Densley-packed analyte layers and detection methods
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 of sequencing a plurality of analytes disposed at high density on a surface of a substrate, comprising:
(a) performing a plurality of cycles of probe binding to a substrate comprising a surface, wherein said surface comprises a plurality of analytes immobilized adjacent to said surface, wherein at least two analytes of said plurality of analytes have a center-to-center distance of less than about 500 nanometers (nm), wherein a cycle of said plurality of cycles comprises:
(i) contacting said plurality of analytes with a plurality of probes, wherein a probe of said plurality of probes comprises a detectable label;
(ii) imaging a field of said surface with an optical system to detect an optical signal from each probe of said plurality of probes brought in contact with said plurality of analytes, thereby detecting a plurality of optical signals in said field for said cycle;
(b) determining a peak location of an analyte of said plurality of analytes from each of said plurality of optical signals by applying an optical distribution model to one or more overlaid optical signals of said plurality of optical signals; and
(c) identifying said analyte from said detectable label of said plurality of probes at said peak location across said plurality of cycles.
2. The method of claim 1 , wherein said plurality of optical signals is overlapping.
3. The method of claim 1 , further comprising overlaying said peak location of each optical signal of said plurality of optical signals to determine a relative position of each detected probe on said surface.
4. The method of claim 3 , further comprising resolving said optical signals in each image of said field using said determined relative position and a resolving function.
5. The method of claim 4 , wherein said resolving function comprises deconvolution.
6. The method of claim 1 , wherein said plurality of analytes comprises a plurality of DNA concatemers.
7. The method of claim 1 , wherein said plurality of analytes comprises proteins or peptides.
8. The method of claim 1 , wherein said plurality of analytes is immobilized on said surface at an average density of more than 3 molecules per square micron.
9. The method of claim 1 , wherein said center-to-center distance is due to, at least in part, a hydrophobic interaction between one or more adjacent analytes and an analyte of said plurality of analytes.
10. The method of claim 9 , wherein said hydrophobic interaction is due to zwitterionic features of said plurality of analytes.
11. The method of claim 9 , wherein said hydrophobic interaction is due to a repellant or attractive substance provided to said plurality of analytes.