Highly-multiplexed fluorescent imaging

Abstract

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 - 26 . (canceled) 
     
     
         27 . A system for analyzing a sample, comprising a processor and a computer-readable medium comprising instructions that, when executed by the processor, cause the controller to analyze the sample, wherein the analysis comprises:
 (a) labeling the sample with a plurality of capture agents;   (b) specifically hybridizing a first sub- set of a plurality of labeled nucleic acid probes with the sample;   (c) reading the sample to obtain an image showing binding pattern for each of the probes hybridized in step (b);   (d) inactivating or removing the labels that are associated with the sample in (b); and   (e) repeating steps (b) and (c) multiple times with a different sub-set of the plurality of the labeled nucleic acid probes, each repeat followed by step (d) except for the final repeat.   
     
     
         28 . The system of  claim 27 , further comprising a sample well, wherein the sample well comprises sample. 
     
     
         29 . The system of  claim 27 , further comprising an auto-sampler. 
     
     
         30 . The system of  claim 29 , wherein the auto-sampler is configured to label the sample with the plurality of capture agents. 
     
     
         31 . The system of  claim 27 , wherein the hybridizing of (b) is for a period of about 2 minutes. 
     
     
         32 . The system of  claim 27 , wherein step (e) comprises repeating steps (b) and (c) 2 to 20 times. 
     
     
         33 . The system of  claim 27 , wherein the plurality of capture agents is at least 10 capture agents. 
     
     
         34 . The system of  claim 27 , wherein each of the subsets is independently 2 to 4 labeled nucleic acid probes. 
     
     
         35 . The system of  claim 27 , wherein the probes are removed in step (d) by denaturation. 
     
     
         36 . The system of  claim 35 , wherein the probes are removed in step (d) by incubating the sample in 70% to 90% formamide for a period of at least 1 minute, followed by a wash.