IP Library › Granted Patent US 12,239,986
Granted Patent B2
US 12,239,986 · App. 17/898,802 · Granted Mar 4, 2025

Flow cell with enhanced well imaging resolution

Inventor: Simon Prince (Carlsbad, CA)
Assignee: ILLUMINA, INC.
B01L3/502784H04N23/45B01L2200/16B01L2300/0654B01L2300/0877H04N23/67
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Quick Facts
Patent No.
US 12,239,986
App. No.
17/898,802
Granted
Mar 4, 2025
Kind
B2
Abstract

Resolution of images used in processes such as sequencing by synthesis may be increased by structuring sites that would emit signals in the images to have different elevations. Differences in focus caused by these differences in elevation may be used to filter out background illumination, thereby providing an image in which in focus sites may be resolved even though the separation between any site and its nearest neighbor may be below the diffraction limit of the light that would be emitted.

Claims (44)

1. A system comprising:

a flow cell comprising a plurality of reaction sites a fluid inlet port and a fluid outlet port, wherein:

the plurality of reaction sites comprises a plurality of sets of reaction sites;

for each set of reaction sites from the plurality of sets of reaction sites:

that set of reaction sites is a subset of the plurality of reaction sites;

that set of reaction sites has a corresponding elevation relative to a bottom of the flow cell;

each reaction site comprised by that set of reaction sites is disposed on either a bottom surface of a corresponding well or a surface above the bottom surface of the corresponding well, and is located at that set of reaction sites' corresponding elevation relative to the bottom of the flow cell; and

no reaction site from any set of reaction sites other than that set of reaction sites is located at that set of reaction sites' corresponding elevation relative to the bottom of the flow cell;

a set of cameras; and

a processor connected to the set of cameras and programmed to:

receive a plurality of unfiltered images captured by the set of cameras, wherein the plurality of unfiltered images comprises, for each set of reaction sites from the plurality of sets of reaction sites, an image corresponding to that set of reaction sites; and

for each set of reaction sites from the plurality of sets of reaction sites, determine a derived image corresponding to that set of reaction sites, wherein the derived image corresponding to that set of reaction sites is based on using differences in focus between images from the plurality of unfiltered images to remove signals from reaction sites not comprised by that set of reaction sites while retaining signals from reaction sites comprised by that set of reaction sites;

wherein:

each set of reaction sites from the plurality of sets of reaction sites is disjoint with all other sets of reaction sites from the plurality of sets of reaction sites; and

for each image from the plurality of unfiltered images, that image depicts:

light emitted from reaction sites comprised by the set of reaction sites corresponding to that image; and

light emitted from reaction sites not comprised by the set of reaction sites corresponding to that image.

2. The system of claim 1 , wherein:

for each reaction site from the plurality of reaction sites, that reaction site has a center;

for each reaction site in the plurality of reaction sites, there is an adjacent reaction site in the plurality of reaction sites, wherein a distance between the center of that reaction site and the center of the adjacent reaction site is less than a diffraction limit for a wavelength of light used in obtaining the plurality of unfiltered images; and

for each set of reaction sites from the plurality of sets of reaction sites, for each reaction site in that set of reaction sites, there is a neighboring reaction site in that set of reaction sites, wherein a distance between the center of that reaction site and the center of the neighboring reaction site is greater than the diffraction limit for the wavelength of light used in obtaining the plurality of unfiltered images.

3. The system of claim 1 , wherein, for each set of reaction sites from the plurality of sets of reaction sites, the set of cameras comprises a corresponding camera focused with a focal plane at that set of reaction sites' corresponding elevation relative to the bottom of the flow cell.

4. The system of claim 3 , wherein the system comprises a one or more beam splitters to direct signals from the plurality of reaction sites to the cameras from the set of cameras.

5. The system of claim 1 , wherein the processor is programmed to:

cause a first camera from the set of cameras to capture a first unfiltered image, wherein the first unfiltered image corresponds to a first set of reaction sites from the plurality of sets of reaction sites, and wherein the first camera is focused on a first focal plane at the first set of reaction sites' corresponding elevation relative to the bottom of the flow cell when it is caused to capture the first unfiltered image;

refocus the first camera on a second focal plane at the second set of reaction sites' corresponding elevation from the bottom of the flow cell; and

cause the first camera to capture a second unfiltered image, wherein the second unfiltered image corresponds to the second set of reaction sites, and wherein the first camera is focused on a second focal plane at the second set of reaction sites' corresponding elevation from the bottom of the flow cell when it is caused to capture the second unfiltered image.

6. The system of claim 1 , wherein:

the set of cameras comprises a line scan camera;

the line scan camera comprises a plurality of sets of sensors, wherein, for each set of sensors from the plurality of sets of sensors:

that set of sensors has a corresponding set of reaction sites from the plurality of sets of reaction sites; and

that set of sensors is focused on a focal plane at its corresponding set of reaction sites' corresponding elevation relative to the bottom of the flow cell; and

the processor is programmed to cause the line scan camera to:

capture a first unfiltered image using a first set of sensors, wherein the first set of sensors corresponds to a first set of reaction sites from the plurality of sets of reaction sites; and

capture a second unfiltered image using a second set of sensors, wherein the second set of sensors corresponds to a second set of reaction sites from the plurality of sets of reaction sites.

7. The system of claim 1 , wherein:

each set of reaction sites from the plurality of sets of reaction sites has a corresponding point spread function; and

for each set of reaction sites from the plurality of sets of reaction sites, determining the derived image corresponding to that set of reaction sites comprises, for each other set of reaction sites from the plurality of sets of reaction sites, removing signals from reaction sites comprised by that other set of reaction sites using the unfiltered image corresponding to that other set of reaction sites and the point spread function corresponding to that other set of reaction sites.

8. The system of claim 7 , wherein, for at least one set of reaction sites from the plurality of sets of reaction sites, the corresponding point spread function for that set of reaction sites is different from the corresponding point spread functions for all other sets of reaction sites from the plurality of sets of reaction sites.

9. The system of claim 7 , wherein, for at least one set of reaction sites from the plurality of sets of reaction sites, the corresponding point spread function for that set of reaction sites is the same as the corresponding point spread function for at least one other set of reaction sites from the plurality of sets of reaction sites.

10. The system of claim 1 , wherein the plurality of sets of reaction sites consists of two sets of reaction sites.

11. The system of claim 1 , wherein the plurality of sets of reaction sites comprises three or more sets of reaction sites.

12. The system of claim 1 , wherein, for each set of reaction sites from the plurality of sets of reaction sites, for each reaction site comprised by that set of reaction sites, that reaction site is located in a corresponding well comprised by the flow cell.

13. The system of claim 1 , wherein, for each set of reaction sites from the plurality of sets of reaction sites, for each reaction site comprised by that set of reaction sites, that reaction site is located on a corresponding post comprised by the flow cell.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 24, 2023
From: PRINCE, SIMON
To: ILLUMINA, INC.
Reel/Frame 063098/0542 →
Continuity (2)
Provisional Application 63239061 · Aug 31, 2021
Related Publication 20230070459A1 · Mar 9, 2023
References Cited (122)
US 5130238A · Malek et al. · 1992 [cited by applicant]
US 5185243A · Ullman et al. · 1993 [cited by applicant]
US 5223414A · Zarling et al. · 1993 [cited by applicant]
US 5455166A · Walker · 1995 [cited by applicant]
US 5573907A · Carrino et al. · 1996 [cited by applicant]
US 5599675A · Brenner · 1997 [cited by applicant]
US 5641658A · Adams et al. · 1997 [cited by applicant]
US 5679524A · Nikiforov et al. · 1997 [cited by applicant]
US 5750341A · Macevicz · 1998 [cited by applicant]
US 6210891B1 · Nyren et al. · 2001 [cited by applicant]
US 6214587B1 · Dattagupta et al. · 2001 [cited by applicant]
US 6258568B1 · Nyren · 2001 [cited by applicant]
US 6266459B1 · Walt et al. · 2001 [cited by applicant]
US 6274320B1 · Rothberg · 2001 [cited by applicant]
US 6355431B1 · Chee et al. · 2002 [cited by applicant]
US 6770441B2 · Dickinson et al. · 2004 [cited by applicant]
US 6859570B2 · Walt et al. · 2005 [cited by applicant]
US 7057026B2 · Barnes et al. · 2006 [cited by applicant]
US 7115400B1 · Adessi et al. · 2006 [cited by applicant]
US 7195872B2 · Agrawal et al. · 2007 [cited by applicant]
US 7211414B2 · Hardin et al. · 2007 [cited by applicant]
US 7244559B2 · Rothberg · 2007 [cited by applicant]
US 7315019B2 · Turner et al. · 2008 [cited by applicant]
US 7323305B2 · Leamon et al. · 2008 [cited by applicant]
US 7329492B2 · Hardin et al. · 2008 [cited by applicant]
US 7329860B2 · Feng et al. · 2008 [cited by applicant]
US 7622294B2 · Walt et al. · 2009 [cited by applicant]
US 7741463B2 · Gormley et al. · 2010 [cited by applicant]
US 7940282B2 · Milanfar et al. · 2011 [cited by applicant]
US 8345144B1 · Georgiev et al. · 2013 [cited by applicant]
US 8749694B2 · Georgiev et al. · 2014 [cited by applicant]
US 8759037B2 · Rigatti et al. · 2014 [cited by applicant]
US 8778848B2 · Lin et al. · 2014 [cited by applicant]
US 8778849B2 · Bowen et al. · 2014 [cited by applicant]
US 8895249B2 · Shen et al. · 2014 [cited by applicant]
US 8906320B1 · Eltoukhy et al. · 2014 [cited by applicant]
US 8951781B2 · Reed et al. · 2015 [cited by applicant]
US 8965076B2 · Garcia et al. · 2015 [cited by applicant]
US 9012022B2 · George et al. · 2015 [cited by applicant]
US 9096899B2 · Eltoukhy et al. · 2015 [cited by applicant]
US 9193996B2 · Buermann et al. · 2015 [cited by applicant]
US 9485432B1 · Medasani et al. · 2016 [cited by applicant]
US 9500846B2 · Betzig et al. · 2016 [cited by applicant]
US 9512422B2 · Barnard et al. · 2016 [cited by applicant]
US 9574226B2 · Gormley et al. · 2017 [cited by applicant]
US 9696534B2 · Shroff et al. · 2017 [cited by applicant]
US 9800856B2 · Venkataraman et al. · 2017 [cited by applicant]
US 20020055100A1 · Kawashima et al. · 2002 [cited by applicant]
US 20030103021A1 · Young et al. · 2003 [cited by applicant]
US 20030160181A1 · Corson et al. · 2003 [cited by applicant]
US 20040002090A1 · Mayer · 2004 [cited by applicant]
US 20040166593A1 · Nolte et al. · 2004 [cited by applicant]
US 20040175843A1 · Roitman et al. · 2004 [cited by applicant]
US 20050191698A1 · Chee et al. · 2005 [cited by applicant]
US 20060057729A1 · Moon et al. · 2006 [cited by applicant]
US 20070134784A1 · Halverson et al. · 2007 [cited by applicant]
US 20070259365A1 · Hah et al. · 2007 [cited by applicant]
US 20080009420A1 · Schroth · 2008 [cited by applicant]
US 20090186777A1 · Lee et al. · 2009 [cited by applicant]
US 20110059865A1 · Smith et al. · 2011 [cited by applicant]
US 20130065794A1 · Law et al. · 2013 [cited by applicant]
US 20130096034A1 · Lebl et al. · 2013 [cited by applicant]
US 20130144217A1 · Ross · 2013 [cited by applicant]
US 20140243224A1 · Barnard et al. · 2014 [cited by applicant]
US 20150293021A1 · Finkelstein et al. · 2015 [cited by applicant]
US 20160019693A1 · Silbersweig · 2016 [cited by applicant]
US 20170274374A1 · Bowen · 2017 [cited by examiner]
US 20210222238A1 · Chen et al. · 2021 [cited by applicant]
CN 109491176A · 2019 [cited by applicant]
EP 0320308B1 · 1993 [cited by applicant]
EP 0336731B1 · 1994 [cited by applicant]
EP 0439182B1 · 1996 [cited by applicant]
EP 2374902A1 · 2011 [cited by applicant]
EP 2675047A1 · 2013 [cited by applicant]
WO WO1989012696A1 · 1898 [cited by applicant]
WO WO1989009835A1 · 1989 [cited by applicant]
WO WO1989010977A1 · 1989 [cited by applicant]
WO WO1990001069A1 · 1990 [cited by applicant]
WO WO1991006678A1 · 1991 [cited by applicant]
WO WO1997013633A1 · 1997 [cited by applicant]
WO WO1998059066A1 · 1998 [cited by applicant]
WO WO2003101618A1 · 2003 [cited by applicant]
WO WO2004018497A2 · 2004 [cited by applicant]
WO WO2005010145A2 · 2005 [cited by applicant]
WO WO2007123744A2 · 2007 [cited by applicant]
WO WO2010039147A1 · 2010 [cited by applicant]
WO WO2011105679A2 · 2011 [cited by applicant]
WO WO2013154770A1 · 2013 [cited by applicant]
WO WO2014142841A1 · 2014 [cited by applicant]
WO WO2014197096A2 · 2014 [cited by applicant]
WO WO2022256226A1 · 2022 [cited by applicant]
WO WO2023287617A1 · 2023 [cited by applicant]
International Search Report and Written Opinion dated Dec. 23, 2022, for International Application No. PCT/US2022/041208, 10 pages. [cited by applicant]
Bains, William, and Geoff C. Smith. “A novel method for nucleic acid sequence determination.” [cited by applicant]
Bentley, David R., et al. “Accurate whole human genome sequencing using reversible terminator chemistry.” [cited by applicant]
Dean, Frank B., et al. “Comprehensive human genome amplification using multiple displacement amplification.” [cited by applicant]
Dressman, Devin, et al. “Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations.” [cited by applicant]
Drmanac, Snezana, et al. “Accurate sequencing by hybridization for DNA diagnostics and individual genomics.” [cited by applicant]
Fodor, Stephen PA, et al. “Light-directed, spatially addressable parallel chemical synthesis.” [cited by applicant]
Haugland, Richard P., “Molecular Probes Handbook of Fluorescent Probes and Research Chemicals”, Sixth Edition, 1996. [cited by applicant]
Joos, Beda, Herbert Kuster, and Richard Cone. “Covalent attachment of hybridizable oligonucleotides to glass supports.” [cited by applicant]
Khandjian, Edouard W. “UV crosslinking of RNA to nylon membrane enhances hybridization signals.” [cited by applicant]
Korlach, Jonas, et al. “Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures.” [cited by applicant]
Korlach, J., Turner, S.W. (2013). Zero-Mode Waveguides. In: Roberts, G.C.K. (eds) Encyclopedia of Biophysics. Springer, Berlin, Heidelberg. [cited by applicant]
Lage, José M., et al. “Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array—CGH.” [cited by applicant]
Lakowicz, Joseph R., ed. [cited by applicant]
Levene, Michael J., et al. “Zero-mode waveguides for single-molecule analysis at high concentrations.” [cited by applicant]
Lizardi, Paul M., et al. “Mutation detection and single-molecule counting using isothermal rolling-circle amplification.” [cited by applicant]
Lundquist, Paul M., et al. “Parallel confocal detection of single molecules in real time.” [cited by applicant]
McNally, James G., et al. “Three-dimensional imaging by deconvolution microscopy.” [cited by applicant]
Oroskar, A. A., et al. “Detection of immobilized amplicons by ELISA-like techniques.” [cited by applicant]
Ronaghi, Mostafa, Mathias Uhlén, and Pål Nyrén. “A sequencing method based on real-time pyrophosphate.” [cited by applicant]
Ronaghi, Mostafa, et al. “Real-time DNA sequencing using detection of pyrophosphate release.” [cited by applicant]
Ronaghi, Mostafa. “Pyrosequencing sheds light on DNA sequencing.” [cited by applicant]
Shendure, Jay, et al. “Accurate multiplex polony sequencing of an evolved bacterial genome.” [cited by applicant]
Sibarita, Jean-Baptiste. “Deconvolution microscopy.” [cited by applicant]
Smith, Steven B., Laura Finzi, and Carlos Bustamante. “Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads.” [cited by applicant]
Taylor, D. M., H. Morgan, and C. D'silva. “Characterization of chemisorbed monolayers by surface potential measurements.” [cited by applicant]
Vincent, Myriam, Yan Xu, and Huimin Kong. “Helicase-dependent isothermal DNA amplification.” [cited by applicant]
Walker, G. T., et al. “A chemiluminescent DNA probe test based on strand displacement amplification.” [cited by applicant]
Walker, G. Terrance, et al. “Strand displacement amplification—an isothermal, in vitro DNA amplification technique.” [cited by applicant]
International Search Report and Written Opinion dates Aug. 18, 2017, for International Application No. PCT/US2017/024578, 11 pages. [cited by applicant]