Spatial-dependent analysis of biological material from intact tissue samples
Biological research requires isolation and analysis of material, for example, RNA, DNA and protein, from tissue samples. The methods and compositions described herein allow for high resolution imaging of large and intact tissue samples, and subsequent isolation of material in a precise and location dependent-manner. The methods and compositions described herein may be used, for example, for biomarker discovery, identification of cell populations, pathology analysis, and generation of expression data in specific regions of interest.
1 . A method for 3-dimensional expression profiling of an intact tissue or tissue sample, comprising:
(a) providing a three-dimensional intact tissue or tissue sample;
(b) clearing the sample;
(c) contacting the tissue or tissue sample with a photo-activatable label;
(d) subjecting a region of the tissue or tissue sample to a multi-photon laser, thereby labeling the region;
(e) imaging the labeled region to create an image;
(f) isolating the labeled region from the tissue or tissue sample;
(g) determining a 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 3-dimensional expression profile of the intact tissue or tissue sample.
2 . The method of claim 1 , wherein the region is a cell, a subcellular compartment, an aggregate, or a secreted aggregate.
3 . The method of claim 2 , wherein the subcellular compartment is a nucleus.
4 . The method of claim 1 , wherein
the RNA composition is determined by sequencing, single cell RNA sequencing (scRNAseq), or SPLITseq, the protein composition is determined by mass spectrometry, and/or the DNA composition is determined by DNA sequencing.
5 . The method of claim 1 , wherein the photo-activatable label comprises one or more of:
(a) a detectable moiety;
(b) a light emitting moiety;
(c) fluorescent moiety;
(d) a chemiluminescent moiety:
(e) a bioluminescent moiety;
(f) an electrochemiluminescent moiety;
(g) a fluorophore;
(h) an antibody or a functional derivative thereof;
(i) a tag;
(i) an affinity tag;
(k) an epitope tag;
( 1 ) a fluorescent tag;
(m) an oligonucleotide tag; and
(n) a biotin tag.
6 . The method of claim 1 , wherein the sample clearing process comprises dehydrating the sample and transferring the sample into a medium with a refraction index:
(a) similar to the tissue;
(b) matching the tissue; or
(c) between about 1.3 and about 1.6.
7 . The method of claim 6 , wherein the medium comprises a solution of Benzyl Alcohol, Benzyl Benzoate (BABB) or derivative thereof, with or without one or more of triethylamine, diphenyl ether, dibenzyl ether, α-tocopherol, and/or N,N,N′,N′-Tetrakis (2-hydroxypropyl) ethylenediamine Quadrol.
8 . The method of claim 7 , wherein the medium comprises a BABB solution.
9 . The method of claim 1 , wherein the photo-activatable label is hydrophobic.
10 . The method of claim 1 , wherein the photo-activatable label comprises a phenyl azide group, an ortho-hydroxyphenyl azide group, a meta-hydroxyphenyl azide group, a tertrafluorophenyl azide group, an ortho-nitrophenyl azide group, a meta-nitrophenyl azide group, a diazirine group, an azido-methylcoumarin group, or a psoralen group.
11 . The method of claim 1 , wherein the multi-photon laser is a two-photon laser or a three-photon laser.
12 . The method of claim 1 , wherein the tissue or tissue sample is a fixed tissue or fixed tissue sample.
13 . The method of claim 1 , wherein the sample is dehydrated by a tert-butanol solution or a tert-butanol solution comprising trimethylamine, tetrahydrofuran, ethanol, or methanol.
14 . The method of claim 1 , wherein the labeled region is isolated by FACS sorting for the label.
15 . The method of claim 1 , wherein the isolated labeled region is a single nucleus or a single cell.