Nucleic acid sequencing systems

Abstract

Methods and systems for sequencing a nucleic acid molecule are described that comprise imaging a first surface and an axially-displaced second surface using a compensation-free optical system, the system comprising an objective lens and at least one image sensor, wherein said optical system has a numerical aperture (NA) of less than 0.6 and a field-of-view (FOV) of greater than 1.0 mm2; and) processing the images of the first surface and the axially-displaced second surface to correct for optical aberration such that the images of the first surface and the axially-displaced second surface have substantially the same optical resolution.

What is claimed is: 
     
         1 . A system for sequencing a nucleic acid molecule comprising:
 a) an optical system comprising an objective lens and at least one image sensor, wherein said optical system has a numerical aperture (NA) of less than 0.6, and is configured to acquire images of a first surface and an axially-displaced second surface; and   b) a processor programmed to:
 i) process images of the first surface and the axially-displaced second surface to correct for optical aberration such that the images of the first surface and the axially-displaced second surface have substantially the same optical resolution; and 
 ii) detect a fluorescently-labeled composition comprising the nucleic acid molecule, or a complement thereof, disposed on the first surface or the axially-displaced second surface to determine an identity of a nucleotide in the nucleic acid molecule. 
   
     
     
         2 . The system of  claim 1 , wherein the images of the first surface and the axially-displaced second surface are acquired without moving an optical compensator into an optical path between said objective lens and said at least one image sensor. 
     
     
         3 . The system of  claim 1 , wherein the images of the first surface and the axially-displaced second surface are acquired by just refocusing the optical system. 
     
     
         4 . The system of  claim 1 , wherein the imaging system has a numerical aperture of greater than 0.3. 
     
     
         5 . The system of  claim 1 , wherein the first surface and axially-displaced second surface comprise two surfaces of a flow cell. 
     
     
         6 . The system of  claim 5 , wherein said two surfaces of the flow cell are coated with a hydrophilic coating layer, and wherein said hydrophilic coating layer further comprises labeled nucleic acid colonies disposed thereon at a surface density of >10,000 nucleic acid colonies/mm 2 . 
     
     
         7 . The system of  claim 1 , wherein said optical system comprises 1, 2, 3, or 4 imaging channels configured to detect nucleic acid colonies disposed on at least one of the first surface or axially-displaced second surface that have been labeled with 1, 2, 3, or 4 distinct detectable labels. 
     
     
         8 . The system of  claim 1 , wherein the system comprises at least one tube lens positioned between the objective lens and the at least one image sensor, and wherein the at least one tube lens is configured to correct an imaging performance metric for imaging a first interior surface of a flow cell and a second interior surface of the flow cell. 
     
     
         9 . The system of  claim 8 , wherein the flow cell has a wall thickness of at least 700 μm and a gap between the first interior surface and the second interior surface of at least 50 μm. 
     
     
         10 . The system of  claim 8 , wherein the optical system comprises two or more tube lenses which are designed to provide optimal imaging performance at two or more fluorescence wavelengths.