (BABB) or a derivative thereof, with or without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the vehicle comprises a solution of benzyl alcohol with one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the vehicle comprises a solution of benzyl alcohol without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the vehicle comprises a solution of benzyl alcohol with one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or quadrol. In some embodiments, the vehicle comprises a solution of benzyl benzoate (BABB) or a derivative thereof, with 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, free of 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 listed components may 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 matching that of the tissue. In some embodiments, for the compositions provided herein, the medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB), or a derivative thereof, with or without 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 with 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 without 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 without 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, with 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, free of 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 listed components may be explicitly excluded.

[0057] In some embodiments, the cleared tissue provided herein comprises labeled cells. In some embodiments, the cells are labeled by multiphoton technology. In some embodiments, the photoactivatable label comprises a detectable moiety. In some embodiments, the detectable moiety comprises a luminescent moiety. In some embodiments, the luminescent moiety is a fluorescent moiety, a chemiluminescent moiety, a bioluminescent moiety, or an electrochemiluminescent moiety. In some embodiments, the luminescent moiety is a fluorescent moiety. In some embodiments, the luminescent moiety is a chemiluminescent moiety. In some embodiments, the luminescent moiety is a bioluminescent moiety. In some embodiments, the luminescent moiety is an electrochemiluminescent moiety. In some embodiments, the fluorescent moiety comprises a fluorophore. In some embodiments, the photoactivatable label comprises a tag. In some embodiments, the tag is selected from the group comprising an affinity tag, an epitope tag, a fluorescent tag, an oligonucleotide tag, or a biotin tag. 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 moiety 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] For cleared tissues provided herein, in some embodiments, 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 listed labels may be explicitly excluded. Purpose

[0059] The methods and compositions described herein can be used in any application where three-dimensional tissue analysis is desirable. The following uses are examples only and are not intended to be limiting.

[0060] In some embodiments, the compositions and methods described herein can be used to detect heterogeneity in tumors or tumor samples (e.g., biopsies). A patient-derived tumor sample can be cleared as described herein. The tumor sample can then be contacted with a photoactivatable label, and one or more regions of interest can be subjected to a multiphoton laser (e.g., a two-photon laser). The regions of interest can 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 can be imaged at any step or multiple steps before isolation or analysis, such as before or after clearing, before or after contact with the photoactivatable label, etc. The 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 protein within various regions can 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 lack thereof) in a sample, such as a tumor sample.For example, as described herein, one or more regions of the sample can be labeled, imaged, and isolated.The isolated region can be analyzed, for example, for RNA and / or protein 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, as described herein, one or more regions of a sample can be labeled, imaged, and isolated based on proximity to blood vessels.The isolated region can be analyzed, for example, for RNA and / or protein expressed (preferentially expressed) by one or more cells of interest.Similarly, metastasis or suspected metastasis regions can be analyzed using these methods.

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

[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 is composed of multiple layers / regions of the dorsal root ganglion, and each layer or region contains different cell types.The compositions and methods described herein can help identify the differences (and / or similarities) between cells in various layers.Therefore, 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, such as expression (e.g., RNA or protein) or mutation (e.g., DNA) data, about regions of interest in a tissue or sample after analysis of the tissue or sample by 3D histology.

[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, regions of interest can be labeled within a resolution of 1.6 x 1.6 x 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 variations in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [Example]

[0068] Those skilled in the art will appreciate that the descriptions of making and using the particles described herein are for illustrative purposes only, and that the disclosure is not limited by these examples. Example 1. Tissue dehydration and clearing

[0069] Dehydration and tissue clearing

[0070] To harvest organs free of residual blood, mice were perfused with warm PBS / heparin (5 U / ml; 50–100 ml / mouse at constant pressure (gravity flow or perfusion system; 1–1.5 mHO (Leica))) followed by PFA 2% (Electron Microscopy Sciences, diluted in PBS). Depending on sample permeability, removed organs were post-fixed in PFA overnight at 4 °C. Samples were then post-fixed in 30% and 50% tert-butanol (pH = 9.5, RT), followed by sequential 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 law of diffusion in a 24-hour-incubated mouse brain, which served as a reference; T = (1 / [2D])r). 2, D = diffusion coefficient (estimated from mouse brain dimensions and incubation time = 0.00015349), r = distance to nearest center of sample), and finally cleared in BABB (benzyl alcohol:benzyl benzoate in a 1:2 volume ratio, pH = 9.5, 30 °C). After the final clearing step, organs can be stored in BABB solution for at least one year at 4 °C. An example of a cleared mouse brain after this protocol, with approximately 24 h of incubation per step, can be seen in Figure 1.

[0071] Stability Experiments

[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. Cells were seeded onto Millipore EZ slides, fixed with 4% PFA, and then processed according to the above protocol, with a 15-minute incubation time per step. For mounting, BABB was used as the mounting medium, and a glass coverslip was used to cover the cells. Cells were then imaged using a Leica SP8 upright laser scanning microscope with wavelengths matching the excitation / emission spectrum of the fluorescent protein. 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 the appropriate compound dissolved in BABB (12.5 μg / ml) for the time dictated by Fick's law of diffusion at room temperature (RT) or overnight at 4 °C. Successful conjugation of compounds to the site of interest was achieved by using a 10 μs pixel dwell time at 512 x 512 resolution with a 20x lens (Leica HCX APO L 20x / 0.95 IMM) and 75 mW laser power at 700 nm. Based on the composition of photobiotin (biotin-linker-phenylazide; Sigma A1935-1MG), photoactivatable Pacific Blue, Cy3, Cy5, and Alexa 488 were synthesized, readily soluble in pH-adjusted BABB at 1 mg / ml (stock concentration). Immediately after photolabeling of the samples, the samples were washed (by transferring the sample to each solution) with BABB (pH = 9.5, 30°C) and then with 100% tert-butanol (pH = 9.5, 30°C) for times determined by Fick's law, followed by a final washing step in 100% DMSO (pH = 9.5, 30°C), and then stored at 4°C in 100% DMSO until further processing. Example 3. Isolation and purification of photolabeled nuclei

[0074] All equipment was treated with RNAse. Photolabeled tissue stored in DMSO was rehydrated by incubating in PBS supplemented with RNAse inhibitor (Takara) on a shaker at 20°C for 30 minutes. The tissue was minced into smaller pieces than 1 mm and transferred to lysis buffer (975 μl of HBSS containing divalent cleavage, 10 μl of proteinase K, 10 μl of RNAse-free DNAse, and 0.2 U / ml of RNAse inhibitor). After 15 minutes of incubation at 30°C, the tissue pieces were transferred to a Dounce homogenizer and homogenized with 5–10 strokes (avoiding bubbles) after adding 1 ml of HBSS+ solution (HBSS, 3% BSA (fatty acid-free), and 0.2 U / ml of RNAse inhibitor). The homogenate was filtered through a pre-wetted 70 μm filter and spun at 300 rcf for 3–5 minutes. The supernatant was discarded, and the pellet was resuspended in HBSS+, triturated with a 200 μl tip to disrupt the nuclear clumps, pelleted at 300 rcf, resuspended in 1 ml of HBSS+, mixed with an equal volume of cold 25% Optiprep (Sigma-Aldrich), and placed on ice. A gradient was prepared in a 15 ml tube on ice by adding 2 ml of 40% Optiprep to the bottom, pipetting 2 ml of 25% Optiprep to the top, and carefully layering the nuclei mixture on top. The tube was then spun at 2500 rcf for 20 minutes in a pre-cooled swinging bucket rotor. Nuclei located at the interface of the 25% and 40% Optiprep solutions (which may appear transparent with a brownish hue, beneath a fluffy, opaque layer of debris) were collected and resuspended in 3 volumes of HBSS+, then spun at 250 rcf for 10 minutes. The pellet was then washed with HBSS+, resuspended in HBSS+, and stained for biotin incorporation by adding an appropriate nuclear label for FACS (PI or DAPI) or a streptavidin-linked dye. After a 5-minute incubation at RT, the sample was spun down and resuspended in HBSS+. For sorting, single nuclei (using the nuclear label) and unlabeled controls were prepared, which were treated identically to the samples except for being exposed to the light used for photoactivation and gating of the photolabeled nuclei. Example 4. Image acquisition and analysis

[0075] Cleared specimens were mounted on insect pins (Austerlitz) fixed to an inert silicone rubber surface (Momentive, RTV615), completely covered with BABB, and imaged using a Leica SP8 microscope equipped with a white light laser and Leica BABB immersion lenses: HCX PL FLUOTAR 5x / 0.15IMM for low resolution and HCX APO L 20x / 0.95IMM for high resolution. The acquired Leica image containers were converted into Imaris containers (Imaris File Converter 9.1.2, Bitplane) and transferred to a power workstation (Dual Xeon E5-2687W v4, 1 TB 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 adapthresh function in Matlab (MathWorks) 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 cords and sorted for dye uptake. Libraries were prepared according to the SPLiT-seq method, and RNA was sequenced using a MiSeq instrument. Figure 7A shows the plot of the number of unique barcodes against the total number of barcodes detected. Two sublibraries were then mixed at a 2:1 ratio (fixed:cleared) (nuclei isolated from fixed but not cleared tissue and nuclei isolated from fixed and cleared tissue). As shown in Figure 7B, TPM counts were plotted for both libraries, demonstrating a linear correlation, indicating that the clearing process does not alter sequencing results. Figure 7C shows a random region from mouse chromosome 2 with reads generated from fixed but not cleared, and fixed and cleared mouse spinal cords. Example 6. 3D analysis of DNA / RNA / protein composition in mouse lungs

[0077] Appropriately prepared mouse lung sections are subjected to a clearing protocol, imaged, and a photoactivatable compound is added in situ (without moving the tissue). Regions of interest are then identified using previously generated, preprogrammed, and photoactivated data. The tissue is then unmounted, washed, and rehydrated. The tissue can then be processed by extracting nuclei using a Dounce homogenizer or by targeting and isolating nuclei, which are then stained 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 a streptavidin-binding dye, and sorted for dye uptake. Libraries are prepared according to the SPLiT-seq method, and RNA is sequenced using an appropriate method. Alternatively, RNA can be analyzed by any method, including RT-PCR, electrophoresis, etc.

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

[0079] For protein analysis, mass spectrometry readings (e.g., liquid chromatography / mass spectrometry) or other suitable protein analysis methods can be used to determine the composition of the protein into which the PA compound is incorporated. Other protein analysis methods may include electrophoresis, protein blotting, Edman degradation or other protein sequencing, etc. Example 7. 3D analysis of DNA / RNA / protein composition in the spinal cord

[0080] Figure 8A is a cross-section of a mouse spinal cord showing different regions of the spinal cord, with the dorsal layers shown as colored circles. In the magnified panel, the area that underwent photoactivation is highlighted. Note that in this example, the region of the PA was 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 x 0.89 μm x 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 of the nuclear counterstain DAPI, and the X-axis shows the PA dye (Cy3-PA). Boxes indicate single nuclei negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right).

[0082] Figure 8C shows a FACS plot of cleared, 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 negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). Example 8. SPLiT-seq of cleared nuclei from spinal cord

[0083] To demonstrate that cleared tissue can be analyzed by SPLiT-seq, mouse spinal cords were 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 demonstrated the expression of markers for the expected cell types present in the cleared tissue, including neurons, oligodendrocytes, and astrocytes. RNA expression profiles were determined based on an RNA expression database. Figure 10A shows an nFeature plot (left) and an nCount plot (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.

Claims

1. A method for labeling a region of a tissue or tissue sample, comprising: (a) providing a three dimensional tissue or tissue sample; (b) clearing the sample; (c) contacting the tissue or tissue sample with a photoactivatable label; and (d) subjecting a region of said tissue or tissue sample to a multiphoton laser, thereby labeling said region.

2. The method of claim 1, wherein the region comprises a cell, an intracellular compartment, an aggregate or a secreted aggregate.

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

4. A method according to any one of claims 1 to 3, wherein the photoactivatable label comprises a detectable moiety or tag.

5. A method according to any one of claims 1 to 3, wherein the sample transparency process comprises dehydrating the sample and transferring the sample to a medium having a refractive index similar or matching that of the tissue.

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

7. The method of claim 1, further comprising isolating the labeled region or a portion of the labeled region from the tissue or tissue sample.

8. Further comprising analyzing the composition of the isolated labeled region to generate analytical data; wherein analyzing includes determining the DNA, RNA and / or protein composition of said isolated targeted region; and 8. The method of claim 7, wherein optionally RNA is analyzed by sequencing or SPLITseq, proteins are analyzed by mass spectrometry, and / or DNA is analyzed by DNA sequencing.

9. The method of claim 8, further comprising imaging the tissue or tissue sample prior to isolating the labeled region to create an image, the method further comprising combining the image with the analysis data to create a three-dimensional compositional map of the region.

10. A method for three-dimensional expression profiling of an intact tissue or tissue sample, comprising: (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 said tissue or tissue sample to a multiphoton laser, thereby labeling said region; (e) imaging the labeled area to generate an image; (f) isolating the target region from the tissue or tissue sample; (g) determining the DNA, RNA and / or protein composition of the isolated targeted region; (h) combining said image with said DNA, RNA and / or protein composition of said isolated labeled regions to generate a three-dimensional expression profile of said intact tissue or tissue sample.

11. The method of claim 10, wherein the region is a cell, an intracellular compartment, an aggregate or a secreted aggregate.

12. The RNA composition, protein composition, and / or DNA composition of the isolated labeled region is determined; and Optionally, 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.

13. The method of any one of claims 10 to 12, wherein the photoactivatable label comprises a detectable moiety or tag.

14. A method according to any one of claims 10 to 13, wherein the sample transparency process comprises dehydrating the sample and transferring the sample to a medium having a refractive index similar or matching that of the tissue.

15. The method of claim 10, wherein the photoactivatable label is hydrophobic.

16. A method for isolating a single cell or nucleus in a tissue or tissue sample, comprising: (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, thereby labeling said cells; (e) dissociating the labeled cells from the tissue or tissue sample; and (f) isolating said labeled cells or nuclei.

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

18. The method of claim 16 or 17, wherein the photoactivatable label comprises a detectable moiety or tag.

19. A method according to any one of claims 16 to 18, wherein the sample transparency process comprises dehydrating the sample and transferring the sample to a medium having a refractive index similar or matching that of the tissue.

20. The method of claim 16, wherein the photoactivatable label is hydrophobic.

21. The method of any one of claims 16 to 20, further comprising isolating the labeled cells or nuclei.

22. The method of claim 21, further comprising analyzing the isolated labeled cells or nuclei. wherein analyzing comprises determining the DNA composition, RNA composition and / or protein composition of said isolated labeled cells or nuclei; 22. The method of claim 21, 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.

23. The method of any one of claims 1 to 22, further comprising contacting the 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.

24. A method for detecting a tissue sample comprising: a tissue sample; a medium having a refractive index similar to or matching that of the tissue; and a photoactivatable label; wherein the tissue comprises a labeled region comprising a cell, a subcellular compartment, an aggregate, or a secreted aggregate; The composition, wherein the cells are labeled by multiphoton techniques.

25. A cleared tissue sample comprising labeled cells, a photoactivatable label, and a medium having a refractive index that substantially matches the refractive index of the tissue.

Images