Sequencing and high resolution imaging
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.
1. A method for processing or analyzing a plurality of analytes, comprising:
a. providing said plurality of analytes on a substrate, wherein said plurality of analytes is provided on said substrate at a density, wherein a minimum effective pitch between an analyte of said plurality of analytes and an adjacent analyte of said plurality of analytes is less than λ/(2*NA), wherein a plurality of optical signals obtained by an optical imaging system have a wavelength λ, wherein said plurality of optical signals is generated from a plurality of probes over a plurality of cycles of said plurality of probes binding to one or more analytes of said plurality of analytes, and wherein NA comprises a numerical aperture of said optical imaging system;
b. obtaining said plurality of optical signals from said plurality of probes over said plurality of cycles of said plurality of probes binding to said one or more analytes of said plurality of analytes, wherein at least a subset of said plurality of optical signals overlap;
c. processing said plurality of optical signals to identify a position of said one or more analytes of said plurality of analytes; and
d. using said position to identify said one or more analytes of said plurality of analytes.
2. The method of claim 1 , wherein (b) further comprises overlaying said plurality of optical signals from said plurality of probes over said plurality of cycles of said plurality of probes binding to said one or more analytes of said plurality of analytes to generate an overlay of said plurality of optical signals, and wherein (c) further comprises applying an optical distribution model to said overlay of said plurality of optical signals to determine said position of said one or more analytes of said plurality of analytes.
3. The method of claim 2 , wherein said optical distribution model comprises a Gaussian distribution.
4. The method of claim 1 , wherein said processing of said plurality of optical signals comprises applying a resolving function to an image of said plurality of optical signals.
5. The method of claim 4 , wherein said resolving function comprises nearest neighbor variable regression.
6. The method of claim 4 , wherein said resolving function comprises removing interfering optical signals from one or more neighboring analytes using a center-to-center distance between said one or more neighboring analytes.
7. The method of claim 1 , wherein said plurality of analytes is provided on said substrate at a density of at least 1 molecule per square micrometer.
8. The method of claim 7 , wherein said plurality of analytes is provided on said substrate at a density of at least 4 molecules per square micrometer.
9. The method of claim 7 , wherein said plurality of analytes is provided on said substrate at a density of at least 6 molecules per square micrometer.
10. The method of claim 7 , wherein said plurality of analytes is provided on said substrate at a density of at least 8 molecules per square micrometer.
11. The method of claim 7 , wherein said plurality of analytes is provided on said substrate at a density of at least 12 molecules per square micrometer.
12. The method of claim 1 , wherein said optical imaging system comprises a resolution of one pixel per 250 nanometers or lower.
13. The method of claim 1 , wherein an oversampled image is generated from said plurality of optical signals with a higher pixel density from a field image for said plurality of cycles of said plurality of probes binding to said one or more analytes of said plurality of analytes provided on said substrate.
14. 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.
15. The method of claim 14 , wherein said plurality of probes comprises a labelled nucleotide.
16. The method of claim 1 , wherein said plurality of probes comprise a detectable label that is indirectly bound to, hybridized to, conjugated to, or covalently linked to said plurality of probes.
17. The method of claim 16 , wherein said detectable label comprises a fluorophore or a chemiluminescent molecule.
18. The method of claim 1 , wherein said wavelength of light of said plurality of optical signals is 400 nanometers (nm)-700 nm.
19. The method of claim 1 , wherein said N. A. is 0.2-2.0.
20. The method of claim 1 , wherein said plurality of optical signals comprises a fluorescent signal.
21. The method of claim 20 , wherein said nucleic acid molecule comprises 1 kilo-base (kb) 100 kb.
22. The method of claim 1 , wherein at least one analyte of said plurality of analytes comprises a nucleic acid molecule.
23. The method of claim 1 , wherein at least one analyte of said plurality of analytes comprises a protein or polypeptide.
24. The method of claim 1 , wherein said position is a relative position with respect to another analyte of said one or more analytes of said plurality of analytes.