Processing high density analyte arrays
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. A method for processing or analyzing one or more analytes of a plurality of analytes, comprising:
(a) providing said plurality of analytes immobilized adjacent to a substrate at a density of at least 1 molecule per square micrometer (μm 2 ), wherein at least 10% of adjacent analytes of said plurality of analytes are separated from one another by a center-to-center distance of less than or equal to 500 nanometers (nm);
(b) obtaining a plurality of optical signals over a plurality of cycles comprising a plurality of probes binding to said one or more analytes of said plurality of analytes immobilized adjacent to said substrate; and
(c) processing at least one optical signal of said plurality of optical signals to distinguish an analyte of said plurality of analytes from another analyte of said plurality of analytes separated by less than said center-to-center distance of less than 500 nm, to identify said analyte of said plurality of analytes.
2. The method of claim 1 , wherein (b) further comprises overlaying said at least one optical signal of said plurality of optical signals with an additional optical signal of said plurality of optical signals to generate an overlay of said optical signal and said additional optical signal.
3. The method of claim 2 , wherein (c) further comprises applying an optical distribution model to said overlay to identify said one or more analytes of said plurality of analytes.
4. The method of claim 2 , further comprising using said overlay to generate an oversampled image identifying said at least one optical signal.
5. The method of claim 1 , wherein said at least one optical signal is obtained by an optical imaging system having a resolution of less than or equal to one pixel per 250 nanometers.
6. The method of claim 1 , wherein said one or more analytes comprises a nucleic acid molecule.
7. The method of claim 6 , wherein said nucleic acid molecule comprises at least 1 kilo-base (kb).
8. The method of claim 6 , wherein said nucleic acid molecule is a concatemer.
9. The method of claim 1 , wherein said one or more analytes comprises a protein or polypeptide.
10. The method of claim 1 , wherein said plurality of optical signals comprises a fluorescent signal.
11. The method of claim 1 , wherein said plurality of probes comprises a labelled nucleotide, an aptamer, an antibody, a polypeptide, an oligonucleotide, or any combination thereof.
12. The method of claim 1 , wherein said plurality of probes comprises a detectable label that is indirectly bound to, hybridized to, conjugated to, or covalently linked to a probe of said plurality of probes.
13. The method of claim 12 , wherein said plurality of probes comprises a labelled nucleotide.
14. The method of claim 12 , wherein said detectable label comprises a fluorophore or a chemiluminescent moiety.
15. The method of claim 1 , wherein said density is at least 2 molecules/μm 2 .
16. The method of claim 1 , wherein said density is at least 4 molecules/μm 2 .
17. The method of claim 1 , wherein said density is at least 6 molecules/μm 2 .
18. The method of claim 1 , wherein said density is at least 8 molecules/μm 2 r.
19. The method of claim 1 , wherein said density is at least 10 molecules/μm 2 .
20. The method of claim 1 , wherein said density is at least 12 molecules/μm 2 .
21. The method of claim 1 , wherein said density is at least 15 molecules/μm 2 .
22. The method of claim 1 , wherein said density is at least 20 molecules/μm 2 .
23. The method of claim 1 , wherein said substrate is a flow cell.
24. The method of claim 1 , wherein said plurality of signals is obtained by an optical imaging system at a resolution of at least one pixel per 300 nm.
25. The method of claim 1 , wherein said center-to-center distance is less than or equal to 400 nm.
26. The method of claim 1 , wherein said center-to-center distance of less than 300 nm.
27. The method of claim 1 , wherein said center-to-center distance of less than 200 nm.
28. The method of claim 1 , further comprising determining a position of said one or more analytes with an accuracy of within 10 nm root mean square (RMS).
29. The method of claim 28 , wherein said position is a relative position.
30. The method of claim 1 , wherein said processing comprises deconvolving said plurality of optical signals to distinguish said analyte from said another analyte.