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 and intact tissue samples, and subsequent isolation of materials in a precise and location-dependent manner. The methods and compositions described herein can be used, for example, in biomarker discovery, identification of cell populations, pathological analysis, and generation of expression data in specific regions of interest.

Description

[0001] Cross-referencing of related patent applications

[0002] 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, each of which is incorporated herein by reference in its entirety and for all purposes. Background Technology

[0003] In most molecular biology-based tissue analysis methods, the spatial context of the analyzed material is lost. Techniques known in the art that combine sequence analysis with spatial information have limitations, such as low sequencing depth, sample volume limitations, analysis of single tissue sections, or time-consuming readouts, among other constraints. Currently, to our knowledge, no available method combines separation precision with analytical depth. The methods and compositions described herein address these and other problems in the art. Summary of the Invention

[0004] This technology generally relates to methods and compositions for three-dimensional labeling of cells or other regions of tissue or tissue samples. The method involves imaging a transparent tissue or sample that has been labeled by contacting a target region (or cell) with an appropriate label, and then subjecting the target region (or cell) to a multiphoton laser. The labeled region can then be isolated from the tissue or sample and analyzed, such as, but not limited to, DNA sequencing, RNA sequencing, proteomics analysis, epigenetic analysis, immunohistochemical analysis, chromosome conformation capture, or immunofluorescence analysis. Various methods for isolating the target labeled region (or cell) can be used, such as, but not limited to, laser-assisted microdissection, fluorescence-assisted cell sorting (FACS), magnetically activated cell sorting (MACS), or buoyancy-activated cell sorting (BACS). Such analyses can provide information about discrete regions of the tissue or sample.

[0005] In one aspect, a method for labeling a region of tissue or tissue sample is provided. The method may include: a) providing a three-dimensional tissue or tissue sample; b) transparentizing the sample; c) contacting the tissue or tissue sample with a photoactivated label; and d) subjecting a region of the tissue or tissue sample to a multiphoton laser beam, thereby labeling the region.

[0006] On the other hand, a method for three-dimensional expression profiling analysis of intact tissue or tissue samples is provided. The method may include: a) providing a three-dimensional intact tissue or tissue sample; b) transparentizing the sample; c) contacting the tissue or tissue sample with a photoactivated label; d) subjecting a region of the tissue or tissue sample to a multiphoton laser to label that region; e) imaging the labeled region to create an image; f) isolating the labeled region from the tissue or tissue sample; g) determining the DNA, RNA, and / or protein composition of the isolated labeled region; and h) combining the image with the DNA, RNA, and / or protein composition of the isolated labeled region to create a three-dimensional expression profile of the intact tissue or tissue sample.

[0007] In one aspect, a method is provided for separating individual cells or nuclei from a tissue or tissue sample. The method may include: a) providing a three-dimensional tissue or tissue sample containing target cells or nuclei; b) transparentizing the sample; c) contacting the tissue or tissue sample with a photoactivated label; d) subjecting the cells or nuclei to a multiphoton laser to label the cells or nuclei; e) dissociating the labeled nuclei from the tissue or tissue sample; and f) separating the labeled cells or nuclei.

[0008] In another aspect, a composition is provided. The composition may include a tissue sample, a culture medium having a refractive index similar to or matching that of the tissue, and a photoactivated label.

[0009] On the one hand, transparent tissue samples are provided. Transparent tissue samples may include photoactivated labels and a culture medium with a refractive index that substantially matches the refractive index of the tissue. Attached Figure Description

[0010] Figure 1 The mouse brain is shown before (left) and after (right) tissue transparency.

[0011] Figure 2 This is a series of fluorescence microscopy images showing the stability of different fluorescent proteins in cleared tissues (bottom row) or control tissues (top row). In this image, 293T cells were transfected with plasmids that drive the expression of EGFP, mKate2, tdTomato, or Venus under the control of the CMV promoter.

[0012] Figures 3A to 3B This is a diagram illustrating an embodiment of using two-photon microscopy to label target cells or cell nuclei in transparent tissue using photoreactive labeling. Figure 3A This diagram illustrates molecules labeled with photoreactive groups and biotin tags when exposed to light with wavelengths between 260 nm and 475 nm. Figure 3BThis diagram illustrates the advantages of using two-photon microscopy (right) to label target cells or nuclei compared to conventional confocal microscopy (left). Conventional confocal microscopy results in photoactivation of the entire area of ​​tissue exposed to light (red) and photoactivated compounds (green). In contrast, two-photon microscopy allows for more precise photoactivation and targeting of the target site (red star), thus avoiding non-specific labeling of non-target tissue.

[0013] Figure 4 An overview of an exemplary analysis using two-photon labeling technology is presented. Tissues with three-dimensional (3D) cellular structures are transparentized, immersed in a photoactivated dye (e.g., photobiotin), and labeled using a two-photon laser (e.g., such as...). Figure 3B (as shown), and then the labeled cell nuclei are separated for analysis, such as single-cell analysis (e.g., SPLiT-seq).

[0014] Figure 5 The polynucleotide length profiles of the treated samples after reverse transcription without EDC treatment are shown after three days at room temperature (RT) or 4°C (4C).

[0015] Figures 6A to 6C An overview of methods for spatial dependence analysis of biological materials from intact tissue samples is presented. Figure 6A An example of organizational transparency is shown. Figure 6A The process includes the labeling (Figure A) and labeling (Figures B through E), as well as the separation of photolabeled cell nuclei (Figure F). The resulting samples can be analyzed using SPLiT seq libraries prepared from the separated photolabeled cell nuclei (Figures G and H), followed by data analysis (I). Additionally (or alternatively), DNA from the separated tissues can be analyzed. Figure 6B ) or protein ( Figure 6C ).

[0016] Figures 7A to 7C The RNA quality of formalin-fixed and cleared samples was demonstrated by SPLiT-seq analysis. Figure 7A This is a graph showing the unique count (log2(UMI)) of each barcode relative to the barcode level (barcode log10 scale). Figure 7B It is a scatter plot, showing the log2 (TPM) of the fixed sample on the y-axis and the log2 (TPM) of the transparent sample on the x-axis (TPM: per million transcripts). Figure 7C An exemplary genome browser window is shown, displaying cDNA coverage of a 72kb fragment from mouse (mm10) chromosome 2 (top), and comparing fixed and cleared (center) samples with fixed (bottom) samples. Figures 7A to 7CAs shown, the spinal cord was photoactivated in Cy3-PA. Cell nuclei were extracted and sorted for labeling incorporation. SPLiT-seq libraries were generated and sequenced using HiSeq.

[0017] Figures 8A to 8C Spinal cord tissue labeled with two-photon markers is shown. Figure 8A This is a cross-sectional view of the mouse spinal cord, showing different regions of the spinal cord, with the dorsal layer indicated by colored circles. In the magnified smaller images, areas that have undergone light activation are highlighted. Figure 8B This is a FACS image of cleared but not photoactivated cell nuclei isolated from the mouse spinal cord. The Y-axis shows the signal of DAPI (a nuclear counterstain), while the X-axis shows the PA dye used in this experiment: Cy3-PA. Boxes indicate whether a single cell nucleus is negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right). Figure 8C It is a transparent and photoactivated cell nucleus isolated from the mouse spinal cord (e.g. Figure 9A The FACS plot (shown) shows the signal of DAPI (a nuclear counterstain), while the X-axis shows the PA dye used in this experiment: Cy3-PA. Boxes indicate whether a single cell nucleus is negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right).

[0018] Figures 9A to 9C Exemplary compounds used in the methods of the present invention are shown. Figure 9A Examples of photoreactive compounds that can be used for photolabeling are shown. Figure 9B Exemplary modifications of aryl azides induced by ultraviolet (UV) light are shown. Figure 9C Examples of synthesized photoreactive compounds are shown.

[0019] Figures 10A to 10B The expression profile of cleared nuclei from the spinal cord is shown using SPLiT-seq analysis. Figure 10A This is a graph showing the expression profile of cell types (the expression levels of genes associated with the cell type shown). Figure 10B The graph is generated using UMAP from the data in Figure 10.

[0020] Figure 11 This is an example cartoon illustrating how multiple reuse allows for multiple labeling events. Tissue can be exposed to a first dye (dye 1), and then the first target area can be two-photon labeled (left inset). The same tissue can then be contacted with a second dye (dye 2) (optionally after washing), and then the second target area can be two-photon labeled (middle inset). If desired, the same tissue can be contacted with a third dye (dye 3) (optionally after washing), and then the third target area can be two-photon labeled (right inset).

[0021] Figures 12A to 12C Examples of application areas for target compounds used for photoactivation throughout immobilization are presented. Examples of application areas include tumor heterogeneity, inflammatory tumors / immune deserts, and the proximal vascular system. Figure 12A ), to discover and label sparse cell populations ( Figure 12B ), generating expressive data and 3D histology ( Figure 12C ). Detailed Implementation

[0022] After reading this specification, it will become apparent to those skilled in the art how to implement this disclosure in various alternative embodiments and applications. However, not all various embodiments of the invention will be described herein. It should be understood that the embodiments presented herein are by way of example only and are not restrictive. Therefore, the detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the disclosure as described herein.

[0023] Before disclosing and describing this technology, it should be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or their uses, as these may, of course, vary. Furthermore, it should be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting.

[0024] The detailed descriptions divided into sections for convenience only, and the disclosure found in any section, may be combined with the content in other sections. For the convenience of the reader, headings or subheadings may be used in this specification, which are not intended to affect the scope of this disclosure.

[0025] definition

[0026] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In this specification and the following claims, several terms will be referenced that are defined as having the following meanings:

[0027] 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 also intended to include the plural forms unless the context clearly indicates otherwise.

[0028] "Optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the possibility that the event or situation will occur and the possibility that it will not occur.

[0029] The term “about” used before numerical designations (e.g., temperature, time, amount, concentration, and other such designations, including ranges) indicates an approximate value that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue between them. Preferably, the term “about” when used with respect to dosage means that the dosage may vary by + / - 10%.

[0030] The terms "comprising" or "including" are intended to indicate that a composition and method include the listed elements but do not exclude other elements. When used to define a composition and method, "consistently of..." means that other elements that are of any significance to the combination are excluded for the purposes stated herein. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the essential and novel features of the claimed invention. "Constitutes of..." means excluding portions other than trace elements of other components and substantial method steps. Each of these transitional terms is within the scope of this disclosure.

[0031] method

[0032] In one aspect, a method for labeling a region of tissue or a tissue sample is provided. The method may include: (a) providing a three-dimensional tissue or tissue sample; (b) transparentizing the sample; (c) contacting the tissue or tissue sample with a photoactivated label; and (d) subjecting a region of the tissue or tissue sample to a multiphoton laser to label the region. In one embodiment, the multiphoton laser is a two-photon laser. In another embodiment, the multiphoton laser is a three-photon laser.

[0033] In embodiments of the methods provided herein, the region includes cells, subcellular compartments, aggregates, or secreted aggregates. In embodiments, the region includes cells. In embodiments, the region includes subcellular compartments. In embodiments, the region includes aggregates. In embodiments, the region includes secreted aggregates. In embodiments, the subcellular compartment is the cell nucleus.

[0034] In some embodiments, the method further includes imaging the tissue or tissue sample. In some embodiments, the method further includes imaging the tissue. In some embodiments, the method further includes imaging the tissue sample. In some embodiments, the tissue or tissue sample is fixed tissue or a fixed tissue sample. In some embodiments, the tissue is fixed tissue. In some embodiments, the tissue is a fixed tissue sample. In some embodiments, the tissue sample is fixed tissue. In some embodiments, the tissue sample is a fixed tissue sample.

[0035] In the methods provided herein, in embodiments, the photoactivated label includes a detectable portion. In embodiments, the detectable portion includes a luminescent portion. In embodiments, the luminescent portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion. In embodiments, the luminescent portion is a fluorescent portion. In embodiments, the luminescent portion is a chemiluminescent portion. In embodiments, the luminescent portion is a bioluminescent portion. In embodiments, the luminescent portion is an electrochemiluminescent portion. In embodiments, the fluorescent portion includes a fluorophore. In embodiments, one of the detectable portions includes an antibody or a functional derivative thereof. In embodiments, the photoactivated label includes a tag. In embodiments, the tag is selected from the group consisting of affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags. In embodiments, the tag is an affinity tag. In embodiments, the tag is an epitope tag. In embodiments, the tag is a fluorescent tag. In embodiments, the tag is an oligonucleotide tag. In embodiments, the tag is a biotin tag.

[0036] In embodiments of the methods provided herein, the sample clearing process includes dehydrating the sample and transferring it to a culture medium having a refractive index similar to or matching that of the tissue. In embodiments, the refractive index is between about 1.3 and about 1.6. In embodiments, the refractive index is about 1.3. In embodiments, the refractive index is about 1.325. In embodiments, the refractive index is about 1.35. In embodiments, the refractive index is about 1.375. In embodiments, the refractive index is about 1.4. In embodiments, the refractive index is about 1.425. In embodiments, the refractive index is about 1.45. In embodiments, the refractive index is about 1.475. In embodiments, the refractive index is about 1.5. In embodiments, the refractive index is about 1.525. In embodiments, the refractive index is about 1.55. In embodiments, the refractive index is about 1.575. In embodiments, the refractive index is about 1.6. In 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 range, including the endpoints, or any range between any of the values.

[0037] In the examples, the culture medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB), or a derivative thereof, with or without triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a solution of benzyl alcohol, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a solution of benzyl alcohol, without triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a solution of benzyl benzoate (BABB), or a derivative thereof, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a solution of benzyl benzoate (BABB), or a derivative thereof, without triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a BABB solution.

[0038] As described above, the method provided herein includes, in some embodiments, a sample clearing process that includes dehydrating the sample. In some embodiments, the sample is dehydrated using a tert-butanol solution. In some embodiments, the tert-butanol solution includes trimethylamine, tetrahydrofuran, ethanol, or methanol. In some embodiments, the tert-butanol solution includes trimethylamine. In some embodiments, the tert-butanol solution includes tetrahydrofuran. In some embodiments, the tert-butanol solution includes ethanol. In some embodiments, the tert-butanol solution includes methanol.

[0039] In the embodiments of the method provided herein, the photoactivated label is hydrophobic. In the embodiments of the method provided herein, the photoactivated label includes a phenyl azide group, an o-hydroxyphenyl azide group, a m-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an o-nitrophenyl azide group, a m-nitrophenyl azide group, a diazidopropylidin group, an azoxymethylcoumarin group, or a psoralen group. In the embodiments, the photoactivated label includes a phenyl azide group. In the embodiments, the photoactivated label includes an o-hydroxyphenyl azide group. In the embodiments, the photoactivated label includes a m-hydroxyphenyl azide group. In the embodiments, the photoactivated label includes a tetrafluorophenyl azide group. In the embodiments, the photoactivated label includes an o-nitrophenyl azide group. In the embodiments, the photoactivated label includes a m-nitrophenyl azide group. In the embodiments, the photoactivated label includes a diazidopropylidin group. In the embodiments, the photoactivated label includes an azoxymethylcoumarin group. In the embodiments, the photoactivated label includes a psoralen group. In the embodiments, the photoactivated label includes an aryl azide group.

[0040] In embodiments, the method further includes isolating a labeled region or a portion of a labeled region from tissue or a tissue sample. In embodiments, the method further includes isolating a labeled region from tissue. In embodiments, the method further includes isolating a portion of a labeled region from tissue. In embodiments, the method further includes isolating a labeled region from a tissue sample. In embodiments, the method further includes isolating a portion of a labeled region from a tissue sample. In embodiments, the labeled region or portion is isolated by FACS sorting for the label. In embodiments, the method further includes analyzing the composition of the isolated labeled region to generate analytical data. In embodiments, the analysis includes determining the DNA, RNA, and / or protein composition of the isolated labeled region. In embodiments, the analysis includes determining the DNA, RNA, and protein composition of the isolated labeled region. In embodiments, the analysis includes determining the DNA, RNA, or protein composition of the isolated labeled region. In embodiments, the analysis includes determining the DNA composition of the isolated labeled region. In embodiments, the analysis includes determining the RNA composition of the isolated labeled region. In embodiments, the analysis includes determining the protein composition of the isolated labeled region. In embodiments, RNA is analyzed using SPLITseq.

[0041] In the embodiments of the method provided herein, the tissue or tissue sample is imaged to create an image prior to the separation of the labeled region, and further includes combining the image with analytical data to create a three-dimensional composition map of the region.

[0042] In one aspect, a method is provided for three-dimensional expression profiling analysis of intact tissue or tissue samples. The method may include: (a) providing a three-dimensional intact tissue or tissue sample; (b) transparentizing the sample; (c) contacting the tissue or tissue sample with a photoactivated label; (d) subjecting a region of the tissue or tissue sample to a multiphoton laser to label the region; (e) imaging the labeled region to create an image; (f) isolating the labeled region from the tissue or tissue sample; (g) determining the DNA, RNA, and / or protein composition of the isolated labeled region; and (h) combining the image with the DNA, RNA, and / or protein composition of the isolated labeled region to create a three-dimensional expression profile of the intact tissue or tissue sample. In an embodiment, the multiphoton laser is a two-photon laser. In an embodiment, the multiphoton laser is a three-photon laser. In an embodiment, the region is a cell, a subcellular compartment, an aggregate, or a secreted aggregate. In an embodiment, the region is a cell. In an embodiment, the region is a subcellular compartment. In an embodiment, the region is an aggregate. In an embodiment, the region is a secreted aggregate. In this embodiment, the isolated labeled region is a single cell nucleus. In this embodiment, the RNA composition of the isolated labeled region is determined.

[0043] In one aspect, a method is provided for separating individual cells or cell nuclei from a tissue or tissue sample. The method may include: (a) providing a three-dimensional tissue or tissue sample; (b) transparentizing the sample; (c) contacting the tissue or tissue sample with a photoactivated label; (d) subjecting cells to a multiphoton laser to label the cells; (e) dissociating the labeled cells from the tissue or tissue sample; and (f) separating the labeled cells or cell nuclei. In one embodiment, the multiphoton laser is a two-photon laser. In another embodiment, the multiphoton laser is a three-photon laser.

[0044] In some embodiments, the method further includes imaging the tissue or tissue sample. In some embodiments, the method further includes imaging the tissue. In some embodiments, the method further includes imaging the tissue sample. In some embodiments, the tissue or tissue sample is fixed tissue or a fixed tissue sample. In some embodiments, the tissue is fixed tissue. In some embodiments, the tissue is a fixed tissue sample. In some embodiments, the tissue sample is fixed tissue. In some embodiments, the tissue sample is a fixed tissue sample.

[0045] In the methods provided herein, in embodiments, the photoactivated label includes a detectable portion. In embodiments, the detectable portion includes a luminescent portion. In embodiments, the luminescent portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion. In embodiments, the luminescent portion is a fluorescent portion. In embodiments, the luminescent portion is a chemiluminescent portion. In embodiments, the luminescent portion is a bioluminescent portion. In embodiments, the luminescent portion is an electrochemiluminescent portion. In embodiments, the fluorescent portion includes a fluorophore. In embodiments, the detectable portion includes an antibody or a functional derivative thereof. In embodiments, the photoactivated label includes a tag. In embodiments, the tag is selected from the group consisting of affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags. In embodiments, the tag is an affinity tag. In embodiments, the tag is an epitope tag. In embodiments, the tag is a fluorescent tag. In embodiments, the tag is an oligonucleotide tag. In embodiments, the tag is a biotin tag.

[0046] In the embodiments of the methods provided herein, the sample clearing process includes dehydrating the sample and transferring it to a culture medium having a refractive index similar to or matching that of the tissue. In the embodiments, the culture medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB), or a derivative thereof, with or without triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the embodiments, the culture medium comprises a solution of benzyl alcohol, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the embodiments, the culture medium comprises a solution of benzyl alcohol, without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the embodiments, the culture medium comprises a solution of benzyl benzoate (BABB), or a derivative thereof, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, and does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a BABB solution.

[0047] In the embodiments of the method provided herein, the sample is dehydrated using a tert-butanol solution. In the embodiments, the tert-butanol solution includes trimethylamine, tetrahydrofuran, ethanol, or methanol. In the embodiments, the tert-butanol solution includes trimethylamine. In the embodiments, the tert-butanol solution includes tetrahydrofuran. In the embodiments, the tert-butanol solution includes ethanol. In the embodiments, the tert-butanol solution includes methanol. In the embodiments, the photoactivation label is hydrophobic.

[0048] In the embodiments of the method provided herein, the photoactivated label includes a phenyl azide group, an o-hydroxyphenyl azide group, a m-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an o-nitrophenyl azide group, a m-nitrophenyl azide group, a diazidopropylidin group, an azoxymethylcoumarin group, or a psoralen group. In the embodiments, the photoactivated label includes a phenyl azide group. In the embodiments, the photoactivated label includes an o-hydroxyphenyl azide group. In the embodiments, the photoactivated label includes a m-hydroxyphenyl azide group. In the embodiments, the photoactivated label includes a tetrafluorophenyl azide group. In the embodiments, the photoactivated label includes an o-nitrophenyl azide group. In the embodiments, the photoactivated label includes a m-nitrophenyl azide group. In the embodiments, the photoactivated label includes a diazidopropylidin group. In the embodiments, the photoactivated label includes an azoxymethylcoumarin group. In the embodiments, the photoactivated label includes a psoralen group. In the embodiments, the photoactivated label includes an aryl azide group. In this embodiment, the photoactivated marker includes Figure 9A The group shown.

[0049] In embodiments, the method provided herein further includes separating labeled cells or cell nuclei. In embodiments, the method further includes separating labeled cells. In embodiments, the method further includes separating labeled cell nuclei. In embodiments, labeled cells or cell nuclei are separated by fluorescent activated cell sorting (FACS) for a label. In embodiments, labeled cells are separated by FACS for a label. In embodiments, cell nuclei are separated by FACS for a label. In embodiments, labeled cells or cell nuclei are separated by magnetic activated cell sorting (MACS) for a label. In embodiments, labeled cells are separated by MACS for a label. In embodiments, labeled cell nuclei are separated by MACS for a label. In embodiments, labeled cells or cell nuclei are separated by buoyancy activated cell sorting (BACS) for a label. In embodiments, labeled cells are separated by BACS for a label. In embodiments, labeled cell nuclei are separated by BACS for a label. In this embodiment, labeled cells or cell nuclei are separated by affinity-based column purification (e.g., affinity chromatography) targeting the label. In this embodiment, labeled cells are separated by affinity-based column purification targeting the label. In this embodiment, labeled cell nuclei are separated by affinity-based column purification targeting the label.

[0050] In embodiments, the method provided herein further includes analyzing isolated labeled cells or cell nuclei. In embodiments, the method further includes analyzing isolated labeled cells. In embodiments, the method further includes analyzing isolated labeled cell nuclei. In embodiments, the analysis includes determining the DNA, RNA, and / or protein composition of the isolated labeled cells or cell nuclei. In embodiments, the analysis includes determining the DNA, RNA, and protein composition of the isolated labeled cells or cell nuclei. In embodiments, the analysis includes determining the DNA, RNA, or protein composition of the isolated labeled cells or cell nuclei. In embodiments, the analysis includes determining the DNA composition of the isolated labeled cells or cell nuclei. In embodiments, the analysis includes determining the RNA composition of the isolated labeled cells or cell nuclei. In embodiments, the analysis includes determining the protein composition of the isolated labeled cells or cell nuclei. In embodiments, the analysis includes determining the DNA composition of the isolated labeled cells. In embodiments, the analysis includes determining the RNA composition of the isolated labeled cells. In embodiments, the analysis includes determining the protein composition of the isolated labeled cells. In embodiments, the analysis includes determining the DNA composition of the isolated labeled cell nuclei. In embodiments, the analysis includes determining the RNA composition of the isolated labeled cell nuclei. In this embodiment, the analysis includes determining the protein composition of isolated, labeled cell nuclei. In this embodiment, RNA is analyzed using single-cell RNA sequencing (scRNAseq). In this embodiment, RNA is analyzed using Splitseq.

[0051] In embodiments of the method provided herein, the method further includes contacting a tissue or tissue sample with a second photoactivated label and subjecting a second cell or nucleus in the tissue or tissue sample to a multiphoton laser to label the second cell or nucleus. See, for example... Figure 11 In one embodiment, the multiphoton laser is a two-photon laser. In another embodiment, the multiphoton laser is a three-photon laser.

[0052] In the embodiments of the methods provided herein, the tissue or tissue sample is a whole organ, a tumor, or an animal. In the embodiments, the tissue or tissue sample is a whole organ. In the embodiments, the tissue or tissue sample is a tumor. In the embodiments, the tissue or tissue sample is an animal. In the embodiments, the tissue is a whole organ. In the embodiments, the tissue is a tumor. In the embodiments, the tissue is an animal. In the embodiments, the tissue sample is a whole organ. In the embodiments, the tissue sample is a tumor. In the embodiments, the tissue sample is an animal.

[0053] In the embodiments of the method provided herein, the wavelength of each photon in the two-photon laser is between approximately 500 nm and approximately 1100 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 500 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 505 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 510 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 515 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 520 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 525 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 530 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 535 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 540 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 545 nm. In the embodiments, the wavelength of each photon in the two-photon laser is approximately 550 nm. In one embodiment, the wavelength of each photon in the two-photon laser is approximately 555 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 560 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 565 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 570 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 575 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 580 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 585 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 590 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 595 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 600 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 605 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 610 nm. In one embodiment, the wavelength of each photon in the two-photon laser is approximately 615 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 620 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 625 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 630 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 635 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 640 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 645 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 650 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 655 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 660 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 665 nm.In one embodiment, the wavelength of each photon in the two-photon laser is approximately 670 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 675 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 680 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 685 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 690 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 695 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 700 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 705 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 710 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 715 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 720 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 725 nm. In one embodiment, the wavelength of each photon in the two-photon laser is approximately 730 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 735 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 740 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 745 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 750 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 755 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 760 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 765 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 770 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 775 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 780 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 785 nm. In one embodiment, the wavelength of each photon in the two-photon laser is approximately 790 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 795 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 800 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 805 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 810 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 815 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 820 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 825 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 830 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 835 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 840 nm.In one embodiment, the wavelength of each photon in the two-photon laser is approximately 845 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 850 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 855 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 860 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 865 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 870 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 875 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 880 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 885 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 890 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 895 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 900 nm. In one embodiment, the wavelength of each photon in the two-photon laser is approximately 905 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 910 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 915 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 920 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 925 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 930 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 935 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 940 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 945 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 950 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 955 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 960 nm. In one embodiment, the wavelength of each photon in the two-photon laser is approximately 965 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 970 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 975 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 980 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 985 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 990 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 995 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1000 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1005 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1010 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1015 nm.In one embodiment, the wavelength of each photon in the two-photon laser is approximately 1020 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1025 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1030 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1035 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1040 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1045 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1050 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1055 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1060 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1065 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1070 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1075 nm. In one embodiment, the wavelength of each photon in the two-photon laser is approximately 1080 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1085 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1090 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1095 nm. In another embodiment, the wavelength of each photon in the two-photon laser is approximately 1100 nm. In another embodiment, the wavelengths of each photon in the two-photon laser are approximately 905 nm, approximately 910 nm, approximately 915 nm, approximately 920 nm, approximately 925 nm, approximately 930 nm, approximately 935 nm, approximately 940 nm, approximately 945 nm, approximately 950 nm, approximately 955 nm, approximately 960 nm, approximately 965 nm, approximately 970 nm, approximately 975 nm, approximately 980 nm, approximately 985 nm, approximately 990 nm, approximately 995 nm, and approximately 1000 nm. The wavelengths are approximately 1005 nm, 1010 nm, 1015 nm, 1020 nm, 1025 nm, 1030 nm, 1035 nm, 1040 nm, 1045 nm, 1050 nm, 1055 nm, 1060 nm, 1065 nm, 1070 nm, 1075 nm, 1080 nm, 1085 nm, 1090 nm, 1095 nm, or 1100 nm. The wavelength can be any value or subrange within the range, including endpoints, or any range between any of the stated values.

[0054] In the embodiments of the method provided herein, the wavelength of each photon in the three-photon laser is between approximately 690 nm and approximately 2500 nm. In the embodiments of the method provided herein, the wavelength of each photon in the three-photon laser is approximately 690 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 695 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 700 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 705 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 710 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 715 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 720 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 725 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 730 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 735 nm. In the embodiments of the three-photon laser, the wavelength of each photon is approximately 740 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 745 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 750 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 755 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 760 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 765 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 770 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 775 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 780 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 785 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 790 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 795 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 800 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 805 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 810 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 815 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 820 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 825 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 830 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 835 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 840 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 845 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 850 nm.In one embodiment, the wavelength of each photon in the three-photon laser is approximately 855 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 860 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 865 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 870 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 875 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 880 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 885 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 890 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 895 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 905 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 910 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 915 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 920 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 925 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 930 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 935 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 940 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 945 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 950 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 955 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 960 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 965 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 970 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 975 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 980 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 985 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 990 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 995 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1000 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1005 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1010 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1015 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1020 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1025 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1030 nm.In one embodiment, the wavelength of each photon in the three-photon laser is approximately 1035 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1040 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1045 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1050 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1055 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1060 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1065 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1070 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1075 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1080 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1085 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1090 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 1095 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1100 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1105 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1110 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1115 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1120 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1125 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1130 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1135 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1140 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1145 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1150 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 1155 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1160 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1165 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1170 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1175 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1180 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1185 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1190 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 1195 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2000 nm.In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2005 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2010 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2015 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2020 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2025 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2030 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2035 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2040 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2045 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2050 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2055 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2060 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2065 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2070 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2075 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2080 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2085 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2090 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2100 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2105 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2110 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2115 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2120 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2125 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2130 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2135 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2140 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2145 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2150 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2155 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2160 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2165 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2170 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2175 nm.In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2180 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2185 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2190 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2195 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2200 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2205 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2210 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2215 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2220 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2225 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2230 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2235 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2240 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2245 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2250 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2255 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2260 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2265 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2270 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2275 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2280 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2285 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2290 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2295 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2300 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2305 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2310 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2315 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2320 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2325 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2330 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2335 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2340 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2345 nm.In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2350 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2350 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2355 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2360 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2365 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2370 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2375 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2380 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2385 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2390 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2395 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2400 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2405 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2410 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2415 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2420 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2425 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2430 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2435 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2440 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2445 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2450 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2455 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2460 nm. In one embodiment, the wavelength of each photon in the three-photon laser is approximately 2465 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2470 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2475 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2480 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2485 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2490 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2495 nm. In another embodiment, the wavelength of each photon in the three-photon laser is approximately 2500 nm.

[0055] Composition

[0056] In one aspect, a composition is provided. The composition may include a tissue sample, a culture medium having a refractive index similar to or matching that of the tissue, and a photoactivated label. In embodiments, the tissue includes a labeled region. In embodiments, the region includes cells, subcellular compartments, aggregates, or secreted aggregates. In embodiments, the region includes cells. In embodiments, the region includes subcellular compartments. In embodiments, the region includes aggregates. In embodiments, the region includes secreted aggregates. In embodiments, the cells are labeled using a multiphoton method. In embodiments, the cells are labeled using a two-photon method. In embodiments, the cells are labeled using a three-photon method. In embodiments, the photoactivated label includes a detectable portion. In embodiments, the detectable portion includes a luminescent portion. In embodiments, the luminescent portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion. In embodiments, the luminescent portion is a fluorescent portion. In embodiments, the luminescent portion is a chemiluminescent portion. In embodiments, the luminescent portion is a bioluminescent portion. In embodiments, the luminescent portion is an electrochemiluminescent portion. In embodiments, the fluorescent portion includes a fluorophore.

[0057] For the compositions provided herein, in the embodiments, the photoactivated label includes a tag. In the embodiments, the tag is selected from the group consisting of affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags. In the embodiments, the tag is an affinity tag. In the embodiments, the tag is an epitope tag. In the embodiments, the tag is a fluorescent tag. In the embodiments, the tag is an oligonucleotide tag. In the embodiments, the tag is a biotin tag. In the embodiments, the detectable portion is an antibody or a functional derivative thereof. In the embodiments, the photoactivated label is hydrophobic.

[0058] For the compositions provided herein, in the embodiments, the photoactivated label includes a phenyl azide group, an o-hydroxyphenyl azide group, a m-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an o-nitrophenyl azide group, a m-nitrophenyl azide group, a diazidopropylidin group, an azoxymethylcoumarin group, or a psoralen group. In the embodiments, the photoactivated label includes a phenyl azide group. In the embodiments, the photoactivated label includes an o-hydroxyphenyl azide group. In the embodiments, the photoactivated label includes a m-hydroxyphenyl azide group. In the embodiments, the photoactivated label includes a tetrafluorophenyl azide group. In the embodiments, the photoactivated label includes an o-nitrophenyl azide group. In the embodiments, the photoactivated label includes a m-nitrophenyl azide group. In the embodiments, the photoactivated label includes a diazidopropylidin group. In the embodiments, the photoactivated label includes an azoxymethylcoumarin group. In the embodiments, the photoactivated label includes a psoralen group.

[0059] For the compositions provided herein, in the examples, the culture 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 the examples, the culture medium comprises a solution of benzyl alcohol, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a solution of benzyl alcohol, without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a solution of benzyl benzoate (BABB), or a derivative thereof, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a BABB solution. In the examples, one or more of the listed compositions may be explicitly excluded.

[0060] In one aspect, a cleared tissue sample is provided. The cleared tissue sample may include a photoactivated label and a culture medium having a refractive index substantially matching that of the tissue. In embodiments, for the cleared tissue provided herein, the culture medium comprises a solution of benzyl alcohol, benzyl benzoate (BABB), or a derivative thereof, with or without triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In embodiments, the culture medium comprises a solution of benzyl alcohol, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In embodiments, the culture medium comprises a solution of benzyl alcohol, without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In embodiments, the culture medium comprises a solution of benzyl benzoate (BABB), or a derivative thereof, containing one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a solution of benzyl benzoate (BABB) or a derivative thereof, and does not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and / or Quadrol. In the examples, the culture medium comprises a BABB solution. In the examples, one or more of the listed compositions may be explicitly excluded.

[0061] In an embodiment, the transparent tissue provided herein comprises labeled cells. In an embodiment, the cells are labeled using a multiphoton method. In an embodiment, the photoactivated label includes a detectable portion. In an embodiment, the detectable portion includes a luminescent portion. In an embodiment, the luminescent portion is a fluorescent portion, a chemiluminescent portion, a bioluminescent portion, or an electrochemiluminescent portion. In an embodiment, the luminescent portion is a fluorescent portion. In an embodiment, the luminescent portion is a chemiluminescent portion. In an embodiment, the luminescent portion is a bioluminescent portion. In an embodiment, the luminescent portion is an electrochemiluminescent portion. In an embodiment, the fluorescent portion comprises a fluorophore. In an embodiment, the photoactivated label includes a tag. In an embodiment, the tag is selected from the group consisting of affinity tags, epitope tags, fluorescent tags, oligonucleotide tags, or biotin tags. In an embodiment, the tag is an affinity tag. In an embodiment, the tag is an epitope tag. In an embodiment, the tag is a fluorescent tag. In an embodiment, the tag is an oligonucleotide tag. In an embodiment, the tag is a biotin tag. In an embodiment, the detectable portion is an antibody or a functional derivative thereof. In an embodiment, the photoactivated label is hydrophobic. In an embodiment, one or more of the listed tags may be explicitly excluded.

[0062] For the transparent tissues provided herein, in embodiments, the photoactivated markers include phenyl azide groups, o-hydroxyphenyl azide groups, m-hydroxyphenyl azide groups, tetrafluorophenyl azide groups, o-nitrophenyl azide groups, m-nitrophenyl azide groups, diazidopropylidin groups, azidomethylcoumarin groups, or psoralen groups. In embodiments, the photoactivated markers include phenyl azide groups. In embodiments, the photoactivated markers include o-hydroxyphenyl azide groups. In embodiments, the photoactivated markers include m-hydroxyphenyl azide groups. In embodiments, the photoactivated markers include tetrafluorophenyl azide groups. In embodiments, the photoactivated markers include o-nitrophenyl azide groups. In embodiments, the photoactivated markers include m-nitrophenyl azide groups. In embodiments, the photoactivated markers include diazidopropylidin groups. In embodiments, the photoactivated markers include azidomethylcoumarin groups. In embodiments, the photoactivated markers include psoralen groups. In embodiments, one or more of the listed markers may be explicitly excluded.

[0063] use

[0064] The methods and compositions described herein can be used in any application requiring three-dimensional tissue analysis. The following uses are illustrative only and are not intended to be limiting.

[0065] In the embodiments, the compositions and methods described herein can be used to detect heterogeneity in tumors or tumor samples (e.g., biopsies). Tumor samples from patients can be cleared as described herein. The tumor sample can then be contacted with a photoactivated label and subjected to one or more target regions by a multiphoton laser (e.g., a two-photon laser). Target regions may include individual regions within the tumor, as well as “normal” cells adjacent to the tumor. 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 or more steps prior to isolation or analysis, such as before or after clearing, before or after contact with the photoactivated label, etc. The DNA, RNA, and / or protein profiles determined from the analysis can be combined with imaging to provide a detailed map of the tumor. Analysis of tumor DNA, RNA, and / or proteins in different regions can allow the construction of a three-dimensional evolutionary model of tumor heterogeneity.

[0066] In the embodiments, the compositions and methods described herein can be used to identify immune cells (or the absence of immune cells) in a sample (e.g., a tumor sample). For example, one or more regions of the sample can be labeled, imaged, and isolated as described herein. The isolated regions can be analyzed, for example, for RNA and / or proteins expressed (preferred) by one or more target immune cells.

[0067] In the embodiments, the compositions and methods described herein can be used to identify the location of blood vessels in a sample (e.g., a tumor sample) and / or to 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 separated based on proximity to blood vessels. The separated regions can be analyzed, for example, for RNA and / or proteins expressed (preferentially expressed) by one or more target cells. Similarly, these methods can be used to analyze metastatic or suspected metastatic regions.

[0068] In the embodiments, the compositions and methods described herein can be used to locate sparse cell populations. Certain cell types are found in small numbers in certain tissues and / or are scattered in discrete regions within the tissue. The compositions and methods described herein can help identify / locate those cells or regions within a tissue.

[0069] In the embodiments, the compositions and methods described herein can be used to analyze specific cell types in a sample. For example, but not limited to, the spinal cord consists of multiple layers / regions of the dorsal root ganglia, each layer or region containing different cell types. The compositions and methods described herein can help identify differences (and / or similarities) between cells in various layers. Therefore, any tissue composed of multiple cell types can be analyzed.

[0070] In the embodiments, the compositions and methods described herein can be used to generate data, such as data on the expression (e.g., RNA or protein) or mutation (e.g., DNA) of target regions in a tissue or sample after 3D histological analysis of the tissue or sample.

[0071] In the embodiments, the compositions and methods described herein can be used to label and extract precise target regions within tissues or samples. For example, target regions can be labeled at a resolution of 1.6 x 1.6 x 3 μm (using a 20x lens).

[0072] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes thereof will be suggested to those skilled in the art and will be included within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0073] Example

[0074] Those skilled in the art will understand that the description of the preparation and use of the particles described herein is for illustrative purposes only, and this disclosure is not limited to such description.

[0075] Example 1. Dehydration and transparentization of tissues

[0076] Dehydration and tissue transparency

[0077] To harvest organs free of residual blood, mice were perfused with warm PBS / heparin (5 U / ml; 50–100 ml per mouse at steady pressure (gravity flow or Perfusion One system; 1–1.5 mH2O (Leica)) followed by perfusion with 2% PFA (Electron Microscopy Sciences, diluted in PBS). Depending on the permeability of the sample, the harvested organs were fixed overnight at 4°C in PFA. Each sample was incubated for X hours in 30% and 50% tert-butanol (pH = 9.5, RT) (where X is the time determined by Fick's diffusion law, with a reference of 24 hours for mouse brain incubation; T = (1 / [2D])r). 2 D = diffusion coefficient (inferred from mouse brain size and incubation time = 0.00015349), r = nearest distance to the center of the sample), followed by clearing in 70%, 80%, 96%, and 100% tert-butanol (pH = 9.5, 30°C), and finally in BABB (benzyl alcohol: benzyl benzoate, volume ratio 1:2, pH = 9.5, 30°C). After the final clearing step, the organ can be stored in BABB solution at 4°C for at least one year. Examples of cleared mouse brains incubated for approximately 24 hours per step following this protocol can be obtained. Figure 1Found it.

[0078] Stability test

[0079] To demonstrate that the above protocol did not alter cell stability and integrity, HEK293 cells were transfected with plasmids encoding EGFP, mKate2, tdTomato, or Venus, respectively. Cells were seeded on Millipore EZ slides, fixed with 4% PFA, and then treated according to the above protocol, with each incubation lasting 15 minutes. For mounting, BABB was used as the mounting medium, and glass coverslips were used to cover the cells. Cells were then imaged using a Leica SP8 vertical laser scanning microscope at wavelengths matched to the excitation / emission spectra of the fluorescent proteins. Control slides were not cleared and mounted using VectaShield mounting medium. Exemplary results from these stability experiments are shown in... Figure 2 middle.

[0080] Example 2. Two-photon labeling of tissues

[0081] To target the spatially confined activation of photoreactive compounds deep within tissues, samples were incubated with suitable compounds dissolved in BABB (12.5 μg / ml) at room temperature (RT) for the time described by Fick's diffusion law or overnight at 4°C. Using a pixel dwell time of 10 μs, a resolution of 512x512, a 20x lens (Leica HCX APO L 20x / 0.95 IMM), and a laser power of 75 mW at 700 nm, the compound was successfully conjugated to the target site under the lens. Based on the composition of photobiotin (biotin-linker-phenylazine; Sigma A1935-1MG), photoactivated aquamarine, Cy3, Cy5, and Alexa 488 were synthesized, readily soluble in pH-adjusted BABB (1 mg / ml (stock solution concentration)). Immediately after photolabeling, the samples were washed in BABB (pH = 9.5, 30°C) for the time determined by Fick's Law (by transferring the samples to the appropriate solutions), then washed in 100% tert-butanol (pH = 9.5, 30°C), followed by a final wash in 100% DMSO (pH = 9.5, 30°C), and then stored in 100% DMSO at 4°C until further processing.

[0082] Example 3. Isolation and purification of cell nuclei by light labeling

[0083] All equipment was treated with RNase. Photolabeled tissues stored in DMSO were rehydrated by incubation in PBS with an RNase inhibitor (Takara) on a shaker at 20°C for 30 minutes. Tissue fragments <1 mm were cut and transferred to lysis buffer (containing 975 μl divalent HBSS, 10 μl proteinase K, 10 μl RNase-free DNAse, and 0.2 U / ml RNase inhibitor). After incubation at 30°C for 15 minutes, the tissue fragments were transferred to a Dounce homogenizer, and 1 ml of HBSS+ solution (HBSS, 3% BSA (fatty acid-free), and 0.2 U / ml RNase inhibitor) was added before homogenization 5 to 10 times (avoiding air bubbles). The homogenate was filtered through a pre-wetted 70 μm filter and rotated at 300 rcf for 3 to 5 minutes. Discard the supernatant, resuspend the precipitate in HBSS+, grind with a 200 μl tip to break up the nucleus clumps, precipitate at 300 rcf, resuspend in 1 ml HBSS+, and then mix with an equal volume of cold 25% Optiprep (Sigma-Aldrich) before placing on ice. On ice, prepare a gradient in a 15 ml tube by adding 2 ml of 40% Optiprep to the bottom, transferring 2 ml of 25% Optiprep to the top, and carefully covering the nucleus mixture on top. Then rotate the tube at 2500 rcf for 20 min in a pre-chilled float-bucket rotor. Collect the nuclei located at the interface of the 25% and 40% Optiprep solutions (located under a fluffy, opaque layer of debris, possibly brownish and transparent) and resuspend in 3 volumes of HBSS+ before rotating at 250 rcf for 10 min. The precipitate was then washed with HBSS+, resuspended in HBSS+, and stained with an appropriate FACS (PI or DAPI) nuclear marker or streptavidin-linked dye to incorporate biotin. After incubation at room temperature for 5 minutes, the sample was centrifuged and resuspended in HBSS+. An unlabeled control was prepared for sorting, treated identically to the sample except for exposure to light for photoactivation and gating for both single nuclei (using nuclear markers) and light-labeled nuclei.

[0084] Example 4. Image Acquisition and Analysis

[0085] The transparent sample was mounted on an insect needle (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 laser and Leica BABB immersion lenses (HCX PLFLUOTAR 5x / 0.15IMM lens for low resolution, HCX APO L 20x / 0.95IMM lens for high resolution). The acquired Leica image containers were converted to Imaris containers (Imaris FileConverter 9.1.2, Bitplane) and transferred to a power workstation (dual Xeon E5-2687W v4, 1TB memory, GeForce Titan (Pascal)) for image analysis using Imaris 9.3.1 (Bitplane). Signal intensity was compensated using non-signal channels when necessary, and the image data was deconvolved using the adapthresh function in Matlab (MathWorks) or Huygens (Scientific Volume Imaging BV).

[0086] Example 5. SPLiT-seq of cleared nuclei from the spinal cord

[0087] Cleared nuclei were isolated from Cy3-PA-labeled mouse spinal cord, dye incorporation was classified, libraries were prepared using the SPLiT seq method, and RNA was sequenced using a MiSeq machine. Figure 7A The graph shows the count of unique barcodes versus the total number of detected barcodes. Two sub-libraries (nuclei isolated from fixed but not transparent tissue and nuclei isolated from both fixed and transparent tissue) were then mixed at a 2:1 ratio (fixed:transparent). Figure 7B As shown, the TPM counts for the two libraries were plotted and a linear correlation was displayed, indicating that the transparency process does not change the sequencing results. Figure 7C The image shows random regions from mouse chromosome 2, with readings generated from both fixed but not transparent and fixed and transparent mouse spinal cords.

[0088] Example 6. 3D Analysis of DNA / RNA / Protein Components in Mouse Lungs

[0089] A clearing protocol was used to clarify and image well-prepared mouse lung slices, and a photoactivating compound was added in situ (without tissue movement). Simultaneously, target regions were identified using previously generated data, photoactivation was programmed, and photoactivation was performed. The tissue was then removed, washed, and rehydrated. The tissue could then be processed by extracting cell nuclei using a Dounce homogenizer and then targeting and separating nuclei stained with the relevant compound (e.g., streptavidin when luciferin was used as the photoactivating compound). The cleared nuclei were isolated from the light-labeled lung, stained with a streptavidin-conjugated dye, and classified for dye incorporation. The library was prepared according to the SPLiT seq method, and RNA was sequenced using an appropriate method. Alternatively, RNA can be analyzed by any method, including RT-PCR, electrophoresis, etc.

[0090] The process is similar for DNA sequencing; after isolating and sorting the cell nucleus, the DNA can be sequenced using appropriate methods. DNA can also be analyzed using, for example, PCR, electrophoresis, or any other suitable method.

[0091] For protein analysis, mass spectrometry readout (e.g., liquid chromatography / mass spectrometry) or other suitable protein analysis methods can be used to determine the composition of proteins incorporating PA compounds. Other protein analysis methods may include electrophoresis, Western blotting, Edman degradation, or other protein sequencing.

[0092] Example 7. 3D Analysis of DNA / RNA / Protein Components in the Spinal Cord

[0093] Figure 8A This is a cross-sectional view of the mouse spinal cord, showing different regions of the spinal cord, with the dorsal layer indicated by colored circles. In the magnified smaller image, areas subjected to light activation are highlighted. Note that in this example, the PA region is not further refined—this is possible because resolution is related to the optical resolution of the lens used. For example, a 20x lens would provide a resolution of 0.89μm x 0.89μm x 1.6μm.

[0094] Figure 8B FACS plots of cleared but not photoactivated cell nuclei isolated from the mouse spinal cord using the procedure described herein are shown. The Y-axis shows the signal of DAPI (a nuclear counterstain), while the X-axis shows the PA dye (Cy3-PA). Boxes indicate individual cell nuclei that are negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right).

[0095] Figure 8C This shows a transparent and photoactivated cell nucleus (e.g., isolated from the mouse spinal cord) Figure 9AThe image shows an FACS plot. The Y-axis represents the signal of DAPI (a nuclear counterstain), while the X-axis represents the PA dye (Cy3-PA). Boxes indicate whether a single cell nucleus is negative for incorporated Cy3 (left) or positive for incorporated Cy3 (right).

[0096] Example 8. SPLiTseq of cleared nuclei from the spinal cord

[0097] To demonstrate that cleared tissue can be analyzed using SPLiT-seq, mouse spinal cord was cleared as described in Example 1. Cell nuclei were isolated from the cleared tissue and analyzed using SPLiT-seq. Figure 10A and 10B As shown, the SPLiT-seq expression profile reveals the expression of markers of the expected cell types present in clear tissue, including neurons, oligodendrocytes, and astrocytes. The RNA expression profile was determined based on an RNA expression database. Figure 10A The nFeature plot (left) and nCount plot (right) show RNA expression levels. Figure 10B The UMAP diagram showing RNA expression levels is presented.

[0098] References

[0099] 1.Olson,E.,Levene,M.and Torres,R.(2016).Multiphoton microscopy withclearing for three dimensional histology of kidney biopsies.Biomedical OpticsExpress,7(8),p.3089.

[0100] 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 spatialresolution.Science,363(6434),pp.1463-1467.

[0101] 3.Schwarz,M.K.,Scherbarth,A.,Sprengel,R.,Engelhardt,J.,Theer,P.,&Giese,G.(2015).Fluorescent-protein stabilization and high-resolution imagingof cleared,intact mouse brains.PLoS ONE,10,1–26。

[0102] 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-celltranscriptional states.Science,361(6400),p.eaat5691。

Claims

1. A method for marking a region of tissue or a tissue sample, comprising: a) Provide three-dimensional tissue or tissue samples; b) Make the sample transparent; c) Contact the tissue or tissue sample with the photoactivated label; d) Image the tissue or tissue sample to create an image; e) Exposing a region of the tissue or tissue sample to multiphoton laser light to mark the region; f) Separate the labeled region or a portion of the labeled region from the tissue or tissue sample; g) Analyze the composition of the separated labeled regions to generate analytical data; h) Combine the image with the analysis data to create a three-dimensional composition map of the region.

2. The method according to claim 1, wherein the multiphoton laser is a two-photon laser or a three-photon laser.

3. The method according to claim 1 or 2, wherein the region comprises a cell, a subcellular compartment, an aggregate, or a secreted aggregate.

4. The method according to claim 3, wherein the subcellular compartment is the cell nucleus.

5. The method according to any one of claims 1 to 4, wherein the tissue or tissue sample is a fixed tissue or a fixed tissue sample.

6. The method according to any one of the preceding claims, wherein the photoactivated label comprises one or more of the following: (a) Detectable portion; and (b) Label.

7. The method of claim 6, wherein the detectable portion comprises: (i) The luminescent part; Or (ii) antibodies or their functional derivatives.

8. The method according to claim 7, wherein the light-emitting portion is selected from: (i) Fluorescent portion; (ii) Chemiluminescence component; (iii) Bioluminescent components; and (iv) Electrochemiluminescence component.

9. The method of claim 8, wherein the fluorescent portion is a fluorophore.

10. The method of claim 6, wherein the label is selected from: (i) Affinity label; (ii) Tablet labels; (iii) Fluorescent tags; (iv) Oligonucleotide tags; and (v) Biotin label.

11. The method according to any one of claims 1 to 10, wherein the sample clearing process comprises dehydrating the sample and transferring the sample to a culture medium having a refractive index similar to or matching that of the tissue.

12. The method of claim 11, wherein the refractive index is between 1.3 and 1.

6.

13. The method according to claim 11, wherein the culture medium comprises a solution of benzyl alcohol, benzyl benzoate, BABB or a derivative thereof, and may or may not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or Quadrol.

14. The method of claim 11, wherein the culture medium comprises a BABB solution.

15. The method according to any one of claims 1 to 14, wherein the sample is dehydrated by a tert-butanol solution.

16. The method of claim 15, wherein the tert-butanol solution comprises trimethylamine, tetrahydrofuran, ethanol, or methanol.

17. The method according to any one of claims 1 to 16, wherein the photoactivated label is hydrophobic.

18. The method according to any one of claims 1 to 17, wherein the photoactivated label comprises a phenyl azide group, an o-hydroxyphenyl azide group, a m-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an o-nitrophenyl azide group, a m-nitrophenyl azide group, a diazidopropylidin group, an azidemethylcoumarin group, or a psoralen group.

19. The method of claim 18, wherein the photoactivated label comprises an aryl azide group.

20. The method according to any one of claims 1-19, wherein the marked region or portion is separated by FACS sorting for the marked area.

21. The method according to any one of claims 1-20, wherein the analysis comprises determining the DNA, RNA and / or protein composition of the isolated labeled region.

22. The method of claim 21, wherein the RNA composition is analyzed by sequencing.

23. The method of claim 21, wherein the RNA composition is analyzed by SPLIT-seq.

24. The method of claim 21, wherein the RNA composition is analyzed by single-cell RNA sequencing (scRNAseq).

25. The method according to any one of claims 21-24, wherein the protein composition is analyzed by mass spectrometry.

26. The method according to any one of claims 21-25, wherein the DNA composition is analyzed by DNA sequencing.

27. A method for three-dimensional expression profiling analysis of intact tissues or tissue samples, comprising: a) Provide a complete three-dimensional tissue or tissue sample; b) Make the sample transparent; c) Contact the tissue or tissue sample with the photoactivated label; d) Subjecting a region of the tissue or tissue sample to multiphoton laser light to mark the region; e) Image the marked area to create an image; f) Isolate the labeled region from the tissue or tissue sample; g) Determine the DNA, RNA, and / or protein composition of the isolated labeled regions; and h) Combine the image with the DNA, RNA and / or protein composition of the isolated labeled regions to create a three-dimensional expression profile of the intact tissue or tissue sample.

28. The method according to claim 27, wherein the multiphoton laser is a two-photon laser or a three-photon laser.

29. The method according to claim 27 or 28, wherein the region is a cell, a subcellular compartment, an aggregate, or a secreted aggregate.

30. The method according to any one of claims 27 to 29, wherein the separated labeled region is a single cell nucleus or a single cell.

31. The method according to any one of claims 27 to 30, wherein the RNA composition of the isolated labeled region is determined.

32. The method of claim 31, wherein the RNA composition is determined by sequencing.

33. The method of claim 31, wherein the RNA composition is determined by SPLIT-seq.

34. The method according to any one of claims 27 to 33, wherein the protein composition of the isolated labeled region is determined.

35. The method of claim 34, wherein the protein composition is determined by mass spectrometry.

36. The method according to any one of claims 27 to 35, wherein the DNA composition of the isolated labeled region is determined.

37. The method of claim 36, wherein the DNA composition is determined by DNA sequencing.

38. The method according to any one of claims 27 to 37, wherein the tissue or tissue sample is a fixed tissue or a fixed tissue sample.

39. The method according to any one of claims 27 to 38, wherein the photoactivated marker comprises one or more of the following: (a) Detectable portion; or (b) Tag.

40. The method of claim 39, wherein the detectable portion comprises: (i) the luminous part; or (ii) Antibodies or their functional derivatives.

41. The method of claim 40, wherein the light-emitting portion is selected from: (i) Fluorescent portion; (ii) Chemiluminescence component; (iii) Bioluminescent components; and (iv) Electrochemiluminescence component.

42. The method of claim 41, wherein the fluorescent portion is a fluorophore.

43. The method of claim 39, wherein the label is selected from: (i) Affinity label; (ii) Tablet labels; (iii) Fluorescent tags; (iv) Oligonucleotide tags; and (v) Biotin label.

44. The method according to any one of claims 27 to 43, wherein the sample clearing process comprises dehydrating the sample and transferring the sample to a culture medium having a refractive index similar to or matching that of the tissue.

45. The method according to claim 44, wherein the culture medium comprises a solution of benzyl alcohol, benzyl benzoate, BABB or a derivative thereof, and may or may not contain one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol and / or Quadrol.

46. ​​The method of claim 44, wherein the culture medium comprises a BABB solution.

47. The method according to any one of claims 27 to 46, wherein the sample is dehydrated by a tert-butanol solution.

48. The method of claim 47, wherein the tert-butanol solution comprises trimethylamine, tetrahydrofuran, ethanol, or methanol.

49. The method according to any one of claims 27 to 48, wherein the photoactivated label is hydrophobic.

50. The method according to any one of claims 27 to 49, wherein the photoactivated label comprises a phenyl azide group, an o-hydroxyphenyl azide group, a m-hydroxyphenyl azide group, a tetrafluorophenyl azide group, an o-nitrophenyl azide group, a m-nitrophenyl azide group, a diazidopropylidin group, an azide-methylcoumarin group, or a psoralen group.

51. The method of claim 50, wherein the photoactivated label comprises an aryl azide group.

52. The method of claim 30, wherein the labeled cells or nuclei are separated by FACS sorting for the label.

53. The method according to any one of claims 1 to 52, further comprising contacting the tissue or tissue sample with a second photoactivated label and subjecting a second cell or nucleus in the tissue or tissue sample to a multiphoton laser to label the second cell or nucleus.

54. The method according to any one of claims 1 to 53, wherein the tissue or tissue sample is a whole organ, a tumor, or an animal.

55. The method according to any one of claims 1 to 54, wherein the wavelength of each photon in the two-photon laser is between 175 nm and 350 nm.

56. The method according to any one of claims 1 to 55, wherein the tissue or tissue sample is derived from a tumor.

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