SEQUENCING AND HIGH RESOLUTION IMAGING
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. There have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy or polynucleotide sequencing applications.
1 .- 20 . (canceled)
21 . A method of identifying an analyte, comprising:
(a) binding a first probe to a first analyte to generate a first optical signal;
(b) detecting said first optical signal with an imaging system, wherein said imaging system detects said first optical signal from said substrate with a resolution of up to about 300 nanometers per pixel of said imaging system; and
(c) using said detected first optical signal to identify said first analyte.
22 . The method of claim 21 , further comprising binding a second probe to a second analyte to generate a second optical signal.
23 . The method of claim 22 , wherein said first probe is bound to said first analyte in a first cycle, and wherein said second probe is bound to said second analyte in a second cycle.
24 . The method of claim 22 , wherein said detecting comprises detecting said first optical signal and said second optical signal with said imaging system.
25 . The method of claim 22 , wherein said first optical signal and said second optical signal are overlapping.
26 . The method of claim 22 , wherein said first analyte and said second analyte are different analytes.
27 . The method of claim 22 , wherein said first analyte and said second analyte are the same analyte.
28 . The method of claim 22 , wherein said first optical signal or said second optical signal comprises a fluorescent optical signal.
29 . The method of claim 22 , wherein said first analyte or said second analyte comprises a nucleic acid molecule.
30 . The method of claim 29 , wherein said nucleic acid molecule comprises at least 1 kilo-base (kb).
31 . The method of claim 22 , wherein said first analyte or said second analyte comprises a protein or a polypeptide.
32 . The method of claim 22 , wherein said first probe or said second probe comprises a labelled nucleotide, an aptamer, an antibody, a polypeptide, an oligonucleotide, or any combination thereof.
33 . The method of claim 22 , wherein said first analyte or said second analyte is immobilized to said substrate.
34 . The method of claim 22 , wherein said first analyte or said second analyte comprises a plurality of analytes.
35 . The method of claim 34 , wherein said plurality of analytes are provided on said substrate at a density of at least 2 molecules/μm 2 .
36 . The method of claim 34 , wherein said plurality of analytes are provided on said substrate at a density of at least 4 molecules/μm 2 .
37 . The method of claim 34 , wherein said plurality of analytes are provided on said substrate at a density of at least 8 molecules/μm 2 .
38 . The method of claim 22 , wherein said first optical signal or said second optical signal comprises a sequence of optical signals.
39 . The method of claim 38 , wherein said sequence of optical signals are determined or obtained over a plurality of cycles.