High-performance fluorescence imaging module for genomic testing assays

Abstract

Optical system designs for high performance fluorescence imaging methods and systems are disclosed [Solution] A fluorescence imaging system design is described that provides a larger field of view, increased spatial resolution, improved modulation transmission and image quality, higher spatial sampling frequency, faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., of different fields of view), and improved imaging system duty cycle, thus enabling higher throughput image acquisition and analysis for genomic and other imaging applications.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 076,361, filed September 9, 2020, and U.S. Provisional Patent Application No. 62 / 962,723, filed January 17, 2020, each of which is incorporated by reference in its entirety. [Background technology]

[0002] In a typical fluorescence-based genomic testing assay, such as genotyping or nucleic acid sequencing (using either real-time, cyclic, or stepwise reaction schemes), dye molecules attached to tethered nucleic acid molecules on a substrate are excited using an excitation light source, and fluorescent photon signals are generated at one or more spatially localized locations on the substrate, which are then imaged through an optical system onto an image sensor. An analytical process then analyzes the image, locates the labeled molecules (or clonally amplified clusters of molecules) on the substrate, and quantifies the fluorescent photon signals in terms of wavelength and spatial coordinates, which can then be correlated to the extent to which a specific chemical reaction, such as a hybridization event or base addition event, occurred at a specified location on the substrate. Imaging-based methods offer massive parallelism and multiplexing capabilities, helping to reduce the cost and accessibility of such technologies. However, detection errors resulting from, for example, excessively dense packing of labeled molecules (or clonally amplified clusters of molecules) within a small area of ​​the substrate surface, or detection errors resulting from low contrast-to-noise ratio (CNR) in the image, can lead to errors in attributing fluorescent signals to the correct molecules (clonally amplified clusters of molecules). Summary of the Invention

[0003] Disclosed herein is an imaging system configured to image a first interior surface and a second interior surface of a flow cell, the imaging system including: a) an objective lens; b) at least one image sensor; and c) at least one tube lens disposed in an optical path between the objective lens and the at least one image sensor, the optical system having a numerical aperture (NA) of less than 0.6 and a 1.0 mm aperture. 2 and the at least one tube lens is configured to modify imaging performance such that images of the first interior surface of the flow cell and the second interior surface of the flow cell have substantially the same optical resolution.

[0004] In some embodiments, the flow cell has a wall thickness of at least 700 μm and a liquid-filled gap of at least 50 μm between the first and second interior surfaces. In some embodiments, images of the first and second interior surfaces are acquired without moving an optical corrector into the optical path between the objective lens and the at least one image sensor. In some embodiments, the imaging system has a numerical aperture (NA) of less than 0.6. In some embodiments, the imaging system has a numerical aperture (NA) of greater than 0.3. In some embodiments, the imaging system has a 1.5 mm 2In some embodiments, the imaging system has a field of view (FOV) greater than 100 nm. In some embodiments, the optical resolution of the images of the first and second internal surfaces is diffraction-limited across the entire field of view (FOV). In some embodiments, the at least one tube lens comprises, in order, an asymmetric convex-convex lens, a convex-plano lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the imaging system comprises two or more tube lenses designed to provide optimal imaging performance for the first and second internal surfaces at two or more fluorescent wavelengths. In some embodiments, the imaging system further comprises a focusing mechanism configured to refocus the optical system between acquiring images of the first and second internal surfaces. In some embodiments, the imaging system is configured to image two or more fields of view on at least one of the first or second internal surfaces. In some embodiments, the first and second internal surfaces of the flow cell are coated with a hydrophilic coating layer, the hydrophilic coating layer having a thickness of 1 mm. 2The imaging system further comprises labeled nucleic acid colonies disposed thereon at a surface density of greater than 10,000 nucleic acid colonies per surface. In some embodiments, images of the first interior surface or the second interior surface acquired using an imaging system exhibit a contrast-to-noise ratio (CNR) of at least 5 when the nucleic acid colonies are labeled with cyanine dye 3 (Cy3), the imaging system comprising a dichroic mirror and bandpass filter set optimized for Cy3 emission, and the images are acquired under non-signal saturating conditions while the surfaces are immersed in 25 mM ACES, pH 7.4 buffer. In some embodiments, the imaging system comprises one, two, three, or four imaging channels configured to detect nucleic acid colonies disposed on at least one of the two different surfaces labeled with one, two, three, or four different detectable labels. In some embodiments, the imaging system is used to monitor avidity sequencing, nucleotide base pairing sequencing, nucleotide binding sequencing, or nucleotide incorporation sequencing on at least one of the first and second internal surfaces, and to detect bound or incorporated nucleotide bases. In some embodiments, the imaging system is used to perform nucleic acid sequencing. In some embodiments, the imaging system is used to determine the genotype of a sample, where determining the genotype of the sample includes preparing nucleic acid molecules extracted from the sample for sequencing and then sequencing the nucleic acid molecules. In some embodiments, the at least one image sensor includes pixels having pixel dimensions selected such that the spatial sampling frequency for the imaging system is at least twice the optical resolution of the imaging system. In some embodiments, the combination of the objective lens and the at least one tube lens is configured to optimize a modulation transfer function in the spatial frequency range of 700 cycles per mm to 1100 cycles per mm at the sample plane.In some embodiments, the at least one tube lens is designed to correct modulation transfer function (MTF), defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast-to-noise ratio (CNR), or any combination thereof, at one or more specified spatial frequencies for the combination of the objective lens and the at least one tube lens.

[0005] Also disclosed herein is a method for sequencing a nucleic acid molecule, the method comprising: a) imaging a first surface and an axially displaced second surface using an optical system including an objective lens and at least one image sensor, the optical system having a numerical aperture (NA) of less than 0.6 and a resolution of 1.0 mm. 2 wherein images of the first surface and the axially displaced second surface having a field of view (FOV) larger than 100 nm and having substantially the same optical resolution are acquired without moving an optical corrector into an optical path between the objective lens and the at least one image sensor; and b) detecting a fluorescently labeled composition comprising a nucleic acid molecule disposed on the first surface or the axially displaced second surface, or a complement thereof, to determine the identity of a nucleotide in the nucleic acid molecule.

[0006] In some embodiments, a focusing mechanism is utilized to refocus the optical system between acquiring images of the first surface and the axially displaced second surface. In some embodiments, the method further includes imaging two or more fields of view at at least one of the first surface or the axially displaced second surface. In some embodiments, the first surface and the axially displaced second surface comprise two surfaces of a flow cell. In some embodiments, the two surfaces of the flow cell are coated with a hydrophilic coating layer. In some embodiments, the hydrophilic coating layer is 1 mm thick. 2The optical system further comprises labeled nucleic acid colonies disposed thereon at a surface density of greater than 10,000 nucleic acid colonies per surface. In some embodiments, an image of one of the two surfaces acquired using the optical system exhibits a contrast-to-noise ratio (CNR) of at least 5 when the nucleic acid colonies are labeled with cyanine dye 3 (Cy3), the optical system comprising a dichroic mirror and bandpass filter set optimized for Cy3 emission, and the image is acquired under non-signal saturation conditions while the surface is immersed in 25 mM ACES, pH 7.4 buffer. In some embodiments, the optical system comprises one, two, three, or four imaging channels configured to detect nucleic acid colonies disposed on at least one of the first surface and the axially displaced second surface labeled with one, two, three, or four different detectable labels. In some embodiments, at least one image sensor comprises pixels having pixel dimensions selected such that the spatial sampling frequency for the optical system is at least twice the optical resolution of the optical system. In some embodiments, the optical system includes at least one tube lens positioned between the objective lens and the at least one image sensor, the at least one tube lens configured to modify imaging performance metrics for imaging the first interior surface of the flow cell and the second interior surface of the flow cell. In some embodiments, the flow cell has a wall thickness of at least 700 μm and a gap of at least 50 μm between the first interior surface and the second interior surface. In some embodiments, the at least one tube lens comprises, in order, an asymmetric convex-convex lens, a convex-plano lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescent wavelengths. In some embodiments, the combination of the objective lens and the tube lens is configured to optimize a modulation transfer function in the mid- to high-spatial frequency range.In some embodiments, the imaging performance metrics include a measurement of the modulation transfer function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast-to-noise ratio (CNR), or any combination thereof. In some embodiments, the optical resolution of the images of the first surface and the axially displaced second surface is diffraction-limited across the entire field of view (FOV). In some embodiments, sequencing the nucleic acid molecules further includes performing avidity sequencing, nucleotide base pairing sequencing, nucleotide binding sequencing, or nucleotide incorporation sequencing on at least one of the first surface and the axially displaced second surface, and detecting the bound or incorporated nucleotide bases. In some embodiments, the method further includes determining the genotype of the sample, wherein determining the genotype of the sample comprises preparing the nucleic acid molecules for sequencing and then sequencing the nucleic acid molecules.

[0007] Disclosed herein is an imaging system configured to image two different axially displaced surfaces, the imaging system including an objective lens and at least one image sensor, the imaging system having a numerical aperture (NA) of less than 0.6 and a resolution of 1.0 mm. 2 and wherein the imaging system is capable of acquiring images of two different axially displaced surfaces with substantially the same optical resolution without moving an optical corrector into the optical path between the objective lens and the at least one image sensor.

[0008] In some embodiments, the imaging system has a numerical aperture greater than 0.3. In some embodiments, the imaging system further includes a focusing mechanism used to refocus the optical system between acquiring images of the two different axially displaced surfaces. In some embodiments, the imaging system is configured to image two or more fields of view on at least one of the two different axially displaced surfaces. In some embodiments, the two different axially displaced surfaces include two surfaces of a flow cell. In some embodiments, the two different surfaces of the flow cell are coated with a hydrophilic coating layer, and the hydrophilic coating layer is 1 mm thick. 2 The imaging system further comprises labeled nucleic acid colonies disposed thereon at a surface density of greater than 10,000 nucleic acid colonies per surface. In some embodiments, the imaging system comprises one, two, three, or four imaging channels configured to detect nucleic acid colonies disposed on at least one of the two different surfaces labeled with one, two, three, or four different detectable labels. In some embodiments, the at least one image sensor comprises pixels having pixel dimensions selected such that the spatial sampling frequency for the imaging system is at least twice the optical resolution of the imaging system. In some embodiments, the imaging system comprises at least one tube lens positioned between the objective lens and the at least one image sensor, the at least one tube lens configured to modify imaging performance metrics for imaging the first interior surface of the flow cell and the second interior surface of the flow cell. In some embodiments, the flow cell has a wall thickness of at least 700 μm and a gap of at least 50 μm between the first and second interior surfaces. In some embodiments, the imaging system comprises two or more tube lenses designed to provide optimal imaging performance at two or more fluorescent wavelengths. In some embodiments, the optical resolution of the images of the two different axially displaced surfaces is diffraction limited over the entire field of view (FOV).

[0009] Disclosed herein is a method for sequencing nucleic acid molecules, the method comprising: a) imaging a first surface and an axially displaced second surface using an uncorrected optical system including an objective lens and at least one image sensor, the optical system having a numerical aperture (NA) of less than 0.6 and a resolution of 1.0 mm. 2 a) imaging a first surface and an axially displaced second surface having a field of view (FOV) greater than 100 nm; b) processing images of the first surface and the axially displaced second surface to correct for optical aberrations so that the images of the first surface and the axially displaced second surface have substantially the same optical resolution; and c) detecting a fluorescently labeled composition comprising a nucleic acid molecule disposed on the first surface or the axially displaced second surface, or a complement thereof, to determine the identity of a nucleotide in the nucleic acid molecule.

[0010] In some embodiments, images of the first surface and the axially displaced second surface are acquired without moving an optical corrector into the optical path between the objective lens and the at least one image sensor. In some embodiments, images of the first surface and the axially displaced second surface are acquired only by focusing light onto an optical system. In some embodiments, the method further comprises imaging two or more fields of view at at least one of the first surface or the axially displaced second surface. In some embodiments, the first surface and the axially displaced second surface comprise two surfaces of a flow cell. In some embodiments, the two surfaces of the flow cell are coated with a hydrophilic coating layer. In some embodiments, the hydrophilic coating layer is 1 mm thick. 2The optical system further comprises labeled nucleic acid colonies disposed thereon at a surface density of greater than 10,000 nucleic acid colonies per surface. In some embodiments, an image of one of the two surfaces acquired using the optical system exhibits a contrast-to-noise ratio (CNR) of at least 5 when the nucleic acid colonies are labeled with cyanine dye 3 (Cy3), the optical system comprising a dichroic mirror and bandpass filter set optimized for Cy3 emission, and the image is acquired under non-signal saturation conditions while the surface is immersed in 25 mM ACES, pH 7.4 buffer. In some embodiments, the optical system comprises one, two, three, or four imaging channels configured to detect nucleic acid colonies disposed on at least one of the first surface and the axially displaced second surface labeled with one, two, three, or four different detectable labels. In some embodiments, at least one image sensor comprises pixels having pixel dimensions selected such that the spatial sampling frequency for the optical system is at least twice the optical resolution of the optical system. In some embodiments, the optical system includes at least one tube lens positioned between the objective lens and the at least one image sensor, the at least one tube lens configured to modify imaging performance metrics for imaging the first interior surface of the flow cell and the second interior surface of the flow cell. In some embodiments, the flow cell has a wall thickness of at least 700 μm and a gap of at least 50 μm between the first interior surface and the second interior surface. In some embodiments, the at least one tube lens comprises, in order, an asymmetric convex-convex lens, a convex-plano lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the combination of the objective lens and the tube lens is configured to optimize a modulation transfer function in the mid- to high-spatial frequency range.In some embodiments, the imaging performance metrics include a measurement of the modulation transfer function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast-to-noise ratio (CNR), or any combination thereof. In some embodiments, the optical resolution of the images of the first surface and the axially displaced second surface is diffraction-limited across the entire field of view (FOV). In some embodiments, sequencing the nucleic acid molecules further includes performing avidity sequencing, nucleotide binding sequencing, or nucleotide incorporation sequencing on at least one of the first surface and the axially displaced second surface, and detecting the bound or incorporated nucleotide bases. In some embodiments, the method further includes determining the genotype of the sample, wherein determining the genotype of the sample comprises preparing the nucleic acid molecules for sequencing and then sequencing the nucleic acid molecules.

[0011] Disclosed herein is a system for sequencing nucleic acid molecules, the system comprising: a) an optical system including an objective lens and at least one image sensor, the optical system having a numerical aperture (NA) of less than 0.6 and a resolution of 1.0 mm; 2 and b) a processor programmed to: i) process the images of the first surface and the axially displaced second surface to correct for optical aberrations so 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 a nucleic acid molecule disposed on the first surface or the axially displaced second surface, or a complement thereof, to determine the identity of a nucleotide in the nucleic acid molecule.

[0012] In some embodiments, images of the first surface and the axially displaced second surface are acquired without moving an optical corrector into the optical path between the objective lens and the at least one image sensor. In some embodiments, images of the first surface and the axially displaced second surface are acquired only by refocusing the optical system. In some embodiments, the imaging system has a numerical aperture greater than 0.3. In some embodiments, the first surface and the axially displaced second surface comprise two surfaces of a flow cell. In some embodiments, the two surfaces of the flow cell are coated with a hydrophilic coating layer, and the hydrophilic coating layer is 1 mm thick. 2 The optical system further comprises labeled nucleic acid colonies disposed thereon at a surface density of greater than 10,000 nucleic acid colonies per surface. In some embodiments, the optical system comprises one, two, three, or four imaging channels configured to detect nucleic acid colonies disposed on at least one of the first surface or the axially displaced second surface labeled with one, two, three, or four different detectable labels. In some embodiments, the at least one image sensor comprises pixels having pixel dimensions selected such that the spatial sampling frequency for the optical system is at least twice the optical resolution of the optical system. In some embodiments, the system comprises at least one tube lens positioned between the objective lens and the at least one image sensor, the at least one tube lens configured to modify imaging performance metrics for imaging the first interior surface of the flow cell and the second interior surface of the flow cell. In some embodiments, the flow cell has a wall thickness of at least 700 μm and a gap of at least 50 μm between the first and second interior surfaces. In some embodiments, the optical system comprises two or more tube lenses designed to provide optimal imaging performance at two or more fluorescent wavelengths.

[0013] Disclosed herein is a fluorescence imaging system comprising: a) at least one light source configured to provide excitation light within one or more specified wavelength ranges; and b) an objective configured to collect fluorescence that arises from within a specified field of view of a sample plane upon exposure of the sample plane to the excitation light, wherein the numerical aperture of the objective is at least 0.3, the working distance of the objective is at least 700 μm, and the field of view is at least 2 mm. 2 and c) at least one image sensor, wherein fluorescence collected by the objective lens is imaged onto the image sensor, and pixel dimensions for the image sensor are selected such that a spatial sampling frequency for the fluorescence imaging system is at least twice the optical resolution of the fluorescence imaging system.

[0014] In some embodiments, the numerical aperture is at least 0.75. In some embodiments, the numerical aperture is at least 1.0. In some embodiments, the working distance is at least 850 μm. In some embodiments, the working distance is at least 1,000 μm. In some embodiments, the field of view is at least 2.5 mm. 2 In some embodiments, the field of view is at least 3 mm 2In some embodiments, the spatial sampling frequency is at least 2.5 times the optical resolution of the fluorescence imaging system. In some embodiments, the spatial sampling frequency is at least 3 times the optical resolution of the fluorescence imaging system. In some embodiments, the system further includes an XYZ translation stage such that the system is configured to acquire a series of two or more fluorescence images in an automated manner, with each image in the series being acquired for a different field of view. In some embodiments, the position of the sample plane is simultaneously adjusted in the X, Y, and Z directions to match the position of the focal plane of the objective lens between acquisition of images at different fields of view. In some embodiments, the time required for simultaneous adjustment in the X, Y, and Z directions is less than 0.4 seconds. In some embodiments, the system further includes an autofocus mechanism configured to adjust the position of the focal plane before acquiring images of the different field of view if the error signal indicates that the difference in the positions of the focal plane and the sample plane in the Z direction is greater than a specified error threshold. In some embodiments, the specified error threshold is 100 nm. In some embodiments, the specified error threshold is 50 nm. In some embodiments, the system includes three or more image sensors, and the system is configured to image fluorescence in each of three or more wavelength ranges onto a different image sensor. In some embodiments, the difference in the position of the focal plane for each of the three or more image sensors and the sample plane is less than 100 nm. In some embodiments, the difference in the position of the focal plane for each of the three or more image sensors and the sample plane is less than 50 nm. In some embodiments, the total time required to reposition the sample plane, adjust focus if necessary, and acquire an image is less than 0.4 seconds per field of view. In some embodiments, the total time required to reposition the sample plane, adjust focus if necessary, and acquire an image is less than 0.3 seconds per field of view.

[0015] Further disclosed in the present specification is a fluorescence imaging system for double-sided imaging of a flow cell, the fluorescence imaging system including: (a) an objective lens configured to collect fluorescence arising from within a specified field of view of a sample plane within the flow cell; and (b) at least one tube lens positioned between the objective lens and at least one image sensor, wherein the at least one tube lens is configured to modify imaging performance metrics for the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an internal surface of the flow cell, the flow cell having a wall thickness of at least 700 μm and a gap of at least 50 μm between its upper and lower internal surfaces, wherein the imaging performance metrics are substantially the same for imaging the upper or lower internal surface of the flow cell without moving an optical corrector in or out of the optical path between the flow cell and the at least one image sensor, without moving one or more optical elements of the tube lens along the optical path, and without moving one or more optical elements of the tube lens in or out of the optical path.

[0016] In some embodiments, the objective lens is a commercially available microscope objective lens. In some embodiments, the commercially available microscope objective lens has a numerical aperture of at least 0.3. In some embodiments, the objective lens has a working distance of at least 700 μm. In some embodiments, the objective lens is modified to compensate for a 0.17 mm coverslip thickness (or flow cell wall thickness). In some embodiments, the fluorescence imaging system further includes an electro-optic phase plate positioned adjacent to the objective lens and between the objective lens and the tube lens, the electro-optic phase plate providing correction for optical aberrations caused by filling the gap between the upper and lower internal surfaces of the flow cell with a liquid. In some embodiments, at least one tube lens is a conjugated lens comprising three or more optical components. In some embodiments, at least one tube lens is a conjugated lens comprising four optical components. In some embodiments, the four optical components comprise, in order, a first asymmetric convex-convex lens, a second convex-plano lens, a third asymmetric concave-concave lens, and a fourth asymmetric convex-concave lens. In some embodiments, the at least one tube lens is configured to modify an imaging performance metric for the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an interior surface of a flow cell having a wall thickness of at least 1 mm. In some embodiments, the at least one tube lens is configured to modify an imaging performance metric for the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an interior surface of a flow cell having a gap of at least 100 μm. In some embodiments, the at least one tube lens is configured to modify an imaging performance metric for the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an interior surface of a flow cell having a gap of at least 200 μm.In some embodiments, the system includes a single objective lens, two tube lenses, and two image sensors, where each of the two tube lenses is designed to provide optimal imaging performance at a different fluorescent wavelength. In some embodiments, the system includes a single objective lens, three tube lenses, and three image sensors, where each of the three tube lenses is designed to provide optimal imaging performance at a different fluorescent wavelength. In some embodiments, the system includes a single objective lens, four tube lenses, and four image sensors, where each of the four tube lenses is designed to provide optimal imaging performance at a different fluorescent wavelength. In some embodiments, the objective lens or at least one tube lens design is configured to optimize a modulation transfer function in a mid-to-high spatial frequency range. In some embodiments, the imaging performance metric includes a measurement of the modulation transfer function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof. In some embodiments, the difference in the imaging performance metric for imaging the upper and lower internal surfaces of the flow cell is less than 10%. In some embodiments, the difference in imaging performance metrics for imaging the upper and lower internal surfaces of the flow cell is less than 5%. In some embodiments, the use of at least one tube lens provides at least equal or better improvement in imaging performance metrics for double-sided imaging compared to imaging performance metrics for a conventional system including an objective lens, a motion actuation corrector, and an image sensor. In some embodiments, the use of at least one tube lens provides at least a 10% improvement in imaging performance metrics for double-sided imaging compared to imaging performance metrics for a conventional system including an objective lens, a motion actuation corrector, and an image sensor.

[0017] Disclosed herein is an illumination system for use in image-based solid-phase genotyping and sequencing applications, the illumination system comprising: a) a light source; and b) a liquid light guide configured to collect light emitted by the light source and deliver it to a specified field of illumination on a support surface comprising a tethered biopolymer.

[0018] In some embodiments, the illumination system further comprises a focusing lens. In some embodiments, the specified field of illumination is at least 2 mm 2 In some embodiments, the light delivered to the specified field of illumination is of uniform intensity across a specified field of view for an imaging system used to acquire an image of the support surface. In some embodiments, the specified field of view is at least 2 mm 2 In some embodiments, the light delivered to the specified field of illumination is of uniform intensity across the specified field of view when the coefficient of variation (CV) of the light intensity is less than 10%. In some embodiments, the light delivered to the specified field of illumination is of uniform intensity across the specified field of view when the coefficient of variation (CV) of the light intensity is less than 5%. In some embodiments, the light delivered to the specified field of illumination has a speckle contrast value of less than 0.1. In some embodiments, the light delivered to the specified field of illumination has a speckle contrast value of less than 0.05.

[0019] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term in this specification and a term in a cited document, the term in this specification shall control. [Brief explanation of the drawings]

[0020] The novel features of the invention are set forth with particularity in the appended claims. For a better understanding of the features and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0021] [Figure 1A] Non-limiting examples of dual-sided imaging support structures are illustrated schematically to show sample sites for imaging by the imaging systems disclosed herein: Figure 1A: Imaging of the front and rear interior surfaces of a flow cell; Figure 1B: Imaging of the front and rear exterior surfaces of a substrate. [Figure 1B] Non-limiting examples of dual-sided imaging support structures are illustrated schematically to show sample sites for imaging by the imaging systems disclosed herein: Figure 1A: Imaging of the front and rear interior surfaces of a flow cell; Figure 1B: Imaging of the front and rear exterior surfaces of a substrate. [Figure 2A]

[0023] Figure 2A illustrates a non-limiting example of a multi-channel fluorescence imaging module including a dichroic beam splitter for transmitting an excitation light beam to a sample and for receiving and redirecting the resulting fluorescence emission by reflection to four detection channels configured for detection of fluorescence emission at four different respective wavelengths or wavelength bands. Figure 2B illustrates a bottom isometric view. [Figure 2B]

[0023] Figure 2A illustrates a non-limiting example of a multi-channel fluorescence imaging module including a dichroic beam splitter for transmitting an excitation light beam to a sample and for receiving and redirecting the resulting fluorescence emission by reflection to four detection channels configured for detection of fluorescence emission at four different respective wavelengths or wavelength bands. Figure 2B illustrates a bottom isometric view. [Figure 3A]2A and 2B, including a dichroic beam splitter for transmitting an excitation light beam to a sample and for receiving and reflecting the resulting fluorescence emission, thereby redirecting it to four detection channels configured for detection of fluorescence emission at four different respective wavelengths or wavelength bands. FIG. 3A: Top view. FIG. 3B: Side view. [Figure 3B] 2A and 2B, including a dichroic beam splitter for transmitting an excitation light beam to a sample and for receiving and reflecting the resulting fluorescence emission, thereby redirecting it to four detection channels configured for detection of fluorescence emission at four different respective wavelengths or wavelength bands. FIG. 3A: Top view. FIG. 3B: Side view. [Figure 4] 1 is a graph illustrating dichroic filter performance of incidence versus beam angle. [Figure 5] 1 is a graph illustrating the relationship between beam footprint size and beam angle of incidence on a dichroic filter. [Figure 6A] 6A-6B are schematic diagrams illustrating exemplary configurations of dichroic filters and detection channels of a multi-channel fluorescence imaging module, where the dichroic filters have reflective surfaces tilted such that the angle between the incident beam (e.g., the angle at the center) of the dichroic filter and the reflective surface is less than 45°. (A) and (B) are detailed diagrams illustrating the angles of incidence (AOI) of the beams on the dichroic reflector. [Figure 6B] 6A-6B are schematic diagrams illustrating exemplary configurations of dichroic filters and detection channels of a multi-channel fluorescence imaging module, where the dichroic filters have reflective surfaces tilted such that the angle between the incident beam (e.g., the angle at the center) of the dichroic filter and the reflective surface is less than 45°. (A) and (B) are detailed diagrams illustrating the angles of incidence (AOI) of the beams on the dichroic reflector. [Figure 7] Graphs are provided illustrating improved dichroic filter performance corresponding to the imaging module configurations illustrated in FIGS. 6A and 6B. [Figure 8] Graphs are provided illustrating improved dichroic filter performance corresponding to the imaging module configurations illustrated in FIGS. 6A and 6B. [Figure 9A] Graphs are provided illustrating reduced surface deformation as a result of the imaging module configuration of Figures 6A and 6B. Figure 9A: Illustrates the effect of fold angle on image quality degradation caused by adding 1 wave of PV spherical power to the last mirror. Figure 9B: Illustrates the effect of fold angle on image quality degradation caused by adding 0.1 wave of PV spherical power to the last mirror. [Figure 9B] Graphs are provided illustrating reduced surface deformation as a result of the imaging module configuration of Figures 6A and 6B. Figure 9A: Illustrates the effect of fold angle on image quality degradation caused by adding 1 wave of PV spherical power to the last mirror. Figure 9B: Illustrates the effect of fold angle on image quality degradation caused by adding 0.1 wave of PV spherical power to the last mirror. [Figure 10A] Graphs are provided illustrating improved excitation filter performance (e.g., sharper transitions between passbands and surrounding stopbands) as a result of using s-polarization of the excitation beam. Figure 10A: Transmission spectra for an exemplary bandpass dichroic filter at 40 and 45 degree angles of incidence, where the incident beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. Figure 10B: By changing the orientation of the light source relative to the dichroic filter, the incident beam becomes s-polarized relative to the plane of the dichroic filter, resulting in a substantially sharper edge between the passband and stopband. [Figure 10B]Graphs are provided illustrating improved excitation filter performance (e.g., sharper transitions between passbands and surrounding stopbands) as a result of using s-polarization of the excitation beam. Figure 10A: Transmission spectra for an exemplary bandpass dichroic filter at 40 and 45 degree angles of incidence, where the incident beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. Figure 10B: By changing the orientation of the light source relative to the dichroic filter, the incident beam becomes s-polarized relative to the plane of the dichroic filter, resulting in a substantially sharper edge between the passband and stopband. [Figure 11A] 11A and 11B illustrate the modulation transfer function (MTF) of an exemplary dual-sided imaging system disclosed herein with a numerical aperture (NA) of 0.3. [Figure 11B] 11A and 11B illustrate the modulation transfer function (MTF) of an exemplary dual-sided imaging system disclosed herein with a numerical aperture (NA) of 0.3. [Figure 12A] 12A and 12B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.4. [Figure 12B] 12A and 12B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.4. [Figure 13A] 13A and 13B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.5. [Figure 13B] 13A and 13B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.5. [Figure 14A] 14A and 14B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.6. [Figure 14B] 14A and 14B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.6. [Figure 15A] 15A and 15B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.7. [Figure 15B] 15A and 15B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.7. [Figure 16A] 16A and 16B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.8. [Figure 16B] 16A and 16B illustrate the MTF of an exemplary dual-sided imaging system disclosed herein with a NA of 0.8. [Figure 17A] 17A and 17B provide plots of calculated Strehl ratios for imaging the surface of a second flow cell through the surface of a first flow cell. Figure 17A: Plots of the Strehl ratio for imaging the surface of a second flow cell through the surface of a first flow cell as a function of the thickness of the intervening fluid layer (height of the fluid channel) for different objective lenses and / or optical system numerical apertures. Figure 17B: Plots of the Strehl ratio as a function of numerical aperture for imaging the surface of a first flow cell and the surface of a second flow cell through an intervening layer of water having a thickness of 0.1 mm. [Figure 17B]17A and 17B provide plots of calculated Strehl ratios for imaging the surface of a second flow cell through the surface of a first flow cell. Figure 17A: Plots of the Strehl ratio for imaging the surface of a second flow cell through the surface of a first flow cell as a function of the thickness of the intervening fluid layer (height of the fluid channel) for different objective lenses and / or optical system numerical apertures. Figure 17B: Plots of the Strehl ratio as a function of numerical aperture for imaging the surface of a first flow cell and the surface of a second flow cell through an intervening layer of water having a thickness of 0.1 mm. [Figure 18] 1 provides a schematic diagram of a dual wavelength excitation / four channel emission fluorescence imaging system of the present disclosure. [Figure 19]

[0033] Figure 1 provides an optical ray tracing diagram for an objective lens design designed to image a surface on the opposite side of a 0.17 mm thick coverslip. [Figure 20] 19 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface on the opposite side of a 0.17 mm thick coverslip. [Figure 21] 19 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface on the opposite side of a 0.3 mm thick coverslip. [Figure 22] 19 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface separated from a surface on the other side of a 0.3 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 23] 19 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface on the opposite side of a 1.0 mm thick coverslip. [Figure 24]19 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface separated from a surface on the other side of a 1.0 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 25]

[0047] Figure 19 provides a ray tracing diagram for a tube lens design that, when used in conjunction with the objective illustrated in Figure 19, provides for improved double-sided imaging through a 1 mm thick coverslip. [Figure 26] 26 provides a plot of the modulation transfer function for the objective and tube lens combination illustrated in FIG. 25 as a function of spatial frequency when used to image a surface on the opposite side of a 1.0 mm thick coverslip. [Figure 27]

[0043] Figure 26 provides a plot of the modulation transfer function for the objective and tube lens combination illustrated in Figure 25 as a function of spatial frequency when used to image a surface separated from a surface on the other side of a 1.0 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 28] Figure 1 provides a ray tracing diagram for the disclosed tube lens design (left) optimized to provide high-quality double-sided imaging performance. Because the tube lens is no longer infinity corrected, a properly designed null lens (right) can be used in combination with the tube lens to correct for non-infinity corrected tube lenses for manufacturing and inspection purposes. [Figure 29] 1 illustrates one non-limiting example of a single capillary flow cell with two fluidic adapters. [Figure 30] 1 illustrates one non-limiting example of a flow cell cartridge designed to hold two capillaries and including a chassis, fluidic adapters, and optionally other components. [Figure 31]One non-limiting example of a system is illustrated that includes a single capillary flow cell connected to various fluid flow control components, where the single capillary is compatible with attachment to a microscope stage or custom imaging device for use in various imaging applications. [Figure 32] 1 illustrates one non-limiting example of a system including a capillary flow cell cartridge with an integrated diaphragm valve to reduce or minimize dead volume and conserve constant key reagents. [Figure 33] 1 illustrates one non-limiting example of a system including a capillary flow cell, a microscope setup, and a temperature control mechanism. [Figure 34] 1 illustrates one non-limiting example of temperature control of a capillary flow cell through the use of a metal plate placed in contact with the cartridge of the flow cell. [Figure 35] 1 illustrates one non-limiting approach for temperature control of a capillary flow cell that includes a non-contact thermal control mechanism. [Figure 36A] Illustrated are non-limiting examples of fabrication of a flow cell device: Figure 36A shows the preparation of a one-piece glass flow cell; Figure 36B shows the preparation of a two-piece glass flow cell; and Figure 36C shows the preparation of a three-piece glass flow cell. [Figure 36B] Illustrated are non-limiting examples of fabrication of a flow cell device: Figure 36A shows the preparation of a one-piece glass flow cell; Figure 36B shows the preparation of a two-piece glass flow cell; and Figure 36C shows the preparation of a three-piece glass flow cell. [Figure 36C] Illustrated are non-limiting examples of fabrication of a flow cell device: Figure 36A shows the preparation of a one-piece glass flow cell; Figure 36B shows the preparation of a two-piece glass flow cell; and Figure 36C shows the preparation of a three-piece glass flow cell. [Figure 37A]

[00137] Figures 37A and 37B illustrate non-limiting examples of glass flow cell designs. Figure 37A shows a one-piece glass flow cell design. Figure 37B shows a two-piece glass flow cell design. Figure 37C shows a three-piece glass flow cell design. [Figure 37B]

[00137] Figures 37A and 37B illustrate non-limiting examples of glass flow cell designs. Figure 37A shows a one-piece glass flow cell design. Figure 37B shows a two-piece glass flow cell design. Figure 37C shows a three-piece glass flow cell design. [Figure 37C]

[00137] Figures 37A and 37B illustrate non-limiting examples of glass flow cell designs. Figure 37A shows a one-piece glass flow cell design. Figure 37B shows a two-piece glass flow cell design. Figure 37C shows a three-piece glass flow cell design. [Figure 38] 1 illustrates the visualization of cluster amplification in the capillary lumen. [Figure 39] 1 provides a non-limiting example of a block diagram for a sequencing system as disclosed herein. [Figure 40] As disclosed herein, a non-limiting example of a flow chart for a method of sequencing is provided. [Figure 41] As disclosed herein, a schematic, non-limiting example is provided for a structured lighting system. [Figure 42] 1 provides a non-limiting example of a flowchart for acquiring and processing structured illumination images of a surface of a flow cell as disclosed herein. [Figure 43A]

[0013] Figures 43A-43B provide non-limiting schematic diagrams of multiplexed readheads as disclosed herein. Figure 43A: A side view of a multiplexed readhead in which individual microfluorometers are configured to image a common surface, e.g., the interior surface of a flow cell. Figure 43B: A top view of a multiplexed readhead illustrating the imaging paths acquired by individual microfluorometers of the multiplexed readhead. [Figure 43B]

[0013] Figures 43A-43B provide non-limiting schematic diagrams of multiplexed readheads as disclosed herein. Figure 43A: A side view of a multiplexed readhead in which individual microfluorometers are configured to image a common surface, e.g., the interior surface of a flow cell. Figure 43B: A top view of a multiplexed readhead illustrating the imaging paths acquired by individual microfluorometers of the multiplexed readhead. [Figure 44A]

[0013] Figures 44A-44B provide non-limiting schematic diagrams of multiplexed readheads as disclosed herein. Figure 44A: A side view of a multiplexed readhead in which a first subset of the plurality of individual microfluorometers is configured to image a first surface, e.g., a first interior surface of a flow cell, and a second subset of the plurality of individual microfluorometers is configured to image a second surface, e.g., a second interior surface of a flow cell. Figure 44B: A top view of the multiplexed readhead of Figure 44A illustrating the imaging paths acquired by the individual microfluorometers of the multiplexed readhead. [Figure 44B]

[0013] Figures 44A-44B provide non-limiting schematic diagrams of multiplexed readheads as disclosed herein. Figure 44A: A side view of a multiplexed readhead in which a first subset of the plurality of individual microfluorometers is configured to image a first surface, e.g., a first interior surface of a flow cell, and a second subset of the plurality of individual microfluorometers is configured to image a second surface, e.g., a second interior surface of a flow cell. Figure 44B: A top view of the multiplexed readhead of Figure 44A illustrating the imaging paths acquired by the individual microfluorometers of the multiplexed readhead. DETAILED DESCRIPTION OF THE INVENTION

[0022] There is a need for fluorescence imaging methods and systems that provide improved optical resolution and improved image quality for genomics applications, leading to corresponding improvements in the accuracy of genomic testing. Disclosed herein are optical system designs for high-performance fluorescence imaging methods and systems that may provide any one or more of improved optical resolution (including high-performance optical resolution), improved image quality, and high throughput for fluorescence imaging-based genomics applications. The disclosed optical illumination and imaging system designs may provide any one or more of the following advantages: improved dichroic filter performance, improved uniformity of the dichroic filter frequency response, improved excitation beam filtering, larger field of view, improved spatial resolution, improved modulation transmission, improved contrast-to-noise ratio, and improved image quality, higher spatial sampling frequencies, faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., of different fields of view), improved imaging system duty cycle, and higher throughput image acquisition and image analysis.

[0023] In some examples, improved imaging performance, for example, in double-sided (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or cover slip) thickness >700 μm) and flow channels (e.g., fluid channel height or thickness of 50-200 μm), may be achieved using novel objective lens designs that correct for optical aberrations introduced by the thick cover slip and / or the imaging surface opposite the fluid channel from the objective lens.

[0024] In some examples, improved imaging performance, for example, for double-sided (flow cell) imaging applications including the use of thick flow cell walls (e.g., wall (or cover slip) thickness >700 μm) and flow channels (e.g., fluid channel height or thickness of 50-200 μm), may be achieved even when using commercially available, off-the-shelf objective lenses by using novel tube lens designs that, unlike conventional microscope tube lenses that simply form an image at an intermediate image plane, induce correction of optical aberrations due to the thick flow cell walls and / or intervening fluid layer in combination with the objective lens.

[0025] In some examples, improved imaging performance, for example in multi-channel (e.g., two-color or four-color) imaging applications, may be achieved by using multiple tube lenses, one for each imaging channel, where each tube lens design is optimized for the particular wavelength range used in that imaging channel.

[0026] In some examples, improved imaging performance, for example, for double-sided (flow cell) imaging applications, may be achieved by using an electro-optic phase plate in combination with an objective lens to correct for optical aberrations induced by a layer of fluid separating the top (near) and bottom (far) inner surfaces of the flow cell. In some examples, this design approach may correct for vibrations introduced by, for example, a motion-actuated corrector that moves in and out of the optical path depending on which surface of the flow cell is being imaged.

[0027] Various multichannel fluorescence imaging module designs are disclosed, which can include illumination and imaging optical paths that include a folded optical path (one or more beam splitters or combiners, such as a dichroic beam splitter or combiner) that directs an excitation light beam to an objective lens and directs emission light transmitted through the objective lens to multiple detection channels. Some particularly advantageous features of the fluorescence imaging modules described herein include the specification of dichroic filter angles of incidence that result in sharper and / or more uniform transitions between the passband and stopband wavelength regions of the dichroic filter. Such filters may be included within a folded optical system and may also include a dichroic beam splitter or combiner. Further advantageous features of the disclosed imaging optical designs may include the position and orientation of one or more excitation light sources and one or more detection optical paths relative to the objective lens and the dichroic filter that receives the excitation beam. The excitation beam may also be linearly polarized, and the linear polarization may be oriented such that s-polarized light is incident on the dichroic reflective surface of the dichroic filter. Such a feature may potentially improve excitation beam filtering and / or reduce wavefront errors introduced into the emitted light beam by surface deformations of the dichroic filter. The fluorescence imaging modules described herein may or may not include any of these features and may or may not include any of these advantages.

[0028] Further described herein are devices and systems that are configured to analyze a large number of different nucleic acid sequences, for example, by imaging the array of immobilized nucleic acid molecules or amplified nucleic acid clusters formed on the surface of a flow cell.The devices and systems described herein are also useful for, for example, performing sequencing for comparative genomics, tracking gene expression, performing microRNA sequence analysis, characterizing epigenomics, aptamers, and phage display libraries, and performing other sequencing applications.The devices and systems disclosed herein include various combinations of optical, mechanical, fluidic, thermal, electrical, and computing devices / aspects. Advantages offered by the disclosed flow cell devices, cartridges, and systems include, but are not limited to: (i) reduced complexity and cost of manufacturing the devices and systems, (ii) significantly reduced consumable costs (e.g., compared to the costs of currently available nucleic acid sequencing systems), (iii) compatibility with typical flow cell surface functionalization methods, (iv) flexible flow control when combined with microfluidic components, such as syringe pumps and diaphragm valves, and (v) flexible system throughput.

[0029] Disclosed herein are capillary flow cell devices and capillary flow cell cartridges comprised of prefabricated, disposable, single-lumen (e.g., single fluid flow channel) or multi-lumen capillaries, which may also include a fluidic adapter, a cartridge chassis, one or more integrated fluid flow control components, or any combination thereof. Also disclosed herein are capillary flow cell-based systems, which may include one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell cartridges (or microfluidic cartridges), a fluid flow controller module, a temperature control module, an imaging module, or any combination thereof.

[0030] Design features of some of the disclosed capillary flow cell devices, cartridges, and systems include, but are not limited to, (i) a single flow channel configuration; (ii) sealed, reliable, and repeatable switching between reagent flows that can be performed with a simple load / unload mechanism, such that the fluidic interface between the system and capillary is securely sealed, thereby facilitating capillary replacement and system reuse and enabling precise control of reaction conditions such as reagent concentration, pH, and temperature; (iii) interchangeable single-fluid flow channel devices or capillary flow cell cartridges containing multiple flow channels that can be used interchangeably to provide flexible system throughput; and (iv) compatibility with a variety of detection methods, such as fluorescence imaging.

[0031] Although the disclosed capillary flow cell devices and systems, capillary flow cell cartridges, capillary flow cell-based systems, microfluidic devices and cartridges, and microfluidic chip-based flow cell systems are described primarily in the context of their use for nucleic acid sequencing applications, various aspects of the disclosed devices and systems can be applied not only to nucleic acid sequencing but also to any other type of chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. It should be understood that different aspects of the disclosed methods, devices, and systems can be evaluated individually, collectively, or in combination with one another. While discussed herein primarily in the context of fluorescence imaging (including, for example, fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, etc.), those skilled in the art will appreciate that many of the disclosed optical design approaches and features are applicable to other imaging modules, such as bright-field imaging, dark-field imaging, and phase-contrast imaging.

[0032] Definitions: Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0033] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" is intended to encompass "and / or" unless specifically stated otherwise.

[0034] As used herein, a number followed by the term "about" refers to a number that is plus or minus 10% of that number. When used in the context of a range, the term "about" refers to a range of minus 10% of the lowest value and plus 10% of the highest value.

[0035] As used herein, the terms "imaging module," "imaging unit," "imaging system," "optical imaging module," "optical imaging unit," and "optical imaging system" are used interchangeably and may include components or subsystems of larger systems, which may also include fluidics modules, temperature control modules, translation stages, robotics fluid distribution and / or microplate handling, processors or computers, instrument control software, data analysis, display software, etc.

[0036] As used herein, a "detection channel" refers to an optical path (and / or optical components therein) within an optical system configured to deliver an optical signal arising from a sample to a detector. In some examples, a detection channel may be configured to perform spectroscopic measurements, e.g., monitoring of fluorescent signals or other optical signals using a detector such as a photomultiplier tube. In some examples, a "detection channel" may also be an "imaging channel," i.e., an optical path (and / or optical components therein) within an optical system configured to capture an image and deliver the image to an image sensor.

[0037] As used herein, "detectable label" may refer to any of a variety of detectable labels or tags known to those skilled in the art. Examples include, but are not limited to, chromophores, fluorophores, quantum dots, upconverting fluorophores, luminescent or chemiluminescent molecules, radioisotopes, magnetic nanoparticles, or mass tags. In some examples, preferred labels may include fluorophores.

[0038] As used herein, "excitation wavelength" refers to the wavelength of light used to excite a fluorescent indicator (e.g., a fluorophore or dye molecule) to produce fluorescence. While an excitation wavelength is commonly specified as a single wavelength, e.g., 620 nm, it will be understood by those skilled in the art that it refers to a wavelength range centered around the specified wavelength or the bandpass of an excitation filter. For example, in some instances, light of a specified excitation wavelength includes light at the specified wavelength ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or more. In some instances, the excitation wavelength used may or may not coincide with the absorption peak maximum of the fluorescent indicator.

[0039] As used herein, "emission wavelength" refers to the wavelength of light emitted by a fluorescent indicator (e.g., a fluorophore or dye molecule) upon excitation with light of an appropriate wavelength. While an emission wavelength is commonly specified as a single wavelength, e.g., 670 nm, it will be understood by those skilled in the art that it refers to a wavelength range centered around the specified wavelength or the bandpass of an emission filter. In some examples, light of a specified emission wavelength includes light at the specified wavelength ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or more. In some examples, the emission wavelength used may or may not coincide with the emission peak maximum of the fluorescent indicator.

[0040] As used herein, fluorescence is "specific" when it arises from a fluorophore annealed or otherwise tethered to the surface, such as a fluorescently labeled nucleic acid sequence, that has a region of reverse complementarity to, and is annealed to, a corresponding segment of an oligonucleotide adaptor on the surface. This fluorescence is contrasted with fluorescence arising from a fluorophore that is not tethered to the surface by such an annealing process, or, in some cases, background fluorescence of the surface.

[0041] As used herein, a "nucleic acid" (also referred to as a "nucleic acid molecule," "polynucleotide," "oligonucleotide," "ribonucleic acid (RNA)," or "deoxyribonucleic acid (DNA)") is a linear polymer of two or more nucleotides linked by covalent internucleoside bonds, or variants or functional fragments thereof. In naturally occurring examples of nucleic acids, the internucleoside linkages are typically phosphodiester linkages. However, other examples optionally include other internucleoside linkages, such as phosphorothiolate linkages, which may or may not include phosphate groups. Nucleic acids include double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA / RNA hybrids, peptide-nucleic acids (PNAs), hybrids of PNAs with DNA or RNA, and may also include other types of nucleic acid modifications.

[0042] As used herein, "nucleotide" refers to a nucleotide, a nucleoside, or an analog thereof. In some cases, the nucleotide is an N-glycoside or C-glycoside of a purine base or a pyrimidine base (e.g., a deoxyribonucleoside containing 2-deoxy-D-ribose or a ribonucleoside containing D-ribose). Examples of other nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, etc.

[0043] Fluorescence imaging viewed as an information pipeline: A useful abstraction of the role that fluorescence imaging systems play in typical genomic assay technologies (including nucleic acid sequencing applications) is as an information pipeline, where photon signals enter at one end of the pipeline, e.g., at the objective lens used for imaging, and location-specific information about the fluorescent signal emerges at the other end of the pipeline, e.g., at the image sensor. As more information is sent through this pipeline, some content is inevitably lost during this transmission process and not recovered. An example of this is when labeled molecules (or clonal amplified clusters of molecules) are too numerous within a small area of ​​the substrate surface to be clearly resolved in the image, making it difficult for the image sensor to distinguish between photon signals originating from neighboring clusters of molecules, thereby increasing the likelihood that signals will be attributed to the wrong cluster, leading to detection errors.

[0044] Optical Imaging Module Design: The goal of designing the optical imaging module is therefore to maximize the flow of information content through this detection pipeline and minimize detection errors. Some key design elements that need to be addressed in the design process are:

[0045] 1) Matching the physical feature density of the substrate surface to the overall image quality of the optical imaging system and the pixel sampling frequency of the image sensor used. Mismatches in these parameters may result in lost or even erroneous information being generated, for example, spatial aliasing if the pixel sampling frequency is less than twice the optical resolution limit.

[0046] 2) Matching the size of the imaged area to the overall image quality and focus quality across the field of view of the optical imaging system.

[0047] 3) Matching the optical collection efficiency, modulation transfer capability, and image sensor performance characteristics of the optical system design to the expected fluorescence photon flux and dye efficiency (related to the dye extinction coefficient and fluorescence quantum yield) for the input excitation photon flux, while accounting for background signal and system noise.

[0048] 4) Maximizing the separation of spectral components to reduce crosstalk between fluorescence imaging channels.

[0049] 5) Effective synchronization of the image acquisition process by repositioning the sample or optical components between image captures of different fields of view minimizes downtime (or maximizes the duty cycle) of the imaging system and therefore maximizes the overall throughput of the image capture process.

[0050] This disclosure addresses each of the design elements outlined above and describes a systematic method for creating component-level specifications for imaging systems.

[0051] Improved optical resolution and image quality to improve or maximize information transmission and throughput: One non-limiting design practice may begin with the optical resolution required to distinguish two adjacent features specified in terms of the number of line pairs per mm (lp / mm), X, and convert that to a corresponding numerical aperture (NA) requirement. The numerical aperture requirement can then be used to evaluate the resulting modulation transfer function and its impact on image contrast.

[0052] The standard modulation transfer function (MTF) describes the spatial frequency response to image contrast (modulation) transmitted through an optical system; image contrast decreases as a function of spatial frequency and increases as the NA increases. This function limits the contrast / modulation that can be achieved for a given NA. Furthermore, wavefront errors can negatively affect the MTF; therefore, it is desirable to improve or optimize optical system designs using the true system MTF instead of that predicted by a diffraction-limited optical system. As used herein, MTF refers to the MTF of the entire system (including the complete optical path from the coverslip to the image sensor), although design practice may primarily consider the MTF of the objective lens.

[0053] The target to be imaged is a series of high-density "spots" on a surface (either randomly distributed or patterned), and in genomic testing applications, the minimum modulation transmission value required by downstream analysis to resolve two adjacent spots and distinguish between four possible states (e.g., on-off, on-on, off-on, and off-off) can be determined. For example, assume the spots are small enough and approximated as point-like sources of light. If the detection task is to determine whether two adjacent spots separated by a distance, d, are on or off (in other words, bright or dark), and the contrast-to-noise ratio (CNR) of the fluorescence signal arising from the spots in the sample plane (object plane) is C, then sample Assuming that, under ideal conditions, the CNR of the read signal for two adjacent spots in the image sensor plane, C image , is C image =C sample *Can be closely approximated as MTF(1 / d), where MTF(1 / d) is the MTF value when spatial frequency = (1 / d).

[0054] In a typical design, the value of C may need to be at least 4 so that simple thresholding can be used to avoid misclassification of the fluorescent signal.image Assuming a Gaussian distribution of fluorescent signal intensities around a mean value of >4, the expected error in accurately classifying a fluorescent signal (e.g., on or off) is <0.035%. By using proprietary high-CNR sequencing and surface chemistry as described in U.S. Patent Application No. 16 / 363,842, the sample plane CNR (C) of clonally amplified clusters of labeled oligonucleotide molecules tethered to the top surface of a substrate can be measured. sample ) values ​​of over 12 (or even much higher numbers) can be achieved when measured in low density fields (i.e., when the surface density of clusters or spots is low), where the MTF has a value approaching 100%. sample >12, and target a classification error rate <0.1% (hence, C image >4), and in some implementations, a minimum value for M(1 / d) can be determined as M(1 / d) = 4 / 12 ~ 33%. Thus, a modulation transfer function threshold of at least 33% may be used to preserve the information content of the transmitted image.

[0055] A design practice can relate the minimum separation distance, d, of two features or spots to the optical resolution requirement (as described and specified above for X (lp / mm)) as d = (1 mm) / X, i.e., d is the minimum separation distance between two features or spots that can be fully resolved by the optical system. In some designs disclosed herein, the goal of the design analysis is to increase or maximize the relative information transmission, and this design criterion can be relaxed to d = (1 mm) / X / A, where 2 > A > 1. For the same optical resolution of X lp / mm, the value of d, i.e., the minimum resolvable spot separation distance at the sample plane, is reduced, allowing for the use of higher feature densities.

[0056] A design practice is to use the Nyquist criterion to determine the minimum spatial sampling frequency at the sample plane, where the spatial sampling frequency is S>=2*X (and where X is the optical resolution of the imaging system specified in terms of X lp / mm, as above). When the spatial sampling frequency of the system is close to the Nyquist criterion, as is often the case, an imaging system resolution above S results in aliasing because the high frequency information resolved by the optical system cannot be adequately sampled by the image sensor.

[0057] In some of the designs disclosed herein, an oversampling scheme based on the relationship S=B*Y (where B>=2 and Y is the MTF limit of the true optical system) may be used to further improve the information transfer capacity of the imaging system. As shown above, X (lp / mm) corresponds to the smallest practical non-zero (>33%) modulation transmission value, where Y (lp / mm) is the optical resolution limit such that modulation at Y (lp / mm) is zero. Thus, in the disclosed designs, Y (lp / mm) may advantageously be significantly greater than X. For values ​​of B>=2, the disclosed designs oversample the sample object frequency X, i.e., S>=B*Y>2*X.

[0058] The above relationship can be used to determine the system magnification and may provide an upper bound for the image sensor pixel size. The selection of the image sensor pixel size is matched to the optical quality of the system and the spatial sampling frequency required to reduce aliasing. A lower bound for the image sensor pixel size can be determined based on the photon throughput, since smaller pixels have a higher relative noise contribution.

[0059] However, other design approaches are possible. For example, reducing the NA below 0.6 (e.g., 0.5 or less) may result in an increased depth of field. Such an increased depth of field may enable double-sided imaging, where two surfaces at different depths can be imaged simultaneously, with or without refocusing. As discussed above, reducing the NA may reduce optical resolution. In some implementations, the use of higher excitation beam power, e.g., 1 W or more, may be utilized to generate a strong signal. Also, inherently high-contrast samples (i.e., including sample surfaces exhibiting a strong foreground signal and dramatically reduced background signal) may be used to facilitate the acquisition of high-contrast-to-noise ratio (CNR) images, e.g., images with CNR values ​​>20, thereby providing improved signal discrimination for base calling, such as in nucleic acid sequencing applications. In some optical system designs disclosed herein, a sample support structure, such as a flow cell with a hydrophilic surface, is used to reduce background noise.

[0060] In various implementations, a large field of view (FOV) is provided by the disclosed optical systems. For example, an FOV of greater than 2 mm or 3 mm may be provided in some optical imaging systems, including, for example, an objective lens and a tube lens. In some cases, the optical imaging system provides a low magnification, for example, a magnification of less than 10x. Such reduced magnification can facilitate large FOV designs in some implementations. Despite the reduced magnification, the optical resolution of such systems may still be sufficient, or a detector array with a small pixel size or pitch may be used. In some implementations, an image sensor including a pixel size smaller than twice the optical resolution provided by the optical imaging system (e.g., the objective lens and tube lens) may be used to satisfy the Nyquist theorem.

[0061] Other designs are also possible. In some optical designs configured to provide double-sided imaging capable of simultaneously imaging two surfaces at different depths, the optical imaging system (e.g., objective lens and / or tube lens) is configured to reduce optical aberrations for imaging the two surfaces (e.g., two planes) at the two respective depths compared to other locations (e.g., other planes) at other depths. In addition, the optical imaging system may be configured to reduce aberrations for imaging the two surfaces (e.g., two planes) at the two respective depths through a transparent layer (such as a glass layer (e.g., a coverslip)) on the sample support structure and through a solution (e.g., an aqueous solution) containing or in contact with the sample on at least one of the two surfaces.

[0062] Multi-channel Fluorescence Imaging Modules and Systems: In some examples, the imaging modules or systems disclosed herein may include fluorescence imaging modules or systems. In some examples, the fluorescence imaging systems disclosed herein may include a single fluorescence excitation light source (to provide excitation light at a single wavelength or within a single excitation wavelength range) and an optical path configured to deliver the excitation light to a sample (e.g., fluorescently tagged nucleic acid molecules or clusters thereof disposed on a substrate surface). In some examples, the fluorescence imaging systems disclosed herein may include a single fluorescence emission imaging detection channel, e.g., an optical path configured to collect fluorescence emitted by the sample and deliver an image of the sample (e.g., an image of the substrate surface on which fluorescently tagged nucleic acid molecules or clusters thereof are disposed) to an image sensor or other light detection device. In some examples, the fluorescence imaging system may include an optical path configured to deliver two, three, four, or more than four fluorescence excitation lights and / or excitation light at two, three, four, or more than four excitation wavelengths (or within two, three, four, or more than four excitation wavelength ranges). In some examples, the fluorescence imaging systems disclosed herein may include two, three, four, or more than four fluorescence emission imaging detection channels configured to collect fluorescence emitted by the sample at two, three, four, or more emission wavelengths (or within two, three, four, or more emission wavelength ranges) and deliver an image of the sample (e.g., an image of a substrate on which fluorescently tagged nucleic acid molecules or clusters thereof are disposed) to two, three, four, or more image sensors or other light detection devices.

[0063] Dual-Sided Imaging: In some examples, imaging systems, including the fluorescence imaging systems disclosed herein, may be configured to acquire high-resolution images of a single sample support structure or substrate surface. In some examples, imaging systems, including the fluorescence imaging systems disclosed herein, may be configured to acquire high-resolution images of two or more sample support structures or substrate surfaces (e.g., two or more surfaces of a flow cell). In some examples, the high-resolution images provided by the disclosed imaging systems may be configured to monitor reactions occurring on two or more surfaces of a flow cell (e.g., nucleic acid hybridization reactions, amplification reactions, and / or sequencing reactions) as various reagents flow through the flow cell or around the flow cell substrate. Figures 1A and 1B provide schematic diagrams of such dual-sided support structures. Figure 1A shows a dual-sided support structure, such as a flow cell, including an internal flow channel through which analytes or reagents can flow. Flow channels may be formed between first and second layers, between upper and lower layers, and / or between front and rear layers, such as a first plate and a second plate, an upper plate and a lower plate, and / or a front and rear plate, as shown. One or more of the plates may include a glass plate, such as a coverslip. In some implementations, the layers include borosilicate glass, quartz, or plastic. The interior surfaces of these upper and lower layers provide flow channel walls that help restrict the flow of analytes or reagents through the flow channels of the flow cell. In some designs, these interior surfaces are planar. Similarly, the upper and lower layers may be planar. In some designs, at least one additional layer (not shown) is disposed between the upper and lower layers. This additional layer may have one or more interrupted paths therein, which may define one or more flow channels and help control the flow of analytes or reagents within the flow channels. Further discussion of sample support structures, such as flow cells, can be found below.

[0064] 1A schematically illustrates multiple fluorescent sampling sites on the first and second, upper and lower, and / or front and rear interior surfaces of a flow cell. In some implementations, sample-binding reactions may occur at these sites, which may result in fluorescent light being emitted from these sites. (Note that FIG. 1A is schematic and not to scale; e.g., the size and spacing of the fluorescent sampling sites may be smaller than shown.)

[0065] FIG. 1B illustrates another double-sided support structure having two surfaces containing fluorescent sampling sites to be imaged. The sample support structure includes a substrate having first and second, upper and lower, and / or front and rear exterior surfaces. In some designs, these exterior surfaces are planar. In various implementations, analytes or reagents are flowed across these first and second exterior surfaces. FIG. 1B also illustrates a schematic representation of multiple fluorescent sampling sites on the first and second, upper and lower, and / or front and rear exterior surfaces of the sample support structure. In some implementations, reactions may occur at these sites that bind the sample, thereby causing fluorescence to be emitted from these sites. (Note that FIG. 1B is schematic and not to scale; for example, the size and spacing of the fluorescent sampling sites may be smaller than shown.)

[0066] In some examples, the fluorescence imaging modules and systems described herein may be configured to image fluorescence sampling sites on a first surface and a second surface at various distances from the objective lens. In some designs, only one of the first surface or the second surface is in focus at a time. Thus, in such designs, one of the surfaces is imaged at a first time and the other surface is imaged at a second time. The focus of the fluorescence imaging module may be changed after imaging one surface to image the other surface with equivalent optical resolution when the images of the two surfaces are not simultaneously in focus. In some designs, an optical correction element may be introduced into the optical path between the sample support structure and the image sensor to image one of the two surfaces. The depth of field in such fluorescence imaging configurations may not be large enough to include both the first and second surfaces. In some implementations of the fluorescence imaging modules described herein, both the first and second surfaces may be imaged at the same time, i.e., simultaneously. For example, the fluorescence imaging module may have a depth of field large enough to include both surfaces. In some examples, such an increased depth of field can be provided by, for example, reducing the numerical aperture of the objective lens (or microscope objective lens), as discussed in more detail below.

[0067] As shown in FIGS. 1A and 1B, the imaging optics (e.g., an objective lens) may be positioned at an appropriate distance (e.g., a distance corresponding to the working distance) from the first and second surfaces to form a focused image of the first and second surfaces on the image sensor of the detection channel. As shown in the examples of FIGS. 1A and 1B, the first surface may be between the objective lens and the second surface. For example, as shown, the objective lens may be positioned above both the first and second surfaces, and the first surface may be positioned above the second surface. The first and second surfaces may be at different depths, for example. The first and second surfaces may be at different distances from any one or more of the fluorescence imaging module, the illumination imaging module, the imaging optics, or the objective lens. The first and second surfaces may be separated from each other, and the first surface may be spaced above the second surface. In the example shown, the first and second surfaces are planar surfaces separated from each other along a direction perpendicular to the first and second planes. Also, in the illustrated example, the objective lens has an optical axis, and the first surface and the second surface are separated from each other along the optical axis. Similarly, the separation between the first surface and the second surface may correspond to a longitudinal distance, such as along the optical path of the excitation beam and / or along the optical axis through the fluorescence imaging module and / or the objective lens. Thus, these two surfaces may be separated from each other by a distance in the longitudinal direction (Z), which may be along the central axis of the excitation beam and / or the optical axis of the objective lens and / or the fluorescence imaging module. This separation may correspond, for example, to a flow channel in a flow cell in some implementations.

[0068] In various designs, the objective lens (perhaps in combination with another optical component, e.g., a tube lens) has a depth of field and / or depth of focus at least as large as the longitudinal (Z-direction) separation between the first and second surfaces. The objective lens may thus, alone or in combination with additional optical components, simultaneously form focused images of both the first and second surfaces on the image sensors of one or more detection channels, where these images have equivalent optical resolution. In some implementations, the imaging module may or may not require refocusing to capture images of both the first and second surfaces with equivalent optical resolution. In some implementations, correction optics may not need to be moved in or out of the optical path of the imaging module to form focused images of the first and second surfaces. Similarly, in some implementations, one or more optical elements (e.g., lens elements) of the imaging module (e.g., objective lens and / or tube lens) do not need to be moved longitudinally along the first and / or second optical paths (e.g., along the optical axis of the imaging optics) to form a focused image of the first surface, compared to the position of the one or more optical elements when used to form a focused image of the second surface. However, in some implementations, the imaging module includes an autofocus system configured to simultaneously focus on both the first and second surfaces. In various implementations, the sample is focused to sufficiently resolve the sampling sites, which are closely spaced together in a lateral direction (e.g., the X and Y directions). Thus, in various implementations, no optical elements enter the optical path between the sample support structure (e.g., the translation stage supporting the sample support structure) and the image sensor (or photodetector array) to form focused images of the fluorescent sampling sites on the first surface of the sample support structure and the second surface of the sample support structure for at least one detection channel.Similarly, in various implementations, optical corrections are not used to form focused images of the fluorescent sampling sites on the first surface of the sample support structure on the image sensor or photodetector array, and the optical corrections are not the same as the optical corrections used to form focused images of the fluorescent sampling sites on the second surface of the sample support structure on the image sensor or photodetector array. Additionally, in certain implementations, optical elements in the optical path of at least one detection channel between the sample support structure (e.g., a translation stage supporting the sample support structure) and the image sensor are not adjusted differently to form focused images of the fluorescent sampling sites on the first surface of the sample support structure than to form focused images of the fluorescent sampling sites on the second surface of the sample support structure. Similarly, in various implementations, optical elements in the optical path of at least one detection channel between the sample support structure (e.g., a translation stage supporting the sample support structure) and the image sensor are moved a different amount or in a different direction on the image sensor to form focused images of the fluorescent sampling sites on the first surface of the sample support structure on the image sensor than to form focused images of the fluorescent sampling sites on the second surface of the sample support structure on the image sensor. Any combination of features is possible. For example, in some implementations, focused images of the upper and lower interior surfaces of the flow cell can be obtained without moving an optical corrector in or out of the optical path between the flow cell and at least one image sensor, and without moving one or more optical elements of the imaging system (e.g., the objective lens and / or the tube lens) along the optical path (e.g., the optical axis) therebetween. For example, focused images of the upper and lower interior surfaces of the flow cell can be obtained without moving one or more optical elements of the tube lens in or out of the optical path or along the optical path (e.g., the optical axis) therebetween.

[0069] Any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at two locations, such as two planes corresponding to two surfaces on a flow cell or other sample support structure, e.g., where the fluorescent sample site is located. Any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at a location or plane selected relative to another location or plane, such as the first and second surfaces containing the fluorescent sample site on a double-sided flow cell. For example, any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at two depths or planes located at different distances from the objective lens compared to aberrations associated with planes at other depths or other distances from the objective lens. For example, optical aberrations may be smaller for imaging the first and second surfaces than elsewhere in a region extending from about 1 mm to about 10 mm from the objective lens. Additionally, any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may, in some instances, be configured to correct optical aberrations induced by transmission of emitted light through one or more portions of the sample support structure, such as one of the surfaces to which the sample is adhered, as well as a layer possibly containing a solution in contact with the sample. This layer (e.g., a coverslip or a wall of a flow cell) may comprise, for example, glass, quartz, plastic, or other transparent material having a refractive index that introduces optical aberrations.

[0070] Thus, imaging performance may be substantially the same when imaging the first and second surfaces. For example, the optical transfer function (OTF) and / or modulation transfer function (MTF) may be substantially the same for imaging the first and second surfaces. Either or both of these transfer functions may be within 20%, 15%, 10%, 5%, 2.5%, or 1% of each other, for example, at one or more specified spatial frequencies or when averaged over a range of spatial frequencies, or within any range formed by any of these values. Thus, imaging performance metrics may be substantially the same for imaging the upper or lower interior surface of a flow cell without moving an optical corrector in or out of the optical path between the flow cell and the at least one image sensor and without moving one or more optical elements of the imaging system (e.g., the objective lens and / or the tube lens) along the optical path (e.g., the optical axis) therebetween. For example, the imaging performance metric may be substantially the same for imaging the upper or lower interior surface of the flow cell without moving one or more optical elements of the tube lens in or out of the optical path or along the optical path (e.g., optical axis) therebetween. Additional discussion of MTF is included below and in U.S. Provisional Patent Application No. 62 / 962,723, filed January 17, 2020, which is incorporated herein by reference in its entirety.

[0071] Those skilled in the art will appreciate that the disclosed imaging modules or systems may, in some examples, be freestanding optical systems designed to image a sample or substrate surface. In some examples, they may include one or more processors or computers. In some examples, they may include one or more software packages providing instrument control and / or image processing functions. In some examples, in addition to optical components such as light sources (e.g., solid-state lasers, dye lasers, diode lasers, arc lamps, tungsten-halogen lamps, etc.), lenses, prisms, mirrors, dichroic reflectors, beam splitters, optical filters, optical passband filters, light guides, optical fibers, apertures, and image sensors (e.g., complementary metal-oxide semiconductor (CMOS) image sensors and cameras, charge-coupled device (CCD) image sensors and cameras, etc.), they may also include mechanical and / or opto-mechanical components such as XY translation stages, XYZ translation stages, piezoelectric focusing mechanisms, electro-optic phase plates, etc. In some instances, they may function as modules, components, subassemblies, or subsystems of larger systems designed, for example, for genomics applications (e.g., genetic testing applications and / or nucleic acid sequencing applications).For example, in some instances, they may function as modules, components, subassemblies, or subsystems of larger systems that further include light-tight and / or other environmentally controlled enclosures, temperature control modules, flow cells and cartridges, fluidics control modules, fluid dispensing robotics components, cartridge and / or microplate handling (pick-and-place) robotics components, one or more processors or computers, one or more local and / or cloud-based software packages (e.g., instrument / system control software packages, image processing software packages, data analysis software packages), data storage modules, data communication modules (e.g., Bluetooth, WiFi, intranet, or internet communication hardware and associated software), display modules, etc. These additional components of larger systems, e.g., systems designed for genomics applications, are discussed in more detail below.

[0072] 2A and 2B show a non-limiting example of an illumination and imaging module (100) for multi-channel fluorescence imaging. The illumination and imaging module (100) includes an objective lens (110), an illumination source (115), multiple detection channels (120), and a first dichroic filter (130), which may include a dichroic reflector and a beam splitter. Some designs may include an autofocus system, which may include, for example, an autofocus laser (102) that projects a spot whose size is monitored to determine when the imaging system is in focus. Some or all components of the illumination and imaging module (100) may be coupled to a base plate (105).

[0073] The illumination or light source (115) may include any suitable light source configured to generate light at least at a desired excitation wavelength (discussed in more detail below). The light source may be a broadband source that emits light within one or more excitation wavelength ranges (or bands). The light source may be a narrowband source that emits light within one or more narrow wavelength ranges. In some examples, the light source may generate a single isolated wavelength (or line) or multiple isolated wavelengths (or lines) corresponding to the desired excitation wavelengths. In some examples, the lines may have several very narrow bandwidths. Exemplary light sources that may be suitable for use in the illumination source (115) include, but are not limited to, incandescent filaments, xenon arc lamps, mercury lamps, light-emitting diodes, laser sources such as laser diodes or solid-state lasers, or other types of light sources. As discussed below, in some designs, the light source may include a polarized light source, such as a linearly polarized light source. In some implementations, the light source is oriented so that s-polarized light is incident on one or more surfaces of one or more optical components, such as the dichroic reflective surfaces of one or more dichroic filters.

[0074] The illumination source (115) may further include one or more additional optical components, such as lenses, filters, optical fibers, or any other suitable transmissive or reflective optical components, as needed to output an excitation light beam with appropriate characteristics for the first dichroic filter (130). For example, beam-shaping optical components may be included to receive light, e.g., from a light emitter of the light source, and generate a beam and / or provide desired beam characteristics. Such optical components may include, for example, a collimating lens configured to reduce light divergence and / or increase collimation and / or collimate the light.

[0075] In some implementations, multiple light sources are included in the illumination and imaging module (100). In some such implementations, different light sources may generate light with different spectral characteristics, for example, to excite different fluorescent dyes. In some implementations, the light generated by the different light sources may be directed to coincide and form a combined excitation light beam. This combined excitation light beam may be composed of excitation light beams from each of the light sources. The combined excitation light beam may have more optical power than the individual beams that overlap to form the combined beam. For example, in some implementations including two light sources generating two excitation light beams, the combined excitation light beam formed from the two individual excitation light beams may have an optical power that is the sum of the optical powers of the individual beams. Similarly, in some implementations, three, four, five, or more light sources may be included, and these light sources may each output an excitation light beam that together form a combined beam, and the combined beam may have an optical power that is the sum of the optical powers of the individual beams.

[0076] In some implementations, the light source (115) outputs a large amount of light, sufficient to generate a sufficiently strong fluorescent emission. A stronger fluorescent emission can increase the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the image acquired by the fluorescent imaging module. In some implementations, the output from the light source and / or the excitation light beam derived therefrom (including the composite excitation light beam) may range in power from about 0.5 W to about 5.0 W or more (as discussed in more detail below).

[0077] Referring again to Figures 2A and 2B, a first dichroic filter (130) is positioned relative to the light source to receive light from the light source. The first dichroic filter may include a dichroic mirror, a dichroic reflector, a dichroic beam splitter, or a dichroic beam combiner configured to transmit light in a first spectral region (or wavelength range) and reflect light having a second spectral region (or wavelength range). The first spectral region may include one or more spectral bands, for example, one or more spectral bands in the ultraviolet and blue wavelength ranges. Similarly, the second spectral region may include one or more spectral bands, for example, one or more spectral bands in wavelengths extending from green to red and infrared wavelengths. Other spectral regions or wavelength ranges are also possible.

[0078] In some implementations, the first dichroic filter may be configured to transmit light from the light source to a sample support structure, such as a microscope slide, capillary, flow cell, microfluidic chip, or other substrate or support structure. The sample support structure supports and positions a sample, e.g., a composition containing fluorescently labeled nucleic acid molecules or their complements, relative to the illumination-imaging module (100). Thus, the first optical path extends from the light source to the sample through the first dichroic filter. In various implementations, the sample support structure includes at least one surface on which the sample is disposed or to which the sample is bound. In some examples, the sample may be disposed within or bound to different localized regions or sites on at least one surface of the sample support structure.

[0079] In some examples, the support structure may include two surfaces located at different distances from the objective lens (110) (i.e., at different positions or depths along the optical axis of the objective lens (110)), on which the sample is disposed. As discussed below, for example, a flow cell may include a fluid channel formed at least in part by a first interior surface and a second interior surface (e.g., an upper interior surface and a lower interior surface), and the sample may be disposed on the first interior surface, the second interior surface, or at localized sites on both interior surfaces. The first and second surfaces may be separated by a region corresponding to the fluid channel through which the solution flows, and thus may be at different distances or depths relative to the objective lens (110) of the illumination and imaging module (100).

[0080] An objective lens (110) may be included in the first optical path between the first dichroic filter and the sample. This objective lens may be configured, for example, to have a focal length, a working distance, and / or be positioned to focus light from the light source onto the sample, such as a microscope slide, capillary, flow cell, microfluidic chip, or other substrate or support structure. Similarly, the objective lens (110) may be configured to have an appropriate focal length, a working distance, and / or be positioned to collect light reflected, scattered, and emitted (e.g., fluorescent emission) from the sample and form an image (e.g., a fluorescent image) of the sample.

[0081] In some implementations, the objective lens (110) may include a microscope objective lens, such as an off-the-shelf objective lens. In some implementations, the objective lens (110) may include a custom-made objective lens. Examples of custom-made objective lenses and / or custom-made objective-tube lens combinations are described below and in U.S. Provisional Patent Application No. 62 / 962,723, filed January 17, 2020, which is incorporated herein by reference in its entirety. The objective lens (110) may be designed to reduce or minimize optical aberrations at two locations, such as two planes corresponding to the two surfaces of a flow cell or other sample support structure. The objective lens (110) may be designed to reduce optical aberrations at selected locations or planes, e.g., the first and second surfaces of a double-sided flow cell, relative to other locations or planes in the optical path. For example, the objective lens (110) may be designed to reduce optical aberrations at two depths or two planes at different distances from the objective lens compared to optical aberrations associated with other depths or planes at other distances from the objective lens. For example, in some instances, optical aberrations for imaging the first and second surfaces of the flow cell may be less than those exhibited anywhere in the range of 1 to 10 mm from the front surface of the objective lens. Additionally, the custom-made objective lens (110) may be configured to correct optical aberrations induced by the transmission of fluorescent radiation through one or more portions of the sample support structure, such as, in some instances, a layer comprising one or more of the flow cell surfaces on which the sample is disposed or a layer containing the solution filling the fluid channels of the flow cell. These layers may include, for example, glass, quartz, plastic, or other transparent materials that have refractive indices and may introduce optical aberrations.

[0082] In some implementations, the objective lens (110) may have a numerical aperture (NA) of 0.6 or greater (as discussed in more detail below). Such a numerical aperture may provide reduced focus and / or depth of field, improved background discrimination, and improved image resolution.

[0083] In some implementations, the objective lens (110) may have a numerical aperture (NA) of 0.6 or less (as discussed in more detail below). Such a numerical aperture may provide an increased depth of focus and / or depth of field. Such an increased depth of focus and / or depth of field may increase the ability to image planes separated by the distance separating the first and second surfaces of a double-sided flow cell.

[0084] As discussed above, a flow cell may include a first layer and a second layer, each including a first interior surface and a second interior surface, separated by a fluidic channel through which, for example, an analyte or a reagent can flow. In some implementations, the objective lens (110) and / or the illumination-imaging module (100) may be configured to provide a depth of field and / or depth of focus large enough to image both the first and second interior surfaces of the flow cell with comparable optical resolution, either sequentially by refocusing the imaging module between imaging the first and second surfaces, or simultaneously by ensuring a sufficient depth of field and / or depth of focus. In some examples, the depth of field and / or depth of focus may be as large as or greater than the distance separating the first and second surfaces of the flow cell being imaged, such as the first and second interior surfaces of the flow cell. In some examples, the first and second surfaces, e.g., the first and second interior surfaces of a double-sided flow cell or other sample support structure, may be separated by a distance ranging from about 10 μm to about 700 μm or more (as discussed in more detail below). In some examples, the depth of field and / or depth of focus may therefore range from about 10 μm to about 700 μm or more (as discussed in more detail below).

[0085] In some designs, correction optics (e.g., "optical corrector" or "corrector") may be moved in or out of the optical path of the imaging module, e.g., the optical path through which light collected by the objective lens (110) is delivered to the image sensor, thereby enabling the imaging module to image the first and second surfaces of a double-sided flow cell. The imaging module may be configured to image, for example, the first surface when the correction optics are included in the optical path between the objective lens and an image sensor or photodetector array configured to capture images of the first surface. In such designs, the imaging module may be configured to image the second surface when the correction optics are removed from or not included in the optical path between the objective lens (110) and an image sensor or photodetector array configured to capture images of the second surface. The need for an optical corrector may be more pronounced when using an objective lens (110) with a high numerical aperture (NA), e.g., at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, or even higher. In some implementations, the optical corrector (e.g., optical corrector or corrector) includes an optical element such as a lens, a plate of an optically transparent material such as glass, or, in the case of polarized light beams, a quarter-wave plate or a half-wave plate. Other configurations may be used to allow the first and second surfaces to be imaged at different times. For example, one or more lenses or optical elements may be configured to be translated into, out of, or along the optical path between the objective lens (110) and the image sensor.

[0086] However, in certain designs, the objective lens (110) is configured to provide a sufficiently large depth of focus and / or depth of field so that the first and second surfaces can be imaged with comparable optical resolution without such corrective optics being moved into or out of the optical path of the imaging module, such as between the objective lens and an image sensor or photodetector array. Similarly, in various designs, the objective lens (110) is configured to provide a sufficiently large depth of focus and / or depth of field so that the first and second surfaces can be imaged with comparable optical resolution without the need for optical components to be moved, e.g., without one or more objective lenses or optical components being translated along the optical path of the imaging module, such as between the image sensor or photodetector array. Examples of such objective lenses will be described in more detail below.

[0087] In some implementations, the objective lens (or microscope objective lens) 110 may be configured to have a reduced magnification. For example, the objective lens 110 may be configured so that the fluorescence imaging module has a magnification of less than 2x to less than 10x (as discussed in more detail below). Such reduced magnification may change design constraints so that other design parameters can be achieved. For example, the objective lens 110 may also be configured so that the fluorescence imaging module has a large field of view (FOV) ranging from about 1.0 mm to about 5.0 mm (e.g., in diameter, width, length, or longest dimension), as discussed in more detail below.

[0088] In some implementations, the objective lens (110) may be configured to provide a field of view as shown above to the fluorescence imaging module, such that the FOV has diffraction-limited performance, e.g., less than 0.15 aberration, across at least 60%, 70%, 80%, 90%, or 95% of the field, as discussed in more detail below.

[0089] In some implementations, the objective lens (110) may be configured to provide a field of view as shown above to the fluorescence imaging module, such that the FOV has diffraction-limited performance, e.g., a Strehl ratio of greater than 0.8, over at least 60%, 70%, 80%, 90%, or 95% of the field, as discussed in more detail below.

[0090] 2A and 2B, a first dichroic beam splitter or combiner is positioned in a first optical path between the light source and the sample to illuminate the sample with one or more excitation beams. This first dichroic beam splitter or combiner is present in one or more second optical paths from the sample to different optical channels used to detect fluorescent emissions. Thus, a first dichroic filter (130) couples the first optical path of the excitation beam emitted by the illumination source (115) to the second optical path of the emission light emitted by the sample specimen to various optical channels, where the light is directed to a respective image sensor or photodetector array for capturing images of the sample.

[0091] In various implementations, the first dichroic filter (130), e.g., a first dichroic reflector or beam splitter, has a passband selected to transmit light from the illumination source (115) only within a specific wavelength or, possibly, multiple wavelength bands, including the desired excitation wavelength. For example, the first dichroic beam splitter (130) includes a reflective surface including a dichroic reflector having a spectrally specific transmittance response, i.e., configured to transmit light having at least some of the wavelengths output by the light source that form part of the excitation beam. The spectrally specific transmittance response may be configured to not transmit (e.g., instead reflect) light of one or more other wavelengths, e.g., one or more other fluorescent emission wavelengths. In some implementations, the spectrally specific transmittance response may also be configured to not transmit (e.g., instead reflect) light of one or more other wavelengths output by the light source. Thus, the first dichroic filter (130) may be utilized to select which wavelengths of light output by the light source reach the sample. Conversely, the dichroic reflector in the first dichroic beam splitter (130) has a spectral reflectance response that can reflect light having one or more wavelengths corresponding to the desired fluorescent emission from the sample and reflect light having one or more wavelengths output from the light source intended to reach the sample. Thus, in some implementations, the dichroic reflector has a spectral transmittance that includes one or more passbands that transmit light incident on the sample and one or more stopbands that reflect light outside the passbands, e.g., light at one or more emission wavelengths not intended to reach the sample and possibly light at one or more wavelengths output by the light source. Similarly, in some implementations, the dichroic reflector has a spectral reflectance that includes one or more spectral regions configured to reflect one or more emission wavelengths not intended to reach the sample and possibly one or more wavelengths output by the light source, and one or more regions that transmit light outside these reflection regions.The dichroic reflector included in the first dichroic filter (130) may include a reflective filter, such as an interference filter (e.g., a quarter-wave stack), configured to provide the appropriate spectral transmittance and reflectance distributions. Figures 2A and 2B also show a dichroic filter (105), which may include, for example, a dichroic beam splitter or beam combiner that may be used to direct the autofocus laser (102) through the objective lens and onto the sample support structure.

[0092] While the imaging module (100) shown in Figures 2A and 2B and discussed above is configured such that the excitation beam is transmitted to the objective lens (110) by the first dichroic filter (130), in some designs the illumination source (115) may be positioned relative to the first dichroic filter (130) and / or the first dichroic filter may be configured (e.g., oriented) such that the excitation beam is reflected by the first dichroic filter (130) to the objective lens (110). Similarly, in some such designs, the first dichroic filter (130) is configured to transmit fluorescent emission from the sample, and possibly to transmit light having one or more wavelengths output from the light source that are not intended to reach the sample. As discussed below, a design in which fluorescent emission is transmitted instead of reflected may potentially reduce wavelength errors in the detected emission and / or may have other advantages. In either case, in various implementations, a first dichroic reflector (130) is positioned in the second optical path to receive fluorescent emissions from the sample, at least some of which continues to the detection channel (120).

[0093] 3A and 3B illustrate the optical paths within the multichannel fluorescence imaging module of FIGS. 2A and 2B. In the example shown in FIGS. 2A and 3A, the detection channel (120) is positioned to receive fluorescent emission from the sample specimen that is transmitted by the objective lens (110) and reflected by the first dichroic filter (130). As mentioned above and described in more detail below, in some designs, the detection channel (120) may be positioned to receive a portion of the emitted light that is transmitted by the first dichroic filter rather than reflected. In either case, the detection channel (120) may include optical components for receiving at least a portion of the emitted light. For example, the detection channel (120) may include one or more lenses, such as a tube lens, and one or more image sensors, such as a photodetector array (e.g., a CCD or CMOS sensor array), for imaging or generating a signal based on the received light. The tube lens may include, for example, one or more lens elements configured to form an image of the sample onto a sensor or photodetector array for capturing the image. Further discussion of detection channels is included below and in U.S. Provisional Patent Application No. 62 / 962,723, filed January 17, 2020, which is incorporated herein by reference in its entirety. In some examples, improved optical resolution may be achieved using an image sensor with relatively high sensitivity, small pixels, and a high pixel count, in conjunction with an appropriate sampling scheme, which may include oversampling or undersampling.

[0094] Figures 3A and 3B are ray tracing diagrams showing the optical paths of the illumination and imaging module (100) of Figures 2A and 2B. Figure 3A corresponds to a plan view of the illumination and imaging module (100). Figure 3B corresponds to a side view of the illumination and imaging module (100). The illumination and imaging module (100) shown in these figures includes four detection channels (120). However, it will be understood that the disclosed illumination and imaging modules may equally be implemented in systems including more or less than four detection channels (120). For example, the multi-channel systems disclosed herein may be implemented with as few as one detection channel (120), or as many as two detection channels (120), three detection channels (120), four detection channels (120), five detection channels (120), six detection channels (120), seven detection channels (120), or eight detection channels (120) without departing from the spirit or scope of the present disclosure.

[0095] 3A and 3B, a non-limiting example of an imaging module (100) includes four detection channels (120), a first dichroic filter (130) that reflects a beam of emitted light (150), a second dichroic filter (e.g., a dichroic beam splitter) (135) that splits the beam (150) into a transmitted and reflected portion, and two channel-specific dichroic filters (e.g., dichroic beam splitters) (140) that further split the transmitted and reflected portions of the beam (150) among the individual detection channels (120). The dichroic reflective surfaces in the dichroic beam splitters (135) and (140) for splitting the beam (150) among the detection channels are shown oriented at 45 degrees relative to the axis of the beam (150), or the optical axis of the imaging module. However, as discussed below, angles less than 45 degrees may be used and may provide advantages such as a sharper transition from the passband to the stopband.

[0096] The different detection channels (120) each include an imaging device (124), which may include an image sensor or a photodetector array (e.g., a CCD or CMOS detector array). The different detection channels (120) may further include optical components (126), such as lenses (e.g., one or more tube lenses, each including one or more lens elements), arranged to focus a portion of the emitted light entering the detection channel (120) at a focal plane coinciding with the plane of the photodetector array (124). The optical components (126) (e.g., tube lenses) combined with the objective lens (110) are configured to form an image of the sample on the photodetector array (124) after the sample is bound to the surface to capture an image of the sample, e.g., an image of the surface of a flow cell or other sample support structure. Thus, such an image of the sample may include multiple fluorescent emission spots or regions spanning the spatial extent of the sample support structure, where the sample is emitting fluorescent light. The objective lens (110), together with the optical elements (126) (e.g., a tube lens), may provide a field of view (FOV) that includes a portion of the sample or the entire sample. Similarly, the photodetector arrays (124) of the different detection channels (120) may be configured to capture an image of the entire field of view (FOV) provided by the objective lens and tube lens, or portions thereof. In some implementations, the photodetector arrays (124) of some or all of the detection channels (120) may detect radiation emitted by a sample disposed on the surface of a sample support structure, e.g., a flow cell, or portions thereof, and record electronic data representing that image. In some implementations, the photodetector arrays (124) of some or all of the detection channels (120) may detect features in the radiation emitted by a sample without capturing and / or storing an image of the entire field of view (FOV) provided by the objective lens and optical elements (126) and / or (122) (e.g., elements of a tube lens).In some implementations, as discussed below, the FOV of the imaging module (e.g., provided by the combination of the disclosed objective lens (110) and optical components (126) and / or (122)) may range, for example, from about 1 mm to 5 mm (e.g., in diameter, width, length, or longest dimension). The FOV may be selected, for example, to provide a balance between the magnification and resolution of the imaging module and / or based on one or more characteristics of the image sensor and / or objective lens. For example, a relatively small FOV may be provided in conjunction with a smaller, faster image sensor to achieve high throughput.

[0097] 3A and 3B, in some implementations, the optical component (126) in the detection channel (e.g., a tube lens) may be configured to reduce optical aberrations in images acquired using the optical component (126) in combination with the objective lens (110). In some implementations including multiple detection channels for imaging at different emission wavelengths, the optical components (126) (e.g., tube lenses) for the different detection channels have different designs to reduce aberrations for each emission wavelength, with the particular channel configured to image at that emission wavelength. In some implementations, the optical component (126) (e.g., a tube lens) may be configured to reduce aberrations when imaging a particular surface (e.g., a plane, an object plane, etc.) on the sample support structure containing the fluorescent sample site disposed thereon, compared to other locations (e.g., other planes in the object space). Similarly, in some implementations, the optical component (126) (e.g., a tube lens) may be configured to reduce aberrations when imaging a first surface and a second surface (e.g., a first objective plane and a second objective plane) on a double-sided sample support structure (e.g., a double-sided flow cell) having a fluorescent sample site disposed thereon, compared to other locations (e.g., other planes in the objective space). For example, the optical component (126) (e.g., a tube lens) in the detection channel may be designed to reduce aberrations at two depths or two planes at different distances from the objective lens, compared to aberrations associated with other depths or planes at other distances from the objective lens. For example, optical aberrations may be smaller for imaging the first and second surfaces than elsewhere in a region from about 1 mm to about 10 mm from the objective lens. Additionally, custom optical components (126) (e.g., tube lenses) in the detection channel may, in some embodiments, be configured to correct aberrations induced by transmission of the emitted light through one or more portions of the sample support structure, such as a layer comprising one of the surfaces on which the sample is placed, and possibly a solution adjacent to and in contact with the surface on which the sample is placed. The layer comprising one of the surfaces on which the sample is placed may comprise, for example, glass, quartz, plastic, or other transparent material that has a refractive index and introduces optical aberrations.The optical components (126) of the detection channel (e.g., a tubing channel) may be configured, for example, in some implementations, to correct for optical aberrations induced by the sample support structure, such as a coverslip or flow cell wall, or other sample support structure components, as well as possibly solutions adjacent to and in contact with the surface on which the sample is placed.

[0098] In some implementations, the detection channel optical component (126) (e.g., a tube lens) is configured to have a reduced magnification. The detection channel optical component (126) (e.g., a tube lens) may be configured, for example, so that the fluorescence imaging module has a magnification of less than 10x, as discussed further below. Such reduced magnification may alter design constraints so that other design parameters can be achieved. For example, the optical component (126) (e.g., a tube lens) may also be configured, for example, so that the fluorescence imaging module has a field of view (FOV) of at least 1.0 mm or more (e.g., diameter, width, length, or longest dimension), as discussed further below.

[0099] In some implementations, the optical component (126) (e.g., a tube lens) may be configured to provide the imaging module with the field of view shown above such that the FOV has an aberration of less than 0.15 waves across at least 60%, 70%, 80%, 90%, or 95% of the field, as discussed further below.

[0100] Referring again to Figures 3A and 3B, in various implementations, the sample is positioned at or near the focal position (112) of the objective lens (110). As described above in connection with Figures 2A and 2B, a light source, such as a laser source, provides an excitation beam to the sample to induce fluorescence. At least a portion of the fluorescent emission is collected by the objective lens (110) as emitted light. The objective lens (110) transmits the emitted light to a first dichroic filter (130), which reflects some or all of the emitted light as a beam (150) that enters a second dichroic filter (135) into a different detection channel, each of which includes optics (126) that form an image of the sample (e.g., multiple fluorescent sample sites on the surface of the sample support structure) on a light detection array (124).

[0101] As discussed above, in some implementations, the sample support structure includes a flow cell, such as a double-sided flow cell, having two surfaces (e.g., two interior surfaces, a first surface and a second surface) that include sampling sites that emit fluorescent radiation. These two surfaces may be separated from each other by a distance in the longitudinal direction (Z), which may be along the central axis of the excitation beam and / or the optical axis of the objective lens. This separation may correspond, for example, to a flow channel within the flow cell. Analytes or reagents may be flowed through the flow channel and contact the first and second interior surfaces of the flow cell, which may cause the analytes or reagents to contact the binding composition, resulting in fluorescent radiation being emitted from multiple sites on the first and second interior surfaces. Imaging optics (e.g., the objective lens (110)) may be positioned at an appropriate distance from the sample (e.g., a distance corresponding to the working distance) to form a focused image of the sample on one or more detector arrays (124). As discussed above, in various designs, the objective lens (110) (possibly in combination with the optics (126)) may have a depth of field and / or depth of focus between the first and second surfaces that is at least as large as the longitudinal separation. The objective lens (110) and optics (126) (for each detection channel) can thus simultaneously form images of both the first and second flow cell surfaces on the photodetector array (124), and these images of the first and second surfaces are both in focus and have equivalent optical resolution (or can be focused with only minor refocusing of the objective lens to obtain images of the first and second surfaces with equivalent optical resolution). In various implementations, correction optics do not need to be moved in or out of the optical path of the imaging module (e.g., into or out of the first and / or second optical paths) to form focused images of the first and second surfaces with equivalent optical resolution.Similarly, in various implementations, one or more optical elements (e.g., lens elements) of the imaging module (e.g., the objective lens (110) or the tube lens (126)) do not need to be moved longitudinally along the first optical path and / or the second optical path to form a focused image of the first surface, for example, compared to the position of the one or more optical elements when used to form a focused image of the second surface. In some implementations, the imaging module includes an autofocus system configured to rapidly and continuously refocus the imaging module on the first surface and the second surface so that the images have comparable optical resolution. In some implementations, the objective lens (110) and / or optics (126) may be configured such that both the first and second flow cell surfaces are simultaneously in focus with comparable optical resolution without moving optical correctors in or out of the first and / or second optical paths and without longitudinally moving one or more lens elements (e.g., the objective lens (110) and / or optics (126) (e.g., a tube lens)) along the first and / or second optical paths. In some implementations, images of the first and / or second surfaces, acquired either sequentially (e.g., with refocusing between surfaces) or simultaneously (e.g., without refocusing between surfaces) using the novel objective lens and / or tube lens designs disclosed herein, may be further processed using appropriate image processing algorithms to improve the effective optical resolution of the images so that the images of the first and second surfaces have comparable optical resolution. In various implementations, the sample plane is sufficiently focused to resolve sample sites on the first flow cell surface and / or the second flow cell surface, and the sample sites are closely spaced laterally (e.g., in the X and Y directions).

[0102] As discussed above, dichroic filters may include interference filters that selectively transmit and reflect different wavelengths of light based on the principles of thin-film interference using layers of optical coatings with different refractive indices and specific thicknesses. Thus, the spectral response (e.g., transmission and / or reflection spectra) of dichroic filters implemented within a multichannel fluorescence imaging module may depend, at least in part, on the angle of incidence, or range of angles of incidence, at which light in the excitation and / or emission beams strikes the dichroic filter. Such effects may be particularly important with respect to dichroic filters in the detection light path (e.g., dichroic filters 135 and 140 in Figures 3A and 3B).

[0103] FIG. 4 is a graph illustrating the relationship between the performance of a dichroic filter and the angle of incidence (AOI) of the beam. Specifically, the graph in FIG. 4 illustrates the effect of the angle of incidence on the transition width or spectral span of the dichroic filter, which corresponds to the range of wavelengths over which the spectral response (e.g., the transmission spectrum and / or the reflection spectrum) transitions between the passband and stopband regions of the dichroic filter. Therefore, a transmission edge (or a reflection edge) with a relatively small spectral span (e.g., a small delta λ value in the graph in FIG. 4) corresponds to a sharper transition between the passband and stopband regions, or between the transmission and reflection regions (or vice versa), while a transmission edge (or a reflection edge) with a relatively large spectral span (e.g., a large delta λ value in the graph in FIG. 4) corresponds to a more gradual transition between the passband and stopband regions. In various implementations, a sharper transition between the passband and stopband regions is generally desirable. Additionally, it may also be desirable to have improved consistency or a relatively consistent transition width across all or most of the field of view and / or beam area.

[0104] A fluorescence imaging module in which a dichroic mirror is positioned at 45 degrees relative to the center of the beam axis of the emission height or the optical axis of the light path (e.g., of the objective lens and / or tube lens) can therefore have a transition width of approximately 50 nm for an exemplary dichroic filter, as shown in FIG. 4 . Because the emission light beam is not collimated and has a deviation of several degrees, the fluorescence imaging module may have a range of incidence angles of approximately 5 degrees between opposite sides of the beam. Thus, as shown in FIG. 4 , different portions of the emission light beam may be incident on the channel-splitting dichroic filter at various incidence angles between 40 and 50 degrees. This range of relatively large incidence angles corresponds to a range of transition widths between approximately 40 nm and approximately 62 nm. This range of relatively large incidence angles thereby leads to an increase in the transition width of the dichroic filter in the imaging module. The performance of a multichannel fluorescence imaging module may therefore be improved by providing smaller incidence angles across the entire beam, which results in sharper transmission edges and allows better discrimination between different fluorescence emission bands.

[0105] FIG. 5 is a graph showing the relationship between the beam footprint size (DBS) of a dichroic filter and the angle of incidence of the beam (DBS angle). In some instances, a smaller beam footprint may be desirable. For example, with a small beam footprint, a dichroic filter can be used to split a beam into different wavelength ranges. Using a smaller dichroic filter, in turn, reduces manufacturing costs and facilitates the manufacture of properly level dichroic filters. As shown in FIG. 5, any angle of incidence greater than 0 degrees (i.e., perpendicular to the surface of the dichroic filter) results in an elliptical beam footprint with an area larger than the cross-sectional area of ​​the beam. A 45-degree angle of incidence results in a large beam footprint on the dichroic reflector that is more than 1.4 times the cross-sectional area of ​​the beam when incident at 0 degrees.

[0106] 6A and 6B schematically illustrate non-limiting exemplary configurations of dichroic filters and detection channels in a multichannel fluorescence imaging module, where the dichroic mirror is positioned at an angle of less than 45 degrees relative to the beam axis center of the emitted light or the optical axis of the light path (e.g., of the objective lens and / or tube lens). FIG. 6A depicts an imaging module (500) including multiple detection channels (520a), (520b), (520c), and (520d). FIG. 6B is a detailed view of the portion of the imaging module (500) within the circle (5B) shown in FIG. 6A. As described in more detail, the configurations illustrated in FIGS. 6A and 6B include many aspects that may result in significant improvements over conventional multichannel fluorescence imaging module designs. However, in some examples, the fluorescence imaging modules and systems of the present disclosure may be implemented with one or a subset of the features described with respect to FIGS. 6A and 6B without departing from the spirit or scope of the present disclosure.

[0107] The imaging module (500) depicted in FIG. 6A includes an objective lens (510) and four detection channels (520a), (520b), (520c), and (520d) arranged to receive and / or image emission light transmitted by the objective lens (510). A first dichroic filter (530) is provided to couple the excitation and detection light paths. In contrast to the designs shown in FIGS. 2A and 2B and 3A and 3B, the first dichroic filter (530) (e.g., a dichroic beam splitter or combiner) is configured to reflect light from the light source to the objective lens (510) and sample, and to transmit fluorescent emission from the sample to the detection channels (520a), (520b), (520c), and (520d). The second dichroic filter (535) splits the beam of emitted light into at least two detection channels (520a) and (520b) by transmitting a first portion (550a) and reflecting a second portion (550b). Additional dichroic filters (540a) and (540b) are provided to further split the emitted light. The dichroic filter (540a) transmits at least a portion of the first portion (550a) of the emitted light and reflects a portion (550c) of the emitted light into a third detection channel (520c). The dichroic filter (540b) transmits at least a portion of the second portion (550b) of the emitted light and reflects a portion (550d) of the emitted light into a fourth detection channel (520d). Although the imaging module (500) is depicted with four detection channels, in various embodiments, the imaging module (500) may include more or fewer detection channels, along with a corresponding greater or fewer dichroic filters, thereby providing a portion of the emitted light to each detection channel as needed. For example, in some embodiments, features of the imaging module (500) may be implemented with similar advantages in a simplified imaging module that includes only two detection channels (520a), (520b) and omits the additional dichroic filters (540a), (540b). In some implementations, only one detection channel may be included.Alternatively, three or more detection channels may be used.

[0108] The detection channels 520a, 520b, 520c, and 520d shown in Figure 6A may include some or all of the same or similar components as the detection channels 120 shown in Figures 2A-3B. For example, the different detection channels 520a, 520b, 520c, and 520d may include one or more image sensors or photodetector arrays, and may include transmissive and / or reflective optical components, such as one or more lenses (e.g., tube lenses), that focus light received by the detection channels onto their respective image sensors or photodetector arrays.

[0109] The objective lens (510) is positioned to receive emission light emitted by fluorescence from the sample. In particular, the first dichroic filter (530) is positioned to receive emission light collected and transmitted by the objective lens (510). As discussed above and shown in FIG. 6A, in some designs, an illumination source such as a laser source (e.g., illumination source (115) in FIGS. 2A and 2B) is positioned to provide an excitation beam that is incident on the first dichroic filter (530), whereby the first dichroic filter (530) reflects the excitation beam back to the same objective lens (510), which transmits the emission light, e.g., in an epifluorescence configuration. In some other designs, the illumination source may be directed to the sample by other optical components along a different optical path that do not include the same objective lens (510). In such configurations, the first dichroic filter (530) may be omitted.

[0110] Similarly, as discussed above and shown in FIG. 6A , the detection optics (including, for example, the detection channels (520a), (520b), (520c), (520d) and any optical components along the optical path between the objective lens (510) and the detection channels (520a), (520b), (520c), (520d), such as the dichroic filters (535), (540a), (540b)) may be positioned on the transmission path of the first dichroic filter (530) rather than on the reflection path of the first dichroic filter (530). In one exemplary implementation, the objective lens (510) and the detection optics are positioned such that the objective lens (510) transmits the beam of emitted light (550) directly toward the second dichroic filter (535). The wavefront quality of the emitted light may be degraded by the presence of the first dichroic filter (530) along the path of the beam (550) of emitted light (e.g., by imparting some wavefront error to the beam (550)). However, the wavefront error introduced by a beam transmitted through the dichroic reflector of the dichroic beamsplitter is typically significantly smaller (by orders of magnitude) than the wavefront error of a beam reflected from the dichroic reflective surface of the dichroic beamsplitter. Therefore, the wavefront quality of the emitted light and the resulting imaging quality in a multichannel fluorescence imaging module may be substantially improved by placing detection optics along the transmitted beam path rather than along the reflected beam path of the first dichroic filter (530).

[0111] 6A, within the detection optics of the imaging module 500, dichroic filters 535, 540a, and 540b are provided to split the beam of emitted light 550 among the detection channels 520a, 520b, 520c, and 520d. For example, the dichroic filters 535, 540a, and 540b split the beam 550 based on wavelength so that a first wavelength or wavelength range of emitted light can be received by the first detection channel 520a, a second wavelength or wavelength range of emitted light can be received by the second detection channel 520b, a third wavelength or wavelength range of emitted light can be received by the third detection channel 520c, and a fourth wavelength or wavelength range of emitted light can be received by the fourth detection channel 520d. In some implementations, multiple separate wavelengths or wavelength bands may be received by a detection channel.

[0112] In contrast to the multichannel fluorescence imaging module designs shown in Figures 2A and 2B and 3A and 3B, the imaging module (500) has dichroic filters (535), (540a), and (540b) positioned at angles of incidence less than 45 degrees relative to the central beam axis of the incident beam. As shown in Figure 6B, the different beams (550), (550a), and (550b) have respective central beam axes (552), (552a), and (552b). In various implementations, the central beam axes (552), (552a), and (552b) are at the center of the cross section of the beam perpendicular to the beam propagation direction. These central beam axes (552), (552a), and (552b) may correspond to the optical axes of the objective lenses and / or optical components in the other channels, such as the optical axes of the respective tube lenses. Additional rays 554, 554a, and 554b of each beam 550, 550a, and 550b are shown in FIG. 6B to indicate the diameter of each beam 550, 550a, and 550b. The beam diameter may be defined, for example, as the full width at half the maximum diameter, D4σ (i.e., four times σ, where σ is the standard deviation of the horizontal or vertical marginal distribution of the beam, respectively), or the second moment width, or any other suitable definition of beam diameter.

[0113] The central beam axis (552) of the beam of radiation (550) may serve as a reference point for defining the angle of incidence of the beam (550) onto the second dichroic filter (535). Thus, the "angle of incidence" (AOI) of the beam (550) may be, for example, the angle between the central beam axis (552) of the incident beam (550) and a line N perpendicular to the surface (e.g., a dichroic reflective surface) on which the beam is incident. When the beam of radiation (550) is incident on the dichroic reflective surface of the second dichroic filter (535) at the angle of incidence AOI, the second dichroic filter (535) transmits a first portion (550a) of the radiation (e.g., a portion having a wavelength within the passband region of the second dichroic filter (535)) and reflects a second portion (550b) of the radiation (e.g., a portion having a wavelength within the stopband region of the second dichroic filter (535)). The first portion 550a and the second portion 550b may similarly be described with respect to the central beam axes 552a and 552b, respectively. As referenced above, optical axes may be used alternatively or additionally.

[0114] In the exemplary configuration of Figures 6A and 6B, the second dichroic filter (535) is positioned so that the central beam axis (552) of the beam (550) is incident at a 30-degree angle of incidence. Similarly, the additional dichroic filters (540a) and (540b) are positioned so that the central beam axes (552a) and (552b) of the first and second portions (550a) and (550b) of the beam (550) are also incident at a 30-degree angle of incidence. However, in various implementations, these angles of incidence may be other angles less than 45 degrees. In some examples, for example, as discussed further below, the angles of incidence may range from about 20 degrees to about 45 degrees. Furthermore, the angles of incidence of each of the dichroic filters (535), (540a), and (540b) do not necessarily have to be the same. In some embodiments, some or all of the dichroic filters 535, 540a, and 540b may be positioned such that their incident beams 550, 550a, and 550b have different angles of incidence. As described above, the angles of incidence may be relative to the optical axes of the optical components in the imaging module, such as the objective lens and / or the optical components (e.g., tube lenses) in the detection channels, and the respective dichroic reflective surfaces of the respective dichroic beam splitters. Similar ranges and values ​​for the angles of incidence apply when the optical axis is used to specify an AOI.

[0115] The beams of emitted light 550, 550a, 550b in a fluorescence imaging module system are generally diverging beams. As noted above, the beams of emitted light can have beam deviations large enough that regions of the beam within the beam diameter are incident on the dichroic filter at angles of incidence that differ by up to 5 degrees or more relative to the angle of incidence of the central beam axis and / or the optical axis of the optical component. In some designs, the objective lens 510 may be configured with an F-number or numerical aperture selected, for example, to produce a smaller beam diameter for a given field of view of the microscope. In one example, the F-number or numerical aperture of the objective lens (510) may be selected so that the full diameter of the beams (550), (550a), (550b) is incident on the dichroic filters (535), (540a), (540b) at an angle of incidence that is, for example, within 1 degree, 1.5 degrees, 2 degrees, 2.5 degrees, 3 degrees, 3.5 degrees, 4 degrees, 4.5 degrees, or 5 degrees of the angle of incidence of the central beam axis (552), (552a), (552b).

[0116] In some implementations, the focal length of an objective lens appropriate for producing such a narrow beam diameter may be longer than those typically used in fluorescence microscopes or imaging systems. For example, in some implementations, the focal length of the objective lens may range from 20 mm to 40 mm, as discussed further below. In one example, an objective lens (510) with a focal length of 36 mm may produce a beam (550) characterized by a deviation small enough that light across the entire diameter of the beam (550) is incident on the second dichroic filter (535) at an angle within 2.5 degrees of the angle of incidence of the central beam axis.

[0117] 7 and 8 provide graphs illustrating the improvement in dichroic filter performance due to the imaging module configuration embodiment of FIGS. 6A and 6B (or any of the imaging module configurations disclosed herein). The graph in FIG. 7 is similar to that of FIG. 4 and illustrates the effect of incidence angle on the transition width (e.g., the spectral span of the transmission edge) of a dichroic filter. FIG. 7 illustrates an example in which dichroic filters (e.g., dichroic filters 535, 540a, and 540b) and their dichroic reflective surfaces are oriented so that the incident beam has an incidence angle of 30 degrees rather than 45 degrees. FIG. 7 shows how this reduction in incidence angle significantly improves the sharpness and uniformity of the transition width across the entire beam diameter. For example, a 45-degree incidence angle at the central beam axis results in a series of transition widths ranging from about 40 nm to about 62 nm, while a 30-degree incidence angle at the central beam axis results in a series of transition widths ranging from about 16 nm to about 30 nm. In this example, the average transition width is reduced from about 51 nm to about 23 nm, indicating a sharper transition between the passband and the stopband. Additionally, the variation in transition width across the beam diameter is reduced by nearly 40%, from a range of 22 nm to a range of 14 nm, indicating a more uniform sharpness of the transition across the beam area.

[0118] FIG. 8 illustrates an additional benefit that can be realized in any of the imaging module configurations disclosed herein by selecting an F-number or numerical aperture for the objective lens to reduce beam deviation. In some implementations, longer focal lengths are used. In the example of FIG. 8, the objective lens (510) has a focal length of 36 mm, which, when used with an appropriate numerical aperture (e.g., less than 5), reduces the range of angles of incidence within the beam (550) from 30°±5° to 30°±2.5°. With this design, the range of transition widths may be reduced to between about 19 nm and about 26 nm. When compared to the improved system of FIG. 7, the average transition width is substantially the same (e.g., a spectral span of approximately 23 nm), but the variation in transition width across the beam diameter is further reduced to a range of 7 nm, representing a nearly 70% reduction relative to the transition width range shown in FIG. 4.

[0119] Referring again to FIG. 5, reducing the angle of incidence from 45 degrees to 30 degrees at the central beam axis is even more advantageous because it reduces the beam spot size on the dichroic filter. As shown in FIG. 5, a 45-degree angle of incidence results in a beam footprint on the dichroic filter with an area greater than 1.4 times the cross-sectional area of ​​the beam. However, a 30-degree angle of incidence results in a beam footprint on the dichroic filter with an area only about 1.15 times the cross-sectional area of ​​the beam. Therefore, reducing the angle of incidence at dichroic filters 535, 540a, and 540b from 45 degrees to 30 degrees results in an approximately 18% reduction in the area of ​​the beam footprint on dichroic filters 535, 540a, and 540b. This reduction in beam footprint area allows smaller dichroic filters to be used.

[0120] Referring now to FIGS. 9A-9B together, reducing the angle of incidence from 45 degrees to 30 degrees may also provide performance improvements associated with surface deformations induced by dichroic filters in any of the imaging module configurations disclosed herein, as indicated by improvements in the modulation transfer function. Generally, the amount of surface deformation increases as the area of ​​the optical element increases. When a larger area of ​​the dichroic filter is used, a larger amount of surface deformation is induced, thereby introducing more wavefront error into the beam. FIG. 9A shows the effect of the fold angle on the image quality degradation induced by adding 1 wave of PV of spherical power to the last mirror. FIG. 9B shows the effect of the fold angle on the image quality degradation induced by adding 0.1 waves of peak-to-valley (PV) spherical power to the last mirror. As shown in FIGS. 9A and 9B, reducing the angle of incidence to 30 degrees significantly reduces the effect of surface deformations, achieving near-diffraction-limited performance of the detection optics.

[0121] In some implementations of the imaging modules disclosed herein, the polarization state of the excitation beam may be utilized to further improve the performance of the multichannel fluorescence imaging modules disclosed herein. For example, referring again to FIGS. 2A, 2B, and 6A, some implementations of the multichannel fluorescence imaging modules disclosed herein have an epi-fluorescence configuration in which a first dichroic filter (130) or (530) merges the optical path of the excitation beam with the optical path of the emission beam so that both excitation and emission light are transmitted through the objective lens (110), (510). As discussed above, the illumination source (115) may include a light source, such as a laser source or other source, that provides light to form the excitation beam. In some designs, the light source includes a linearly polarized light source, and the excitation beam may be linearly polarized. In some designs, polarization optics are included to polarize the light and / or rotate the polarization of the light. For example, a polarizer, such as a linear polarizer, may be included in the optical path of the excitation beam to polarize the excitation beam. A retarder, such as a half-wave retarder, or multiple quarter-wave retarders, or other retarders with other amounts of phase difference, may be included in some designs to rotate linearly polarized light.

[0122] When incident on any dichroic filter or other planar interface, the linearly polarized excitation beam may be p-polarized (e.g., with an electric field component parallel to the plane of incidence), s-polarized (e.g., with an electric field component perpendicular to the plane of incidence), or may have a combination of p- and s-polarization states within the beam. The p- or s-polarization state of the excitation beam may be selected and / or changed by selecting the orientation of the illumination source (115) and / or one or more components thereof relative to the first dichroic filter (130), (530) and / or relative to any other surface with which the excitation beam interacts. In some implementations in which the light source outputs linearly polarized light, the light source may be configured to provide s-polarized light. For example, the light source may include an emitter, such as a solid-state laser or laser diode, that can be rotated about its optical axis or central beam axis to direct the linearly polarized light output therefrom. Alternatively, or additionally, a retarder may be used to rotate the linear polarization about the optical axis or central beam axis. As discussed above, in some implementations, a polarizer placed in the path of the excitation beam can polarize the excitation beam, for example, when the light source does not output polarized light. In some designs, for example, a linear polarizer is placed in the path of the excitation beam. This polarizer may be rotated to provide the appropriate orientation of the linear polarization to provide s-polarized light.

[0123] In some designs, linearly polarized light is rotated about the optical axis or central beam axis so that s-polarized light is incident on the dichroic reflector of the dichroic beamsplitter. When s-polarized light is incident on the dichroic reflector of the dichroic beamsplitter, as opposed to when p-polarized light is incident on the dichroic reflector of the dichroic beamsplitter, the transition between the passband and the stopband is sharp.

[0124] As shown in Figures 10A and 10B, the use of p- or s-polarization states of the excitation beam can significantly affect the narrowband performance of any excitation filter, such as the first dichroic filter (130), (530). Figure 10A shows the transmission spectrum between 610 nm and 670 nm for an exemplary bandpass dichroic filter at angles of incidence of 40 and 45 degrees, where the incident beam is linearly polarized and p-polarized with respect to the plane of the dichroic filter. As shown in Figure 10B, changing the orientation of the light source relative to the dichroic filter so that the incident beam is s-polarized with respect to the plane of the dichroic filter results in a substantially sharper edge between the passband and the stopband of the dichroic filter. Therefore, the illumination and imaging modules (100) and (500) disclosed herein may advantageously have the illumination source (115) oriented with respect to the first dichroic filter (130), (530) such that the excitation beam is s-polarized with respect to the plane of the first dichroic filter (130), (530). As discussed above, in some implementations, a polarizer, such as a linear polarizer, may be used to polarize the excitation beam. This polarizer may be rotated to provide an orientation of linearly polarized light corresponding to s-polarized light. Also, as discussed above, in some implementations, other approaches for rotating linearly polarized light may be used. For example, an optical retarder, such as a half-wave retarder or multiple quarter-wave retarders, may be used to rotate the polarization direction. Other arrangements are possible.

[0125] As discussed elsewhere herein, reducing the numerical aperture (NA) of the fluorescence imaging module and / or objective lens may increase the depth of field and allow equivalent imaging of the two surfaces. Figures 11A-16B show how the MTF becomes more similar at the first and second surfaces with 1 mm of glass at low NA compared to high NA.

[0126] 11A and 11B show the MTF at the first surface (FIG. 11A) and the second surface (FIG. 11B) for an NA of 0.3.

[0127] 12A and 12B show the MTF at the first surface (FIG. 12A) and the second surface (FIG. 12B) for an NA of 0.4.

[0128] 13A and 13B show the MTF at the first surface (FIG. 13A) and the second surface (FIG. 13B) for an NA of 0.5.

[0129] 14A and 14B show the MTF at the first surface (FIG. 14A) and the second surface (FIG. 14B) for an NA of 0.6.

[0130] 15A and 15B show the MTF at the first surface (FIG. 15A) and the second surface (FIG. 15B) for an NA of 0.7.

[0131] Figures 16A and 16B show the MTF for a first surface (Figure 16A) and a second surface (Figure 16B) for an NA of 0.8, where the first and second surfaces in each of these figures correspond to, for example, the upper and lower surfaces of a flow cell.

[0132] 17A-17B provide plots of calculated Strehl ratios (i.e., the ratio of the peak light intensity collected by the optical system to the peak light intensity collected by an ideal optical system and a point light source) for imaging a second flow cell surface through a first flow cell surface. FIG. 17A shows plots of the Strehl ratio for imaging a second flow cell surface through a first flow cell surface as a function of the thickness of the intervening fluid layer (fluid channel height) for different objective and / or optical system numerical apertures. As shown, the Strehl ratio decreases as the separation between the first and second surfaces increases. At one of the surfaces, and therefore as the separation between the two surfaces increases, image quality will degrade. The decrease in imaging performance of the second surface with increasing separation distance between the two surfaces is reduced for imaging systems with smaller numerical apertures compared to those with larger numerical apertures. Figure 17B shows a plot of the Strehl ratio as a function of numerical aperture for imaging a second flow cell surface through a first flow cell surface and an intervening layer of water with a thickness of 0.1 mm. The loss in imaging performance at higher numerical apertures may be due to the increase in optical aberrations induced by the fluid for second-surface imaging. As the numerical aperture increases, the increase in optical aberrations introduced by the fluid for second-surface imaging significantly degrades image quality. However, lowering the numerical aperture of an optical system generally reduces the achievable resolution. This loss in image quality can be at least partially offset by increasing the contrast-to-noise ratio at the sample plane (or object plane), for example, by using chemistries for nucleic acid sequencing applications that enhance the fluorescence emission of labeled nucleic acid clusters and / or reduce background fluorescence emission. In some examples, a sample support structure including, for example, a hydrophilic substrate material and / or a hydrophilic coating may be used. In some cases, such a hydrophilic substrate and / or hydrophilic coating may reduce background noise.Additional discussion regarding sample support structures, hydrophilic surfaces, and hydrophilic coatings, as well as methods for improving contrast-to-noise ratio, for example, for nucleic acid sequencing applications, can be found below.

[0133] In some implementations, any one or more of the fluorescence imaging system, illumination imaging module (100), imaging optics (e.g., optics (126)), objective lens, and / or tube lens may be configured to have a reduced magnification, such as a magnification of less than 10x, as discussed further below. Such reduced magnification may accommodate design constraints so that other design parameters can be achieved. For example, any one or more of the fluorescence microscope, illumination imaging module (100), imaging optics (e.g., optics (126)), objective lens, or tube lens may also be configured so that the fluorescence imaging module has a large field of view (FOV), e.g., a field of view of at least 3.0 mm or more (e.g., in diameter, width, height, or longest dimension), as discussed further below. Any one or more of the fluorescence imaging system, illumination imaging module (100), imaging optics (e.g., optics (126)), objective lens, and / or tube lens may be configured to provide the fluorescence microscope with a field of view such that the FOV has an aberration of less than, e.g., 0.1 waves across at least 80% of the field. Similarly, any one or more of the fluorescence imaging system, illumination imaging module (100), imaging optics (e.g., optics (126)), objective lens, and / or tube lens may be configured such that the fluorescence imaging module has such an FOV and is diffraction limited or is diffraction limited over such an FOV.

[0134] As discussed above, in various implementations, a large field of view (FOV) is provided by the disclosed optical system. In some implementations, achieving an increased FOV is facilitated in part by the use of a larger image sensor or photodetector array. The photodetector array may have an active area with a diagonal of at least 15 mm or more, for example, as discussed further below. As discussed above, in some implementations, the disclosed optical imaging system provides a reduced magnification, for example, less than 10x, which may facilitate large FOV designs. Despite the reduced magnification, the optical resolution of the imaging module may still be sufficient, or a detector array with a small pixel size or pitch may be used. The pixel size and / or pitch may be, for example, about 5 μm or less, as discussed in more detail below. In some implementations, the pixel size is less than twice the optical resolution provided by the optical imaging system (e.g., the objective lens and tube lens) to satisfy the Nyquist theorem. Accordingly, the pixel dimension and / or pitch for the image sensor may be such that the spatial sampling frequency of the imaging module is at least twice the optical resolution of the imaging module. For example, the spatial sampling frequency of the photodetector array may be at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, or at least 5 times the spatial sampling frequency of the fluorescence imaging module (e.g., the illumination imaging module, the objective lens and tube lens, the objective lens and optics (126) in the detection channel, the sample support structure configured to support the sample support stage or optics between the stage and the photodetector array), or in a range between any of these values.

[0135] Although a wide range of features are discussed herein with respect to fluorescence imaging modules, any of the features and designs described herein may be applied to other types of optical imaging systems, including, without limitation, bright-field imaging and dark-field imaging, and may also apply to luminescence imaging or phosphorescence imaging.

[0136] Dual-wavelength excitation / four-channel imaging system: Figure 18 shows a dual-wavelength excitation / four-channel imaging system for dual-wavelength imaging applications. The dual-wavelength excitation / four-channel imaging system includes an objective lens and a tube lens combination that are scanned perpendicular to the optical axis to provide large-area imaging, e.g., tile several images to create a composite image with a full field of view (FOV) that is much larger than that of each individual image. The system includes two excitation light sources, e.g., lasers or laser diodes operating at different wavelengths and an autofocus laser. The two excitation light beams and the autofocus laser beam are combined using a series of mirrors and / or dichroic reflectors and delivered to the upper or lower interior surface of the flow cell through the objective lens. Fluorescence emitted by a labeled oligonucleotide (or other biomolecule) tethered to one of the flow cell surfaces is collected by the objective lens, transmitted through the tube lens, and directed to one of four imaging sensors according to the wavelength of the emitted light through a series of intermediate dichroic reflectors. Autofocus laser light reflected from the flow cell surface is collected by the objective lens, transmitted through a tube lens, and directed to the autofocus sensor by a series of intermediate dichroic reflectors. The system allows precise focus to be maintained while the objective lens / tube lens combination is scanned in a direction perpendicular to the objective lens's optical axis (e.g., by adjusting the relative distance between the flow cell surface and the objective lens using a precision linear actuator, translation stage, or microscope turret-mounted focusing mechanism to lower or minimize the reflected light spot size on the autofocus image sensor). Dual-wavelength excitation, used in combination with four-channel (i.e., four-wavelength) imaging capability, provides high-throughput images of the upper (near) and lower (far) interior surfaces of the flow cell.

[0137] Multiple optical read heads:

[0138] In some examples, miniaturized versions of any of the imaging modules described herein may be assembled to create a multiple read head that can be translated in one or more directions parallel to the sample surface, e.g., the interior surface of a flow cell, thereby simultaneously imaging several sections of the surface. A non-limiting example of a multiple read head was recently disclosed in U.S. Published Patent Application No. 2020 / 0139375 A1.

[0139] In some examples, for example, the miniaturized imaging module may include a "microfluorometer" including an illumination or excitation light source such as an LED or laser diode (or the tip of an optical fiber connected to an external light source), one or more lenses to collimate or focus the illumination or excitation light, one or more dichroic reflectors, one or more optical filters, one or more mirrors, beam splitters, prisms, apertures, etc., one or more objective lenses, one or more custom-made tube lenses to enable double-sided imaging with minimal focusing, as described elsewhere herein, one or more image sensors, or any combination thereof. In some examples, the miniaturized imaging module (e.g., a "microfluorometer") may further include an autofocus mechanism, a microprocessor, power and data transfer connectors, a light-tight housing, etc. The resulting miniaturized imaging module may therefore include an integrated imaging package or unit having a small form factor. In some examples, the shortest dimension (e.g., width or diameter) of the miniaturized imaging module may be less than 5 cm, less than 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, less than 1.2 cm, less than 1 cm, less than 0.8 cm, or less than 0.6 cm. In some examples, the longest dimension (e.g., height or length) of the miniaturized imaging module may be less than 16 cm, less than 14 cm, less than 12 cm, less than 10 cm, less than 9 cm, less than 8 cm, less than 7 cm, less than 5 cm, less than 5 cm, less than 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, less than 1.2 cm, or less than 1 cm. In some examples, one or more individual miniaturized imaging modules in the multiplexed readhead may include an autofocus mechanism.

[0140] In some examples, the multiplexed readheads described herein may include assemblies of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 miniaturized imaging modules or microfluorometers held in fixed positions relative to each other. In some examples, the optical design specifications and performance characteristics of the individual miniaturized imaging modules or microfluorometers, such as numerical aperture, field of view, depth of field, image resolution, etc., may be the same as those described elsewhere herein for other versions of the disclosed imaging modules. In some examples, the multiple individual miniaturized imaging modules may be arranged in a linear arrangement including 1, 2, 3, 4, or more than 4 rows and / or columns. In some examples, the multiple individual miniaturized imaging modules may be arranged in, for example, a hexagonal close pack arrangement. In some examples, the multiple miniaturized imaging modules may be arranged in a circular or spiral arrangement, a randomly distributed arrangement, or any other arrangement known to one of skill in the art.

[0141] 43A-43B provide non-limiting schematic diagrams of a multiplexed readhead as disclosed herein. FIG. 43A shows a side view of a multiplexed readhead in which two rows of individual microfluorometers (as viewed from the end) with common optical design specifications, such as numerical aperture, field of view, working distance, etc., are configured to image a common surface, such as the first interior surface of a flow cell. FIG. 43B shows a top view of the same multiplexed readhead, illustrating the overlap of imaging paths acquired by the individual microfluorometers of the multiplexed readhead as the readheads are translated relative to the flow cell (or vice versa). In some instances, the individual fields of view for the individual microfluorometers may overlap, as shown in FIG. 43B. In some instances, they may not overlap. In some instances, the multiplexed readhead may be designed to align with and image predetermined features within the flow cell, such as individual fluidic channels.

[0142] 44A-44B provide non-limiting schematic diagrams of a multiplexed readhead, where a first subset of a plurality of individual miniaturized imaging modules is configured to image a first sample plane, e.g., a first interior surface of a flow cell, and a second subset of a plurality of individual miniaturized imaging modules is configured to simultaneously image a second sample plane, e.g., a second interior surface of a flow cell. FIG. 44A shows a side view of the multiplexed readhead, where a first subset of individual microfluorometers is configured to image a first interior surface, e.g., a top interior surface, of a flow cell, and a second subset is configured to image a second surface, e.g., a second interior surface, or a bottom interior surface of a flow cell. FIG. 44B shows a top view of the multiplexed readhead of FIG. 44A illustrating the imaging paths acquired by the individual microfluorometers of the multiplexed readhead. Furthermore, in some instances, the individual fields of view for the individual microfluorometers in a given subset may overlap. In some instances, they may not overlap. In some examples, the multiplexed read head may be designed such that the individual miniaturized imaging modules of the first and second subsets are aligned with and image predetermined features within the flow cell, such as individual fluid channels.

[0143] Improved or optimized objective and / or tube lenses for use with thicker coverslips: Existing design practices include optimizing image quality when acquiring images through thin (e.g., <200 μm thick) microscope coverslips using objective lens designs and / or commonly available off-the-shelf microscope objectives. When used to image both sides of a fluidic channel or flow cell, the extra height of the gap between the two surfaces (i.e., the fluidic channel height; typically about 50 μm to 200 μm) introduces optical aberrations in images captured for the non-optimal side of the fluidic channel, thereby causing a reduction in optical resolution. This is primarily because the additional gap height is significant compared to the optimal coverslip thickness (typical fluidic channel or gap heights of 50–200 μm vs. coverslip thicknesses <200 μm). Another common design practice is to utilize an additional “corrector” lens in the optical path when imaging is to be performed on the non-optimal side of a fluidic channel or flow cell. This "corrector" lens and the mechanisms required to move it in and out of the optical path so that either side of the flow cell can be imaged further increase system complexity and imaging system downtime, and can degrade image quality due to vibrations, etc.

[0144] In the present disclosure, the imaging system is designed to be compatible with consumer flow cells containing thicker coverslips or flow cell walls (thickness >= 700 μm). The objective lens design is improved or optimized for coverslips equal to the true coverslip thickness plus half the effective gap thickness (e.g., 700 μm + 1 / 2 the fluid channel (gap) height). This design significantly reduces the impact of the gap height on the image quality of the two sides of the fluid channel and balances the optical quality for images of the two surfaces because the gap height is small relative to the total thickness of the coverslip, thus reducing its impact on optical quality.

[0145] Additional benefits of using a thicker coverslip include improved thickness tolerances during manufacturing and a reduced likelihood that the coverslip will deform due to thermal and mounting-induced loads. Coverslip thickness tolerances and deformations have a detrimental effect on the imaging quality of both the top and bottom surfaces of the flow cell.

[0146] To further improve double-sided imaging quality for sequencing applications, our optical system design focuses on improving or optimizing the MTF (e.g., through improving or optimizing the objective and / or tube lens design) in the mid-to-high spatial frequency range, which is best suited to imaging and resolving small spots or clusters.

[0147] Improved or optimized tube lens designs for use in combination with commercially available, off-the-shelf objective lenses: For low-cost sequencing instrument designs, the use of commercially available, off-the-shelf objective lenses is sometimes preferred due to their relatively low price. However, as noted above, low-cost, off-the-shelf objective lenses are mostly optimized for use with thin coverslips, approximately 170 μm thick. In some examples, the disclosed optical systems may utilize tube lens designs that compensate for thicker flow cell coverslips while enabling high image quality for both interior surfaces of a flow cell for double-sided imaging applications. In some examples, the tube lens designs disclosed herein enable high-quality imaging of both interior surfaces of a flow cell without moving an optical corrector in or out of the optical path between the flow cell and the image sensor, without moving one or more optical elements or tube lens components along the optical path, and without moving one or more optical elements or tube lens components in or out of the optical path.

[0148] Figure 19 provides an optical ray tracing diagram for a low-light objective that has been improved or optimized for imaging the surface of the opposing side of a 0.17 mm thick coverslip. The modulation transfer function plot for this objective, shown in Figure 20, demonstrates near-diffraction-limited imaging performance when used with one designed for a 0.17 mm thick coverslip.

[0149] Figure 21 provides a plot of the modulation transfer function of the same objective shown in Figure 19 as a function of spatial frequency used to image the surface of the opposing side of a 0.3 mm thick coverslip. The relatively small deviation in MTF values ​​over the spatial frequency range of about 100 to about 800 lines / mm (or cycles / mm) indicates that the image quality obtained when using a 0.3 mm thick coverslip is still reasonable.

[0150] Figure 22 provides a plot of the modulation transfer function of the same objective shown in Figure 19 as a function of spatial frequency when used to image a surface separated from the opposing surface of a 0.3 mm thick coverslip by a 0.1 mm thick layer of liquid (i.e., under the types of conditions encountered in double-sided imaging of a flow cell when imaging distant surfaces). As can be seen in the plot in Figure 22, imaging performance degrades as indicated by the deviation of the MTF curve from the ideal diffraction-limited case over the spatial frequency range from about 50 lp / mm to about 900 lp / mm.

[0151] Figures 23 and 24 show plots of the modulation transfer function as a function of spatial frequency for the upper (near) interior surface (Figure 23) and the lower (far) interior surface (Figure 24) of a flow cell when imaged using the objective shown in Figure 19 through a 1.0 mm thick coverslip, and when the upper and lower interior surfaces are separated by a 1.0 mm thick layer of liquid. As can be seen, imaging performance is significantly degraded for both surfaces.

[0152] Figure 25 provides a ray tracing diagram of a tube lens design that, when used in conjunction with the objective shown in Figure 19, results in improved double-sided imaging through a 1 mm thick coverslip. The optical design (700), including the compound objective lens (lens elements (702), (703), (704), (705), (706), (707), (708), (709), and (710)) and the tube lens (lens elements (711), (712), (713), and (714)), is improved or optimized for use with thick coverslips (or walls), for example, flow cells including coverslips (or walls) greater than 700 μm and fluidic channels at least 50 μm thick, and transmits images of the interior surfaces from the flow cell (701) to the image sensor (715) with dramatically improved optical quality and higher CNR.

[0153] In some examples, the tube lens (or tube lens assembly) may include at least two optical lens elements, at least three optical lens elements, at least four optical lens elements, at least five optical lens elements, at least six optical lens elements, at least seven optical lens elements, at least eight optical lens elements, at least nine optical lens elements, at least ten optical lens elements, or more, where the number of optical lens elements, the surface shape of each element, and the order in which they are arranged in the assembly are improved or optimized to correct for optical aberrations induced by the thick walls of the flow cell, and in some cases, allow for the use of commercially available, off-the-shelf objective lenses while still maintaining high-quality, double-sided imaging capabilities.

[0154] In some examples, as shown in FIG. 25, the tube lens assembly may include, in order, a first asymmetric convex-convex lens (711), a second convex-plano lens (712), a third asymmetric concave-concave lens (713), and a fourth asymmetric convex-concave lens (714).

[0155] Figures 26 and 27 show plots of the modulation transfer function as a function of spatial frequency for the upper (near) interior surface (Figure 26) and the lower (far) interior surface (Figure 27) of a flow cell when imaged using the objective lens (modified for a 0.17 mm coverslip) and tube lens combination shown in Figure 25 through a 1.0 mm thick coverslip, and when the upper and lower interior surfaces are separated by a 1.0 mm thick layer of liquid. As can be seen, the imaging performance achieved is nearly what would be expected for a diffraction-limited optical design.

[0156] 28 provides a ray tracing diagram of a tube lens design (left) of the present disclosure that has been improved or optimized to provide high-quality double-sided imaging performance. Because the tube lens is no longer infinity corrected, a properly designed null lens (right) may be used in combination with the tube lens to compensate for the infinity uncorrected tube lens for manufacturing and testing purposes.

[0157] Imaging channel-specific tube lens adaptation or optimization: In imaging system designs, both the objective lens and the tube lens can be improved or optimized in the same wavelength region for all imaging channels. Typically, the same objective lens is shared by all imaging channels (see, for example, FIG. 18), and each imaging channel uses the same tube lens or has tube lenses that share the same design.

[0158] In some examples, the imaging systems disclosed herein may further include a tube lens for each imaging channel, where the tube lens is individually adapted or optimized for a particular imaging channel to improve image quality, e.g., reduce or minimize distortion and field curvature, and improve depth-of-field (DOF) performance for each channel. Because the wavelength range (or bandpass) for each particular imaging channel is much narrower than the combined wavelength range for all channels, wavelength-specific or channel-specific adaptation or optimization of the tube lenses used in the disclosed systems can result in significant improvements in imaging quality and performance. This channel-specific adaptation or optimization can result in improved image quality for both the top and bottom surfaces of a flow cell in double-sided imaging applications.

[0159] Dual-sided imaging without fluid in the flow cell: For optimal imaging performance of both the upper and lower interior surfaces of the flow cell, a motion-actuated corrector is generally required to correct for optical aberrations induced by flow in the flow cell (which typically involves a fluid layer thickness of approximately 50-200 μm). In some examples of the disclosed optical system designs, the upper interior surface of the flow cell may be imaged with fluid present in the flow cell. Once the sequencing chemistry cycle is complete, the fluid may be extracted from the flow cell for imaging of the lower interior surface. Thus, in some examples, image quality of the lower surface is maintained even without the use of a corrector.

[0160] Correction of optical aberrations and / or vibrations using electro-optic phase plates: In some examples, bi-directional image quality can be improved without the need to remove fluid from the flow cell by using an electro-optic phase plate (or other corrective lens) in combination with the objective lens to offset optical aberrations induced by the presence of fluid. In some examples, an electro-optic phase plate (or lens) may be used to eliminate vibrations resulting from the mechanical motion of a motion-actuated corrector, providing faster image acquisition times and sequencing cycle times for genome sequencing applications.

[0161] Improved contrast-to-noise ratio (CNR), field of view (FOV), spectral separation, and timing design to enhance or maximize information transfer and throughput: Another way to enhance or maximize information transfer in imaging systems designed for genomics applications is to increase the size of the field of view (FOV) and reduce the time required to image a particular FOV. Typical large NA optical imaging systems have a resolution of approximately 1 mm. 2 While it may be common to acquire images of a field of view over an area of ​​2 mm, currently disclosed imaging system designs use large FOV objectives with long working distances. 2 This is specified to enable imaging of an area of ​​more than 100m.

[0162] In some cases, the disclosed imaging systems are designed for use in combination with a unique low-binding substrate surface and a DNA amplification process that reduces fluorescent background arising from various confounding signals, including, but not limited to, non-specific adsorption of fluorescent dyes to the substrate surface, non-specific nucleic acid amplification products (e.g., nucleic acid amplification products that arise on the substrate surface in areas between spots or features corresponding to clonal amplification clusters of nucleic acid molecules (i.e., specifically amplified colonies)), non-specific nucleic acid amplification products that may arise within amplified colonies, phased and pre-phased nucleic acid strands, etc. The use of a low-binding substrate surface and a DNA amplification process that reduces fluorescent background, when used in combination with the disclosed optical imaging systems, can significantly reduce the time required to image each FOV.

[0163] The presently disclosed system design may further reduce the required imaging time through improvements or optimization of the imaging sequence, where multiple channels of fluorescence images are acquired simultaneously or with overlapping timing, and where spectral separation of the fluorescence signals is designed to reduce crosstalk between the fluorescence detection channels and between the excitation light and the fluorescence signal.

[0164] The presently disclosed system design may further reduce the required imaging time through improved or optimized scanning motion sequences. In a typical approach, an XY translation stage is used to move the target FOV to a position below the objective lens, an autofocus step is performed where the optimal focus position is determined, the objective lens is moved in the Z direction to determine the focus position, and an image is acquired. A series of fluorescence images is acquired by cycling through a series of target FOV positions. From the perspective of the information transmission duty cycle, information is transmitted only during the fluorescence image acquisition portion of the cycle. In the presently disclosed imaging system design, a single-step motion is performed in which all axes (X, Y, and Z) are repositioned simultaneously, and an autofocus step is used to check for focus position errors. An additional Z-direction motion is commanded only if the focus position error (i.e., the difference between the focal plane position and the sample plane position) exceeds a certain limit (e.g., a specified error threshold). This approach, coupled with high-speed XY motion, increases the system's duty cycle and, therefore, increases the imaging throughput per unit of image.

[0165] Furthermore, by matching the design's light collection efficiency, modulation transfer function, and image sensor performance characteristics to the expected fluorescence photon flux given the input excitation photon flux and dye efficiency (related to the dye excitation coefficient and fluorescence quantum yield), while taking into account background signal and system noise characteristics, the time required to acquire a high-quality (high contrast-to-noise ratio (CNR) image) may be reduced or minimized.

[0166] Efficient image acquisition, combined with improved or optimized translation stage step and settling times, leads to faster imaging times (i.e., total time required per field of view) and higher throughput imaging system performance.

[0167] Along with a large FOV and fast image acquisition duty cycle, the disclosed designs may also include specifications for specific image flatness, chromatic focus performance between fluorescence detection channels, sensor flatness, image distortion, and focus quality.

[0168] Chromatic focus performance is further improved by individually aligning the image sensors for the different fluorescence detection channels so that the best focal planes for each detection channel overlap. The design goal is to ensure that images across 90% or more of the field of view are acquired within ±100 nm (or less) of each channel's best focal plane, thereby increasing or maximizing the transfer of individual spot intensity signals. In some examples, the disclosed designs further ensure that images across 99% or more of the field of view are acquired within ±150 nm (or less) of each channel's best focal plane, and that images across the entire field of view are acquired within ±200 nm (or less) of each imaging channel's best focal plane.

[0169] Illumination Light Path Design: Another factor for improving the signal-to-noise ratio (SNR), improving the contrast-to-noise ratio (CNR), and / or increasing throughput is increasing the illumination power intensity at the sample. In some examples, the disclosed imaging system may include an illumination path design that utilizes a high-power laser or laser diode coupled with a liquid light guide. The liquid light guide eliminates the optical speckle inherent in coherent light sources such as lasers and laser diodes. Additionally, the coupling optics are designed in a way that underfills the entrance aperture of the liquid light guide. Underfilling the liquid light guide entrance aperture reduces the effective numerical aperture of the illumination beam entering the objective lens and therefore improves the light delivery efficiency through the objective lens onto the sample plane. With this design innovation, illumination power intensities up to 3x that of conventional designs can be achieved over a large field of view (FOV).

[0170] By taking advantage of the angle-dependent distinction between s-polarized and p-polarized light, in some examples, the illumination beam polarization may be oriented to reduce the amount of backscattered and back-reflected illumination that reaches the imaging sensor. Structured illumination system: In some examples, the disclosed imaging modules and systems may include a structured illumination optical design to increase the effective spatial resolution of the imaging system, thereby enabling the use of higher surface densities of clonally amplified target nucleic acid sequences (clusters) on the flow cell surface for improved sequencing throughput. Structured illumination microscopy (SIM) utilizes spatially structured (i.e., periodic) light patterns for illumination of the sample plane and relies on the generation of interference patterns known as Moiré fringes. Several images are acquired under slightly different illumination conditions by shifting and / or rotating the structured illumination pattern to create Moiré fringes. Mathematical deconvolution of the resulting interference signal enables the reconstruction of super-resolution images with up to approximately two-fold improvement in spatial resolution over that achieved using diffraction-limited imaging optics. [Lutz (2011), "Biological Imaging by Superresolution Light Microscopy," Comprehensive Biotechnology (Second Ed.), vol. 1, pp. 579-589, Elsevier; Feiner-Gracia, et al. (2018), "15 - Advanced Optical Microscopy Techniques for the Investigation of Cell-Nanoparticle Interactions," Smart Nanoparticles for Biomedicine: Micro and Nano Technologies, pp. 219-236, Elsevier; Nylk, et al. (2019), "Light-Sheet Fluorescence Microscopy With Structured Light," Neurophotonics and Biomedical Spectroscopy, pp. 477-501, Elsevier]. An example of a structured illumination microscopy imaging system was recently disclosed by Hong, U.S. Patent Application Publication No. 2020 / 0218052.

[0171] 41 provides a non-limiting schematic diagram of an imaging system (4100) including a branched structure illumination optical design as disclosed herein. The first branch (or arm) of the illumination optical path of the system (4100) includes, for example, a light source (light emitter) (4110A), an optical collimator (4120A) for collimating the light emitted by the light source (4110A), a diffraction grating (4130A) at a first orientation relative to the optical axis, a rotating window (4140A), and a lens (4150A). The second branch of the illumination optical path of the system (4100) includes, for example, a light source (4110B), an optical collimator (4120B) for collimating the light emitted by the light source (4110B), a diffraction grating (4130B) at a second orientation relative to the optical axis, a rotating window (4140B), and a lens (4150B). Diffraction gratings (4130A) and (4130B) allow the projection of a pattern of optical interference fringes onto the sample plane.

[0172] In some examples, light sources 4110A and 4110B may be incoherent light sources (e.g., including one or more light emitting diodes (LEDs)) or coherent light sources (e.g., including one or more lasers or laser diodes). In some examples, light sources 4110A and 4110B may include optical fibers coupled to, for example, LEDs, lasers, or laser diodes that output beams that are then collimated by respective collimator lenses 4120A and 4120B. In some examples, light sources 4110A and 4110B may output light of the same wavelength. In some examples, light sources 4110A and 4110B may output light of different wavelengths. Either light source 4110A or 4110B may be configured to output light of any wavelength and / or wavelength range, as described elsewhere herein. During imaging, light sources (4110A) and (4110B) may be switched on or off, for example, using a high-speed shutter (not shown) placed in the light path or by pulsing the light source at a predetermined frequency.

[0173] In the example shown in Figure 41, the first illumination arm of the system (4100) includes a vertical grating (4130A) used to project a grating pattern of a first orientation (e.g., a vertical optical fringe pattern) onto a sample plane, e.g., a first interior surface (4188) of a flow cell (4187), and the second illumination arm includes a horizontal grating (4130B) used to project a grating pattern of a second orientation (e.g., a horizontal optical fringe pattern) onto the sample plane (4188). Advantageously, in this non-limiting example, the diffraction grating of the imaging system (4100) does not need to be mechanically rotated or translated during imaging, which may provide improved imaging speed, system reliability, and system reproducibility. In some examples, the diffraction gratings (4130A) and / or (4130B) may be rotatable about their respective optical axes such that the angle between the optical fringe patterns projected onto the sample plane is adjustable.

[0174] As shown in FIG. 41, in some examples, diffraction gratings 4130A and 4130B may be transmissive diffraction gratings including multiple diffractive elements (e.g., parallel slits or grooves) formed in a glass substrate or other suitable surface. In some examples, the gratings may be implemented as phase gratings, providing a periodic variation in the refractive index of the grating material. In some examples, the spacing for the grooves or features may be selected to diffract light at an appropriate angle and / or to accommodate the smallest resolvable feature size of the imaging sample for operation of imaging system 4100. In other examples, the diffraction gratings may be reflective diffraction gratings.

[0175] In the example shown in FIG. 41 , the orientations of the vertical and horizontal optical interference fringe patterns are offset by approximately 90 degrees. In other examples, other orientations of the diffraction gratings may be used to create the approximately 90-degree offset. For example, the diffraction gratings may be oriented so that they project optical interference fringe patterns that are offset by ±45 degrees from the x-axis or y-axis of the sample plane (4188) (e.g., the first interior flow cell surface). The configuration of the imaging system (4100) shown in FIG. 41 may be particularly advantageous for sample support surfaces (e.g., the interior surface (4188) of the flow cell (4187)) that include regularly patterned features arranged on a rectangular grid, because improved image resolution using a structured illumination approach can be achieved using only two perpendicular grating orientations (e.g., a vertical grating orientation and a horizontal grating orientation).

[0176] Diffraction gratings 4130A and 4130B may be configured to diffract the input illumination beam into a series of maximum intensities by constructive interference according to the following relationship in the example system 4100: m=order=d sin(θ) / λ

[0177] where d = distance between the slits or grooves of the diffraction grating, θ = angle of incidence of the illumination light relative to the normal to the surface of the diffraction grating, λ = wavelength of the illumination light, and m = integer value corresponding to the maximum intensity of the diffracted light, e.g., m = 0, ±1, ±2, etc. In some examples, a specific order of light of the diffracted illumination light, e.g., the first order (m = ±1), may be projected onto the sample plane (e.g., the internal flow cell surface (4188)). In some examples, for example, a vertical grating (4130A) may diffract a collimated light beam into first order diffracted beams (±1 orders) that are focused onto the sample plane at a first orientation, and a horizontal grating (4130B) may diffract the collimated light beam into first order diffracted beams that are focused onto the sample plane at a second orientation. In some examples, the zeroth order beam and / or all other higher order beams (e.g., m=±2 or higher) may be blocked, i.e., filtered out, from the illumination pattern projected onto the sample plane (4188) using a beam blocking element (not shown), such as, for example, an order filter that may be inserted in the optical path after the diffraction grating.

[0178] Each branch of the structured radiation system in the example of (4100) includes an optical phase modulator or phase shifter (4140A) and (4140B) to phase-shift the diffracted light transmitted or reflected by each of the diffraction gratings (4130A) and (4130B). During structured imaging, the optical phase of each diffracted beam may be shifted by some fraction (e.g., ½, ½, ¼, etc.) of the pitch (X) of each interference fringe of the structured pattern. In the example of FIG. 41, the phase modulators (4140A) and (4140B) may be implemented, for example, as rotating optical phase plates actuated by a rotary or other actuator mechanism to rotate and modulate the optical path length of each diffracted beam. For example, the optical phase plate (4140A) may be rotated around a vertical axis to shift the image projected onto the sample plane (4188) by the vertical grating (4130A) to the left or right, and the optical phase plate (4140B) may be rotated around a horizontal axis to shift the image projected onto the sample plane (4188) by the horizontal grating (4130B) vertically.

[0179] In other implementations, other types of phase modulators may be used that change the optical path length of the diffracted light (e.g., an optical wedge mounted on a linear translation stage). Additionally, while optical phase modulators 4140A and 4140B are shown as being positioned after diffraction gratings 4130A and 4130B, in other implementations they may be positioned at other locations in the illumination light path. In some examples, a single optical phase modulator may be manipulated in two different directions to generate different optical interference fringe patterns, or the position of a single optical phase modulator may be adjusted using a single motion to simultaneously adjust the path length of both arms of the illumination light path.

[0180] In the example shown in FIG. 41 , optical component (4160) may be used to combine light from the two illumination light paths. Optical component (4160) may include, for example, a partially silvered mirror, a dichroic mirror (depending on the wavelengths of the power output by light sources (4110) and (4110B)), a mirror containing a pattern of holes or a patterned reflective coating such that light from the two arms of the illumination system combines in a lossless or nearly lossless manner (e.g., without significant loss of optical power other than a small amount of absorption by the reflective coating), a polarizing beam splitter (if light sources (4110A) and (4110B) are configured to generate polarized light), etc. Optical component (4160) may be positioned such that light of desired diffraction orders reflected or transmitted by each of the diffraction gratings is spatially resolved and light of undesired orders is blocked. In some examples, optical component (4160) may pass first-order light output by the first illumination light path and may reflect first-order light output by the second illumination light path. In some examples, the structured illumination pattern on the sample surface (4188) may be switched from a vertical orientation (e.g., using diffraction grating (4130A)) to a horizontal orientation (e.g., using diffraction grating (4130B)) by turning each light source on or off or by opening or closing an optical shutter in the light path for the light source. In other examples, the structured illumination pattern may be switched by using an optical switch to change the illumination light path used to illuminate the sample plane.

[0181] Referring again to Figure 41, a lens (4170), a semi-reflective or dichroic mirror (4180), and an objective lens (4185) may be used to focus the structured illumination light onto a sample surface (4188) (e.g., a first interior surface of a flow cell (4187)). Light emitted, reflected, or scattered by the sample surface (4188) is then collected by the objective lens (4185), transmitted through the mirror (4180), and imaged by an image sensor or camera (4195). As noted, mirror (4180) may be a dichroic mirror for reflecting structured illumination light received from each branch of the illumination light path into objective lens (4185) for projection onto sample plane (4188), and for passing light emitted by sample plane (4188) (e.g., fluorescent light emitted at a different wavelength than the excitation light) for imaging onto image sensor (4195).

[0182] In some examples, the system (4100) may optionally include a custom-made tube lens (4190), as described elsewhere herein, so that the focal point of the imaging system can be shifted from the first interior surface (4188) to the second interior surface (4189) of the flow cell (4187), enabling double-sided imaging with minimal adjustment. In some examples, the lens (4170) may include a custom-made tube lens, as described elsewhere herein, so that the focal point of the illumination light path can be shifted from the first interior surface (4188) to the second interior surface (4189) of the flow cell (4187), enabling double-sided imaging with minimal adjustment. In some examples, the lens (4170) may be implemented to couple along the optical axis to adjust the focus of the structured illumination pattern on the sample plane. In some examples, the system (4100) may include an autofocus mechanism (not shown) for adjusting the focus of the illumination light and / or the focus of the image at the plane of the image sensor (4195). In some examples, the system (4100) shown in Figure 41 may provide high optical efficiency due to the lack of a polarizer in the optical path. The use of unpolarized light may or may not significantly affect the contrast of the illumination pattern, depending on the numerical aperture of the objective lens (4185).

[0183] For purposes of brevity, some optical components of imaging system (4100) may have been omitted from FIG. 41 and the preceding discussion. While system (4100) is shown in this non-limiting example as a single-channel detection system, in other examples it may be implemented as a multi-channel detection system (e.g., using two different image sensors and appropriate optics, as well as light sources emitting at two different wavelengths). Furthermore, while the illumination light path of system (4100) is shown in this non-limiting example as including two branches, in some examples it may be implemented as including, for example, three branches, four branches, or more than four branches, each of which includes a diffraction grating at a fixed orientation or an adjustable relative orientation with respect to each other.

[0184] In some examples, alternative illumination path designs may be used to create structured illumination. For example, in some examples, a single large rotating optical phase modulator may be placed after the optical component (4160) and used in place of the optical phase modulators (4140A) and (4140B), thereby modulating the phase of both diffracted beams output by the vertical and horizontal diffraction gratings (4130A) and (4130B). In some examples, the rotation axis of the single rotating optical corrector may be offset 45 degrees (or at another angle) from the optical axis of each of the vertical and horizontal diffraction gratings, instead of being parallel to the optical axis of one of the gratings, thereby enabling phase shifts along both illumination directions. In some examples, the single rotating optical phase modulator may be replaced by, for example, a wedge-shaped optical component that rotates about the vertical beam axis.

[0185] In another alternative illumination path design, the diffraction gratings (4130A) and (4130B) may be mounted on respective linear stages such that they can be translated to change the optical path length (and therefore the phase) of light reflected or transmitted by the diffraction gratings (4130A) and (4130B). The axes of motion of the linear stages may be perpendicular or offset from their respective diffraction gratings to provide translation of the diffraction gratings' interference fringe patterns along the sample plane (4188). Suitable translation stages may be, for example, crossed roller bearing stages, linear motors, high-precision linear encoders, and / or linear actuators to provide precise linear translation of the diffraction gratings.

[0186] FIG. 42 provides a non-limiting example of a workflow for acquiring and processing images using structured illumination to improve the spatial resolution of an imaging system. In some examples, the workflow shown in FIG. 42 may be performed to image the entire sample plane (e.g., the interior surface of a flow cell by image tiling) or to image a single area of ​​a larger sample plane. The vertical (4130A) and horizontal (4130B) diffraction gratings of the system (4100) shown in FIG. 41 may be used to project illumination light interference fringes onto the sample plane with different known orientations and / or different known phase shifts. For example, the imaging system (4100) may use the vertical grating (4130A) and horizontal grating (4130B) to generate horizontal and vertical illumination patterns, respectively, while the optical phase modulators (4140A) and (4140B) may be set to three different positions to generate the three phase shifts shown for each orientation.

[0187] In operation, a first illumination condition (e.g., a particular orientation and phase-shift setting of the diffraction grating) may be used to project a grating optical interference pattern onto a sample plane, e.g., a flow cell surface. After capturing an image using the first illumination condition, one or more additional images (e.g., 1, 2, 3, 4, 5, 6, or more than 6 additional images acquired using one or more phase-shift illumination patterns) may be acquired. If the imaging system includes a second branch of the illumination light path, the image acquisition process may be repeated using a second illumination condition (e.g., a second particular orientation of the diffraction grating and phase-shift setting) as the starting point, and the image acquisition process may be repeated. In some examples, images may be acquired for at least three different orientations of the diffraction grating (e.g., spaced 60 degrees apart from each other) using at least five different phase-shift optical interference fringe patterns. If no more images are acquired using different orientations of the diffraction grating or phase-shifted illumination light fringe patterns, an image reconstruction algorithm may be used to acquire the images and generate a reconstructed super-resolution image. In some examples, images may be acquired for at least 1, 2, 3, 4, 5, 6, or more than 6 orientations of the diffraction grating, using at least 1, 2, 3, 4, 5, 6, or more than 6 different phase-shifted illumination light fringe patterns at each orientation.

[0188] A potential drawback of acquiring multiple images for use in reconstructing a single, super-resolution image is the time required to adjust the orientation and / or relative phase shift of the projected optical interference fringe pattern, the exposure time required to acquire each image, and downstream image processing. Therefore, optical designs that minimize the time required to change the orientation and relative phase of the diffraction grating, along with highly efficient image reconstruction algorithms, are preferred. In some instances, for example, fewer images may be required to reconstruct a super-resolution image of a flow cell surface containing discrete, fluorescently labeled clusters of amplified target nucleic acid sequences tethered to a low-nonspecific binding surface, as described elsewhere herein, than would typically be required to reconstruct a higher-resolution image of a conventional sample, such as a stained tissue sample.

[0189] 42, the cycle described above may be repeated for different areas of a given flow cell surface, for example, if the image is tiled to create a higher resolution image of the entire flow cell surface. In some instances, for example, if a second flow cell surface is to be imaged, the cycle described above may be repeated after adjusting the focus of the imaging system.

[0190] Other super-resolution imaging techniques: In some examples, the disclosed imaging systems may include alternative super-resolution imaging techniques, such as photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), and / or stochastic optical reconstruction microscopy (STORM) [see, e.g., Lutz, et al. (2011), "Biological Imaging by Superresolution Light Microscopy," Comprehensive Biotechnology (Second Ed.), vol. 1, pp. 579-589, Elsevier], which are based on statistical curve-fitting of the intensity distribution observed in an image of a single molecule's point spread function (PSF) to a Gaussian distribution function. The Gaussian distribution function is then used to define the molecule's position in the sample plane with much greater precision than is possible with classical resolution limits. The same approach may be used to image small, dispersed subsets of fluorescently labeled molecules, such as low-nonspecific binding surfaces on a sample support or clonal amplification clusters of target nucleic acid sequences tethered to the interior surface of a flow cell.

[0191] The spatial accuracy or resolution achieved using these methods depends on the number of photons collected from a molecule before it is photobleached and the background noise level [Lutz, et al. (2011), ibid.]. Positional accuracy of 1-2 nm has been demonstrated when background noise is negligible and collection of at least 10,000 photons per molecule is possible. In some instances, for example, using the avidity-based sequencing approach described elsewhere herein, polymer-nucleotide conjugates containing multiple fluorescent labels (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 labels per conjugate) may be used to ensure high photon counts, optionally in combination with low nonspecific binding surfaces as disclosed elsewhere herein, to ensure very low background signals, facilitating the use of these super-resolution imaging techniques for genetic testing and sequencing applications. Spatial accuracy or resolution decreases with decreasing number of collected photons, but when only moderate numbers of photons are collected, positional accuracy or resolution of 20 nm is possible. In some cases, a 10-fold or better improvement in lateral spatial resolution may be achieved. In some cases, image resolutions of better than 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm, or 10 nm may be achieved.

[0192] The second essential principle for this class of imaging is that only a small number of spatially separated fluorescent molecules within the sample are imaged at a given time.

[0193] In some instances, the ability to control the fluorescence emission of small, dispersed subsets of fluorescent molecules in the sample plane is key to facilitating super-resolution imaging. In the case of fluorescence photoactivation localization microscopy (FPALM) and photoactivation localization microscopy (PALM), for example, the use of photoactivatable green fluorescent protein (PA-GFP) as a label allows for the controlled induction of fluorescent subsets in a sample using short pulses of 405 nm light to photoconvert PA-GFP from a dark, non-fluorescent state to an excitable fluorescent state at 488 nm, thereby resulting in a spatially separated subset of fluorescent molecules that can be imaged [Lutz, et al. (2011), ibid]. In the case of stochastic optical reconstruction microscopy (STORM), for example, the photon switching properties of the cyanine dye pair Cy5-Cy3 may be used in a similar manner to enable the stochastic induction of Cy5 fluorescence from a small subset of molecules in a sample at a given time, e.g., a small subset of molecules that are spatially separated by at least a few resolution units. In some examples, for example, when combined with avidity-directed sequencing, as described elsewhere herein, the polymer-nucleotide conjugate may comprise a photoactivatable green fluorescent protein (PA-GFP), or a subdomain, or portion thereof. In some examples, the polymer-nucleotide conjugate may comprise a mixture of conjugates in which a first portion is labeled, for example, with a Cy3 label, and a second portion is labeled, for example, with a Cy5 label. In some examples, the polymer-nucleotide conjugate may comprise a mixture of, for example, a Cy3 label and a Cy5 label within the same conjugate.

[0194] Super-resolution images are reconstructed from the sum of Gaussian fits from all molecules or features (e.g., identified nucleic acid clusters) imaged in a time stack of acquired images [Lutz, et al. (2011), ibid], where intensity corresponds to the uncertainty in the position of each molecule or subset of molecules. Inherent in this type of dataset is the ability to render images with different localization accuracies or resolutions. In some examples, imaging modules including total internal reflection fluorescence (TIRF) optical imaging designs may be advantageous in implementing the use of these super-resolution imaging techniques because the evanescent wave used to excite fluorescence is limited to less than 200 nm from the sample support or flow cell surface in the axial dimension, thereby suppressing background fluorescence signals. In some examples, the imaging system may include a higher numerical aperture objective lens utilized in other imaging module designs disclosed herein. The use of a higher numerical aperture objective lens may facilitate the implementation of evanescent wave excitation and highly efficient photon capture from fluorescent probes. In some examples, wide-field imaging using single-photon sensitive EM-CCD cameras, or other types of image sensors, may allow for simultaneous imaging of many molecules or subsets of molecules (e.g., nucleic acid sequence clusters) per frame.

[0195] In some instances, the data acquisition time required to obtain sufficient images with adequate feature definition and resolution may be reduced by improvements in the sensitivity and speed of imaging systems through the use of avidity sequencing reagents and low non-specific binding surfaces, as well as improved image reconstruction algorithms, as disclosed herein, to increase signal while reducing or eliminating background.

[0196] Assessing Image Quality: For any of the optical imaging design embodiments disclosed herein, imaging performance or quality may be assessed using any of a variety of performance metrics known to those skilled in the art. Examples include, but are not limited to, measurements of the modulation transfer function (MTF) of one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof.

[0197] In some examples, the disclosed optical designs for double-sided imaging (e.g., the disclosed objective lens designs, tube lens designs, use of an electro-optic phase plate in combination with an objective lens, etc., alone or in combination) may provide significant improvements to both the upper (near) and lower (far) interior surfaces of the flow cell, such that the difference in imaging performance metrics for imaging the upper and lower interior surfaces of the flow cell is less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%, either alone or in combination, for the imaging performance metrics listed above.

[0198] In some examples, the disclosed optical designs for double-sided imaging (e.g., the disclosed tube lens designs, the use of an electro-optic phase plate in combination with an objective lens, etc.) may provide significant improvements in image quality, such that image quality performance metrics for double-sided imaging provide an improvement in imaging performance for double-sided imaging of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% compared to conventional systems that include, for example, an objective lens, a motion actuation corrector (which is moved in and out of the optical path when imaging the near or far interior surface of the flow cell), and an image sensor for any of the imaging performance metrics listed above, either alone or in combination. In some examples, a fluorescence imaging system that includes one or more of the disclosed tube lens designs provides at least the same improvement, or better, in the imaging performance metrics for double-sided imaging compared to the imaging performance of conventional systems that include an objective lens, a motion actuation corrector, and an image sensor. In some examples, a fluorescence imaging system including one or more of the disclosed tube lens designs provides an improvement in imaging performance for double-sided imaging of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to the imaging performance of a conventional system including an objective lens, a motion actuation corrector, and an image sensor.

[0199] Imaging module specifications: Excitation Light Wavelength: In any of the disclosed optical imaging module designs, the light source of the disclosed imaging module may generate visible light, such as green light and / or red light. In some examples, the light source, alone or in combination with one or more optical components, such as an excitation optical filter and / or a dichroic beam splitter, may generate excitation light at approximately 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. Those skilled in the art will recognize that the excitation wavelength may have any value within this range, for example, within approximately 620 nm.

[0200] Excitation Light Bandwidth: In any of the disclosed optical imaging module designs, the light source, alone or in combination with one or more optical components, such as an excitation optical filter and / or a dichroic beam splitter, may generate light at a particular excitation wavelength within a bandwidth of ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or more. One skilled in the art will recognize that the excitation bandwidth may have any value within this range, for example, about ±18 nm.

[0201] Light Source Power Output: In any of the disclosed optical imaging module designs, the output from the light source and / or the excitation light beam derived therefrom (including the composite excitation light beam) may range in power from about 0.5 W to about 5.0 W or more (as discussed in more detail below). In some examples, the power of the output from the light source and / or the excitation light beam derived therefrom may be at least 0.5 W, at least 0.6 W, at least 0.7 W, at least 0.8 W, at least 1 W, at least 1.1 W, at least 1.2 W, at least 1.3 W, at least 1.4 W, at least 1.5 W, at least 1.6 W, at least 1.8 W, at least 2.0 W, at least 2.2 W, at least 2.4 W, at least 2.6 W, at least 2.8 W, at least 3.0 W, at least 3.5 W, at least 4.0 W, at least 4.5 W, or at least 5.0 W. In some implementations, the power of the output from the light source and / or the excitation light beam derived therefrom (including the composite excitation light beam) may be at most 5.0W, at most 4.5W, at most 4.0W, at most 3.5W, at most 3.0W, at most 2.8W, at most 2.6W, at most 2.4W, at most 2.2W, at most 2.0W, at most 1.8W, at most 1.6W, at most 1.5W, at most 1.4W, at most 1.3W, at most 1.2W, at most 1.1W, at most 1W, at most 0.8W, at most 0.7W, at most 0.6W, or at most 0.5W. Any of the lower and higher values ​​recited within this paragraph may be combined to form ranges included within the present disclosure, for example, in some examples, the output from the light source and / or the power of the excitation light beam derived therefrom (including the composite excitation light beam) may range from about 0.8 W to about 2.4 W. One of ordinary skill in the art will recognize that the output from the light source and / or the power of the excitation light beam derived therefrom (including the composite excitation light beam) may have any value within this range, for example, about 1.28 W.

[0202] Light Source Output Power and CNR: In some implementations of the disclosed optical imaging module designs, the output power from the light source and / or the excitation light beam derived therefrom (including the composite excitation light beam), in combination with a suitable sample, is sufficient to provide, in an image acquired by the illumination imaging module, a contrast-to-noise ratio (CNR) of at least 5, at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, or at least 50, or more, or any CNR within any range formed by any of these values.

[0203] Fluorescence Emission Bands: In some examples, the disclosed fluorescence optical imaging modules may be configured to detect fluorescence emissions produced by any of a variety of fluorophores known to those skilled in the art. Examples of fluorescent dyes suitable for use in genotyping and nucleic acid sequencing applications (e.g., by attachment to nucleotides, oligonucleotides, or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, cyanine, and cyanine derivatives, including cyanine dye-3 (Cy3), cyanine dye-5 (Cy5), cyanine dye-7, etc. (Cy7).

[0204] Fluorescence Emission Wavelength: In any of the disclosed optical imaging module designs, the detection or imaging channels of the disclosed optical systems may include one or more optical components, such as emission optical filters and / or dichroic beam splitters, configured to collect emitted light at approximately 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. One skilled in the art will recognize that the emission wavelength may have any value within this range, such as approximately 825 nm.

[0205] Fluorescence Emission Optical Bandwidth: In any of the disclosed optical imaging module designs, the detection or imaging channels may include one or more optical components, such as emission optical filters and / or dichroic beam splitters, configured to collect light of a particular emission wavelength within a bandwidth of + / -2 nm, + / -5 nm, + / -10 nm, + / -20 nm, + / -40 nm, + / -80 nm, or greater. One skilled in the art will recognize that the excitation bandwidth may have any value within this range, for example, about 18 nm.

[0206] Numerical Aperture: In some examples, the numerical aperture of the objective lens and / or optical imaging module (e.g., including the objective lens and / or tube lens) in any of the disclosed optical system designs may range from about 0.1 to about 1.4. In some examples, the numerical aperture may be at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, or at least 1.4. In some examples, the numerical aperture may be at most 1.4, at most 1.3, at most 1.2, at most 1.1, at most 1.0, at most 0.9, at most 0.8, at most 0.7, at most 0.6, at most 0.5, at most 0.4, at most 0.3, at most 0.2, or at most 0.1. Any of the lower and higher values ​​recited within this paragraph may be combined to form a range within this disclosure, e.g., in some examples, the numerical aperture may range from about 0.1 to about 0.6. One of ordinary skill in the art will recognize that the numerical aperture may have any value within this range, e.g., about 0.55.

[0207] Optical Resolution: In some examples, depending on the numerical aperture of the objective lens and / or optical system (e.g., including the objective lens and / or tube lens), the minimum resolvable spot (or feature) separation distance at the sample plane achieved by any of the disclosed optical system designs may range from about 0.5 μm to about 2 μm. In some examples, the minimum resolvable spot separation distance at the sample plane may be at least 0.5 μm, at least 0.6 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, at least 1.2 μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or at least 1.0 μm. In some examples, the minimum resolvable spot separation distance may be at most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most 0.7 μm, at most 0.6 μm, or at most 0.5 μm. Any of the lower and higher values ​​recited within this paragraph may be combined to form a range included within the present disclosure; for example, in some examples, the minimum resolvable spot separation distance may range from about 0.8 μm to about 1.6 μm. One of ordinary skill in the art will recognize that the minimum resolvable spot separation distance may have any value within this range, such as about 0.95 μm.

[0208] Optical Resolution of First and Second Surfaces at Different Depths: In some examples, use of the novel objective and / or tube lens designs disclosed herein may provide comparable optical resolution for a first and second surface (e.g., the upper and lower interior surfaces of a flow cell) in any of the optical modules or optical systems disclosed herein, with or without the need for refocusing between acquisition of images of the first and second surfaces. In some examples, the optical resolution of the images thus acquired of the first and second surfaces may be within 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, or 1% of each other, or any value within this range.

[0209] Magnification: In some examples, the magnification of the objective lens and / or tube lens and / or optical system (e.g., including the objective lens and / or tube lens) in any of the disclosed optical configurations may range from about 2x to about 20x. In some examples, the optical system magnification may be at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least 10x, at least 15x, or at least 20x. In some examples, the optical system magnification may be up to 20x, up to 15x, up to 10x, up to 9x, up to 8x, up to 7x, up to 6x, up to 5x, up to 4x, up to 3x, or up to 2x. Any of the lower and higher values ​​listed within this paragraph may be combined to form ranges included within the present disclosure; for example, in some examples, the optical system magnification may range from about 3x to about 10x. Those skilled in the art will recognize that the optical system magnification may have any value within this range, for example, about 7.5x.

[0210] Objective Lens Focal Length: In some implementations of the disclosed optical designs, the focal length of the objective lens may range from 20 mm to 40 mm. In some examples, the focal length of the objective lens may be at least 20 mm, at least 25 mm, at least 30 mm, at least 35 mm, or at least 40 mm. In some examples, the focal length of the objective lens may be up to 40 mm, up to 35 mm, up to 30 mm, up to 25 mm, or up to 20 mm. Any of the lower and higher values ​​listed in this paragraph may be combined to form a range included within the present disclosure; for example, in some examples, the focal length of the objective lens may range from 25 mm to 35 mm. One of ordinary skill in the art will recognize that the focal length of the objective lens may have any value within the range of values ​​specified above, for example, about 37 mm.

[0211] Objective Lens Working Distance: In some implementations of the disclosed optical designs, the objective lens working distance may range from about 100 μm to 30 mm. In some examples, the working distance may be at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1 mm, at least 2 mm, at least 4 mm, at least 6 mm, at least 8 mm, at least 10 mm, at least 15 mm, at least 20 mm, at least 25 mm, or at least 30 mm. In some examples, the working distance may be up to 30 mm, up to 25 mm, up to 20 mm, up to 15 mm, up to 10 mm, up to 8 mm, up to 6 mm, up to 4 mm, up to 2 mm, up to 1 mm, up to 900 μm, up to 800 μm, up to 700 μm, up to 600 μm, up to 500 μm, up to 400 μm, up to 300 μm, up to 200 μm, or up to 100 μm. Any of the lower and higher values ​​listed within this paragraph may be combined to form a range included within the present disclosure; for example, in some examples, the working distance of the objective lens may range from 500 μm to 2 mm. One skilled in the art will recognize that the working distance of the objective lens may have any value within the range of values ​​specified above, for example, about 1.25 mm.

[0212] Objective Lens Optimized for Imaging Through Thick Coverslips: In some examples of the disclosed optical designs, the objective lens design may be improved or optimized for coverslips of different flow cell thicknesses. For example, in some examples, the objective lens may be designed for optimal optical performance with coverslips that are about 200 μm to about 1,000 μm thick. In some examples, the objective lens may be designed for optimal performance with coverslips that are at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, or at least 1,000 μm thick. In some examples, the objective lens may be designed for optimal performance with coverslips that are up to 1,000 μm, up to 900 μm, up to 800 μm, up to 700 μm, up to 600 μm, up to 500 μm, up to 400 μm, up to 300 μm, or up to 200 μm thick. Any of the lower and higher values ​​recited in this paragraph may be combined to form ranges included within the present disclosure; for example, in some examples, the objective lens may be designed for optimal optical performance with coverslips that may range from about 300 μm to about 900 μm. One skilled in the art will appreciate that the objective lens may be designed for optimal optical performance with coverslips that are any value within this range, such as about 725 μm.

[0213] Depth of Field and Depth of Focus: In some examples, the depth of field and / or depth of focus for any of the disclosed imaging module (e.g., including the objective lens and / or tube lens) designs may range from about 10 μm to about 800 μm, or more. In some examples, the depth of field and / or depth of focus may be at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 125 μm, at least 150 μm, at least 175 μm, at least 200 μm, at least 250 μm, at least 300 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, or more. In some examples, the depth of field and / or depth of focus may be at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 250 μm, at most 200 μm, at most 175 μm, at most 150 μm, at most 125 μm, at most 100 μm, at most 75 μm, at most 50 μm, at most 40 μm, at most 30 μm, at most 20 μm, at most 10 μm, or less. Any of the lower and higher values ​​recited within this paragraph may be combined to form ranges included within the present disclosure; for example, in some examples, the depth of field and / or depth of focus may range from about 100 μm to about 175 μm. Those skilled in the art will recognize that the depth of field and / or depth of focus may have any value within the range of values ​​specified above, for example, about 132 μm.

[0214] Field of View (FOV): In some implementations, the FOV of any of the disclosed imaging module designs (e.g., provided by the combination of the objective lens and detection channel optics (e.g., tube lens)) may range from, for example, about 1 mm to 5 mm (e.g., in diameter, width, length, or longest dimension). In some examples, the FOV may be at least 1.0 mm, at least 1.5 mm, at least 2.0 mm, at least 2.5 mm, at least 3.0 mm, at least 3.5 mm, at least 4.0 mm, at least 4.5 mm, or at least 5.0 mm (e.g., in diameter, width, length, or longest dimension). In some examples, the FOV may be up to 5.0 mm, up to 4.5 mm, up to 4.0 mm, up to 3.5 mm, up to 3.0 mm, up to 2.5 mm, up to 2.0 mm, up to 1.5 mm, or up to 1.0 mm (e.g., in diameter, width, length, or longest dimension). Any of the lower and higher values ​​recited within this paragraph may be combined to form a range included within the present disclosure, e.g., in some examples, the FOV may range from about 1.5 mm to about 3.5 mm (e.g., in diameter, width, length, or longest dimension). One of ordinary skill in the art will recognize that the FOV may have any value within the above-specified range of values, e.g., about 3.2 mm (e.g., in diameter, width, length, or longest dimension).

[0215] Field of View (FOV) Area: In some examples of the disclosed optical system designs, the area of ​​the field of view is approximately 2 mm 2 ~about 5mm 2 In some instances, the field of view may be at least 2 mm 2 , at least 3 mm 2 , at least 4 mm 2 , or at least 5 mm 2 In some instances, the field of view may be at most 5 mm 2 , up to 4 mm 2 , up to 3 mm 2 , or up to 2 mm 2Any of the lower and higher values ​​set forth in this paragraph may be combined to form ranges within the disclosure, e.g., in some instances, the field of view may be an area of ​​about 3 mm 2 ~about 4mm 2 Those skilled in the art will appreciate that the area of ​​the field of view may range from any value within this range, for example 2.75 mm 2 . . .

[0216] Objective Lens and / or Tube Lens MTF Optimization: In some examples, the objective lens and / or at least one tube lens design of the disclosed imaging modules and systems is configured to optimize the modulation transfer function over a medium to high spatial frequency range. For example, in some examples, the objective lens and / or at least one tube lens design of the disclosed imaging modules and systems is configured to optimize the modulation transfer function over a spatial frequency range at the sample plane of 500 cycles per mm to 900 cycles per mm, 700 cycles per mm to 1100 cycles per mm, 800 cycles per mm to 1200 cycles per mm, or 600 cycles per mm to 1000 cycles per mm.

[0217] Optical Aberrations and Diffraction-Limited Imaging Performance: In some implementations of any of the optical imaging module designs disclosed herein, the objective lens and / or tube lens may be configured to provide the imaging module with the field of view shown above such that the FOV has less than 0.15 waves of aberration across at least 60%, 70%, 80%, 90%, or 95% of the field. In some implementations, the objective lens and / or tube lens may be configured to provide the imaging module with the field of view shown above such that the FOV has less than 0.1 waves of aberration across at least 60%, 70%, 80%, 90%, or 95% of the field. In some implementations, the objective lens and / or tube lens may be configured to provide the imaging module with the field of view shown above such that the FOV has less than 0.075 waves of aberration across at least 60%, 70%, 80%, 90%, or 95% of the field. In some implementations, the objective lens and / or tube lens may be configured to provide the imaging module with the field of view shown above such that the FOV is diffraction limited over at least 60%, 70%, 80%, 90%, or 95% of the field.

[0218] Incident angle of light beams onto dichroic reflectors, beam splitters, and beam combiners: In some examples of the disclosed optical designs, the incident angle of a light beam entering a dichroic reflector, beam splitter, or beam combiner may range from about 20 degrees to about 45 degrees. In some examples, the incident angle may be at least 20 degrees, at least 25 degrees, at least 30 degrees, at least 35 degrees, at least 40 degrees, or at least 45 degrees. In some examples, the incident angle may be up to 45 degrees, up to 40 degrees, up to 35 degrees, up to 30 degrees, up to 25 degrees, or up to 20 degrees. Any of the lower and higher values ​​listed within this paragraph may be combined to form a range included within the present disclosure; for example, in some examples, the incident angle may range from about 25 degrees to about 40 degrees. One of ordinary skill in the art will recognize that the incident angle may have any value within the range of values ​​specified above, such as about 43 degrees.

[0219] Image Sensor (Photodetector Array) Size: In some examples, the disclosed optical systems may include an image sensor having an active area with a diagonal ranging from about 10 mm to about 30 mm, or more. In some examples, the image sensor may have an active area with a diagonal of at least 10 mm, at least 12 mm, at least 14 mm, at least 16 mm, at least 18 mm, at least 20 mm, at least 22 mm, at least 24 mm, at least 26 mm, at least 28 mm, or at least 30 mm. In some examples, the image sensor may have an active area with a diagonal of up to 30 mm, up to 28 mm, up to 26 mm, up to 24 mm, up to 22 mm, up to 20 mm, up to 18 mm, up to 16 mm, up to 14 mm, up to 12 mm, or up to 10 mm. Any of the lower and higher values ​​recited within this paragraph may be combined to form ranges included within this disclosure, e.g., in some examples, an image sensor may have an active area with a diagonal ranging from about 12 mm to about 24 mm. One of ordinary skill in the art will recognize that an image sensor may have any value within the range of values ​​specified above, e.g., about 28.5 mm.

[0220] Image Sensor Pixel Size and Pitch: In some examples, the pixel size and / or pitch selected for the image sensor used in the disclosed optical system designs may range in at least one dimension from about 1 μm to about 10 μm. In some examples, the pixel size and / or pitch may be at least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, or at least 10 μm. In some examples, the pixel size and / or pitch may be at most 10 μm, at most 9 μm, at most 8 μm, at most 7 μm, at most 6 μm, at most 5 μm, at most 4 μm, at most 3 μm, at most 2 μm, or at most 1 μm. Any of the lower and higher values ​​listed within this paragraph may be combined to form ranges included within the present disclosure; for example, in some examples, the pixel size and / or pitch may range from about 3 μm to about 9 μm. Those skilled in the art will recognize that the pixel size and / or pitch may have any value within this range, for example, about 1.4 μm.

[0221] Oversampling: In some examples of the disclosed optical designs, a spatial oversampling scheme is utilized, where the spatial sampling frequency is at least 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, or 10x the optical resolution X (lp / mm).

[0222] Maximum Translation Stage Speed: In some examples of the disclosed optical imaging modules, the maximum translation stage speed on any one axis may range from about 1 mm / s to about 5 mm / s. In some examples, the maximum translation stage speed may be at least 1 mm / s, at least 2 mm / s, at least 3 mm / s, at least 4 mm / s, or at least 5 mm / s. In some examples, the maximum translation stage speed may be up to 5 mm / s, up to 4 mm / s, up to 3 mm / s, up to 2 mm / s, or up to 1 mm / s. Any of the lower and higher values ​​listed within this paragraph may be combined to form a range included within the present disclosure; for example, in some examples, the maximum translation stage speed may range from about 2 mm / s to about 4 mm / s. One of ordinary skill in the art will recognize that the maximum translation stage speed may have any value within this range, for example, about 2.6 mm / s.

[0223] Maximum Translation Stage Acceleration: In some examples of the disclosed optical imaging modules, the maximum acceleration on any one axis of motion is about 2 mm / sec 2 ~about 10mm / sec 2 In some examples, the maximum acceleration may be at least 2 mm / sec 2 , at least 3 mm / sec 2 , at least 4 mm / sec 2 , at least 5 mm / sec 2 , at least 6 mm / sec 2 , at least 7 mm / sec 2 , at least 8 mm / sec 2 , at least 9 mm / sec 2 , or at least 10 mm / sec 2 In some examples, the maximum acceleration may be up to 10 mm / sec 2 , up to 9mm / sec 2 , up to 8mm / sec 2 , up to 7mm / sec 2 , up to 6mm / sec 2 , up to 5mm / sec 2 , up to 4mm / sec 2 , up to 3mm / sec 2 , or up to 2 mm / sec 2Any of the lower and higher values ​​set forth in this paragraph may be combined to form ranges included within this disclosure, e.g., in some examples, the maximum acceleration may be greater than or equal to about 2 mm / sec. 2 ~about 8mm / sec 2 Those skilled in the art will appreciate that the maximum acceleration may range anywhere within this range, for example, about 3.7 mm / sec. 2 . . .

[0224] Translation Stage Positioning Repeatability: In some examples of the disclosed optical imaging modules, the positioning repeatability for any one axis may range from about 0.1 μm to about 2 μm. In some examples, the positioning repeatability may be at least 0.1 μm, at least 0.2 μm, at least 0.3 μm, at least 0.4 μm, at least 0.5 μm, at least 0.6 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, at least 1.2 μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or at least 2.0 μm. In some examples, the placement repeatability may be at most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most 0.7 μm, at most 0.6 μm, at most 0.5 μm, at most 0.4 μm, at most 0.3 μm, at most 0.2 μm, or at most 0.1 μm. Any of the lower and higher values ​​recited within this paragraph may be combined to form a range included within the present disclosure; for example, in some examples, the placement repeatability may range from about 0.3 μm to about 1.2 μm. One of ordinary skill in the art will recognize that the placement repeatability may have any value within this range, such as about 0.47 μm.

[0225] FOV Repositioning Time: In some examples of the disclosed optical imaging modules, the maximum time required to reposition the sample plane (field of view) relative to the optics, or vice versa, may range from about 0.1 seconds to about 0.5 seconds. In some examples, the maximum repositioning time (i.e., the time for the scan stage to move and settle) may be at least 0.1 seconds, at least 0.2 seconds, at least 0.3 seconds, at least 0.4 seconds, or at least 0.5 seconds. In some examples, the maximum repositioning time may be up to 0.5 seconds, up to 0.4 seconds, up to 0.3 seconds, up to 0.2 seconds, or up to 0.1 seconds. Any of the lower and higher values ​​described within this paragraph may be combined to form a range encompassed within the present disclosure; for example, in some examples, the maximum repositioning time may range from about 0.2 seconds to about 0.4 seconds. One of ordinary skill in the art will recognize that the maximum repositioning time may have any value within this range, such as about 0.45 seconds.

[0226] Error Threshold for Autofocus Correction: In some examples of the disclosed optical imaging module, the error threshold specified to trigger an autofocus correction may range from about 50 nm to about 200 nm. In some examples, the error threshold may be at least 50 nm, at least 75 nm, at least 100 nm, at least 125 nm, at least 150 nm, at least 175 nm, or at least 200 nm. In some examples, the error threshold may be at most 200 nm, at most 175 nm, at most 150 nm, at most 125 nm, at most 100 nm, at most 75 nm, or at most 50 nm. Any of the lower and higher values ​​described within this paragraph may be combined to form a range included within the present disclosure; for example, in some examples, the error threshold may range from about 75 nm to about 150 nm. One of ordinary skill in the art will recognize that the error threshold may have any value within this range, such as about 105 nm.

[0227] Image Acquisition Time: In some examples of the disclosed optical imaging modules, the image acquisition time may range from about 0.001 seconds to about 1 second. In some examples, the image acquisition time may be at least 0.001 seconds, at least 0.01 seconds, at least 0.1 seconds, or at least 1 second. In some examples, the image acquisition time may be up to 1 second, up to 0.1 seconds, up to 0.01 seconds, or up to 0.001 seconds. Any of the lower and higher values ​​recited within this paragraph may be combined to form a range encompassed within the present disclosure; for example, in some examples, the image acquisition time may range from about 0.01 seconds to about 0.1 seconds. One of ordinary skill in the art will recognize that the image acquisition time may have any value within this range, for example, about 0.250 seconds.

[0228] Imaging Time per FOV: In some examples, the imaging time may range from about 0.5 seconds to about 3 seconds per field of view. In some examples, the imaging time may be at least 0.5 seconds, at least 1 second, at least 1.5 seconds, at least 2 seconds, at least 2.5 seconds, or at least 3 seconds per FOV. In some examples, the imaging time may be up to 3 seconds, up to 2.5 seconds, up to 2 seconds, up to 1.5 seconds, up to 1 second, or up to 0.5 seconds per FOV. Any of the lower and higher values ​​described within this paragraph may be combined to form a range encompassed within the present disclosure; for example, in some examples, the imaging time may range from about 1 second to about 2.5 seconds. One of ordinary skill in the art will recognize that the imaging time may have any value within this range, such as about 1.85 seconds.

[0229] Flatness of field: In some examples, images spanning 80%, 90%, 95%, 98%, 99%, or 100% percent of the field of view are acquired within ±200 nm, ±175 nm, ±150 nm, ±125 nm, ±100 nm, ±75 nm, or ±50 nm relative to the best focal plane of each fluorescence (or other imaging mode) detection channel.

[0230] Systems and System Components for Genomics and Other Applications: As noted above, in some implementations, the disclosed optical imaging modules may function as modules, components, subassemblies, or subsystems of larger systems configured to perform, for example, genomics applications (e.g., genetic testing applications and / or nucleic acid sequencing applications), or other chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. Figure 39 provides a non-limiting example block diagram for a sequencing system, for example, as disclosed herein. In addition to one, two, three, four, or more than four imaging modules as disclosed herein (each of which may include one or more illumination light paths and / or one or more detection light paths (e.g., one or more detection channels configured to image fluorescent emissions within a particular wavelength range onto an image sensor)), such systems may include one or more XY translation stages, one or more XYZ translation stages, flow cells or cartridges, fluidic-optical systems and fluid flow control modules, reagent cartridges, temperature control modules, fluid dispensing robotics components, cartridge and / or microplate handling (pick-and-place) robotics components, light-tight enclosures and / or environmental control chambers, one or more processors or computers, data storage modules, data communications modules (e.g., Bluetooth, WiFi, intranet, or internet communications hardware and associated software), display modules, one or more local and / or cloud-based software packages (e.g., instrument / system control software packages, image processing software packages, data analysis software packages), etc., or any combination thereof.

[0231] Translation Stage: In some implementations of the imaging and analysis systems (e.g., nucleic acid sequencing systems) disclosed herein, the systems may include one or more (e.g., 1, 2, 3, 4, or more than four) high-precision XY (or, in some cases, XYZ) translation stages for repositioning one or more sample support structures (e.g., flow cells) relative to one or more imaging modules, e.g., to tile one or more images, each corresponding to a field of view of an imaging module, to reconstruct a composite image of the entire flow cell surface. In some implementations of the imaging and genomics analysis systems (e.g., nucleic acid sequencing systems) disclosed herein, the systems may include one or more (e.g., 1, 2, 3, 4, or more than four) high-precision XY (or, in some cases, XYZ) translation stages for repositioning one or more imaging modules relative to one or more sample support structures (e.g., flow cells), e.g., to tile one or more images, each corresponding to a field of view of an imaging module, to reconstruct a composite image of the entire flow cell surface.

[0232] Suitable translation stages are commercially available from a variety of vendors, e.g., Parker Hannifin. Precision translation stage systems typically include a combination of several components, including, but not limited to, linear actuators, optical encoders, servo and / or step motors, and motor controllers or drive units. High precision and high reproducibility of stage movement are required to ensure accurate and reproducible placement and imaging of, for example, fluorescent signals, when interspersed with the iterative steps of reagent delivery and light detection.

[0233] Accordingly, the systems disclosed herein may include specifying the precision with which the translation stage is configured to position the sample support structure relative to the illumination and / or imaging optics (or vice versa). In one embodiment of the present disclosure, the precision of one or more translation stages is between about 0.1 μm and about 10 μm. In other embodiments, the precision of the translation stage is about 10 μm or less, about 9 μm or less, about 8 μm or less, about 7 μm or less, about 6 μm or less, about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, about 1 μm or less, about 0.9 μm or less, about 0.8 μm or less, about 0.7 μm or less, about 0.6 μm or less, about 0.5 μm or less, about 0.4 μm or less, about 0.3 μm or less, about 0.2 μm or less, or about 0.1 μm or less. In some examples, one skilled in the art will understand that the alignment of the translation stage may lie within any range bounded by either two of these values ​​(e.g., about 0.5 μm to about 1.5 μm). In some examples, the alignment accuracy of the translation stage may have any value within the range of values ​​included in this paragraph, for example, about 0.12 μm.

[0234] Flow Cells, Microfluidic Devices, and Cartridges: The flow cell devices and flow cell cartridges disclosed herein may be used as components of systems designed for various chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. Generally, such systems may include one or more of the disclosed single capillary flow cell devices, multiple capillary flow cell devices, capillary flow cell cartridges, and / or microfluidic devices and cartridges described herein. Additional description of the disclosed flow cell devices and cartridges can be found in PCT Patent Application Publication No. WO 2020 / 118255, which is incorporated herein by reference in its entirety.

[0235] In some examples, the systems disclosed herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more single capillary flow cell devices, multiple capillary flow cell devices, capillary flow cell cartridges, and / or microfluidic devices and cartridges. In some examples, the single capillary flow cell devices, multiple capillary flow cell devices, and / or microfluidic devices and cartridges may be fixed components of the disclosed systems. In some examples, the single capillary flow cell devices, multiple capillary flow cell devices, and / or microfluidic devices and cartridges may be removable and replaceable components of the disclosed systems. In some examples, the single capillary flow cell devices, multiple capillary flow cell devices, and / or microfluidic devices and cartridges may be disposable and consumable components of the disclosed systems.

[0236] In some implementations, the disclosed single capillary flow cell devices (or single capillary flow cell cartridges) include a single capillary, e.g., a glass or fused silica capillary, whose lumen forms a fluid flow path through which reagents or solutions may flow and whose interior surface forms a sample support structure to which a sample of interest is attached or tethered. In some implementations, the disclosed multiple capillary flow cell devices (or multiple capillary flow cell cartridges) may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20 or more capillaries configured to perform analytical techniques further including imaging as a detection method.

[0237] In some examples, one or more capillaries may be packaged within a chassis to form a cartridge that facilitates easy handling, incorporates adapters or connectors for creating external fluid connections, and optionally includes additional integrated features such as reagent reservoirs, waste reservoirs, valves (e.g., microvalves), pumps (e.g., micropumps), etc., or any combination thereof.

[0238] 29 shows a non-limiting example of a single capillary flow cell that includes two fluidic adapters, one attached to each end of a piece of glass capillary, designed to mate with standard OD fluidic tubing to provide convenient, interchangeable fluidic connections to external fluid flow control systems. The fluidic adapters can be attached to the capillaries using any of a variety of techniques known to those skilled in the art, including, but not limited to, press-fitting, adhesive bonding, solvent bonding, laser welding, etc., or any combination thereof.

[0239] Generally, capillaries used in the disclosed capillary flow cell devices and capillary flow cell cartridges have at least one internal, axially aligned fluid flow channel (or "lumen") running the entire length of the capillary. In some examples, a capillary may have two, three, four, five, or more than five internal, axially aligned fluid flow channels (or "lumens").

[0240] Numerous specified cross-sectional shapes of suitable capillaries (or lumens) are consistent with the present disclosure, including, but not limited to, circular, oval, square, rectangular, triangular, rounded square, rounded rectangular, or rounded triangular cross-sectional shapes. In some examples, the capillary (or its lumen) may have any specified cross-sectional dimension or set of dimensions. For example, in some examples, the largest cross-sectional dimension of the capillary lumen (e.g., the diameter if the lumen is circular in shape, or the diagonal if the lumen is square or rectangular in shape) may range from about 10 μm to about 10 mm. In some embodiments, the largest cross-sectional dimension of the capillary lumen may be at least 10 μm, at least 25 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, or at least 10 mm. In some embodiments, the largest cross-sectional dimension of the capillary lumen may be at most 10 mm, at most 9 mm, at most 8 mm, at most 7 mm, at most 6 mm, at most 5 mm, at most 4 mm, at most 3 mm, at most 2 mm, at most 1 mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 75 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any of the lower and higher values ​​recited within this paragraph may be combined to form ranges included within the present disclosure; for example, in some examples, the largest cross-sectional dimension of the capillary lumen may range from about 100 μm to about 500 μm. Those skilled in the art will recognize that the maximum cross-sectional dimension of the capillary lumen may have any value within this range, for example, about 124 μm.

[0241] For example, in some instances where the lumen of one or more capillaries of a flow cell device or cartridge has a square or rectangular cross-section, the distance between a first interior surface (e.g., upper surface or top surface) and a second interior surface (e.g., lower surface or bottom surface), which defines the height or thickness of the gap of the fluid flow channel, may range from about 10 μm to about 500 μm. In some examples, the gap height may be at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, at least 100 μm, at least 125 μm, at least 150 μm, at least 175 μm, at least 200 μm, at least 225 μm, at least 250 μm, at least 275 μm, at least 300 μm, at least 325 μm, at least 350 μm, at least 375 μm, at least 400 μm, at least 425 μm, at least 450 μm, at least 475 μm, or at least 500 μm. In some examples, the gap height may be at most 500 μm, at most 475 μm, at most 450 μm, at most 425 μm, at most 400 μm, at most 375 μm, at most 350 μm, at most 325 μm, at most 300 μm, at most 275 μm, at most 250 μm, at most 225 μm, at most 200 μm, at most 175 μm, at most 150 μm, at most 125 μm, at most 100 μm, at most 90 μm, at most 80 μm, at most 70 μm, at most 60 μm, at most 50 μm, at most 40 μm, at most 30 μm, at most 20 μm, or at most 10 μm. Any of the lower and higher values ​​recited in this paragraph may be combined to form a range included within the present disclosure, e.g., in some examples, the gap height may range from about 40 μm to about 125 μm. One of ordinary skill in the art will recognize that the gap height may have any value within the range of values ​​in this paragraph, e.g., about 122 μm.

[0242] In some examples, the length of one or more capillaries used to fabricate the disclosed capillary flow cell devices or flow cell cartridges may range from about 5 mm to about 5 cm or more. In some examples, the length of one or more capillaries may be less than 5 mm, at least 5 mm, at least 1 cm, at least 1.5 cm, at least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least 4.5 cm, or at least 5 cm. In some examples, the length of one or more capillaries may be up to 5 cm, up to 4.5 cm, up to 4 cm, up to 3.5 cm, up to 3 cm, up to 2.5 cm, up to 2 cm, up to 1.5 cm, up to 1 cm, or up to 5 mm. Any of the lower and higher values ​​recited within this paragraph may be combined to form ranges encompassed within the present disclosure; for example, in some examples, the length of one or more capillaries may range from about 1.5 cm to about 2.5 cm. Those skilled in the art will recognize that the length of one or more capillaries may have any value within this range, for example, about 1.85 cm. In some examples, a device or cartridge may include a plurality of two or more capillaries that are the same length. In some examples, a device or cartridge may include a plurality of two or more capillaries that are different lengths.

[0243] The capillaries used to construct the capillary flow cell device or capillary flow cell cartridge of the present disclosure may be fabricated from any of a variety of materials known to those skilled in the art, including, but not limited to, glass (e.g., borosilicate glass, soda-lime glass, etc.), fused silica (quartz), polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), or, as a more chemically inert alternative, polyetherimide (PEI) and perfluoroelastomer (FFKM), or any combination thereof. PEI is intermediate between polycarbonate and PEEK in terms of cost and chemical compatibility. FFKM is also known as Kalrez.

[0244] One or more materials used to fabricate the capillary are often optically transparent to facilitate use with spectroscopy or imaging-based detection techniques. In some instances, the entire capillary may be optically transparent. Alternatively, in some instances, only a portion of the capillary (e.g., an optically transparent "window") is optically transparent.

[0245] The capillaries used to construct the capillary flow cell devices and capillary flow cell cartridges of the present disclosure can be fabricated using any of a variety of techniques known to those of skill in the art, with the choice of fabrication technique often dependent on the choice of material used, and vice versa. Examples of suitable capillary fabrication techniques include, but are not limited to, extrusion, drawing, precision computer numerical control (CNC) machining and boring, laser photoablation, etc.

[0246] In some implementations, the capillaries used in the capillary flow cell devices and cartridges of the present disclosure can be off-the-shelf commercial products. Examples of commercial suppliers of precision capillary tubing include Accu-Glass (St. Louis, Missouri, precision glass capillary tubing), Polymicro Technologies (Phoenix, Arizona, precision glass and fused silica capillary tubing), Friedrich & Dimmock, Inc. (Millville, New Jersey, custom precision glass capillary tubing), and Drummond Scientific (Broomall, Pennsylvania, OEM glass and plastic capillary tubing).

[0247] Fluidic adapters associated with capillaries of the capillary flow cell devices and cartridges disclosed herein, as well as other components of the capillary flow cell devices or cartridges, may be fabricated using any of a variety of suitable techniques (e.g., extrusion, injection molding, compression molding, precision CNC machining, etc.) and materials (e.g., glass, fused silica, ceramic, metal, polydimethylsiloxane, polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), etc.), where again, the choice of fabrication technique often depends on the choice of materials used, and vice versa.

[0248] Figure 30 provides a non-limiting example of a capillary flow cell cartridge including two glass capillaries, fluidics adapters (in this example, two per capillary), and a cartridge chassis that mates with the capillaries and / or fluidics adapters so that the capillaries are held in a fixed orientation relative to the cartridge. In some examples, the fluidics adapters may be integrated into the cartridge chassis. In some examples, the cartridge may include additional adapters that mate with the capillaries and / or capillary fluidics adapters. As noted elsewhere herein, in some examples, the cartridge may include additional functional components. In some examples, the capillaries are permanently mounted to the cartridge. In some examples, the cartridge chassis is designed to allow one or more capillaries of the flow cell cartridge to be interchangeably removed and replaced. For example, in some examples, the cartridge chassis may include a hinged "clamshell" configuration that allows it to be opened so that one or more capillaries can be removed and replaced. In some examples, the cartridge chassis is configured to be mounted, for example, on the stage of a fluorescence microscope, or in a cartridge holder of a fluorescence imaging module, or within an instrument system of the present disclosure.

[0249] In some examples, the disclosed flow cell devices may include microfluidic devices (or "microfluidic chips") and cartridges, where the microfluidic devices are fabricated by forming fluidic channels in one or more layers of suitable material and include one or more fluidic channels (e.g., "analysis" channels) configured to perform analytical techniques that further include imaging as a detection method. In some implementations, the microfluidic devices or cartridges disclosed herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 fluidic channels (e.g., "analysis" fluidic channels) configured to perform analytical techniques that further include imaging as a detection method. In some examples, the disclosed microfluidic devices may further include additional fluidic channels (e.g., for diluting or mixing reagents), reagent reservoirs, waste reservoirs, adapters for external fluidic connections, etc., to provide "lab-on-a-chip" functionality integrated within the device.

[0250] Non-limiting examples of microfluidic flow cell cartridges include a chip including two or more parallel glass channels formed thereon, a fluidic adapter coupled to the chip, and a cartridge chassis that mates with the chip and / or fluidic adapter such that the chip is positioned in a fixed orientation relative to the cartridge. In some examples, the fluidic adapter may be integrated into the cartridge chassis. In some examples, the cartridge may include an additional adapter that mates with the chip and / or fluidics adapter. In some examples, the chip is permanently mounted to the cartridge. In some examples, the cartridge chassis is designed to allow one or more chips in the flow cell cartridge to be removed and replaced interchangeably. For example, in some examples, the cartridge chassis may include a hinged "clamshell" configuration that allows it to be opened so that one or more chips can be removed and replaced. In some examples, the cartridge chassis is configured to be mounted, for example, on the stage of a microscope system or within a cartridge holder of an imaging system. While only one chip is described in the non-limiting examples, it is understood that more than one chip may be used in a microfluidic flow cell cartridge. The flow cell cartridges of the present disclosure can contain a single microfluidic chip or multiple microfluidic chips. In some examples, a flow cell cartridge can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 microfluidic chips. Packaging one or more microfluidic devices within a cartridge can facilitate easy handling and precise placement of the devices within an optical imaging system.

[0251] Fluid channels in the microfluidic devices and cartridges of the present disclosure can have a variety of cross-sectional geometries, including, but not limited to, circular, oval, square, rectangular, triangular, rounded square, rounded rectangular, or rounded triangular cross-sectional geometries. In some examples, a fluid channel can have any specific cross-sectional dimension or set of dimensions. For example, in some examples, the height (e.g., gap height), width, or maximum cross-sectional dimension (e.g., diagonal, if the fluid channel has a square, rounded square, rectangular, or rounded rectangular cross-section) of a fluid channel can range from about 10 μm to about 10 mm. In some embodiments, the height (e.g., gap height), width, or largest cross-sectional dimension of a fluid channel can be at least 10 μm, at least 25 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, or at least 10 mm. In some embodiments, the height (e.g., gap height), width, or largest cross-sectional dimension of a fluid channel can be at most 10 mm, at most 9 mm, at most 8 mm, at most 7 mm, at most 6 mm, at most 5 mm, at most 4 mm, at most 3 mm, at most 2 mm, at most 1 mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 75 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges within the present disclosure; for example, in some examples, the height (e.g., gap height), width, or largest cross-sectional dimension of a fluid channel may be in the range of about 20 μm to about 200 μm.Those skilled in the art will recognize that the height (eg, gap height), width, or largest cross-sectional dimension of the fluid channel can have any value within this range (eg, about 122 μm).

[0252] In some examples, the length of a fluid channel in a microfluidic device or cartridge of the present disclosure can range from about 5 mm to about 10 cm, or more. In some examples, the length of a fluid channel can be less than 5 mm, at least 5 mm, at least 1 cm, at least 1.5 cm, at least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least 4.5 cm, at least 5 cm, at least 6 cm, at least 7 cm, at least 8 cm, at least 9 cm, or at least 10 cm. In some examples, the length of a fluid channel can be up to 10 cm, up to 9 cm, up to 8 cm, up to 7 cm, up to 6 cm, up to 5 cm, up to 4.5 cm, up to 4 cm, up to 3.5 cm, up to 3 cm, up to 2.5 cm, up to 2 cm, up to 1.5 cm, up to 1 cm, or at least 5 mm. Any of the lower and upper limits described in this paragraph may be combined to form ranges within the present disclosure; for example, in some examples, the length of a fluid channel may range from about 1.5 cm to about 2.5 cm. One of ordinary skill in the art will recognize that the length of a fluid channel may have any value within this range, such as about 1.35 cm. In some examples, a microfluidic device or cartridge may include multiple fluid channels of the same length. In some examples, a microfluidic device or cartridge may include multiple fluid channels of different lengths.

[0253] The disclosed microfluidic devices can include at least one layer of material having one or more fluid channels formed therein. In some examples, a microfluidic chip can include two layers bonded together to form one or more fluid channels. In some examples, a microfluidic chip can include three layers bonded together to form one or more fluid channels. In some examples, a microfluidic fluid channel can have an open top. In some examples, a microfluidic fluid channel can be fabricated in one layer, for example, on the top surface of a lower layer, and sealed by bonding the top surface of the lower layer to the bottom surface of an upper layer of material. In some examples, a microfluidic channel can be fabricated in one layer, for example, as a patterned channel whose depth extends the entire thickness of the layer, and then sandwiched and bonded between two unpatterned layers to seal the fluid channel. In some examples, a microfluidic channel is fabricated by removing a sacrificial layer on the surface of a substrate. This method does not require a large amount of substrate (e.g., glass or silicon wafer) to be removed. Instead, the fluid channel is located on the surface of the substrate. In some examples, microfluidic channels may be fabricated in or on the surface of a substrate and then sealed by depositing a conformal film or layer on the surface of the substrate to create subsurface or buried channels in the chip.

[0254] Microfluidic chips can be fabricated using a combination of microfabrication processes. In order for devices to be microfabricated, substrate materials will typically be selected based on their compatibility with known microfabrication techniques, including photolithography, wet chemical etching, laser ablation, laser irradiation, air ablation techniques, injection molding, embossing, and other techniques. Substrate materials are also generally chosen for their compatibility with all conditions to which the microfluidic device may be exposed, including extremes of pH, temperature, salt concentration, and the application of electromagnetic (e.g., light) or electric fields.

[0255] The microfluidic chips of the present disclosure may be fabricated from any of a variety of materials known to those skilled in the art, including, but not limited to, glass (e.g., borosilicate glass, soda-lime glass, etc.), fused silica (quartz), silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI), and perfluoroelastomer (FFKM) (as a more chemically inert alternative), or any combination thereof. In some preferred examples, the substrate material may include silica-based substrates such as borosilicate glass and quartz, as well as other substrate materials.

[0256] The disclosed microfluidic devices can be fabricated using any of a variety of techniques known to those skilled in the art, with the choice of fabrication technique often depending on the choice of material used, and vice versa. Microfluidic channels on chips can be constructed using techniques suitable for forming micropatterns or microstructures on the surface of a substrate. In some embodiments, the fluidic channels are formed by laser irradiation. In some embodiments, the microfluidic channels are formed by focused femtosecond laser irradiation. In some examples, the microfluidic channels are formed by photolithography and etching, including, but not limited to, chemical etching, plasma etching, or deep reactive ion etching. In some examples, the microfluidic channels are formed using laser etching. In some examples, the microfluidic channels are formed using direct-write lithography techniques. Examples of direct-write lithography include electron beam direct writing and focused ion beam milling.

[0257] In additional preferred examples, the substrate material may comprise a plastic, such as a polymeric material, for example, polymethyl methacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and the like. Such polymeric substrates can be easily patterned or microfabricated using available microfabrication techniques, such as those described above. In some examples, microfluidic chips can be fabricated from polymeric materials, for example, from microfabricated masters, using well-known molding techniques, such as injection molding, embossing, stamping, or polymerization of polymer precursor materials in a mold (see, e.g., U.S. Pat. No. 5,512,131). In some examples, such polymeric substrate materials are preferred due to their ease of manufacture, low cost, and disposability, as well as their general inertness to most extreme reaction conditions. Like flow cell devices made from other materials, e.g., glass, flow cell devices made from these polymeric materials may include engineered surfaces, e.g., derivatized or coated surfaces, to enhance their usefulness in microfluidic systems, as described in more detail below.

[0258] The fluid channels and / or fluid chambers of a microfluidic device are typically fabricated as microscale channels (e.g., trenches, depressions, etc.) in the top surface of a first substrate using the microfabrication techniques described above. The first substrate includes a top surface having a first planar surface and a bottom surface. In microfluidic devices prepared by the methods described herein, a plurality of fluid channels (e.g., trenches and / or depressions) are formed on the first planar surface. In some examples, the fluid channels (e.g., trenches and / or depressions) formed in the first planar surface (prior to bonding to a second substrate) have bottom and side walls, while the top surface remains open. In some examples, the fluid channels (e.g., trenches and / or depressions) formed in the first planar surface (prior to bonding to a second substrate) have bottom and side walls, while the top surface remains closed. In some examples, the fluid channels (e.g., grooves and / or depressions) formed in the first planar surface (prior to bonding to the second substrate) have only side walls and no top or bottom surfaces (i.e., the fluid channels span the entire thickness of the first substrate).

[0259] Fluidic channels and chambers can be sealed by placing a first planar surface of a first substrate in contact with and bonding to a planar surface of a second substrate, forming channels and / or chambers (e.g., interior portions) of the device at the interface of the two components. In some examples, after the first substrate is bonded to the second substrate, the structure can be further placed in contact with and bonded to a third substrate. In some examples, the third substrate can be placed in contact with the side of the first substrate that is not in contact with the second substrate. In some examples, the first substrate is placed between the second and third substrates. In some examples, the second and third substrates can cover and / or seal grooves and depressions or apertures formed in the first substrate, forming channels and / or chambers (e.g., interior portions) of the device at the interface of these components.

[0260] The device may have an opening oriented to be in fluid communication with at least one of the fluid channels and / or fluid chambers formed in an interior portion of the device, thereby forming a fluid inlet and / or a fluid outlet. In some examples, the opening is formed on the first substrate. In some examples, the opening is formed on the first substrate and the second substrate. In some examples, the opening is formed on the first, second, and third substrates. In some examples, the opening is disposed on the top side of the device. In some examples, the opening is disposed on the bottom side of the device. In some examples, the opening is disposed at the first and / or second end of the device, and the channel extends along a direction from the first end to the second end.

[0261] The conditions under which substrates can be bonded together are generally well understood by those skilled in the art, and such bonding of substrates can generally be performed by any of a variety of methods, with the choice of bonding method varying depending on the nature of the substrate materials used. For example, thermal bonding of substrates may be applied to many substrate materials, including glass, silica-based substrates, and some polymer-based substrates. Such thermal bonding techniques typically involve mating the surfaces of the substrates to be bonded under conditions of elevated temperature, and possibly the application of external pressure. The exact temperature and pressure utilized will generally vary depending on the nature of the substrate materials used.

[0262] For example, for silica-based substrate materials, i.e., glass (borosilicate glass, Pyrex™, soda-lime glass, etc.), fused silica (quartz), etc., thermal bonding of the substrates is typically carried out at temperatures ranging from about 500°C to about 1400°C, and preferably from about 500°C to about 1200°C. For example, soda-lime glass is typically bonded at a temperature of about 550°C, while borosilicate glass is typically thermally bonded at or near 800°C. On the other hand, quartz substrates are typically thermally bonded at or near 1200°C. These bonding temperatures are typically achieved by placing the substrates to be bonded in the high temperature of an annealing oven.

[0263] On the other hand, thermally bonded polymer substrates will typically utilize lower temperatures and / or pressures than silica-based substrates to prevent excessive melting of the substrates and / or distortion, e.g., flattening of the interior portions of the device (i.e., fluidic channels or chambers). Typically, such elevated temperatures for bonding polymer substrates can vary from about 80°C to about 200°C, and preferably between about 90°C and about 150°C, depending on the polymer material used. Because the temperatures required for bonding polymer substrates are significantly reduced, such bonding can be performed without the need for the high-temperature ovens typically used for bonding silica-based substrates. This allows for the incorporation of a heat source into a single, integrated bonding system, as described in more detail below.

[0264] Adhesives may be used to bond substrates together by known methods, which typically involve applying a layer of adhesive between the substrates to be bonded and pressing them together until the adhesive sets. A variety of adhesives may be used according to these methods, including, for example, commercially available UV-curable adhesives. Alternative methods may be used to bond substrates together according to the present invention, including, for example, sonic or ultrasonic welding of polymer parts and / or solvent welding.

[0265] Typically, many of the described microfluidic chips or devices will be fabricated simultaneously, for example, using "wafer-scale" fabrication. For example, polymer substrates can be stamped or molded in large, separable sheets that are then laminated and bonded together. Individual devices or bonded substrates can then be separated from the larger sheet by cutting or dicing. Similarly, for silica-based substrates, individual devices can be fabricated from a larger substrate wafer or plate, allowing for higher throughput in the manufacturing process. Specifically, multiple fluid channel structures are fabricated on a first substrate wafer or plate, which is then covered and bonded to a second substrate wafer or plate, and, optionally, further covered and bonded to a third substrate wafer or plate. Individual devices are then separated from the larger substrate using known methods such as sawing, scribing, breaking, etc.

[0266] As noted above, the top or second substrate overlies the bottom or first substrate to seal the various channels and chambers. When performing the bonding process according to the disclosed method, bonding of the first and second substrates may be performed using vacuum and / or pressure to maintain optimal contact between the two substrate surfaces. In particular, the bottom substrate can be maintained in optimal contact with the top substrate, for example, by laminating the flat surface of the bottom substrate with the flat surface of the top substrate and applying a vacuum through holes disposed through the top substrate. Typically, applying a vacuum to the holes in the top substrate is performed by placing the top substrate on a vacuum chuck, which typically includes a mounting platform or surface with an integrated vacuum source. In the case of silica-based substrates, the substrates to be bonded are exposed to an elevated temperature to create an initial bond, so that the bonded substrates can be moved to an annealing oven without shifting relative to each other.

[0267] Alternative bonding systems for incorporation into the devices described herein include, for example, adhesive application systems for applying an adhesive layer between two planar substrates. This may be done by applying the adhesive layer before laminating the substrates together, or by placing a quantity of adhesive on one edge of an adjacent substrate, allowing the wicking action of the two laminated substrates to pull the adhesive across the space between the two substrates.

[0268] In some examples, the overall bonding system can include an automatable system for placing the top and bottom substrates on the mounting surface and aligning them for subsequent bonding. Typically, such a system includes a translation system for moving either the mounting surface or one or more of the top and bottom substrates relative to one another. For example, a robotic system may be used to lift, translate, and place each of the top and bottom substrates, in turn, on the mounting table and within the alignment structure. Following the bonding process, such a system may also remove the finished product from the mounting surface and transport the bonded substrates to subsequent operations, such as a separating or dicing operation, or an oven to anneal the silica-based substrate before placing an additional substrate on top for bonding.

[0269] In some examples, the fabrication of microfluidic chips involves layering or laminating two or more layers of substrates, e.g., patterned and unpatterned polymer sheets, to produce a chip. For example, in a microfluidic device, the microfluidic features of the device are typically created by laser irradiation, etching, or other fabrication features on the surface of the first layer. A second layer is then laminated or bonded to the surface of the first layer to seal these features and provide the fluidic elements of the device, e.g., the fluidic channels.

[0270] As described above, in some examples, one or more capillary flow cell devices or microfluidic chips can be mounted on a cartridge chassis to form a capillary flow cell cartridge or microfluidic cartridge. In some examples, the capillary flow cell cartridge or microfluidic cartridge can further include additional components integrated with the cartridge to provide enhanced performance for specific applications. Examples of additional components that can be integrated into the cartridge include, but are not limited to, adapters or connectors for fluidic connection with other components of the system, fluid flow control components (e.g., miniature valves, miniature pumps, mixing manifolds, etc.), temperature control components (e.g., resistive heating elements, metal plates serving as heat sources or heat sinks, piezoelectric elements (Peltier elements) for heating or cooling, temperature sensors), or optical components (e.g., optical lenses, windows, filters, mirrors, prisms, optical fibers and / or light-emitting diodes (LEDs), or other miniature light sources that can be collectively used to facilitate spectroscopic measurements and / or imaging of one or more capillaries or fluid flow channels).

[0271] Fluidic adapters, cartridge chassis, and other cartridge components may be attached to capillaries, capillary flow cell device(s), microfluidic chip(s) (or fluidic channels within the chip) using a variety of techniques known to those skilled in the art, including, but not limited to, press-fitting, adhesive bonding, solvent bonding, laser welding, etc., or any combination thereof. The inlets and / or outlets of the microfluidic channels within the microfluidic chip are apertures on the top surface of the chip, and the fluidic adapters may be attached or coupled to the inlets and / or outlets of the microfluidic channels within the chip. In some examples, the cartridge may include additional adapters (i.e., in addition to the fluidic adapters) that couple with the chip and / or fluidic adapters and aid in positioning the chip within the cartridge. These adapters may be constructed using similar fabrication techniques and materials as the aforementioned fluidic adapters.

[0272] The cartridge chassis (or "housing") may be made of metal and / or polymer materials, such as aluminum, anodized aluminum, polycarbonate (PC), acrylic (PMMA), or Ultem (PEI), although other materials are not inconsistent with the present disclosure. The housing may be fabricated using CNC machining and / or molding techniques and designed to constrain one or more capillaries or microfluidic chips in a fixed orientation by the chassis, thereby creating one or more independent flow paths. The capillaries or chips may be attached to the chassis, for example, by a compression-fit design or by mating with a compressible adapter made of silicone or fluoroelastomer. In some examples, two or more components of the cartridge chassis (e.g., top and bottom halves) are assembled using, for example, screws, clips, clamps, or other fasteners so that the two halves are separable. In some examples, two or more components of the cartridge chassis are assembled using, for example, adhesives, solvents, laser welding, etc., so that the two or more components are permanently attached.

[0273] Flow Cell Surface Coatings: In some examples, one or more interior surfaces of the capillary lumen or microfluidic channel of a flow cell device of the present disclosure may be coated using any of a variety of surface modification techniques or polymer coatings known to those of skill in the art. In some examples, the coating may be formulated to increase or maximize the number of available binding sites (e.g., adapter / primer sequences of tethered oligonucleotides) on one or more interior surfaces to increase or maximize foreground signals, such as fluorescent signals generated from labeled nucleic acid molecules hybridized to the oligonucleotide adapter / primer sequences. In some examples, the coating may be formulated to reduce or minimize nonspecific binding of fluorophores and other small molecules, or labeled or unlabeled nucleotides, proteins, enzymes, antibodies, oligonucleotides, or nucleic acid molecules (e.g., DNA, RNA, etc.), to reduce or minimize background signals, such as nonspecific binding of labeled biomolecules or autofluorescence of sample support structures. The combination of increased foreground signal and decreased background signal that may be achieved in some instances by using the coatings of the present disclosure may thus result in improved SNR (signal-to-noise ratio) in spectroscopic measurements and improved CNR (contrast-to-noise ratio) in imaging methods.

[0274] As will be explained in more detail below, the disclosed hydrophilic polymer-coated flow cell devices, optionally used in combination with improved hybridization and / or amplification protocols, result in solid-phase bioassay reactions that: (i) exhibit negligible nonspecific binding of proteins and other reaction components (thus reducing or minimizing substrate background), (ii) exhibit negligible nonspecific nucleic acid amplification products, and (iii) provide tunable nucleic acid amplification reactions. While this specification primarily describes nucleic acid hybridization, amplification, and sequencing assays, it will be understood by those skilled in the art that the low-binding supports of the present disclosure can be used in a variety of bioassay formats, including, but not limited to, sandwich immunoassays, enzyme-linked immunosorbent assays (ELISAs), and the like.

[0275] In preferred embodiments, one or more layers of coating material may be applied to the interior surface of the flow cell device, where the number of layers and / or the material composition of each layer are selected to adjust one or more surface properties of the interior surface of the flow cell device, as described in U.S. Patent Application No. 16 / 363,842. Examples of surface properties that may be adjusted include, but are not limited to, the hydrophilicity / hydrophobicity of the surface, the overall coating thickness, the surface density of chemically reactive functional groups, the surface density of grafted linker molecules or oligonucleotide adapters / primers, etc. In some preferred applications, one or more surface properties of the capillary or channel lumen are adjusted to, for example, (i) significantly reduce nonspecific binding of proteins, oligonucleotides, fluorophores, and other molecular components in chemical or biological analytical applications, including solid-phase nucleic acid amplification and / or sequencing applications; (ii) improve the specificity and efficiency of solid-phase nucleic acid hybridization; or (iii) improve the speed, specificity, and efficiency of solid-phase nucleic acid amplification.

[0276] Any of a variety of molecules known to those skilled in the art, including, but not limited to, silanes, amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof, may be used to create one or more chemically modified layers on the interior surface of a flow cell device, where the selection of components used can be varied to alter one or more properties of the support surface, such as, for example, the surface density of functional groups and / or tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the support surface, or the three-dimensional nature (i.e., "thickness") of the support surface.

[0277] The attachment chemistry used to graft the chemically modified first layer onto the interior surface of a flow cell (capillary or channel) generally depends on both the material from which the flow cell device is fabricated and the chemical nature of the layer. In some instances, the first layer may be covalently attached to the interior surface of the flow cell device. In some instances, the first layer may be non-covalently attached, e.g., adsorbed to the surface through non-covalent bonds such as electrostatic interactions, hydrogen bonding, or van der Waals interactions between the surface and molecular components of the first layer. In either case, the substrate surface may be treated prior to attachment or deposition of the first layer. Any of a variety of surface treatment techniques known to those skilled in the art may be used to clean or treat the support surface. For example, glass or silicon surfaces may be acid-cleaned using Piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)) and / or cleaned using oxygen plasma treatment methods.

[0278] Silane chemistry is one non-limiting approach to covalently modifying silanol groups on glass or silicon surfaces and attaching more reactive functional groups (e.g., amine or carboxyl groups), which may be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths, such as C6, C12, or C18 hydrocarbons, or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to create any of the disclosed low-binding support surfaces include, but are not limited to, (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), any of the various PEG silanes (e.g., including molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silane (i.e., containing a free amino functional group), maleimide-PEG silane, biotin-PEG silane, and the like.

[0279] Examples of preferred polymers that can be used to create one or more layers of low nonspecific binding material on any of t...

Claims

1. 1. A method for sequencing a nucleic acid molecule, said method comprising: a) imaging the first surface and the axially displaced second surface using an uncorrected optical system including an objective lens and at least one image sensor, the optical system having a numerical aperture (NA) of less than 0.6 and a resolution of 1.0 mm; ２ and having a field of view (FOV) greater than the optical system includes at least one tube lens positioned between the objective lens and the at least one image sensor, the at least one tube lens configured to modify an imaging performance metric for imaging a first interior surface of a flow cell and a second interior surface of a flow cell, the at least one tube lens including, in order, an asymmetric convex-convex lens, a convex-plano lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens; b) processing the images of the first surface and the axially displaced second surface to correct for optical aberrations so that the images of the first surface and the axially displaced second surface have substantially the same optical resolution; c) detecting a fluorescently labeled composition comprising the nucleic acid molecule disposed on the first surface or the axially displaced second surface, or its complement, to determine the identity of the nucleotides in the nucleic acid molecule.

2. 10. The method of claim 1, wherein the images of the first surface and the axially displaced second surface are acquired without moving an optical corrector into an optical path between the objective lens and the at least one image sensor.

3. The method of claim 1 , wherein the images of the first surface and the axially displaced second surface are acquired solely by refocusing the optical system.

4. The method of claim 1 , further comprising imaging two or more fields of view at at least one of the first surface or the axially displaced second surface.

5. The method of claim 1 , wherein the first surface and the axially displaced second surface comprise two surfaces of a flow cell.

6. The method of claim 5 , wherein the two surfaces of the flow cell are coated with a hydrophilic coating layer.

7. The hydrophilic coating layer is 1 mm ２ 7. The method of claim 6, further comprising labeling nucleic acid colonies disposed thereon at a surface density of greater than 10,000 nucleic acid colonies per well.

8. 8. The method of claim 7, wherein an image of one of the two surfaces acquired using the optical system exhibits a contrast-to-noise ratio (CNR) of at least 5 when nucleic acid colonies are labeled with cyanine dye 3 (Cy3), the optical system including a dichroic mirror and bandpass filter set optimized for Cy3 emission, and the image is acquired under non-signal saturating conditions while the surface is immersed in 25 mM ACES, pH 7.4 buffer.

9. 10. The method of claim 1, wherein the optical system comprises one, two, three, or four imaging channels configured to detect nucleic acid colonies disposed on at least one of a first surface and an axially displaced second surface labeled with one, two, three, or four different detectable labels.

10. The method of claim 1 , wherein the at least one image sensor includes pixels having a pixel size selected such that the spatial sampling frequency for the optical system is at least twice the optical resolution of the optical system.

11. 10. The method of claim 1, wherein the flow cell has a wall thickness of at least 700 μm and a gap of at least 50 μm between the first and second interior surfaces.

12. 10. The method of claim 1, wherein the optical system comprises two or more tube lenses designed to provide optimal imaging performance at two or more fluorescent wavelengths.

13. The method of claim 1 , wherein the objective lens and the tube lens combination are configured to optimize a modulation transfer function in the mid-to-high spatial frequency range.

14. 10. The method of claim 1, wherein the imaging performance metrics include a measure of modulation transfer function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast-to-noise ratio (CNR), or any combination thereof.

15. The method of claim 1 , wherein the optical resolution of the images of the first surface and the axially displaced second surface is diffraction limited over the entire field of view (FOV).

16. 2. The method of claim 1, wherein sequencing the nucleic acid molecule further comprises performing avidity sequencing, nucleotide binding sequencing, or nucleotide incorporation sequencing on at least one of the first surface and the axially displaced second surface, and detecting the bound or incorporated nucleotide bases.

17. 10. The method of claim 1, further comprising determining the genotype of the sample, wherein determining the genotype of the sample comprises preparing the nucleic acid molecule for sequencing and then sequencing the nucleic acid molecule.

18. 1. A system for sequencing a nucleic acid molecule, said system comprising: a) i) an objective lens and at least one image sensor; ii) an optical system including at least one tube lens positioned between the objective lens and the at least one image sensor, the at least one tube lens configured to modify an imaging performance metric for imaging a first interior surface of a flow cell and a second interior surface of a flow cell, the at least one tube lens including, in order, an asymmetric convex-convex lens, a convex-plano lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens, the optical system having a numerical aperture (NA) of less than 0.6 and a 3.0 mm aperture; ２ an optical system having a field of view (FOV) greater than b) a processor, said processor comprising: i) processing the images of the first surface and the axially displaced second surface to correct for optical aberrations so that the images of the first surface and the axially displaced second surface have substantially the same optical resolution; ii) a processor programmed to detect a fluorescently labeled composition comprising a nucleic acid molecule disposed on the first surface or the axially displaced second surface, or a complement thereof, to determine the identity of a nucleotide in the nucleic acid molecule.

19. 20. The system of claim 18, wherein images of the first surface and the axially displaced second surface are acquired without moving an optical corrector into an optical path between the objective lens and the at least one image sensor.

20. 20. The system of claim 18, wherein the images of the first surface and the axially displaced second surface are acquired solely by refocusing the optical system.

21. The system of claim 18, wherein the optical system has a numerical aperture greater than 0.

3.

22. 20. The system of claim 18, wherein the first surface and the axially displaced second surface comprise two surfaces of a flow cell.

23. The two surfaces of the flow cell are coated with a hydrophilic coating layer, and the hydrophilic coating layer has a thickness of 1 mm. ２ 23. The system of claim 22, further comprising labeled nucleic acid colonies disposed thereon at a surface density of greater than 10,000 nucleic acid colonies per surface.

24. 20. The system of claim 18, wherein the optical system comprises one, two, three, or four imaging channels configured to detect nucleic acid colonies disposed on at least one of a first surface or an axially displaced second surface labeled with one, two, three, or four different detectable labels.

25. 20. The system of claim 18, wherein the at least one image sensor includes pixels having a pixel size selected such that the spatial sampling frequency for the optical system is at least twice the optical resolution of the optical system.

26. 20. The system of claim 18, wherein the system includes at least one tube lens positioned between the objective lens and the at least one image sensor, the at least one tube lens configured to modify an imaging performance metric for imaging the first internal surface of the flow cell and the second internal surface of the flow cell.

27. 27. The system of claim 26, wherein the flow cell has a wall thickness of at least 700 μm and a gap of at least 50 μm between the first and second interior surfaces.

28. 27. The system of claim 26, wherein the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescent wavelengths.

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