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. A method for processing or analyzing a plurality of analytes, comprising:
(a) providing said plurality of analytes on a substrate;
(b) obtaining a plurality of optical signals from a plurality of probes over one or more cycles of said plurality of probes binding to one or more analytes of said plurality of analytes;
(c) determining a first peak intensity of a first analyte of said plurality of analytes at a first peak location and determining a second peak intensity of a second analyte of said plurality of analytes at a second peak location, wherein said first peak location and said second peak location are determined with no more than 10 nanometer root mean square error, and wherein said first peak intensity and said second peak intensity at least partially overlap; and
(d) processing said first peak intensity and said second peak intensity to distinguish said first analyte from said second analyte.
2. The method of claim 1 , wherein (b) further comprises configuring an image processing module to overlay said plurality of optical signals from said one or more cycles of said plurality of probes binding to said one or more analytes of said plurality of analytes, and wherein (d) further comprises applying an optical distribution to an optical signal of said overlay of said plurality of optical signal to identify said one or more analytes of said plurality of analytes.
3. The method of claim 2 , wherein said optical distribution comprises a gaussian distribution.
4. The method of claim 1 , further comprising using an image processing module to generate an oversampled image 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 as compared to a field image that is not oversampled.
5. The method of claim 1 , wherein said plurality of analytes is immobilized on 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 on said substrate at a density of at least 4 molecules per square micrometer.
7. The method of claim 1 , wherein said plurality of optical signals is obtained by an optical imaging module 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 1 , wherein at least one optical signal of said plurality of optical signals comprises a fluorescent signal.
10. 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 said plurality of probes.
11. The method of claim 1 , wherein said one or more cycles comprises at least two cycles.
12. A system for processing or analyzing a plurality of analytes, comprising:
(a) a substrate configured to support said plurality of analytes immobilized adjacent thereto;
(b) an optical imaging module configured to obtain a plurality of optical signals from said substrate over a plurality of cycles of a plurality of probes binding to one or more analytes of said plurality of analytes immobilized adjacent to said substrate; and
(c) an image processing module configured to:
(i) determine a first peak intensity of a first analyte of said plurality of analytes at a first peak location and a second peak intensity of a second analyte of said plurality of analytes at a second peak location, wherein said first peak location and said second peak location are determined with no more than 10 nanometer root mean square error, and wherein said first peak intensity and said second peak intensity at least partially overlap; and
(ii) process said first peak intensity and said second peak intensity to distinguish said first analyte from said second analyte.
13. The system of claim 12 , wherein said image processing module is further configured to (iii) overlay said plurality of optical signals from said substrate over said plurality of cycles of said probes binding to said one or more analytes of said plurality of analytes and (iv) apply an optical distribution module to said overlay of said plurality of optical signals to identify said one or more analytes of said plurality of analytes.
14. The system of claim 13 , wherein said optical distribution model comprises a gaussian distribution.
15. The system of claim 12 , wherein said image processing module is further configured to generate an oversampled image from said plurality of optical signals with a higher pixel density from a field image for said plurality of cycles of said probes binding to said one or more analytes of said plurality of analytes as compared to a field image that is not oversampled.
16. The system of claim 12 , wherein said plurality of analytes is immobilized adjacent to said substrate at a density of at least 2 molecules per square micrometer.
17. The system of claim 12 , wherein said plurality of analytes is immobilized adjacent to said substrate at a density of at least 4 molecules per square micrometer.
18. The system of claim 12 , wherein at least one analyte of said plurality of analytes is a nucleic acid molecule.
19. The system of claim 12 , wherein at least one analyte of said plurality of analytes is a protein or polypeptide.
20. The system of claim 12 , wherein at least one optical signal of said plurality of optical signals is a fluorescent signal.