ANALYZING CELL SAMPLES
Disclosed herein, inter alia, are methods and compositions useful for detecting multiple biological targets.
1 . A method of analyzing a sample, the method comprising:
depositing a sample comprising a cell on a solid support;
contacting the sample with a first probe and a second probe, thereby forming a first complex and a second complex, wherein
the first complex comprises the first probe bound to a first organelle, wherein said first probe comprises a first fluorescent dye attached to the first probe via a cleavable linker;
the second complex comprises the second probe bound to a second organelle, wherein said second probe comprises a second fluorescent dye attached to the second probe via a covalent linker;
directing an excitation light to sample and detecting an emission light from the first fluorescent dye; and
cleaving the cleavable linker and the directing an excitation light to sample and detecting an emission light from the second fluorescent dye.
2 . The method of claim 1 , wherein the first fluorescent dye and the second fluorescent dye are spectrally distinct.
3 . The method of claim 1 , wherein the first fluorescent dye and the second fluorescent dye are spectrally identical.
4 . The method of claim 1 , wherein cleaving comprises contacting the cleavable linker with a cleaving agent.
5 . The method of claim 4 , wherein the cleaving agent is a reducing agent.
6 . The method of claim 4 , wherein the cleavable linker is a hydrazone linker, disulfide linker, azido linker, β-glucuronide linker, para-aminobenzyl alcohol (PABA) linker, phosphoramidate linker, maleimide ether linker, or triglycine (Gly-Gly-Gly) linker.
7 . The method of claim 4 , wherein the cleavable linker comprises two or more cleavable sites.
8 . The method of claim 1 , wherein the cleavable linker is a chemically cleavable linker, enzymatically cleavable linker, photo-cleavable linker.
9 . The method of claim 1 , wherein the first organelle is actin and the second organelle is a mitochondria.
10 . The method of claim 1 , wherein the first organelle is an endoplasmic reticulum and the second organelle is a mitochondria.
11 . The method of claim 1 , wherein the sample is frozen tissue.
12 . The method of claim 1 , wherein the sample is Formalin-Fixed Paraffin-Embedded tissue.
13 . The method of claim 1 , wherein the first probe and the second probe are each an antibody.
14 . The method of claim 1 , wherein the first probe comprises phalloidin, wheat germ agglutinin, concanavalin A, or annexin V.
15 . The method of claim 1 , further comprising detecting a nucleic acid molecule in the sample by binding a third probe to the nucleic acid molecule, wherein said third probe is an oligonucleotide, and detecting a sequence of the oligonucleotide.
16 . The method of claim 15 , wherein the nucleic acid molecule is outside of the cell.
17 . The method of claim 1 , further comprising detecting a protein in the sample by binding a third probe to the protein, wherein said third probe is an antibody-oligonucleotide conjugate, and detecting the oligonucleotide of the antibody-oligonucleotide conjugate.
18 . The method of claim 1 , further comprising determining the morphology of the cell based on the detected emissions.
19 . A device configured to analyze a sample, said device comprising
a solid support comprising the sample, wherein the sample comprises a cell;
a first probe bound to a first organelle in the cell, wherein said first probe comprises a first fluorescent dye attached to the first probe via a cleavable linker;
a second probe bound to a second organelle in the cell, wherein said second probe comprises a second fluorescent dye attached to the second probe via a covalent linker;
a light source configured to provide excitation light having a wavelength between 200 nm to 1500 nm; and
an image sensor configured to detect fluorescent emissions from the sample;
wherein the solid support forms part of a flow cell.
20 . The device of claim 19 , wherein the solid support comprises polymerized units of alkoxysilyl methacrylate, alkoxysilyl acrylate, alkoxysilyl methylacrylamide, alkoxysilyl methylacrylamide, or a copolymer thereof.