Densley-packed analyte layers and detection methods

Abstract

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 .- 19 . (canceled) 
     
     
         20 . A method for accurately determining a relative position of analytes deposited on a surface of a densely packed substrate, comprising:
 (a) providing a substrate comprising a surface, wherein the surface is patterned or unpatterned and comprises a plurality of analytes deposited on the surface at discrete locations;   (b) performing a plurality of cycles of probe binding and signal detection on said surface, each cycle comprising:
 (i) contacting said analytes with a plurality of probes from a probe set, wherein said probes comprise a detectable label, wherein each of said probes binds specifically to a target analyte; and 
 (ii) imaging a field of said surface with an optical system to detect a plurality of optical signals from individual probes bound to said analytes at discrete locations on said surface; 
   (c) determining a peak location from each of said plurality of optical signals from images of said field from at least two of said plurality of cycles; and   (d) overlaying said peak locations for each optical signal and applying an optical distribution model at each cluster of optical signals to determine a relative position of each detected analyte on said surface with improved accuracy.   
     
     
         21 . The method of claim  1 , further comprising:
 (e) resolving said optical signals in each field image from each cycle using said determined relative position and a resolving function; and   (f) identifying said detectable labels bound to said deposited analytes for each field and each cycle from said deconvolved optical signals.   
     
     
         22 . The method of claim  1 , wherein one or more analytes of said plurality of analytes are treated with a repellant or attractive substance. 
     
     
         23 . The method of claim (f), wherein said repellant or attractive substance comprises zwitterionic features. 
     
     
         24 . The method of claim (f), wherein said repellant or attractive substance comprises PEG, a polysaccharide, ampholine ampholytes, sulphobetaine, and/or BSA. 
     
     
         25 . The method of claim  1 , wherein said analytes are DNA concatemers. 
     
     
         26 . The method of  claim 24 , wherein said DNA concatemers are hybridized to ssDNA hairs. 
     
     
         27 . The method of claim  1 , wherein said analytes are proteins or peptides. 
     
     
         28 . The method of  claim 21 , further comprising using said detectable label identity for each analyte detected at each cycle to identify a plurality of said analytes on said substrate. 
     
     
         29 . The method of  claim 21 , wherein said resolving comprises removing interfering optical signals from neighboring analytes using a center-to-center distance between said neighboring analytes from said determined relative positions of said neighboring analytes. 
     
     
         30 . The method of  claim 21 , wherein said resolving function comprises deconvolution. 
     
     
         31 . The method of claim  1 , wherein said analytes are single biomolecules. 
     
     
         32 . The method of claim  1 , wherein said analytes deposited on said surface are spaced apart on average less than the diffraction limit of the light emitted by the detectable labels and imaged by the optical system. 
     
     
         33 . The method of claim  1 , wherein the deposited analytes comprises an average center-to-center distance between each analyte and the nearest adjacent analyte of less than 500 nm. 
     
     
         34 . The method of claim  1 , wherein said overlaying said peak locations comprises aligning positions of said optical signal peaks detected in each field for a plurality of said cycles to generate a cluster of optical peak positions for each analyte from said plurality of cycles. 
     
     
         35 . The method of claim  1 , wherein said relative position is determined with an accuracy of within 10 nanometers RMS. 
     
     
         36 . The method of claim  1 , wherein said method resolves optical signals from a surface at a density of about 4 to about 25 analytes per square micron. 
     
     
         37 . A system for determining the identity of a plurality of analytes, comprising
 (a) an optical imaging device configured to image a plurality of optical signals from a field of a substrate over a plurality of cycles of probe binding to analytes deposited on a surface of the substrate, wherein said surface is unpatterned; and   (b) an image processing module, said module configured to:
 (i) determine a peak location from each of said plurality of optical signals from images of said field from at least two of said plurality of cycles; 
 (ii) determine a relative position of each detected analyte on said surface with improved accuracy by applying an optical distribution model to each cluster of optical signals from said plurality of cycles; and 
 (iii) deconvolve said optical signals in each field image from each cycle using said determined relative position and a resolving function. 
   
     
     
         38 . The system of  claim 36 , wherein said image processing module is further configured to determine an identity of said analytes deposited on said surface using said deconvolved optical signals. 
     
     
         39 . The system of  claim 36 , wherein said optical image device comprises a moveable stage defining a scannable area. 
     
     
         40 . The system of  claim 36 , wherein said optical image device comprises a sensor and optical magnification configured to sample a surface of a substrate at below the diffraction limit in said scannable area. 
     
     
         41 . The system of  claim 36 , further comprising a substrate comprising analytes deposited to an unpatterned surface of the substrate at a center-to-center spacing below the diffraction limit. 
     
     
         42 . The system of  claim 36 , wherein said resolving comprises removing interfering optical signals from neighboring analytes using a center-to-center distance between said neighboring analytes to determine said relative positions of said neighboring analytes. 
     
     
         43 .- 79 . (canceled)