US 11,053,540 B1
High performance fluorescence imaging module for genomic testing assay
Steve Xiangling Chen, San Diego, CA (US); Minghao Guo, San Diego, CA (US); Michael Previte, San Diego, CA (US); Chunhong Zhou, San Diego, CA (US); and Derek Fuller, San Diego, CA (US)
Assigned to ELEMENT BIOSCIENCES, INC., San Diego, CA (US)
Filed by Element Biosciences, Inc., San Diego, CA (US)
Filed on Sep. 9, 2020, as Appl. No. 17/16,349.
Claims priority of provisional application 63/076,361, filed on Sep. 9, 2020.
Claims priority of provisional application 62/962,723, filed on Jan. 17, 2020.
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6869 (2018.01); G01N 21/64 (2006.01)
CPC C12Q 1/6869 (2013.01) [G01N 21/6428 (2013.01); G01N 21/6458 (2013.01); G01N 2021/6439 (2013.01)] 30 Claims
 
1. A method of sequencing a nucleic acid molecule, the method comprising:
a) acquiring images of a first surface of a flow cell coated with a first hydrophilic coating layer and an axially-displaced second surface of a flow cell coated with a second hydrophilic coating layer, wherein said first hydrophilic coating layer and said second hydrophilic coating layer comprise labeled nucleic acid colonies disposed thereon at a surface density of greater than 10,000 nucleic acid colonies/mm2, using an optical system which comprises 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 wherein images of said first surface and said axially-displaced second surface having substantially the same optical resolution are acquired without moving an optical compensator into an optical path between said objective lens and said at least one image sensor; and
b) detecting a fluorescently-labeled composition comprising said nucleic acid molecule, or a complement thereof, disposed on said first surface or said axially-displaced second surface to determine an identity of a nucleotide in said nucleic acid molecule, wherein an image of said first surface or said axially-displaced second surface acquired using said optical system shows a contrast to noise ratio (CNR) of at least 5 when:
i) said labeled nucleic acid colonies are labeled with cyanine dye 3 (Cy3);
ii) said optical system comprises a dichroic mirror and bandpass filter set optimized for Cy3 emission; and
iii) said image is acquired under non-signal saturating conditions while said first surface or said axially-displaced second surface is immersed in 25 mM ACES, pH 7.4 buffer.