US-12618108-B2 - Flow cell devices and optical systems for nucleic acid sequencing

Abstract

Fluorescence imaging systems designs, flow cell devices, and methods of are described herein that enable imaging of three or more axially displaced surfaces without using any optical compensators. The optical systems and flow cell devices herein provides higher throughput analysis for genomics and other imaging applications at a lower cost.

Inventors

• Steve Xiangling Chen
• Omid Khandan
• Chiting Chang
• Derek Fuller
• Jordan Neysmith
• Michael Previte
• Francisco Garcia

Assignees

• ELEMENT BIOSCIENCES, INC.

Dates

Publication Date

20260505

Application Date

20250110

Claims (20)

1. 1 . An optical system for sequencing nucleic acids, comprising: an objective lens having a field-of-view (FOV) of greater than 1.0 square millimeters (mm 2 ); an excitation energy source configured to illuminate one or more surfaces of a flow cell; and at least one image sensor configured to acquire one or more images of the one or more surfaces of the flow cell, wherein the one or more surfaces are axially displaced from each other along an optical axis of the optical system, wherein the optical system exhibits a root-mean-square (RMS) wavefront error of less than 0.09λ, wherein λ is a center wavelength of the excitation energy source, wherein the RMS wavefront error is measured by use of a Shack-Hartmann wavefront sensor over the FOV of the objective lens using broadband illumination from the excitation energy source, and wherein the RMS wavefront error is an average of individual RMS wavefront error values for at least three color channels.
2. 2 . The optical system of claim 1 , wherein the RMS wavefront error is for a field of view (FOV) of about 1.5 millimeters (mm) in a direction that is orthogonal to the optical axis.
3. 3 . The optical system of claim 1 , wherein the one or more surfaces comprises four surfaces, and the RMS wavefront error is greater for a fourth surface disposed furthest from the objective lens than a first, second, or third surface of the four surfaces.
4. 4 . The optical system of claim 1 , wherein the RMS wavefront error of the optical system is less than a diffraction limit of the optical system.
5. 5 . The optical system of claim 1 , wherein the optical system has a numerical aperture (NA) of less than 0.7.
6. 6 . The optical system of claim 1 , wherein optical resolution of the optical system is sufficient to resolve two objects coupled to the one or more surfaces of the flow cell, wherein a distance between the two objects is at most about 500 nanometers (nm).
7. 7 . The optical system of claim 1 , wherein the optical system is configured to acquire the one or more images of the one or more surfaces of the flow cell, including sequencing time, in less than 20 mins per square millimeter (mm 2 ).
8. 8 . The optical system of claim 1 , wherein the one or more images of the one or more surfaces of the flow cell are from two or more different color channels.
9. 9 . The optical system of claim 8 , wherein the one or more images of the one or more surfaces of the flow cell are from 4 different color channels.
10. 10 . The optical system of claim 1 , wherein the optical system is configured to complete an image cycle in less than 6 minutes.
11. 11 . The optical system of claim 1 , further comprising the flow cell, wherein the one or more images of the one or more surfaces of the flow cell comprises optical signals emitting from a sample immobilized on the one or more surfaces of the flow cell.
12. 12 . The optical system of claim 1 , wherein the nucleic acids are from a sample, and wherein the sample comprises an in situ sample of a cell, a tissue, or both.
13. 13 . The optical system of claim 1 , wherein the excitation energy source is configured to uniformly illuminate an area of the one or more surfaces of the flow cell that is greater than 1 mm2 with less than 10% variance in illumination power across the illuminated area.
14. 14 . The optical system of claim 1 , wherein the objective lens comprises an optical aperture stop with an adjustable size configured to change numerical aperture (NA) of the optical system.
15. 15 . The optical system of claim 14 , wherein the optical aperture stop is configured to change the NA of the optical system in the range from 0.4 to 0.6.
16. 16 . The optical system of claim 1 , wherein the optical system does not comprise a movable optical compensator.
17. 17 . The optical system of claim 1 , wherein the flow cell has a top or bottom wall thickness of at least 700 micrometers (um) along an axial direction orthogonal to an image plane of the at least one image sensor.
18. 18 . The optical system of claim 1 , wherein the flow cell has a gap of at least 50 um along an axial direction orthogonal to an image plane of the at least one image sensor in a first or second fluidic channel of the flow cell.
19. 19 . The optical system of claim 1 , wherein the flow cell has an interposer of at least 500 um along an axial direction orthogonal to an image plane of the at least one image sensor between a first and second fluidic channel of the flow cell.
20. 20 . A system comprising: the optical system of claim 1 ; and a controller comprising at least one processor.

Description

CROSS-REFERENCE This application is a continuation of International Patent Application No. PCT/US2023/081406, filed Nov. 28, 2023, which claims the benefit of U.S. Provisional Application No. 63/385,386, filed Nov. 29, 2022, which is herein incorporated by reference in its entirety. SEQUENCE LISTING The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled 52933-753_301.XML, created on Feb. 1, 2025, which is 3,031 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. BACKGROUND In typical fluorescence-based genomic testing assays, e.g., genotyping or nucleic acid sequencing (using either real time, cyclic, or stepwise reaction schemes), dye molecules that are attached to nucleic acid molecules tethered on a substrate are excited using an excitation light source, a fluorescence photon signal is generated in one or more spatially-localized positions on the substrate, and the fluorescence is subsequently imaged through an optical system onto an image sensor. An analysis process is then used to analyze the images, find the positions of labeled molecules (or clonally amplified clusters of molecules) on the substrate, and quantify the fluorescence photon signal in terms of wavelength and spatial coordinates, which may then be correlated with the degree to which a specific chemical reaction, e.g., a hybridization event or base addition event, occurred in the specified locations on the substrate. Imaging-based methods provide large scale parallelism and multiplexing capabilities, which help to drive down the cost and accessibility of such technologies. However, detection errors that arise from, for example, overly dense packing of labeled molecules (or clonally-amplified clusters of molecules) within a small region of the substrate surface, or due to low contrast-to-noise ratio (CNR) in the image, may lead to errors in attributing the fluorescence signal to the correct molecules (or clonally amplified clusters of molecules). SUMMARY Described herein are flow cell devices, systems (e.g., optical systems), and methods of using thereof for analyzing biological polymers. In some embodiments, the biological polymers are nucleic acids. In some embodiments, the methods for use of the devices and systems comprise sequencing nucleic acids. The flow cell devices described herein can include multiple axially-displaced fluidic channels and three, four, or even more axially-displaced surfaces facing the channels so that the surfaces advantageously allow more samples (e.g., increased sample volume and/or sample varieties) to be disposed on a single flow cell than traditional flow cells. The optical systems and methods described herein is capable of imaging two, three, four, or even more surfaces of the flow cell devices that are axially-displaced from each other, therefore advantageously achieving improved sequencing throughput within a set system run time. As such, the optical systems and methods herein can increase effectiveness and efficiency of sequencing analysis. The devices, systems, and methods herein can enable imaging of three or more axially-displaced surfaces of the flow cell device without moving any optical compensator into, out of, or along the optical path, therefore providing simpler and more conveniently optical systems that are also less prone to errors due to vibrations. Further, the devices, systems, and methods herein can advantageously allow conveniently switching between imaging traditional flow cells, e.g., with one or dual surfaces, and the multiple surface flow cells herein with three or more axially-displaced surfaces. Such switching does not require adding or removing any optical elements in the system in and out of the optical path, e.g., an optical compensator. The devices, systems, and methods herein also enable imaging with a numerical aperture (NA) of less than 0.6 with sufficient image qualities for accurate sequencing analysis, allow imaging of the multiple axially-displaced surfaces independently, and allow adjustment of the objective lens to alter the NA to a desired value less than 0.6. The devices, systems, and methods herein can reduce reagent consumption needed for sequencing analysis of a same amount of samples than existing flow cell devices and optical systems. In an aspect, the present disclosure provides an optical system for sequencing nucleic acids, comprising: an objective lens having a field-of-view (FOV) of greater than 1.0 square millimeters (mm2); an excitation energy source configured to illuminate one or more surfaces of a flow cell; at least one image sensor configured to acquire one or more images of the one or more surfaces of the flow cell, wherein the one or more surfaces are axially displaced from each other along an optical axis of the optical system, wherein the optical system exhibits a root-mean-square (RMS) w