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 immobilized adjacent to said substrate at a density of at least 1 molecule per square micrometer, wherein at least 10% of said plurality of analytes are separated from one another by less than λ/(2*N.A.), 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 immobilized adjacent to said substrate, and wherein N.A. 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 immobilized adjacent to said substrate; and
(c) processing at least one optical signal of said plurality of optical signals to identify said one or more analytes 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 from 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 at least one optical signal of said plurality of optical signals to identify said one or more analytes of said plurality of analytes.
3. The method of claim 2 , further comprising generating an oversampled image from said at least one optical signal of 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.
4. The method of claim 2 , wherein said optical distribution model comprises a Gaussian distribution.
5. The method of claim 1 , wherein said plurality of analytes is immobilized adjacent to said substrate at a density of at least 2 molecules per square micrometer.
6. The method of claim 1 , wherein said plurality of analytes is immobilized adjacent to said substrate at a density of at least 4 molecules per square micrometer.
7. The method of claim 1 , wherein said at least one optical signal is obtained by said optical imaging system at a resolution of one pixel per 250 nanometers or lower.
8. The method of claim 1 , wherein at least one analyte of said plurality of analytes is a nucleic acid molecule.
9. The method of claim 8 , wherein said nucleic acid molecule comprises 1 kilo-base (kb)-100 kb.
10. The method of claim 1 , wherein at least one analyte of said plurality of analytes is a protein or polypeptide.
11. The method of claim 1 , wherein said plurality of optical signals comprises a fluorescent signal.
12. 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.
13. The method of claim 12 , wherein said plurality of probes comprises a labelled nucleotide.
14. 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.
15. The method of claim 14 , wherein said detectable label comprises a fluorophore or a chemiluminescent molecule.
16. The method of claim 1 , wherein said wavelength λ is 400 nanometers (nm)-700 nm.
17. The method of claim 1 , wherein said N.A. is 0.2-2.0.
18. The method of claim 1 , wherein said plurality of analytes is immobilized adjacent to said substrate at a density of at least 6 molecules per square micrometer.
19. The method of claim 1 , wherein said plurality of analytes is immobilized adjacent to said substrate at a density of at least 8 molecules per square micrometer.
20. The method of claim 1 , wherein said plurality of analytes is immobilized adjacent to said substrate at a density of at least 12 molecules per square micrometer.