Highly-multiplexed fluorescent imaging
Provided herein is a method and system for analyzing a sample. In some embodiments the method makes use of a plurality of capture agents that are each linked to a different oligonucleotide and a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides. The sample is labeled with the capture agents en masse, and sub-sets of the capture agents are detected using iterative cycles using corresponding subsets of the labeled nucleic acid probes.
1. A method for analyzing a sample comprising fixed cells, comprising the following steps, performed in order:
(a) obtaining:
i. a plurality of capture agents that are each linked to a different oligonucleotide; and
ii. a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides of (a)(i);
(b) labeling the sample comprising fixed cells with the plurality of capture agents of (a)(i);
(c) specifically hybridizing a first sub-set of the labeled nucleic acid probes of (a)(ii) with the sample comprising fixed cells, wherein the probes in the first sub-set are distinguishably labeled, to produce labeled probe/oligonucleotide duplexes;
(d) reading the sample comprising fixed cells to obtain an image showing binding pattern for each of the probes hybridized in step (c);
(e) completely removing the probes hybridized in step (c) from the sample comprising fixed cells by denaturation by incubating the sample in 70%-90% of a chemical denaturant in the absence of heating, wherein the chemical denaturant is DMSO or formamide and the denaturation is not by displacing the DNA probes using displacement probes, leaving the plurality of capture agents of (b) and their associated oligonucleotides still bound to the sample comprising fixed cells; and
(f) repeating steps (c) and (d) multiple times with a different sub-set of the labeled nucleic acid probes of (a)(ii), each repeat followed by step (e) except for the final repeat, to produce a plurality of images of the sample comprising fixed cells, each image corresponding to a different sub-set of labeled nucleic acid probes used in (c).
2. The method of claim 1 , wherein the sample is a planar cellular sample.
3. The method of claim 1 , wherein the oligonucleotides of (a)(i) are at least 5 nucleotides in length.
4. The method of claim 1 , wherein the labeled nucleic acid probes of (a)(ii) are at least 5 nucleotides in length.
5. The method of claim 1 , wherein sequences of the oligonucleotides to which the capture agents of (a)(i) are linked are: i. longer than sequences of the labeled nucleic acid probes of (a)(ii) and ii. otherwise identical to one other except for a sub-sequence that is complementary to a single labeled nucleic acid probe of (a)(ii).
6. The method of claim 1 , wherein the probe/oligonucleotide duplexes of (c) have a T m of at least 15° C.
7. The method of claim 1 , wherein the hybridizing of (c) is for a period of time of about 2 minutes.
8. The method of claim 1 , wherein each labeled nucleic acid probe has a sequence selected from SEQ ID NOS: 1-47, or a complement thereof.
9. The method of claim 1 , wherein each oligonucleotide linked to a capture agent is selected from SEQ ID NOS: 48-94, or a complement thereof.
10. The method of claim 1 , wherein the plurality of capture agents is at least 10 capture agents.
11. The method of claim 1 , wherein each different sub-set is independently 2 to 4 labeled nucleic acid probes.
12. The method of claim 1 , wherein the probes are removed in step (e) by incubating the sample in 70% to 90% formamide for a period of at least 1 minute, followed by a wash.
13. The method of claim 1 , wherein step (f) comprises repeating steps (c) and (d) 2 to 20 times.
14. The method of claim 1 , further comprising analyzing at least two of the images.
15. The method of claim 14 , wherein the analyzing comprises comparing or overlaying at least two images of the plurality of images.
16. The method of claim 1 , further comprising overlaying all of the plurality of images to produce an image showing the pattern of binding of all of the plurality capture agents to the planar sample.
17. The method of claim 1 , wherein the labeled nucleic acid probes are fluorescently labeled.
18. The method of claim 1 , wherein reading is done by fluorescence microscopy.
19. The method of claim 1 , wherein the capture agent is antibody or aptamer.
20. The method of claim 1 , wherein the planar sample is a formalin-fixed, paraffin-embedded (FFPE) section.
21. The method of claim 1 , wherein (f) comprises repeating steps (c) and (d) at least 5 times with the different sub-set of the labeled nucleic acid probes of (a)(ii).