High performance fluorescence imaging module for genomic testing assays

Abstract

Fluorescence imaging system designs are described that provide greater field of view, higher spatial resolution, improved modulation transfer 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., 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

[0001] This application is a divisional application of Chinese patent application No. 202180003305.6, filed on January 15, 2021, entitled "High-performance fluorescence imaging module for genome testing" (the corresponding PCT application was filed on January 15, 2021, and has the application number PCT / US2021 / 013696).

[0002] Cross-references

[0003] This application claims the benefits of U.S. Provisional Application No. 63 / 076,361, filed September 9, 2020, and U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020, which are incorporated herein by reference in their entirety. Background Technology

[0004] In typical fluorescence-based genomic assays, such as genotyping or nucleic acid sequencing (using real-time, cyclic, or step-reaction protocols), an excitation light source excites dye molecules linked to nucleic acid molecules tethered to a matrix, generating fluorescent photon signals at one or more spatially located sites on the matrix. The fluorescence is then imaged onto an image sensor via an optical system. Analytical methods are then used to analyze the image, locate the positions of labeled molecules (or clonal amplification clusters of molecules) on the matrix, and quantify the fluorescent photon signals according to wavelength and spatial coordinates. These signals can then be correlated with the extent of a specific chemical reaction (e.g., hybridization or base addition events) occurring at the specified locations on the matrix. Image-based methods offer massive parallelism and multiplexing capabilities, which helps reduce the cost and availability of such technologies. However, detection errors, such as over-density packing of labeled molecules (or clonal amplification clusters of molecules) in small regions on the matrix surface or due to low contrast-to-noise ratio (CNR) in the image, can lead to errors in attributing the fluorescent signal to the correct molecule (or clonal amplification cluster of molecules). Summary of the Invention

[0005] This document discloses an imaging system configured to image a first inner surface and a second inner surface of a flow cell, the imaging system comprising: a) an objective lens; b) at least one image sensor; and c) at least one barrel lens disposed in the optical path between the objective lens and the at least one image sensor; wherein the optical system has a numerical aperture (NA) of less than 0.6 and a diameter greater than 1.0 mm. 2 The field of view (FOV); and wherein the at least one barrel lens is configured to correct imaging performance such that the images of the first inner surface of the flow cell and the second inner surface of the flow cell have substantially the same optical resolution.

[0006] In some embodiments, the flow cell wall thickness is at least 700 μm, and the fluid-filled gap between the first inner surface and the second inner surface is at least 50 μm. In some embodiments, images of the first and second inner surfaces are acquired without moving the optical compensator 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 numerical aperture (NA) of greater than 1.5 mm. 2 The field of view (FOV) is defined. In some embodiments, the optical resolution of the images of the first and second inner surfaces is diffraction-limited across the entire field of view (FOV). In some embodiments, at least one barrel lens sequentially comprises an asymmetric convex-convex lens, a convex-planar lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the imaging system includes two or more barrel lenses designed to provide optimal imaging performance for the first and second inner surfaces at two or more fluorescence wavelengths. In some embodiments, the imaging system further includes a focusing mechanism configured to refocus the optics between acquiring images of the first and second inner 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 inner surfaces. In some embodiments, the first and second inner surfaces of the flow cell are coated with a hydrophilic coating, and said hydrophilic coating further comprises >10,000 nucleic acid colonies / mm². 2The surface density is arranged with labeled nucleic acid colonies. In some embodiments, when the nucleic acid colonies are labeled with cyanine dye 3 (Cy3), images of the first or second inner surface acquired using an imaging system show a contrast-to-noise ratio (CNR) of at least 5. The imaging system includes a dichroic mirror and bandpass filter set optimized for Cy3 emission and acquires images under non-signal saturation conditions while immersing the surface in 25 mM ACES pH 7.4 buffer. In some embodiments, the imaging system includes 1, 2, 3, or 4 imaging channels configured to detect nucleic acid colonies arranged on at least one of the two different surfaces that have been labeled with 1, 2, 3, or 4 different detectable markers. In some embodiments, the imaging system is used to monitor and detect bound or incorporated nucleotide bases on at least one of the first and second inner surfaces by affinity sequencing, nucleotide base pairing sequencing, nucleotide binding sequencing, or nucleotide incorporation reaction sequencing. 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, wherein 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, at least one image sensor includes pixels, the pixel size of which is selected such that the spatial sampling frequency of the imaging system is at least twice the optical resolution of the imaging system. In some embodiments, the combination of the objective lens and at least one tube lens is configured to optimize the modulation transfer function in the sample plane within a spatial frequency range of 700 cycles / mm to 1100 cycles / mm. In some embodiments, for the combination of the objective lens and at least one tube lens, at least one tube lens is designed to correct the modulation transfer function (MTF) in terms of 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.

[0007] This paper also discloses a method for nucleic acid molecular sequencing, the method comprising: a) imaging a first surface and an axially displaced second surface using an optical system, the optical system comprising an objective lens and at least one image sensor, wherein the optical system has a numerical aperture (NA) of less than 0.6 and a diameter greater than 1.0 mm. 2 The field of view (FOV) is obtained, and images of the first surface and the axially shifted second surface with substantially the same optical resolution are acquired without moving the optical compensator into the optical path between the objective lens and the at least one image sensor; b) a fluorescently labeled composition containing nucleic acid molecules or their complementary sequences disposed on the first surface or the axially shifted second surface is detected to determine the identity of nucleotides in the nucleic acid molecules.

[0008] In some embodiments, a focusing mechanism is used 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 on 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. In some embodiments, the hydrophilic coating further comprises >10,000 nucleic acid colonies / mm². 2The surface density is arranged on a labeled nucleic acid colony. In some embodiments, when the nucleic acid colony is labeled with cyanine dye 3 (Cy3), surface images of the two surfaces acquired using the optical system show a contrast-to-noise ratio (CNR) of at least 5. The optical system includes a dichroic mirror and a bandpass filter assembly optimized for Cy3 emission, and acquires images under non-signal saturation conditions while immersing the surface in 25 mM ACES pH 7.4 buffer. In some embodiments, the optical system includes 1, 2, 3, or 4 imaging channels configured to detect nucleic acid colonies labeled with 1, 2, 3, or 4 different detectable markers arranged on at least one of the first surface and the axially displaced second surface. In some embodiments, at least one image sensor includes pixels whose pixel size is selected such that the spatial sampling frequency of the optical system is at least twice the optical resolution of the optical system. In some embodiments, the optical system includes at least one barrel lens positioned between an objective lens and at least one image sensor, wherein said at least one barrel lens is configured to correct imaging performance metrics for imaging a first inner surface and a second inner surface of the flow cell. In some embodiments, the flow cell wall thickness is at least 700 μm, and the gap between the first and second inner surfaces is at least 50 μm. In some embodiments, the at least one barrel lens sequentially comprises an asymmetric convex-convex lens, a convex-planar lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the optical system includes two or more barrel lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the combination of the objective lens and the barrel lens is configured to optimize the modulation transfer function over a medium to high spatial frequency range. In some embodiments, the imaging performance metrics include measurements 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 shifted second surface is diffraction-limited across the entire field of view (FOV). In some embodiments, sequencing of the nucleic acid molecule further includes performing sequencing on at least one of the first surface and the axially shifted second surface by affinity sequencing, sequencing by nucleotide base pairing, sequencing by nucleotide binding, or sequencing by nucleotide incorporation reaction, 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 includes preparing the nucleic acid molecule for sequencing and then sequencing the nucleic acid molecule.

[0009] This document discloses an imaging system configured to image two axially displaced surfaces. The imaging system includes an objective lens and at least one image sensor, wherein the imaging system has a numerical aperture (NA) of less than 0.6 and a focal length greater than 1.0 mm. 2 The field of view (FOV) is such that, without moving the optical compensator into the optical path between the objective lens and the at least one image sensor, the imaging system is able to acquire images of two different axially displaced surfaces with substantially the same optical resolution.

[0010] 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 for refocusing the optics between acquiring images of two distinct 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 distinct axially displaced surfaces. In some embodiments, the two distinct axially displaced surfaces comprise two surfaces of a flow cell. In some embodiments, the two distinct surfaces of the flow cell are coated with a hydrophilic coating, and said hydrophilic coating further comprises >10,000 nucleic acid colonies / mm². 2 The surface density of the surface is arranged with labeled nucleic acid colonies. In some embodiments, the imaging system includes 1, 2, 3, or 4 imaging channels configured to detect nucleic acid colonies labeled with 1, 2, 3, or 4 different detectable markers arranged on at least one of the two different surfaces. In some embodiments, at least one image sensor includes pixels whose pixel size is selected such that the spatial sampling frequency of the imaging system is at least twice the optical resolution of the imaging system. In some embodiments, the imaging system includes at least one tube lens positioned between the objective lens and at least one image sensor, and said at least one tube lens is configured to correct imaging performance metrics for imaging a first inner surface and a second inner surface of the flow cell. In some embodiments, the flow cell wall thickness is at least 700 μm, and the gap between the first inner surface and the second inner surface is at least 50 μm. In some embodiments, the imaging system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the optical resolution of the images of the two different axially displaced surfaces is diffraction-limited across the entire field of view (FOV).

[0011] This article discloses a method for nucleic acid molecular sequencing, the method comprising: a) imaging a first surface and an axially displaced second surface using an uncompensated optical system, the uncompensated optical system comprising an objective lens and at least one image sensor, wherein the optical system has a numerical aperture (NA) of less than 0.6 and a diameter greater than 1.0 mm.2 a) the field of view (FOV); b) processing images of the first surface and the axially shifted second surface to correct optical aberrations so that the images of the first surface and the axially shifted second surface have substantially the same optical resolution; and c) detecting fluorescently labeled compositions containing nucleic acid molecules or their complementary sequences disposed on the first surface or the axially shifted second surface to determine the identity of nucleotides in the nucleic acid molecules.

[0012] In some embodiments, images of the first surface and the axially displaced second surface are acquired without moving the optical compensator 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 by simply refocusing the optical system. In some embodiments, the method further includes imaging two or more fields of view on 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. In some embodiments, the hydrophilic coating further comprises >10,000 nucleic acid colonies / mm². 2The surface density is arranged on a labeled nucleic acid colony. In some embodiments, when the nucleic acid colony is labeled with cyanine dye 3 (Cy3), surface images of the two surfaces acquired using the optical system show a contrast-to-noise ratio (CNR) of at least 5. The optical system includes a dichroic mirror and a bandpass filter group optimized for Cy3 emission, and acquires images under non-signal saturation conditions while immersing the surface in 25 mM ACES pH 7.4 buffer. In some embodiments, the optical system includes 1, 2, 3, or 4 imaging channels configured to detect nucleic acid colonies labeled with 1, 2, 3, or 4 different detectable markers arranged on at least one of the first surface and the axially displaced second surface. In some embodiments, at least one image sensor includes pixels whose pixel size is selected such that the spatial sampling frequency of the optical system is at least twice the optical resolution of the optical system. In some embodiments, the optical system includes at least one barrel lens positioned between an objective lens and at least one image sensor, wherein said at least one barrel lens is configured to correct imaging performance metrics for imaging a first inner surface and a second inner surface of the flow cell. In some embodiments, the flow cell wall thickness is at least 700 μm, and the gap between the first and second inner surfaces is at least 50 μm. In some embodiments, the at least one barrel lens sequentially comprises an asymmetric convex-convex lens, a convex-planar lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the optical system includes two or more barrel lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the combination of the objective lens and the barrel lens is configured to optimize the modulation transfer function over a medium to high spatial frequency range. In some embodiments, the imaging performance metrics include measurements 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 shifted second surface is diffraction-limited across the entire field of view (FOV). In some embodiments, sequencing of the nucleic acid molecule further includes performing affinity sequencing, nucleotide binding sequencing, or nucleotide incorporation reaction sequencing on at least one of the first surface and the axially shifted 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 includes preparing the nucleic acid molecule for sequencing and then sequencing the nucleic acid molecule.

[0013] This document discloses a system for nucleic acid molecular sequencing, the system comprising: a) an optical system including an objective lens and at least one image sensor, wherein the optical system has a numerical aperture (NA) of less than 0.6 and a focal length greater than 1.0 mm. 2 The image is configured to: a) a field of view (FOV) of a first surface and an axially shifted second surface; b) a processor programmed to: i) process the images of the first surface and the axially shifted second surface to correct optical aberrations so that the images of the first surface and the axially shifted second surface have substantially the same optical resolution; and ii) detect fluorescently labeled compositions containing nucleic acid molecules or their complementary sequences disposed on the first surface or the axially shifted second surface to determine the identity of nucleotides in the nucleic acid molecules.

[0014] In some embodiments, images of the first surface and the axially displaced second surface are acquired without moving the optical compensator 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 by simply 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, and said hydrophilic coating further contains >10,000 nucleic acid colonies / mm². 2 The surface density of the optical system comprises labeled nucleic acid colonies arranged thereon. In some embodiments, the optical system includes one, two, three, or four imaging channels configured to detect nucleic acid colonies labeled with one, two, three, or four different detectable markers arranged on at least one of the first surface or the axially displaced second surface. In some embodiments, at least one image sensor comprises pixels whose pixel size is selected such that the spatial sampling frequency of the optical system is at least twice the optical resolution of the optical system. In some embodiments, the system includes at least one barrel lens positioned between the objective lens and at least one image sensor, and said at least one barrel lens is configured to correct imaging performance metrics for imaging the first inner surface and the second inner surface of the flow cell. In some embodiments, the flow cell wall thickness is at least 700 μm, and the gap between the first inner surface and the second inner surface is at least 50 μm. In some embodiments, the optical system includes two or more barrel lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths.

[0015] The fluorescence imaging system disclosed herein includes: a) at least one light source configured to provide excitation light within one or more specified wavelength ranges; b) an objective lens configured to collect fluorescence generated within a specified field of view from the sample plane after the sample plane has been exposed to the excitation light, wherein the numerical aperture of the objective lens is at least 0.3, the working distance of the objective lens is at least 700 μm, and the area of ​​the field of view is at least 2 mm². 2 ; and c) at least one image sensor, wherein fluorescence collected by the objective lens is imaged onto the image sensor, and wherein the pixel size of the image sensor is selected such that the spatial sampling frequency of the fluorescence imaging system is at least twice the optical resolution of the fluorescence imaging system.

[0016] 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 area is at least 2.5 mm. 2 In some implementations, the field of view area 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 shift platform, such that the system is configured to automatically acquire a series of two or more fluorescence images, wherein each image in the series is acquired for different fields 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 objective focal plane between acquired images for 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 focal plane position before acquiring images for different fields of view if an error signal indicates that the difference between 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 from each of three or more wavelength ranges onto different image sensors. In some embodiments, the difference in position between the focal plane of each of the three or more image sensors and the sample plane is less than 100 nm. In some embodiments, the difference in position between the focal plane of each of the three or more image sensors and the sample plane is less than 50 nm. In some embodiments, the total time required for repositioning the sample plane, adjusting the focus if necessary, and acquiring an image is less than 0.4 seconds per field of view. In some embodiments, the total time required for repositioning the sample plane, adjusting the focus if necessary, and acquiring an image is less than 0.3 seconds per field of view.

[0017] This document also discloses a fluorescence imaging system for double-sided imaging of a flow cell, comprising: a) an objective lens configured to collect fluorescence generated within a designated 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 correct the imaging performance of the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging the inner surface of the flow cell. The flow cell has a wall thickness of at least 700 μm, and the gap between the upper inner surface and the lower inner surface is at least 50 μm. The imaging performance is substantially the same for imaging the upper inner surface or the lower inner surface of the flow cell without moving an optical compensator into 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 into or out of the optical path.

[0018] In some embodiments, the objective lens is a commercially available microscope objective lens. In some embodiments, the numerical aperture of the commercially available microscope objective lens is at least 0.3. In some embodiments, the working distance of the objective lens is at least 700 μm. In some embodiments, the objective lens is calibrated to compensate for a coverslip thickness (or flow cell wall thickness) of 0.17 mm. In some embodiments, the fluorescence imaging system further includes an electro-optic phase plate located near the objective lens and between the objective lens and the tube lens, wherein the electro-optic phase plate provides correction for optical aberrations caused by the gap between the upper and lower inner surfaces of the fluid-filled flow cell. In some embodiments, at least one tube lens is a compound lens comprising three or more optical components. In some embodiments, at least one tube lens is a compound lens comprising four optical components. In some embodiments, the four optical components sequentially comprise a first asymmetric convex-convex lens, a second convex-planar lens, a third asymmetric concave-concave lens, and a fourth asymmetric convex-concave lens. In some embodiments, at least one tube lens is configured to correct the imaging performance of the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging the inner surface of a flow cell having a wall thickness of at least 1 mm. In some embodiments, at least one tube lens is configured to correct the imaging performance of the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging the inner surface of a flow cell having a gap of at least 100 μm. In some embodiments, at least one tube lens is configured to correct the imaging performance of the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging the inner 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, and each of the two tube lenses is designed to provide optimal imaging performance at different fluorescence wavelengths. In some embodiments, the system includes a single objective lens, three tube lenses, and three image sensors, and each of the three tube lenses is designed to provide optimal imaging performance at different fluorescence wavelengths. In some embodiments, the system includes a single objective lens, four barrel lenses, and four image sensors, each of the four barrel lenses being designed to provide optimal imaging performance at different fluorescence wavelengths. In some embodiments, the objective lens or at least one barrel lens is designed to optimize the modulation transfer function over a medium to high spatial frequency range. In some embodiments, imaging performance metrics include measurements 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 imaging performance metrics for imaging the upper and lower inner surfaces of the flow cell is less than 10%.In some embodiments, the difference in imaging performance metrics for imaging the upper and lower inner surfaces of the flow cell is less than 5%. In some embodiments, the use of at least one barrel lens provides at least an equivalent or better improvement in imaging performance metrics for double-sided imaging compared to a conventional system including an objective lens, a motion-actuated compensator, and an image sensor. In some embodiments, the use of at least one barrel lens provides at least a 10% improvement in imaging performance metrics for double-sided imaging compared to a conventional system including an objective lens, a motion-actuated compensator, and an image sensor.

[0019] This document discloses an illumination system for imaging-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 the light to a designated illumination field on the surface of a carrier containing tethered biomacromolecules.

[0020] In some implementations, the lighting system also includes a focusing lens. In some implementations, the designated lighting area has a depth of at least 2mm. 2 The area. In some embodiments, for an imaging system used to acquire an image of a carrier surface, the light delivered to the designated illumination field has a uniform intensity across the entire designated field of view. In some embodiments, the designated field of view has a width of at least 2 mm. 2 The area. In some embodiments, when the coefficient of variation (CV) of light intensity is less than 10%, the light transmitted to the designated illumination field has a uniform intensity across the entire designated field of view. In some embodiments, when the coefficient of variation (CV) of light intensity is less than 5%, the light transmitted to the designated illumination field has a uniform intensity across the entire designated field of view. In some embodiments, the light transmitted to the designated illumination field has a speckle contrast value of less than 0.1. In some embodiments, the light transmitted to the designated illumination field has a speckle contrast value of less than 0.05.

[0021] By incorporating via reference

[0022] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication, patent, or patent application is specifically and individually incorporated by reference in its entirety. In the event of any conflict between the terminology used herein and the terminology in the incorporated references, the terminology used herein shall prevail. Attached Figure Description

[0023] The novel features of the invention are specifically set forth in the appended claims. The features and advantages of the invention can be better understood by referring to the following detailed description of illustrative embodiments utilizing the principles of the invention, in conjunction with the accompanying drawings, in which:

[0024] Figures 1A-1BA non-limiting example of an imaging dual-surface carrier structure is schematically shown for presenting sample sites for imaging via the imaging system disclosed herein. Figure 1A : Illustration of the images of the inner surfaces before and after the flow cell. Figure 1B Illustration of the front and back outer surfaces of the matrix.

[0025] Figures 2A-2B A non-limiting example of a multichannel fluorescence imaging module including a dichroic beam splitter is shown, the dichroic beam splitter being used to transmit an excitation beam to a sample and to receive and redirect fluorescence emission generated by reflection to four detection channels, the four detection channels being configured to detect fluorescence emission at four different corresponding wavelengths or bands. Figure 2A Top isometric view. Figure 2B Isometric view of the bottom.

[0026] Figures 3A-3B It shows Figure 2A and Figure 2B The optical path within the multi-channel fluorescence imaging module includes a dichroic beam splitter, which transmits the excitation beam to the sample and receives and redirects the fluorescence emission generated by reflection to four detection channels. The four detection channels are used to detect fluorescence emission at four different corresponding wavelengths or bands. Figure 3A Top view. Figure 3B Side view.

[0027] Figure 4 This is a graph showing the relationship between the performance of a dichroic filter and the incident beam angle.

[0028] Figure 5 This is a diagram showing the relationship between the beam footprint size and the incident beam angle on a dichroic filter.

[0029] Figures 6A-6B An example configuration of a dichroic filter and detection channel for a multi-channel fluorescence imaging module is schematically shown, wherein the dichroic filter has a tilted reflective surface such that the angle between the incident beam (e.g., the central angle) and the reflective surface of the dichroic filter is less than 45 degrees. Figure 6A : A schematic diagram of a multi-channel fluorescence imaging module including four detection channels. Figure 6B : Shows a detailed view of the angle of incidence (AOI) of the light beam on the dichroic mirror.

[0030] Figure 7 A diagram is provided that shows the corresponding Figure 6A and 6B The imaging module configuration shown in the figure demonstrates improved dichroic filter performance.

[0031] Figure 8A diagram is provided that shows the corresponding Figure 6A and 6B The imaging module configuration shown in the figure demonstrates improved dichroic filter performance.

[0032] Figures 9A-9B A diagram is provided that illustrates the composition of... Figure 6A and 6B The reduced surface deformation is caused by the configuration of the imaging module. Figure 9A The effect of the folding angle on the image quality degradation caused by adding 1 wave of PV spherical power to the last mirror is shown. Figure 9B The effect of the folding angle on the image quality degradation caused by adding 0.1-wave PV spherical power to the last mirror is shown.

[0033] Figures 10A-10B A figure is provided, which shows the improved excitation filter performance (e.g., a steeper transition between the passband and the surrounding stopband) due to the use of s-polarization of the excitation beam. Figure 10A : The transmission spectra of an exemplary bandpass dichroic filter at incident angles of 40 degrees and 45 degrees, wherein 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 is s-polarized relative to the plane of the dichroic filter, resulting in a substantially steeper edge between the passband and the stopband.

[0034] Figures 11A-11B The modulation transfer function (MTF) of an exemplary dual-surface imaging system with a numerical aperture (NA) of 0.3 disclosed herein is shown. Figure 11A : First surface. Figure 11B : Second surface.

[0035] Figures 12A-12B The MTF of an exemplary dual-surface imaging system with an NA of 0.4 disclosed herein is shown. Figure 12A : First surface. Figure 12B : Second surface.

[0036] Figures 13A-13B The MTF of an exemplary dual-surface imaging system with an NA of 0.5 disclosed herein is shown. Figure 13A : First surface. Figure 13B : Second surface.

[0037] Figures 14A-14B The MTF of an exemplary dual-surface imaging system with an NA of 0.6 disclosed herein is shown. Figure 14A : First surface. Figure 14B : Second surface.

[0038] Figures 15A-15BThe MTF of an exemplary dual-surface imaging system with an NA of 0.7 disclosed herein is shown. Figure 15A : First surface. Figure 15B : Second surface.

[0039] Figures 16A-16B The MTF of an exemplary dual-surface imaging system with an NA of 0.8 disclosed herein is shown. Figure 16A : First surface. Figure 16B : Second surface.

[0040] Figures 17A-17B A plot of the calculated Strehl ratio is provided for imaging the surface of the second flow cell through the surface of the first flow cell. Figure 17A : A plot of Strelby ratio for imaging a second flow cell surface through a first flow cell surface as a function of the intermediate fluid layer thickness (fluid channel height) for different objective lenses and / or optical system numerical apertures. Figure 17B The graph shows the Strell ratio as a function of numerical aperture for imaging the surface of the second flow cell through the surface of the first flow cell and the intermediate water layer with a thickness of 0.1 mm.

[0041] Figure 18 A schematic diagram of the dual-wavelength excitation / four-channel emission fluorescence imaging system disclosed herein is provided.

[0042] Figure 19 A ray tracing diagram is provided for an objective lens design designed to image the surfaces of opposite sides of a 0.17 mm thick coverslip.

[0043] Figure 20 Provided for imaging the surfaces of opposite sides of a 0.17 mm thick coverslip, Figure 19 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0044] Figure 21 Provided for imaging the surfaces of opposite sides of a 0.3 mm thick coverslip, Figure 19 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0045] Figure 22 Provided for imaging surfaces separated from the 0.1 mm thick aqueous fluid layer by a 0.3 mm thick coverslip, Figure 19 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0046] Figure 23 Provided for imaging the surfaces of opposite sides of a 1.0 mm thick coverslip, Figure 19The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0047] Figure 24 Provided for imaging surfaces separated from the surface of a 1.0 mm thick coverslip by a 0.1 mm thick aqueous fluid layer. Figure 19 The graph shows the modulation transfer function of the objective lens as a function of spatial frequency.

[0048] Figure 25 Provides ray tracing diagrams for lens barrel design, if compared with... Figure 19 When used in combination with the objective lens shown, the tube lens provides improved double-sided imaging through a 1 mm thick coverslip.

[0049] Figure 26 Provided for imaging the surfaces of opposite sides of a 1.0 mm thick coverslip, Figure 25 The figure shows a graph of the modulation transfer function of the combination of objective lens and tube lens as a function of spatial frequency.

[0050] Figure 27 Provided for imaging surfaces separated from the surface of a 1.0 mm thick coverslip by a 0.1 mm thick aqueous fluid layer. Figure 25 The figure shows a graph of the modulation transfer function of the combination of objective lens and tube lens as a function of spatial frequency.

[0051] Figure 28 A ray-tracing plot is provided for a barrel lens design (left) used in this disclosure, which has been optimized to provide high-quality double-sided imaging performance. Since the barrel lens is no longer infinitely corrected, a suitably designed zero lens (right) can be combined with the barrel lens to compensate for barrel lenses that are not infinitely corrected for manufacturing and testing purposes.

[0052] Figure 29 The illustration shows a non-limiting example of a single capillary flow cell with two fluid adapters.

[0053] Figure 30 The illustration shows a non-limiting example of a flow cell box that includes a base, a fluid adapter, and optional other components, and is designed to accommodate two capillaries.

[0054] Figure 31 The illustration shows a non-limiting example of a system comprising a single capillary flow cell connected to various fluid flow control components, wherein the single capillary is compatible with mounting on a microscope stage or in a custom imaging instrument for a variety of imaging applications.

[0055] Figure 32The illustration shows a non-limiting example of a system that includes a capillary flow cell box with an integrated diaphragm valve to reduce or minimize dead volume and save certain critical reagents.

[0056] Figure 33 The illustration shows a non-limiting example of a system that includes a capillary flow cell, a microscope apparatus, and a temperature control mechanism.

[0057] Figure 34 The illustration shows a non-limiting example of controlling the temperature of a capillary flow cell by using a metal plate placed in contact with the flow cell box.

[0058] Figure 35 The illustration depicts a non-limiting method for temperature control of a capillary flow cell, including a non-contact thermal control mechanism.

[0059] Figures 36A-36C The illustration shows a non-limiting example of the manufacture of a flow cell apparatus. Figure 36A The fabrication of a one-piece glass flow cell is shown. Figure 36B The preparation of a two-piece glass flow cell is shown. Figure 36C The fabrication of a three-piece glass flow cell is shown.

[0060] Figures 37A-37C The illustration shows a non-limiting example of a glass flow cell design. Figure 37A This demonstrates a one-piece glass flow tank design. Figure 37B The design of the two-piece glass flow cell is shown. Figure 37C The design of the three-piece glass flow cell is shown.

[0061] Figure 38 The illustration shows a visualization of cluster amplification in the capillary lumen.

[0062] Figure 39 Non-limiting examples of block diagrams of the sequencing systems disclosed herein are provided.

[0063] Figure 40 Non-limiting examples of flowcharts for the sequencing methods disclosed herein are provided.

[0064] Figure 41 Non-limiting examples of schematic diagrams of the structured lighting systems disclosed herein are provided.

[0065] Figure 42 Non-limiting examples of flowcharts for acquiring and processing structured illuminated images of flow cell surfaces as disclosed herein are provided.

[0066] Figures 43A-43B A non-limiting schematic diagram of a multiplexed read head, as disclosed herein, is provided. Figure 43ASide view of a multiplexed readhead, in which individual microfluorometers are configured to image a common surface (e.g., the inner surface of a flow cell). Figure 43B Top view of the multiplexed readhead, showing the imaging paths acquired by the individual microfluorometers of the multiplexed readhead.

[0067] Figures 44A-44B A non-limiting schematic diagram of a multiplexed read head, as disclosed herein, is provided. Figure 44A Side view of a multiplexed readhead, wherein a first subset of multiple individual microfluorometers is configured to image a first surface (e.g., a first inner surface) of a flow cell, and a second subset of multiple individual microfluorometers is configured to image a second surface (e.g., a second inner surface) of a flow cell. Figure 44B : Figure 44A A top view of the multiplexed readhead, showing the imaging paths acquired by the individual microfluorometers of the multiplexed readhead. Detailed Implementation

[0068] Fluorescence imaging methods and systems are needed to provide increased optical resolution and improved image quality for genomics applications, leading to a corresponding improvement in the accuracy of genomic testing. This paper discloses optical system designs for high-performance fluorescence imaging methods and systems that can provide any one or more of improved optical resolution (including high-performance optical resolution), improved image quality, and higher throughput for fluorescence imaging-based genomics applications. The disclosed optical illumination and imaging system designs can provide any one or more of the following advantages: improved dichroic filter performance, increased dichroic filter frequency response uniformity, improved excitation beam filtering, a larger field of view, increased spatial resolution, improved modulation transfer, contrast-to-noise ratio, 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., images from different fields of view), improved imaging system duty cycle, and higher throughput image acquisition and analysis.

[0069] In some cases, such as for two-sided (flow cell) imaging applications (including the use of thick flow cell walls (e.g., wall (or coverslip) thickness > 700 μm) and fluid channels (e.g., fluid channel height or thickness is 50-200 μm)), improved imaging performance can be achieved using novel objective designs that correct for optical aberrations introduced by imaging the surfaces on the opposite side of the thick coverslip and / or fluid channels to the objective.

[0070] In some cases, such as for two-sided (flow cell) imaging applications (including the use of thick flow cell walls (e.g., wall (or coverslip) thickness > 700 μm) and fluid channels (e.g., the height or thickness of the fluid channels is 50-200 μm)), improved imaging performance can be achieved even when using commercially available off-the-shelf objectives by using novel tube lens designs (different from tube lenses in conventional microscopes that only form images on the intermediate image plane), which, in combination with the objectives, correct for optical aberrations caused by the thick flow cell walls and / or intermediate fluid layer.

[0071] In some cases, such as for multi-channel (e.g., two-color or four-color) imaging applications, improved imaging performance can be achieved by using multiple lens barrels, one lens barrel for one imaging channel, wherein each lens barrel is designed to be optimized for a specific wavelength range used in that imaging channel.

[0072] In some cases, such as for two-sided (flow cell) imaging applications, improved imaging performance can be achieved by using an electro-optic phase plate in combination with the objective lens to compensate for optical aberrations caused by the fluid layer separating the upper (near) inner and lower (far) inner surfaces of the flow cell. In some cases, this design approach can also compensate for vibrations introduced by, for example, motion-actuated compensators that move into or out of the optical path depending on which surface of the flow cell is being imaged.

[0073] Various multichannel fluorescence imaging module designs are disclosed, which may include illumination and imaging optical paths, including folded optical paths (e.g., including one or more beam splitters or beam combiners, such as dichroic beam splitters or beam combiners), which guide an excitation beam to an objective lens and guide emitted light transmitted through the objective lens to multiple detection channels. Some particularly advantageous features of the fluorescence imaging modules described herein include specifying an incident angle for a dichroic filter, resulting in a steeper and / or more uniform transition between the passband and stopband wavelength regions of the dichroic filter. Such a filter may be included within a folded optics and may include a dichroic beam splitter or beam combiner. Further advantageous features of the disclosed imaging optics designs may include the position and orientation of one or more excitation sources and one or more detection optical paths relative to the objective lens and the dichroic filter receiving the excitation beam. The excitation beam may also be linearly polarized, and the linear polarization orientation may allow s-polarized light to be incident on the dichroic reflective surface of the dichroic filter. Such features can potentially improve excitation beam filtering and / or reduce wavefront errors introduced into the emitted beam due to surface deformation of the dichroic filter. The fluorescence imaging module described herein may or may not include any of these features, and may or may not include any of these advantages.

[0074] This document also describes devices and systems configured to analyze a large number of diverse nucleic acid sequences by imaging, for example, arrays of immobilized nucleic acid molecules formed on the surface of a flow cell. The devices and systems described herein can also be used, for example, to perform sequencing of comparative genomes, track gene expression, perform microRNA sequence analysis, epigenomics, aptamer and phage display library characterization, and perform other sequencing applications. The devices and systems disclosed herein encompass various combinations of optical, mechanical, fluidic, thermal, electrical, and computational devices / aspects. The advantages offered by the disclosed flow cell devices, cartridges, and systems include, but are not limited to: (i) reduced fabrication complexity and cost of the devices and systems; (ii) significantly reduced consumable costs (e.g., compared to existing 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.

[0075] This document discloses capillary flow cell devices and capillary flow cell boxes constructed from readily available, disposable single-cavity (e.g., a single fluid flow channel) or multi-cavity capillaries, which may also include a fluid adapter, a box base, one or more integrated fluid flow control components, or any combination thereof. This document also discloses capillary flow cell-based systems that may include one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell boxes (or microfluidic cartridges), a fluid flow controller module, a temperature control module, an imaging module, or any combination thereof.

[0076] Some publicly disclosed capillary flow cell devices, cartridges, and systems have design features including, but not limited to, (i) an integrated flow channel construction; (ii) sealed, reliable, and repeatable switching between reagent flows, which can be achieved through a simple loading / unloading mechanism that reliably seals the fluid interface between the system and the capillary, thereby facilitating capillary replacement and system reuse, and enabling precise control of reaction conditions such as reagent concentration, pH, and temperature; (iii) replaceable single fluid flow channel devices or capillary flow cell cartridges comprising multiple interchangeable flow channels to provide flexible system throughput; and (iv) compatibility with a variety of detection methods, such as fluorescence imaging.

[0077] Although the disclosed capillary flow cell devices and systems, capillary flow cell boxes, capillary flow cell-based systems, microfluidic devices and boxes, and microfluidic chip-based systems are described primarily in the context of their use in 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 analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. It should be understood that different aspects of the disclosed methods, devices, and systems can be understood individually, collectively, or in combination with each other. Although this document is primarily discussed 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 understand that many of the disclosed optical design methods and features can be applied to other imaging modalities, such as bright-field imaging, dark-field imaging, phase-contrast imaging, etc.

[0078] Definitions: Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0079] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include repeated references unless the context clearly indicates otherwise. Unless otherwise stated, any reference to “or” herein is intended to cover “and / or”.

[0080] As used herein, the term “approximately” refers to the number plus or pointing to 10% of that number. In the context of a range, the term “approximately” refers to the range minus 10% of its minimum value and plus 10% of its maximum value.

[0081] As used herein, the phrases “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 a larger system, such as, for example, a fluid module, a temperature control module, a displacement platform, an automatic fluid distribution and / or microplate processing unit, a processor or computer, instrument control software, data analysis and display software, etc.

[0082] As used herein, the term "detection channel" refers to an optical path (and / or optical components therein) within an optical system configured to transmit light signals generated from a sample to a detector. In some cases, a detection channel may be configured to perform spectroscopic measurements, such as using a detector (e.g., a photomultiplier tube) to monitor fluorescence signals or other light signals. In other cases, a "detection channel" may be an "imaging channel," i.e., an optical path (and / or optical components therein) within an optical system configured to capture an image and transmit it to an image sensor.

[0083] As used herein, "detectable marker" can refer to any of a variety of detectable markers or labels known to those skilled in the art. Examples include, but are not limited to, chromophores, fluorophores, quantum dots, upconversion phosphors, luminescent or chemiluminescent molecules, radioactive isotopes, magnetic nanoparticles, mass tags, etc. In some cases, preferred markers may include fluorophores.

[0084] As used herein, the term "excitation wavelength" refers to the wavelength of light used to excite a fluorescent indicator (e.g., a fluorophore or dye molecule) and produce fluorescence. While the excitation wavelength is typically specified as a single wavelength, such as 620 nm, those skilled in the art will understand that this specification refers to a wavelength range centered on the specified wavelength or an excitation filter bandpass. For example, in some cases, the light specified for the excitation wavelength includes light with a specified wavelength ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or greater. In some cases, the excitation wavelength used may or may not coincide with the maximum absorption peak of the fluorescent indicator.

[0085] As used herein, the term "emission wavelength" refers to the wavelength of light emitted by a fluorescent indicator (e.g., a fluorophore or dye molecule) after excitation by light of an appropriate wavelength. While emission wavelengths are typically specified as a single wavelength, such as 670 nm, those skilled in the art will understand that this specification refers to a wavelength range centered on the specified wavelength or an emission filter bandpass. In some cases, light with a specified emission wavelength includes light within ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or greater. In some cases, the emission wavelength used may or may not coincide with the maximum emission peak of the fluorescent indicator.

[0086] As used herein, fluorescence is “specific” if it originates from fluorophores that are annealed or otherwise bound to a surface, such as fluorescently labeled nucleic acid sequences having regions that are anticomplementary to the corresponding segments of oligonucleotide adaptors on the surface and annealed to said corresponding segments. This fluorescence contrasts with fluorescence originating from fluorophores that are not bound to the surface by such an annealing process, or in some cases, are background fluorescence on the surface.

[0087] As used herein, a “nucleic acid” (also known 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 nucleoside bonds, or a variant or functional segment thereof. In natural examples of nucleic acids, the nucleoside bonds are typically phosphodiester bonds. However, other examples optionally contain other nucleoside bonds, such as phosphate thioester bonds, and may or may not contain phosphate groups. Nucleic acids include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA, DNA / RNA hybrids, peptide nucleic acids (PNA), hybrids of PNA with DNA or RNA, and may also include other types of nucleic acid modifications.

[0088] As used herein, “nucleotide” means nucleotide, nucleoside, or analogue thereof. In some cases, a nucleotide is an N- or C-glycoside of a purine or pyrimidine base (e.g., a deoxyribonucleoside containing 2-deoxy-D-ribose or a ribonucleoside containing D-ribose). Examples of other nucleotide analogues include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, etc.

[0089] Fluorescence imaging as an information conduit: A useful summary of the role of fluorescence imaging systems in typical genomics assays (including nucleic acid sequencing applications) is that they act as information conduits, where photon signals enter at one end, such as the objective lens used for imaging, and specific information about the location of the fluorescence signals appears at the other end, such as the location of the image sensor. As more information is transmitted through this conduit, some content is inevitably lost during this transmission and can never be recovered. An example of this is when there are too many labeled molecules (or clonal amplification clusters of molecules) in a small area on the matrix surface to be clearly distinguishable in the image; at the location of the image sensor, it is difficult to distinguish photon signals generated by adjacent molecular clusters, thus increasing the probability of attributing signals to incorrect clusters and leading to detection errors.

[0090] Optical Imaging Module Design: Therefore, the goal of designing the optical imaging module is to maximize the information content flow through the detection pipeline and minimize detection errors. Several key design elements need to be addressed during the design process, including:

[0091] 1) Match the physical feature density on the substrate surface to be imaged with the overall image quality of the optical imaging system and the pixel sampling frequency of the image sensor used. Mismatches in these parameters can lead to information loss or sometimes even erroneous information; for example, spatial aliasing may occur when the pixel sampling frequency is less than twice the optical resolution limit.

[0092] 2) Match the size of the area to be imaged with the overall image quality and focus quality of the optical imaging system across the entire field of view.

[0093] 3) Match the optical collection efficiency, modulation transfer function, and image sensor performance characteristics of the optical system design with the expected fluorescence photon flux and dye efficiency (related to the dye extinction coefficient and fluorescence quantum yield) of the input excitation photon flux, while taking into account the background signal and system noise characteristics.

[0094] 4) To separate spectral contents to the greatest extent possible in order to reduce crosstalk between fluorescence imaging channels.

[0095] 5) By repositioning the sample or optics between image captures in different fields of view, the image acquisition steps are effectively synchronized to minimize the downtime of the imaging system (or maximize the duty cycle) and thus maximize the overall throughput of the image capture process.

[0096] This disclosure describes a systematic approach to addressing each design element outlined above and creating a component-level specification for an imaging system.

[0097] Improved optical resolution and image quality to improve or maximize information transmission and throughput: A non-limiting design practice could begin with optical resolution required to distinguish two adjacent features specified with respect to the number of line pairs X / mm (lp / mm) and convert them to the corresponding numerical aperture (NA). The numerical aperture requirement can then be used to evaluate the impact on modulation transfer function and image contrast.

[0098] The standard modulation transfer function (MTF) describes the spatial frequency response of image contrast (modulation) transmitted through an optical system; image contrast decreases as a function of spatial frequency and increases with increasing NA. This function limits the contrast / modulation that can be achieved for a given NA. Furthermore, wavefront errors can negatively impact the MTF, thus necessitating the use of the true system MTF, rather than diffraction-limited optics predictions, to improve or optimize optical system design. Note that while design practice may primarily consider the objective's MTF, as used herein, the MTF refers to the entire system MTF (including the complete optical path from the coverslip to the image sensor).

[0099] In genomic testing applications, the target for imaging is a high-density array of “dots” (randomly distributed or patterned) on a surface. This allows determining the minimum modulation transfer value required for downstream analysis to distinguish between two adjacent points and differentiate between four possible states (e.g., ON-OFF, ON-ON, OFF-ON, and OFF-OFF). For example, assume the dots are small enough to be approximated as point sources. The detection task is to determine whether two adjacent points separated by a distance d are ON or OFF (in other words, bright or dark), and to determine the contrast-to-noise ratio (CNR) of the fluorescence signal generated by the points on the sample plane (or object plane) as C. 样品 Then, under ideal conditions, the CNR of the readout signals from two adjacent points on the image sensor plane is C 图像 It can be approximated as C 图像 =C 样品 *MTF(1 / d), where MTF(1 / d) is the MTF value at spatial frequency = (1 / d).

[0100] In a typical design, the value of C might need to be at least 4 so that a simple thresholding method can be used to avoid misclassification of the fluorescence signal. Assuming the fluorescence signal intensity follows a Gaussian distribution near the mean, in C... 图像 At >4, the expected error for correctly classifying fluorescence signals (e.g., ON or OFF) is <0.035%. Using proprietary high CNR sequencing and surface chemistry methods (e.g., as described in U.S. Patent Application No. 16 / 363,842), sample-plane CNR (C12 or even higher) for clusters of cloned, labeled oligonucleotide molecules tethered to the matrix surface can be achieved when measuring sparse fields (i.e., when the surface density of clusters or points is low) (where the MTF value is close to 100%). 样品 ) value. Assume the CNR value of the sample plane is C 样品 >12, and the target classification error rate is <0.1% (therefore, C 图像 >4), then in some implementations, the minimum value of M(1 / d) can be determined to be M(1 / d) = 4 / 12 to 33%. Therefore, at least 33% of the modulation transfer function threshold can be used to preserve the information content of the transmitted image.

[0101] Design practice can correlate the minimum separation distance *d* between two features or points with the optical resolution requirement (expressed as X (lp / mm) as described above), where *d* = (1 mm) / X, i.e., *d* is the minimum separation distance between two features or points that can be completely resolved by the optical system. In some designs disclosed herein, where the design analysis aims to increase or maximize the transfer of relevant information, this design criterion can be relaxed to *d* = (1 mm) / X / A, where 2 > A > 1. For the same optical resolution X lp / mm, reducing the value of *d* (the minimum resolvable point separation distance at the sample plane) allows for the use of higher feature densities.

[0102] Design practice uses the Nyquist criterion to determine the minimum spatial sampling frequency at the sample plane, where the spatial sampling frequency S ≥ 2*X (and where X is the optical resolution of the imaging system expressed as X lp / mm as described above). When the system spatial sampling frequency is close to the Nyquist criterion (which is usually the case), an imaging system resolution greater than S will cause aliasing because the optical sensor cannot adequately sample higher frequency information that the optical system can resolve.

[0103] In some designs disclosed herein, an oversampling scheme based on the relationship S = B * Y (where B ≥ 2 and Y is the true MTF limit of the optical system) can be used to further improve the information transfer capability of the imaging system. As mentioned above, X (lp / mm) corresponds to the actual non-zero (>33%) minimum modulation transfer value, while Y (lp / mm) is the limit of optical resolution, and therefore modulation is 0 at Y (lp / mm). Therefore, in the disclosed designs, Y (lp / mm) can 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.

[0104] The above relationships can be used to determine the system magnification and provide an upper limit for the image sensor pixel size. The choice of image sensor pixel size is matched with the system's optical quality and the spatial sampling frequency required to reduce aliasing. The lower limit for the image sensor pixel size can be determined based on photon flux, since the relative noise contribution increases as the pixel size decreases.

[0105] However, other design approaches are also possible. For example, reducing the NA to less than 0.6 (e.g., 0.5 or less) can provide increased depth of field. This increased depth of field enables bi-surface imaging, where two surfaces at different depths can be imaged simultaneously with or without refocusing. As mentioned above, reducing the NA can reduce optical resolution. In some implementations, higher excitation beam power, such as 1 W or higher, can be used to generate a strong signal. Inherently high-contrast samples (i.e., sample surfaces exhibiting strong foreground signals and significantly reduced background signals) can also be used to facilitate the acquisition of high contrast-to-noise ratio (CNR) images, such as those with a CNR value >20, which provides improved signal discrimination for base detection in applications such as nucleic acid sequencing. In some optical system designs disclosed herein, sample carrier structures (e.g., flow cells with hydrophilic surfaces) are used to reduce background noise.

[0106] In various embodiments, the disclosed optical systems provide a large field of view (FOV). For example, some optical imaging systems, including, for example, objective lenses and barrel lenses, can provide an FOV greater than 2 mm or 3 mm. In some cases, the optical imaging system provides a reduced magnification, for example, less than 10x. In some embodiments, this reduced magnification can facilitate a large FOV design. Despite the reduced magnification, the optical resolution of such a system remains sufficient because detector arrays with small pixel sizes or spacing can be used. In some embodiments, an image sensor comprising pixels with a size less than twice the optical resolution provided by the optical imaging system (e.g., objective lenses and barrel lenses) can be used to satisfy the Nyquist theorem.

[0107] Other designs are also possible. In some optical designs, a configuration is made to provide dual-surface imaging, where two surfaces at different depths can be imaged simultaneously. The optical imaging system (e.g., objectives and / or barrel lenses) is configured to reduce optical aberrations to image more of the two surfaces (e.g., two planes) at these two corresponding depths compared to other locations (e.g., other planes) at other depths. Alternatively, the optical imaging system can be configured to reduce aberrations to image the two surfaces (e.g., two planes) at these two corresponding depths through a transmission layer (e.g., a glass layer, e.g., a coverslip) on the sample carrier structure and through a solution (e.g., an aqueous solution) containing the sample or in contact with the sample on at least one of the two surfaces.

[0108] Multichannel fluorescence imaging modules and systems: In some cases, the imaging modules or systems disclosed herein may include fluorescence imaging modules or systems. In some cases, the fluorescence imaging systems disclosed herein may include a single fluorescence excitation source (for providing 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 labeled nucleic acid molecules or clusters thereof disposed on a matrix surface). In some cases, the fluorescence imaging systems disclosed herein may include a single fluorescence emission imaging and detection channel, such as an optical path configured to collect fluorescence emitted by the sample and deliver an image of the sample (e.g., an image of a matrix surface on which fluorescently labeled nucleic acid molecules or clusters thereof are disposed) to an image sensor or other photodetector. In some cases, the fluorescence imaging system may include two, three, four, or more than four fluorescence excitation sources and / or optical paths configured to deliver 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 cases, the fluorescence imaging systems disclosed herein may include two, three, four, or more than four fluorescence emission imaging and detection channels configured to collect fluorescence emitted by a sample at two, three, four, or more than four emission wavelengths (or within two, three, four, or more than four emission wavelength ranges) and deliver images of the sample (e.g., images of a matrix surface on which fluorescently labeled nucleic acid molecules or clusters thereof are disposed) to two, three, four, or more than four image sensors or other photoelectric detection devices.

[0109] Dual-surface imaging: In some cases, the imaging systems disclosed herein, including fluorescence imaging systems, can be configured to acquire high-resolution images of a single sample carrier structure or matrix surface. In some cases, the imaging systems disclosed herein, including fluorescence imaging systems, can be configured to acquire high-resolution images of two or more sample carrier structures or matrix surfaces (e.g., two or more surfaces of a flow cell). In some cases, the high-resolution images provided by the disclosed imaging systems can be used to monitor reactions occurring on two or more surfaces of a flow cell (e.g., nucleic acid hybridization, amplification, and / or sequencing reactions) as various reagents flow through or around the matrix of the flow cell. Figure 1A and Figure 1B A schematic diagram of this dual-surface carrier structure is provided. Figure 1AA dual-surface support structure, such as a flow cell, is illustrated, comprising internal flow channels through which analytes or reagents can flow. Flow channels may be formed between a first and second layer, a top and bottom layer, and / or a front and rear layer, for example, between the first and second plates, the top and bottom plates, and / or the front and rear plates shown. One or more plates may comprise glass plates, such as coverslips. In some embodiments, the layers comprise borosilicate glass, quartz, or plastic. The inner surfaces of these top and bottom layers provide walls for the flow channels, which help restrict the flow of analytes or reagents through the flow channels of the flow cell. In some designs, these inner surfaces are flat. Similarly, the top and bottom layers may be flat. In some designs, at least one additional layer (not shown) is arranged between the top and bottom layers. This additional layer may have one or more channels cut out therein, which help define one or more flow channels and control the flow of analytes or reagents within the flow channels. Further discussion of sample support structures (e.g., flow cells) can be found below.

[0110] Figure 1A The diagram schematically illustrates multiple fluorescent sample sites on the first and second inner surfaces, top and bottom inner surfaces, and / or front and rear inner surfaces of the flow cell. In some embodiments, a reaction can occur at these sites to bind the sample, causing fluorescence to be emitted from these sites (note, Figure 1A This is illustrative and not drawn to scale; for example, the size and spacing of fluorescent sample sites may be smaller than shown.

[0111] Figure 1B Another dual-surface carrier structure with two surfaces is shown, the two surfaces containing fluorescent sample sites to be imaged. The sample carrier structure includes a matrix having a first outer surface and a second outer surface, a top outer surface and a bottom outer surface, and / or a front outer surface and a rear outer surface. In some designs, these outer surfaces are flat. In various embodiments, the analyte or reagent flows through these first and second outer surfaces. Figure 1B The diagram schematically illustrates multiple fluorescent sample sites on the first and second outer surfaces, top and bottom outer surfaces, and / or front and rear outer surfaces of the sample carrier structure. In some embodiments, a reaction can occur at these sites to bind the sample, causing fluorescence to be emitted from these sites (note, Figure 1B This is illustrative and not drawn to scale; for example, the size and spacing of fluorescent sample sites may be smaller than shown.

[0112] In some cases, the fluorescence imaging modules and systems described herein can be configured to image such fluorescent sample sites on a first and a second surface at different distances from the objective lens. In some designs, only one of the first or second surfaces is focused at a time. Thus, in such designs, one of these surfaces is imaged at a first time, and the other surface at a second time. The focus of the fluorescence imaging module can be changed after one of these surfaces has been imaged to image the other surface with comparable optical resolution, since the images of the two surfaces are not focused simultaneously. In some designs, optical compensation elements can be introduced into the optical path between the sample carrier structure and the image sensor to image one of the two surfaces. In such fluorescence imaging configurations, the depth of field may not be large enough to include both the first and second surfaces. In some embodiments of the fluorescence imaging modules described herein, both the first and second surfaces can be imaged simultaneously. For example, the fluorescence imaging module can have a sufficiently large depth of field to include both surfaces. In some cases, this increased depth of field can be provided, for example, by reducing the numerical aperture of the objective lens (or microscope objective), which will be discussed in more detail below.

[0113] like Figure 1A and Figure 1B As shown, imaging optics (e.g., an objective lens) can be positioned at a suitable 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. Figure 1A and Figure 1B As illustrated, a first surface may be located between the objective lens and the second surface. For example, as shown, the objective lens is disposed over both the first and second surfaces, and the first surface is disposed over the second surface. The first and second surfaces are, for example, at different depths. The first and second surfaces are located at different distances from any one or more of the fluorescence imaging module, illumination and imaging module, imaging optics, or objective lens. The first and second surfaces are separated from each other, with the first surface spaced apart above the second surface. In the illustrated example, the first and second surfaces are planar and separated from each other along directions perpendicular to the first and second planes. Similarly, in the illustrated example, the objective lens has an optical axis, and the first and second surfaces are separated from each other along the direction of the optical axis. Likewise, the spacing between the first and second surfaces may correspond to a longitudinal distance, such as along the optical path of the excitation beam and / or along the optical axis passing through the fluorescence imaging module and / or the objective lens. Thus, the two surfaces may be separated from each other by a distance in a longitudinal (Z) direction, which may be along the central axis of the excitation beam and / or the direction of the optical axis of the objective lens and / or the fluorescence imaging module. In some embodiments, this spacing may, for example, correspond to a flow channel within a flow cell.

[0114] In various designs, the objective lens (possibly combined with another optical component, such as a barrel lens) has a depth of field and / or depth of focus that is at least as large as the longitudinal spacing (along the Z direction) between the first and second surfaces. Therefore, the objective lens can simultaneously form focused images of both the first and second surfaces on an image sensor of one or more detection channels, either alone or in combination with other optical components, where these images have comparable optical resolution. In some embodiments, the imaging module may or may not require refocusing to capture images of both the first and second surfaces with comparable optical resolution. In some embodiments, compensation optics do not need to move into or out of the optical path of the imaging module to form focused images of the first and second surfaces. Similarly, in some embodiments, one or more optical elements (e.g., lens elements) in the imaging module (e.g., the objective lens and / or barrel lens) do not need to move longitudinally, for example, 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 said one or more optical elements when forming a focused image of the second surface. However, in some embodiments, the imaging module includes an autofocus system configured to simultaneously focus the first and second surfaces. In various embodiments, the sample is focused to adequately resolve sample sites that are closely spaced together in the lateral directions (e.g., the X and Y directions). Therefore, in various embodiments, in at least one detection channel, no optical element enters the optical path between the sample carrier structure (e.g., between translation stages supporting the sample carrier structure) and the image sensor (or photodetector array) in at least one detection channel to form a focused image of the fluorescent sample sites on the first and second surfaces of the sample carrier structure. Similarly, in various embodiments, no optical compensation is provided for forming a focused image of the fluorescent sample sites on the first surface of the sample carrier structure on the image sensor or photodetector array, which differs from the optical compensation provided for forming a focused image of the fluorescent sample sites on the second surface of the sample carrier structure on the image sensor or photodetector array. In addition, in some embodiments, compared with the focused image of the fluorescent sample sites on the second surface of the sample carrier structure, no optical elements in the optical path between the sample carrier structure (e.g., between translation stages supporting the sample carrier structure) and the image sensor in at least one detection channel are differently adjusted to form a focused image of the fluorescent sample sites on the first surface of the sample carrier structure.Similarly, in some various embodiments, compared to a focused image of a fluorescent sample site on a second surface of the sample carrier structure formed on an image sensor, a focused image of a sample site on a first surface of the sample carrier structure formed on an image sensor is obtained without any optical element moving by a different amount or direction in the optical path between the sample carrier structure (e.g., between translation stages supporting the sample carrier structure) and the image sensor in at least one detection channel. Any combination of features is possible. For example, in some embodiments, focused images of the upper and lower inner surfaces of the flow cell can be obtained without moving an optical compensator into 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., objectives and / or barrel lenses) along the optical path (e.g., optical axis) therebetween. For example, focused images of the upper and lower inner surfaces of the flow cell can be obtained without moving one or more optical elements of the barrel lens into or out of the optical path or without moving one or more optical elements of the barrel lens along the optical path (e.g., optical axis) therebetween.

[0115] Any one or more of the fluorescence imaging module, illumination path, imaging path, objective lens, or barrel lens can be designed to reduce or minimize optical aberrations at two locations (e.g., two planes corresponding to two surfaces on a flow cell or other sample carrier structure, such as where the fluorescent sample site is located). Any one or more of the fluorescence imaging module, illumination path, imaging path, objective lens, or barrel lens can be designed to reduce or minimize optical aberrations at selected locations or planes relative to other locations or planes, such as the first and second surfaces containing the fluorescent sample site on a double-surface flow cell. For example, any one or more of the fluorescence imaging module, illumination path, imaging path, objective lens, or barrel lens can be designed to reduce or minimize optical aberrations at two depths or 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, the optical aberrations used for imaging the first and second surfaces can be smaller than elsewhere in a region ranging from about 1 mm to about 10 mm from the objective lens. Additionally, in some cases, any one or more of the fluorescence imaging module, illumination path, imaging path, objective lens, or tube lens may be configured to compensate for optical aberrations caused by transmission of emitted light through one or more portions of the sample carrier structure (e.g., a layer including one of the surfaces to which the sample is attached and a solution that may be in contact with the sample). This layer (e.g., a coverslip or the wall of a flow cell) may comprise, for example, glass, quartz, plastic, or other transparent materials having a refractive index that introduces optical aberrations.

[0116] Therefore, the imaging performance can be substantially the same when imaging the first and second surfaces. For example, the optical transfer function (OTF) and / or modulation transfer function (MTF) can be substantially the same for imaging the first and second surfaces. One or both of these transfer functions, when averaged at one or more specified spatial frequencies or over a series of spatial frequencies, can be, for example, within 20%, 15%, 10%, 5%, 2.5%, or 1% of each other, or any range formed by any of these values. Therefore, the imaging performance metrics can be substantially the same for imaging the upper or lower inner surface of the flow cell without moving the optical compensator into 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., objective lenses and / or barrel lenses) along the optical path (e.g., optical axis) therebetween. For example, the imaging performance metrics can be substantially the same for imaging the upper or lower inner surface of the flow cell without moving one or more optical elements of the barrel lens into or out of the optical path or along the optical path (e.g., optical axis) therebetween. Further discussion of the MTF is contained below and in U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020, the entire contents of which are incorporated herein by reference.

[0117] Those skilled in the art will understand that, in some cases, the disclosed imaging modules or systems may be stand-alone optical systems designed for imaging sample or matrix surfaces. In some cases, they may include one or more processors or computers. In some cases, they may include one or more software packages providing instrument control and / or image processing functions. In some cases, in addition to optical components such as light sources (e.g., solid-state lasers, dye lasers, diode lasers, arc lamps, halogen tungsten lamps, etc.), lenses, prisms, mirrors, dichroic reflectors, beam splitters, optical filters, bandpass 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 optomechanical components such as XY translation stages, XYZ translation stages, piezoelectric focusing mechanisms, electro-optic phase plates, etc. In some cases, they may serve as modules, components, sub-components, or subsystems of larger systems designed for applications such as genomics (e.g., gene testing and / or nucleic acid sequencing applications). For example, in some cases, they can serve as modules, components, subcomponents, or subsystems of larger systems, which also include opaque and / or other environmental control enclosures, temperature control modules, flow cells and cassettes, fluid control modules, fluid dispensing robots, cassette and / or microplate handling (pick-and-place) robots, 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 related software), display modules, etc., or any combination thereof. These additional components of larger systems (e.g., systems designed for genomics applications) will be discussed in more detail below.

[0118] Figure 2A and Figure 2B A non-limiting example of an illumination and imaging module 100 for multichannel fluorescence imaging is shown. 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 or a beam splitter. In some designs, an autofocusing system may be included, which may include an autofocusing laser 102, for example, projecting a spot whose size is monitored to determine when the imaging system focuses. Some or all components of the illumination and imaging module 100 may be coupled to a substrate 105.

[0119] Illumination or light source 115 may include any suitable light source configured to produce light at least the desired excitation wavelength (discussed in more detail below). The light source may be a broadband light source emitting light within one or more excitation wavelength ranges (or bands). The light source may be a narrowband light source emitting light within one or more narrower wavelength ranges. In some cases, the light source may produce a single isolated wavelength (or line) or multiple isolated wavelengths (or lines) corresponding to the desired excitation wavelength. In some cases, the line may have some very narrow bandwidth. Example light sources suitable for use as illumination source 115 include, but are not limited to, incandescent filaments, xenon arc lamps, mercury vapor lamps, light-emitting diodes, laser sources (e.g., laser diodes), or solid-state lasers or other types of light sources. As described below, in some designs, the light source may include a polarized light source, such as a linearly polarized light source. In some embodiments, the light source is oriented such that s-polarized light is incident on one or more surfaces of one or more optical components, such as the dichroic reflective surface of one or more dichroic filters.

[0120] The illumination source 115 may also include one or more additional optical components, such as lenses, filters, optical fibers, or any other suitable transmissive or reflective optics, to output an excitation beam with suitable characteristics to the first dichroic filter 130. For example, beam-shaping optics may be included to receive light from a light emitter in the light source and generate a beam and / or provide desired beam characteristics. Such optics may, for example, include a collimating lens configured to reduce light divergence and / or increase collimation and / or collimate the light.

[0121] In some embodiments, the illumination and imaging module 100 includes multiple light sources. In some such embodiments, different light sources can produce light with different spectral characteristics, for example, to excite different fluorescent dyes. In some embodiments, light produced by different light sources can be guided to overlap and form a converged excitation beam. This composite excitation beam can be composed of excitation beams from each light source. Compared to a single beam that overlaps to form a composite beam, the composite excitation beam will have greater optical power. For example, in some embodiments including two light sources that produce two excitation beams, the composite excitation beam formed by the two separate excitation beams can have an optical power that is the sum of the optical powers of the individual beams. Similarly, in some embodiments, three, four, five, or more light sources can be included, and these light sources can each output an excitation beam, which together form a composite beam having an optical power equal to the sum of the optical powers of the individual beams.

[0122] In some embodiments, the light source 115 outputs a sufficiently large amount of light to produce sufficiently strong fluorescence emission. Stronger fluorescence emission can increase the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the image acquired by the fluorescence imaging module. In some embodiments, the power output of the light source and / or the excitation beam (including composite excitation beams) obtained therefrom can be in the range of about 0.5W to about 5.0W, or greater (as will be discussed in more detail below).

[0123] Refer again Figure 2A and Figure 2B A first dichroic filter 130 is arranged relative to a light source to receive light therefrom. 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 extending from green to red and infrared wavelengths. Other spectral regions or wavelength ranges are also possible.

[0124] In some embodiments, a first dichroic filter may be configured to transmit light from a light source to a sample carrier structure, such as a microscope slide, capillary, flow cell, microfluidic chip, or other matrix or carrier structure. The sample carrier structure supports and positions a sample, such as a composition containing fluorescently labeled nucleic acid molecules or their complementary sequences, relative to the illumination and imaging module 100. Thus, a first optical path extends from the light source to the sample via the first dichroic filter. In various embodiments, the sample carrier structure includes at least one surface on which a sample is disposed or attached. In some cases, the sample may be disposed in or attached to different local regions or sites on at least one surface of the sample carrier structure.

[0125] In some cases, the carrier structure may include two surfaces at different distances from the objective lens 110 (i.e., at different positions or depths along the optical axis of the objective lens 110), with the sample disposed on the two surfaces. As described below, for example, the flow cell may include fluid channels at least partially formed by first and second (e.g., upper and lower) inner surfaces, and the sample may be disposed at a localized site on the first inner surface, the second inner surface, or both inner surfaces. The first and second surfaces may be separated by regions corresponding to the fluid channels through which the solution flows, and thus at different distances or depths relative to the objective lens 110 of the illumination and imaging module 100.

[0126] Objective 110 may be included in a first optical path between the first dichroic filter and the sample. The objective may be configured, for example, to have a focal length, working distance, and / or be positioned to focus light from a light source onto the sample, such as the surface of a microscope slide, capillary, flow cell, microfluidic chip, or other matrix or carrier structure. Similarly, objective 110 may be configured to have a suitable focal length, working distance, and / or be positioned to collect light reflected, scattered, or emitted from the sample (e.g., fluorescence emission) and form an image of the sample.

[0127] (e.g., fluorescence images).

[0128] In some embodiments, objective 110 may include a microscope objective, such as an off-the-shelf objective. In some embodiments, objective 110 may include a custom objective. Examples of custom objectives and / or custom objective-tube lens combinations are described below and in U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020 (the entire contents of which are incorporated herein by reference). Objective 110 may be designed to reduce or minimize optical aberrations at two locations, such as two planes corresponding to two surfaces of a flow cell or other sample carrier structure. Objective 110 may be designed to reduce optical aberrations at selected locations or planes (e.g., the first and second surfaces of a dual-surface flow cell) relative to other locations or planes in the optical path. For example, objective 110 may be designed to reduce optical aberrations at two depths or planes located at different distances from the objective compared to optical aberrations associated with other depths or planes located at other distances from the objective. For example, in some cases, the optical aberrations of the first and second surfaces used for imaging the flow cell can be smaller than those elsewhere in a region spanning 1 to 10 mm from the front surface of the objective lens. Additionally, the custom objective lens 110 can be configured in some cases to compensate for optical aberrations caused by the transmission of fluorescence emission light through one or more portions of the sample carrier structure (e.g., a layer containing one or more flow cell surfaces on which the sample is disposed, or a layer containing a solution filling the fluid channels of the flow cell). These layers can include, for example, glass, quartz, plastic, or other transparent materials with a refractive index that can introduce optical aberrations.

[0129] In some embodiments, objective lens 110 may have a numerical aperture (NA) of 0.6 or greater (discussed in more detail below). Such a numerical aperture can provide reduced depth of focus and / or depth of field, improved background resolution, and increased imaging resolution.

[0130] In some embodiments, objective lens 110 may have a numerical aperture (NA) of 0.6 or less (discussed in more detail below). Such a numerical aperture can provide increased depth of focus and / or depth of field. This increased depth of focus and / or depth of field can improve the ability to image planes that are spaced apart, for example, the distance separating the first and second surfaces of a bisurface flow cell.

[0131] As described above, the flow cell may include, for example, a first layer and a second layer, each comprising a first inner surface and a second inner surface, separated by a fluid channel through which an analyte or reagent can flow. In some embodiments, the objective lens 110 and / or the illumination and imaging module 100 may be configured to provide a sufficiently large depth of field and / or depth of focus to image the first and second inner surfaces of the flow cell sequentially (by refocusing the imaging module between the first and second surfaces) or simultaneously (by ensuring a sufficiently large depth of field and / or depth of focus) with comparable optical resolution. In some cases, the depth of field and / or depth of focus may be at least equal to or greater than the distance between the first and second surfaces of the flow cell to be imaged (e.g., the first and second inner surfaces of the flow cell). In some cases, the first and second surfaces, such as the first and second inner surfaces of a dual-surface flow cell or other sample carrier structure, may be separated by a distance ranging from about 10 μm to about 700 μm or greater (as will be discussed in more detail below). Therefore, in some cases, the depth of field and / or depth of focus can range from about 10 μm to about 700 μm, or even greater (as will be discussed in more detail below).

[0132] In some designs, compensating optics (e.g., "optical compensator" or "compensator") can be moved into or out of the optical path of the imaging module, for example, through which light collected by objective 110 is transmitted to an image sensor to enable the imaging module to image the first and second surfaces of the bisurface flow cell. The imaging module can be configured to image, for example, the first surface when the compensating optics are included in the optical path between the objective and an image sensor or photodetector array configured to capture an image of the first surface. In such a design, the imaging module can be configured to image the second surface when the compensating optics are removed or not included in the optical path between objective 110 and an image sensor or photodetector array configured to capture the second surface. The need for an optical compensator may be more pronounced when using an objective 110 with a high numerical aperture (NA) value (e.g., for 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 higher). In some embodiments, the optical compensation optics (e.g., optical compensators or compensators) include refractive optical elements (e.g., lenses), optically transparent material plates (e.g., glass), or quarter-wavelength or half-wavelength plates in the case of polarized beams, etc. Other configurations can be employed to enable the first and second surfaces to image at different times. For example, one or more lenses or optical elements can be configured to move in and out of or translate along the optical path between the objective lens 110 and the image sensor.

[0133] However, in some designs, objective 110 is configured to provide a sufficiently large depth of focus and / or depth of field to enable the first and second surfaces to be imaged at comparable optical resolution without requiring such compensating optics to move in and out of the optical path within the imaging module, such as the optical path between the objective and the image sensor or photodetector array. Similarly, in various designs, objective 110 is configured to provide a sufficiently large depth of focus and / or depth of field to enable the first and second surfaces to be imaged at comparable optical resolution without requiring the movement of optics, such as translating one or more lenses or other optical components along the optical path within the imaging module (e.g., the optical path between the objective and the image sensor or photodetector array). Examples of such objectives will be described in more detail below.

[0134] In some embodiments, the objective lens (or microscope objective) 110 may be configured to have a reduced magnification. The objective lens 110 may be configured, for example, to give the fluorescence imaging module a magnification of less than 2x to less than 10x (as will be discussed in more detail below). This reduced magnification can alter design constraints, allowing for the implementation of other design parameters. For example, the objective lens 110 may also be configured to give the fluorescence imaging module a large field of view (FOV), ranging, for example, from about 1.0 mm to about 5.0 mm (e.g., in terms of diameter, width, length, or longest dimension), as will be discussed in more detail below.

[0135] In some embodiments, objective lens 110 may be configured to provide the fluorescence imaging module with the field of view as described above, such that the FOV has diffraction-limited performance, for example, aberrations of less than 0.15 waves in at least 60%, 70%, 80%, 90%, or 95% of the field of view, as will be discussed in more detail below.

[0136] In some embodiments, objective lens 110 may be configured to provide the fluorescence imaging module with the field of view as described above, such that the FOV has diffraction-limited performance, for example, a Strell ratio greater than 0.8 in at least 60%, 70%, 80%, 90%, or 95% of the field of view, as will be discussed in more detail below.

[0137] Refer again Figure 2A and 2B A first dichroic beam splitter or beam combiner is arranged 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 beam combiner is also in one or more second optical paths from the sample to different optical channels for detecting fluorescence emission. Therefore, the first dichroic filter 130 couples the first optical path of the excitation beam emitted by the illumination source 115 and the second optical path of the emitted light emitted by the sample to respective optical channels, in which the light is guided to a corresponding image sensor or photodetector array for capturing an image of the sample.

[0138] In various embodiments, the first dichroic filter 130 (e.g., a first dichroic reflector, beam splitter, or beam combiner) has a passband selected to transmit light from the illumination source 115 only within a specified wavelength band or potentially within multiple wavelength bands (including one or more desired excitation wavelengths). For example, the first dichroic beam splitter 130 includes a reflective surface with a dichroic reflector having a spectral transmittance response, i.e., configured to transmit light of at least some wavelengths emitted by the light source, which forms part of the excitation beam. The spectral transmittance response may be configured not to transmit (e.g., but reflect) light of one or more other wavelengths, such as light of one or more other fluorescent emission wavelengths. In some embodiments, the spectral transmittance response may also be configured not to transmit (e.g., but reflect) light of one or more other wavelengths emitted by the light source. Thus, the first dichroic filter 130 can be used to select which wavelengths or wavelengths of light emitted by the light source reach the sample. Conversely, the dichroic reflector in the first dichroic beam splitter 130 has a spectral reflectivity response that reflects light having one or more wavelengths corresponding to desired fluorescence emission from the sample, and may reflect light from the light source having one or more wavelengths not intended to reach the sample. Thus, in some embodiments, the dichroic reflector has a spectral transmittance comprising one or more passbands for transmitting light to be incident on the sample; and one or more stopbands that reflect light outside the passbands, such as light of one or more emission wavelengths, and possible one or more wavelengths of light from the light source not intended to reach the sample. Similarly, in some embodiments, the dichroic reflector has a spectral reflectivity comprising one or more spectral regions (configured to reflect one or more emission wavelengths and possible one or more wavelengths of light from the light source not intended to reach the sample), and including one or more regions that transmit light outside these reflective 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-wavelength stack) configured to provide an appropriate spectral transmittance and reflectance distribution. Figure 2A and Figure 2B A dichroic filter 105 is also shown, which may include, for example, a dichroic beam splitter or beam combiner, which can be used to guide the autofocusing laser 102 through the objective lens and to the sample carrier structure.

[0139] although Figure 2A and Figure 2BThe imaging module 100 shown and described above is configured such that the excitation beam is transmitted through the first dichroic filter 130 to the objective lens 110. However, in some designs, the illumination source 115 may be arranged 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 fluorescence emission from the sample and may transmit light of one or more wavelengths from the light source output that is not intended to reach the sample. As will be discussed below, the design of transmitting rather than reflecting fluorescence emission can potentially reduce wavefront errors in detected emission and / or may have other advantages. In any case, in various embodiments, the first dichroic reflector 130 is arranged in a second optical path to receive fluorescence emission from the sample, of which at least some continues to reach the detection channel 120.

[0140] Figure 3A and 3B It shows Figure 2A and 2B The optical path within the multi-channel fluorescence imaging module. Figure 2A and Figure 3A In the example shown, detection channel 120 is arranged to receive fluorescence emission from a sample, which is transmitted through objective lens 110 and reflected by first dichroic filter 130. As mentioned above and described in more detail below, in some designs, detection channel 120 may be arranged to receive a portion of the emitted light transmitted rather than reflected by the first dichroic filter. In either case, detection channel 120 may include optics for receiving at least a portion of the emitted light. For example, detection channel 120 may include one or more lenses, such as a barrel lens, and may include one or more image sensors or detectors, such as a photodetector array (e.g., a CCD or CMOS sensor array) for imaging or otherwise generating signals based on the received light. The barrel lens may, for example, include one or more lens elements configured to form an image of the sample onto the sensor or photodetector array to capture its image. Further discussion of detection channels is contained below and in U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020, the entire contents of which are incorporated herein by reference. In some cases, by combining appropriate sampling schemes (including oversampling or undersampling), improved optical resolution can be achieved using image sensors with relatively high sensitivity, small pixels, and high pixel count.

[0141] Figure 3A and 3B It is shown Figure 2A and 2BRay tracing diagram of the optical path of the illumination and imaging module 100. Figure 3A Top view corresponding to the lighting and imaging module 100. Figure 3B The figures show a side view corresponding to 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 module can be equivalently implemented in systems including more or fewer than four detection channels 120. For example, without departing from the spirit or scope of this disclosure, the multi-channel system disclosed herein can be implemented with as few as one detection channel 120, or as many as two, three, four, five, six, seven, eight, or more detection channels 120.

[0142] Figure 3A and 3B A non-limiting example of the imaging module 100 shown includes four detection channels 120, a first dichroic filter 130 (which reflects the emitted beam 150), a second dichroic filter (e.g., a dichroic beam splitter) 135 (which splits the beam 150 into a transmission portion and a reflection portion), and two channel-specific dichroic filters (e.g., dichroic beam splitters) 140 (which further separate the transmission and reflection portions of the beam 150 between the respective detection channels 120). The dichroic reflective surfaces in the dichroic beam splitters 135 and 140 for separating the beam 150 between the detection channels are shown arranged at a 45-degree angle relative to the central beam axis of the beam 150 or the optical axis of the imaging module. However, as described below, angles less than 45 degrees can be used, and advantages such as a steeper transition from the passband to the stopband can be provided.

[0143] Different detection channels 120 include imaging devices 124, which may include image sensors or photodetector arrays (e.g., CCD or CMOS detector arrays). Different detection channels 120 also include optics 126, such as lenses (e.g., one or more barrel lenses, each including one or more lens elements), arranged to focus a portion of the emitted light entering the detection channel 120 onto a focal plane coinciding with the plane of the photodetector array 124. The optics 126 (e.g., barrel lenses) combined with the objective lens 110 are configured to form an image of the sample on the photodetector array 124 to capture an image of the sample, for example, an image of the surface after the sample has been attached to a flow cell or other sample carrier structure. Thus, such an image of the sample may include multiple fluorescence emission points or regions within the spatial extent of the sample carrier structure in which the sample is emitting fluorescence. Together with the optics 126 (e.g., barrel lenses), the objective lens 110 can provide a field of view (FOV) including a portion or the entire sample. Similarly, the photodetector array 124 of different detection channels 120 can be configured to capture an image of the full field of view (FOV) provided by the objective lens and the lens barrel, or a portion thereof. In some embodiments, the photodetector array 124 of some or all detection channels 120 can detect emitted light emitted by a sample disposed on a sample carrier structure, such as the surface of a flow cell, or a portion thereof, and record electronic data representing its image. In some embodiments, the photodetector array 124 of some or all detection channels 120 can detect features in the emitted light emitted by the sample without capturing and / or storing an image of the sample disposed on the surface of the flow cell and / or an image of the full field of view (FOV) provided by the objective lens and optics 126 and / or 122 (e.g., elements of the lens barrel). In some embodiments, the FOV of the disclosed imaging module (e.g., provided by a combination of objective lens 110 and optics 126 and / or 122) can be in the range of, for example, about 1 mm to 5 mm (e.g., in terms of diameter, width, length, or longest dimension), as described below. The field of view (FOV) can 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 instance, a smaller and faster imaging sensor can be combined to provide a relatively small FOV for high throughput.

[0144] Refer again Figure 3A and 3BIn some embodiments, the optics 126 (e.g., a telescope lens) in the detection channel can be configured to reduce optical aberrations in images acquired using the optics 126 in conjunction with the objective lens 110. In some embodiments including multiple detection channels for imaging at different emission wavelengths, the optics 126 (e.g., telescope lenses) for different detection channels have different designs to reduce aberrations at the respective emission wavelengths for which that particular channel is configured for imaging. In some embodiments, the optics 126 (e.g., a telescope lens) can be configured to reduce aberrations compared to other locations (e.g., other planes in object space) when imaging a specific surface (e.g., a plane, object plane, etc.) on a sample carrier structure including a fluorescent sample disposed thereon. Similarly, in some embodiments, the optics 126 (e.g., a telescope lens) can be configured to reduce aberrations compared to other locations (e.g., other planes in object space) when imaging a first surface and a second surface (e.g., a first plane and a second plane, a first object plane and a second object plane, etc.) on a dual-surface sample carrier structure (e.g., a dual-surface flow cell) having fluorescent sample sites disposed thereon. For example, the optics 126 (e.g., a barrel lens) in the detection channel can be designed to reduce aberrations at two depths or 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, the optical aberrations for imaging the first and second surfaces can be smaller compared to other areas in a region from about 1 mm to about 10 mm from the objective lens. Additionally, in some embodiments, the custom optics 126 (e.g., a barrel lens) in the detection channel can be configured to compensate for aberrations caused by transmission of emitted light through one or more portions of the sample carrier structure, such as a layer including one of the surfaces on which the sample is disposed and a solution possibly adjacent to and in contact with the surface on which the sample is disposed. The layer including one of the surfaces on which the sample is disposed can include, for example, glass, quartz, plastic, or other transparent materials having a refractive index and introducing optical aberrations. For example, in some embodiments, the custom optics 126 (e.g., a barrel lens) in the detection channel can be configured to compensate for optical aberrations caused by the sample carrier structure (e.g., a coverslip or flow cell wall) or other sample carrier structure components and a solution possibly adjacent to and in contact with the surface on which the sample is disposed.

[0145] In some embodiments, the optics 126 (e.g., a telescope lens) in the detection channel are configured to have a reduced magnification. The optics 126 (e.g., a telescope lens) in the detection channel may be configured, for example, such that the fluorescence imaging module has a magnification of less than, for example, 10 times, as will be discussed further below. This reduced magnification can alter design constraints, allowing for the implementation of other design parameters. For example, the optics 126 (e.g., a telescope lens) may also be configured to give the fluorescence imaging module a large field of view (FOV), for example, at least 1.0 mm or greater (e.g., in terms of diameter, width, length, or longest dimension), as will be discussed further below.

[0146] In some embodiments, the optics 126 (e.g., a barrel lens) may be configured to provide the fluorescence imaging module with the field of view as described above, such that the FOV has an aberration of less than 0.15 waves in at least 60%, 70%, 80%, 90%, or 95% of the field of view, as will be discussed further below.

[0147] Refer again Figure 3A and 3B In various embodiments, the sample is located at or near the focal point 112 of the objective lens 110. (See above reference...) Figure 2A and 2B The light source, such as a laser source, provides an excitation beam to the sample to induce fluorescence. Objective lens 110 collects at least a portion of the emitted fluorescence as emitted light. Objective lens 110 transmits the emitted light toward a first dichroic filter 130, which reflects some or all of the emitted light as a beam 150, which is incident on a second dichroic filter 135 and reaches different detection channels. Each detection channel includes an optics 126 that forms an image of the sample (e.g., multiple fluorescent sample sites on the surface of a sample carrier structure) on a photodetector array 124.

[0148] As described above, in some embodiments, the sample carrier structure includes a flow cell, such as a dual-surface flow cell, having two surfaces (e.g., two inner surfaces, a first surface and a second surface, etc.) containing sample sites for emitting fluorescence. These two surfaces may be separated from each other in the longitudinal (Z) direction along the central axis of the excitation beam and / or the optical axis of the objective lens. This separation may, for example, correspond to a flow channel within the flow cell. An analyte or reagent may flow through this flow channel and contact the first and second inner surfaces of the flow cell, thereby contacting it with the binding composition, causing fluorescence emission from multiple sites on the first and second inner surfaces. Imaging optics (e.g., objective lens 110) may be positioned at a suitable 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 described above, in various designs, objective lens 110 (possibly in combination with optics 126) may have a depth of field and / or depth of focus at least as large as the longitudinal spacing between the first and second surfaces. Therefore, the objective lens 110 and optics 126 (of each detection channel) can 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 comparable optical resolution (or can be focused with only minor refocusing of the object to obtain images of the first and second surfaces with comparable optical resolution). In various embodiments, the compensating optics do not need to move into 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 comparable optical resolution. Similarly, in various embodiments, one or more optical elements (e.g., lens elements) in the imaging module (e.g., objective lens 110 or optics 126) do not need to move longitudinally, for example, along the first and / or second optical paths to form a focused image of the first surface, compared to the position of the one or more optical elements used to form a focused image of the second surface. In some embodiments, the imaging module includes an autofocus system configured to rapidly and sequentially refocus the imaging module on the first and / or second surfaces, resulting in an image with comparable optical resolution. In some embodiments, the objective lens 110 and / or optics 126 are configured such that both the first and second flow cell surfaces are simultaneously focused with comparable optical resolution without moving the optical compensator into or out of the first and / or second optical paths, and without longitudinally moving one or more lens elements (e.g., objective lens 110 and / or optics 126 (e.g., barrel lens)) along the first and / or second optical paths.In some embodiments, images of the first and / or second surfaces acquired sequentially (e.g., by refocusing between surfaces) or simultaneously (e.g., without refocusing between surfaces) using the novel objective and / or barrel lens designs disclosed herein can be further processed using suitable image processing algorithms to enhance the effective optical resolution of the images, such that the images of the first and second surfaces have comparable optical resolution. In various embodiments, the sample plane is sufficiently focused to resolve sample sites on the first and / or second flow cell surfaces, the sample sites being closely spaced in the lateral direction (e.g., in the X and Y directions).

[0149] As described above, a dichroic filter can include an interference filter that uses optical coatings with different refractive indices and specific thicknesses to selectively transmit and reflect light of different wavelengths based on the principle of thin-film interference. Therefore, the spectral response (e.g., transmission and / or reflection spectra) of a dichroic filter implemented within a multi-channel fluorescence imaging module can depend at least in part on the incident angle or range of incident angles of the excitation beam and / or emission beam incident on the dichroic filter. The dichroic filter relative to the detection optical path (e.g., Figure 3A and Figure 3B The effect may be particularly noticeable with dichroic filters (135 and 140).

[0150] Figure 4 This is a graph showing the relationship between the performance of a dichroic filter and the incident beam angle (AOI). Specifically, Figure 4 The figure illustrates the effect of the incident angle on the transition width or spectral span of a dichroic filter, which corresponds to the wavelength range of the spectral response (e.g., transmission and / or reflection spectra) transitioning between the passband and stopband regions of the dichroic filter. Therefore, a relatively small spectral span (e.g., Figure 4 The small Δλ values ​​in the figure correspond to a steeper transition between the passband and stopband regions or between the transmission and reflection regions (or vice versa), while having a relatively large spectral span (e.g., Figure 4 The transmission edge (or reflection edge) with a large Δ_λ value in the figure corresponds to a less steep transition between the passband and stopband regions. In various implementations, a steeper transition between the passband and stopband regions is generally desirable. Furthermore, an increased or relatively uniform transition width may also be desired across all or most of the field of view and / or beam regions.

[0151] Accordingly, a fluorescence imaging module in which the dichroic mirror is arranged at a 45-degree angle to the optical axis of the emitted light's central beam axis or optical path (e.g., the optical path of the objective lens and / or barrel lens) can have a transition width of approximately 50 nm for an exemplary dichroic filter, such as in Figure 4As shown in the diagram. Because the emitted beam is not collimated and has a certain degree of divergence, the fluorescence imaging module can have an incident angle range of approximately 5 degrees between opposite sides of the beam. Therefore, as... Figure 4 As shown, different portions of the emitted beam can be incident on the dichroic filters of the sub-channels at various incident angles ranging from 40 to 50 degrees. The relatively large range of incident angles corresponds to a transition width range of approximately 40 nm to approximately 62 nm. This relatively large range of incident angles thus leads to an increase in the transition width of the dichroic filters in the imaging module. Therefore, the performance of the multi-channel fluorescence imaging module can be improved by providing a smaller incident angle across the entire beam, resulting in steeper transmission edges and better differentiation of different fluorescence emission bands.

[0152] Figure 5 This is a graph showing the relationship between the beam footprint size (DBS) and the incident beam angle (DBS angle) on a dichroic filter. In some cases, a smaller beam footprint can be desirable. For example, a smaller beam footprint allows the use of a smaller dichroic filter to split the beam into different wavelength ranges. The use of a smaller dichroic filter further reduces manufacturing costs and improves the ease of manufacturing a suitable flat dichroic filter. Figure 5 As shown, any incident angle greater than 0 degrees (perpendicular to the surface of the dichroic filter) will result in an elliptical beam footprint with an area larger than the cross-sectional area of ​​the beam. An incident angle of 45 degrees will result in a large beam footprint on the dichroic reflector, which is more than 1.4 times the cross-sectional area of ​​the beam when incident at 0 degrees.

[0153] Figure 6A and Figure 6B A non-limiting example configuration of a dichroic filter and detection channel in a multi-channel fluorescence imaging module is schematically shown, wherein the dichroic filter is arranged at an angle of less than 45 degrees relative to the optical axis of the central beam axis of the emitted light or the optical axis of the optical path (e.g., the optical path of the objective lens and / or the barrel lens). Figure 6A An imaging module 500 is described, comprising multiple detection channels 520a, 520b, 520c, and 520d. Figure 6B Is it like this? Figure 6A A detailed view of the imaging module 500 within circle 5B shown. As will be described in more detail... Figure 6A and Figure 6B The configurations shown include many aspects that can lead to significant improvements in the design of conventional multichannel fluorescence imaging modules. However, in some cases, without departing from the spirit or scope of this disclosure, the fluorescence imaging modules and systems of this disclosure can be used with respect to... Figure 6A and Figure 6B It can be implemented using one or a subset of the described features.

[0154] Figure 6A The imaging module 500 depicted includes an objective lens 510 and four detection channels 520a, 520b, 520c, and 520d, which are arranged to receive emitted light transmitted by the objective lens 510 and / or image it. A first dichroic filter 530 is provided to connect the excitation and detection optical paths. Figure 2A and Figure 2B as well as Figure 3A and Figure 3B In contrast to the design shown, a first dichroic filter 530 (e.g., a dichroic beam splitter or beam combiner) is configured to reflect light from the light source to the objective lens 510 and the sample, and to transmit fluorescence emission from the sample to detection channels 520a, 520b, 520c, and 520d. A second dichroic filter 535 splits the emitted beam between at least two detection channels 520a, 520b by transmitting a first portion 550a and reflecting a second portion 550b. Additional dichroic filters 540a, 540b are provided to further split the emitted light. Dichroic filter 540a transmits at least a portion of the first portion 550a of the emitted light and reflects a portion 550c to the third detection channel 520c. Dichroic filter 540b transmits at least a portion of the second portion 550b of the emitted light and reflects a portion 550d to the fourth detection channel 520d. Although the imaging module 500 is depicted as having four detection channels, in various embodiments, the imaging module 500 may include more or fewer detection channels and have a correspondingly larger or smaller number of dichroic filters to appropriately provide a portion of the emitted light to each detection channel. For example, in some embodiments, the features of the imaging module 500 can be achieved with similar advantageous effects in a simplified imaging module comprising only two detection channels 520a, 520b and omitting the additional dichroic filters 540a, 540b. In some embodiments, only one detection channel may be included. Alternatively, three or more detection channels may be employed.

[0155] Figure 6A The detection channels 520a, 520b, 520c, and 520d shown may include... Figures 2A-3B Some or all of the same or similar components are present in the detection channels 120 shown. For example, different detection channels 520a, 520b, 520c, 520d may include one or more image sensors or photodetector arrays and may include transmissive optics and / or reflective optics, such as one or more lenses (e.g., barrel lenses), which focus the light received by the detection channel onto their respective image sensors or photodetector arrays.

[0156] Objective lens 510 is arranged to receive emitted light from the sample by fluorescence. Specifically, a first dichroic filter 530 is arranged to receive the emitted light collected and transmitted by objective lens 510. As discussed above and in... Figure 6A As shown, in some designs, lighting sources such as laser sources are arranged (e.g., Figure 2A and Figure 2B An illumination source 115 is used to provide an excitation beam incident on the first dichroic filter 530, such that the first dichroic filter 530 reflects the excitation beam into the same objective lens 510 that transmits the emitted light, for example, in an epifluorescence configuration. In some other designs, the illumination source can be guided to the sample via other optical components along a different optical path that does not include the same objective lens 510. In such a configuration, the first dichroic filter 530 can be omitted.

[0157] Similarly, as discussed above and in Figure 6A As shown, the detection optics (e.g., including 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 dichroic filters 535, 540a, 540b) can be arranged in the transmission path of the first dichroic filter 530, rather than in the reflection path of the first dichroic filter 530. In an exemplary embodiment, the objective lens 510 and the detection optics are arranged such that the objective lens 510 transmits the emitted light beam 550 directly toward the second dichroic filter 535. The presence of the first dichroic filter 530 along the path of the emitted light beam 550 may slightly reduce the wavefront quality of the emitted light (e.g., by introducing some wavefront error into the beam 550). However, the wavefront error introduced by the beam transmitted through the dichroic reflector of the dichroic beam splitter is typically much smaller (e.g., an order of magnitude smaller) than the wavefront error of the beam reflected from the dichroic reflective surface of the dichroic beam splitter. Therefore, by placing the detection optics along the transmitted beam path of the first dichroic filter 530 instead of along the reflected beam path, the wavefront quality of the emitted light and the subsequent imaging quality in the multi-channel fluorescence imaging module can be substantially improved.

[0158] Still referencing Figure 6AAs shown, in the detection optics of the imaging module 500, dichroic filters 535, 540a, and 540b are provided to split the emitted light beam 550 between detection channels 520a, 520b, 520c, and 520d. For example, the dichroic filters 535, 540a, and 540b split the beam 550 based on wavelength, such that a first wavelength or wavelength band of the emitted light can be received by the first detection channel 520a, a second wavelength or wavelength band of the emitted light can be received by the second detection channel 520b, a third wavelength or wavelength band of the emitted light can be received by the third detection channel 520c, and a fourth wavelength or wavelength band of the emitted light can be received by the fourth detection channel 520d. In some embodiments, the detection channels can receive multiple individual wavelengths or wavelength bands.

[0159] and Figure 2A and Figure 2B as well as Figure 3A and Figure 3B In contrast to the multi-channel fluorescence imaging module design shown, imaging module 500 has dichroic filters 535, 540a, and 540b, which are arranged at an incident angle of less than 45 degrees relative to the central beam axis of the incident beam. Figure 6B As shown, different beams 550, 550a, and 550b have their own central beam axes 552, 552a, and 552b. In various embodiments, the central beam axes 552, 552a, and 552b are located at the center of the cross-section of the beam orthogonal to the beam's propagation direction. These central beam axes 552, 552a, and 552b may correspond to the optical axis of the objective lens and / or to optics within separate channels, such as the optical axis of the corresponding barrel lens. Additional rays 554, 554a, and 554b of each beam 550, 550a, and 550b are... Figure 6B The figures are shown to indicate the diameter of each beam 550, 550a, 550b. The beam diameter can be defined as, for example, half the full width of the maximum diameter, D4σ (i.e., 4 times σ, where σ are the standard deviations of the horizontal or vertical edge distribution of the beam, respectively), or the second moment width, or any other suitable definition of the beam diameter.

[0160] The central beam axis 552 of the emitted beam 550 can be used as a reference point to define the angle of incidence of the beam 550 on the second dichroic filter 535. Therefore, the "angle of incidence" (AOI) of the beam 550 can be the angle between the central beam axis 552 of the incident beam 550 and a line N perpendicular to the surface into which the beam is incident (e.g., a dichroic reflective surface). When the emitted beam 550 is incident on the dichroic reflective surface of the second dichroic filter 535 at an angle of incidence AOI, the second dichroic filter 535 transmits a first portion 550a of the emitted light (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 emitted light (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 can each be described similarly with respect to the central beam axes 552a, 552b. As described above, an optical axis may be used alternatively or additionally.

[0161] exist Figure 6A and Figure 6B In this configuration, the second dichroic filter 535 is arranged such that the central beam axis 552 of the beam 550 is incident at an angle of incidence of 30 degrees. Similarly, additional dichroic filters 540a and 540b are arranged such 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 an angle of incidence of 30 degrees. However, in various embodiments, these angles of incidence can be other angles less than 45 degrees. In some cases, for example, the angles of incidence can be in the range of about 20 degrees to about 45 degrees, as will be discussed further below. Moreover, the angles of incidence on each of the dichroic filters 535, 540a, and 540b need not necessarily be the same. In some embodiments, some or all of the dichroic filters 535, 540a, and 540b can be arranged such that their incident beams 550, 550a, and 550b have different angles of incidence. As described above, the angle of incidence can be relative to the optical axis of the optics within the imaging module (e.g., the objective lens and / or the optics in the detection channel (e.g., the barrel lens)) and the dichroic reflective surface in the respective dichroic beam splitter. The same range and values ​​of the angle of incidence apply when the optical axis is used to specify the AOI.

[0162] The emitted light beams 550, 550a, and 550b in the fluorescence imaging module system are typically divergent beams. As described above, the emitted light beams can have a sufficiently large beam divergence so that a region of the beam within the beam diameter is incident on the dichroic filter at an angle of incidence differing by up to 5 degrees or more relative to the central beam axis and / or optical axis of the optics. In some designs, the objective lens 510 can be configured, for example, to have an f-number or numerical aperture, which is selected to produce a small beam diameter for a given field of view of the microscope. In one example, the f-number or numerical aperture of objective lens 510 can be selected such that the entire diameter of beams 550, 550a, 550b is incident on dichroic filters 535, 540a, 540b at an incident angle of, 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 at the incident angle of the central beam axis 552, 552a, 552b.

[0163] In some embodiments, the focal length of the objective lens suitable for producing such a narrow beam diameter can be longer than those typically used in fluorescence microscopes or imaging systems. For example, in some embodiments, the focal length of the objective lens can be in the range of 20 mm to 40 mm, as will be discussed further below. In one example, an objective lens 510 with a focal length of 36 mm can produce a beam 550 characterized by sufficiently small divergence such that light across the entire diameter of the beam 550 is incident on the second dichroic filter 535 at an angle of incidence within 2.5 degrees of the central beam axis.

[0164] Figure 7 and Figure 8 Provided to show due to Figure 6A and 6B A graph showing the improved performance of a dichroic filter by aspects of the imaging module construction (or any imaging module construction disclosed herein). Figure 7 The diagram in the middle is similar to Figure 4 The figure shows the effect of the incident angle on the transition width of the dichroic filter (e.g., the spectral span of the transmission edge). Figure 7 An example is shown in which the orientation of the dichroic filter (e.g., dichroic filters 535, 540a and 540b) and the dichroic reflective surface therein is such that the incident beam has an incident angle of 30 degrees instead of 45 degrees. Figure 7This demonstrates how a reduced incident angle significantly improves the steepness and uniformity of the transition width across the entire beam diameter. For example, while a 45-degree incident angle at the central beam axis results in a transition width range of approximately 40 nm to approximately 62 nm, a 30-degree incident angle at the central beam axis results in a transition width range of approximately 16 nm to approximately 30 nm. In this example, the average transition width decreases from approximately 51 nm to approximately 23 nm, indicating a steeper transition between the passband and stopband. Moreover, the variation in transition width across the entire beam diameter decreases from a range of 22 nm to a range of 14 nm, a reduction of nearly 40%, indicating a more uniform transition steepness across the entire beam region.

[0165] Figure 8 Further advantages can be demonstrated by selecting an appropriate f-number or numerical aperture for the objective lens to reduce beam divergence in any imaging module configuration disclosed herein. In some embodiments, a longer focal length is used. Figure 8 In the example, objective lens 510 has a focal length of 36 mm and an appropriate numerical aperture (e.g., less than 5), reducing the range of incident angles within beam 550 from 30° ± 5° to 30° ± 2.5°. With this design, the range of the transition width can be reduced to approximately 19 nm to approximately 26 nm. When compared with… Figure 7 When compared with the improved system, although the average transition width is essentially the same (e.g., spectral span of approximately 23 nm), the transition width variation across the entire beam diameter is further reduced to the 7 nm range, indicating a significant improvement compared to the improved system. Figure 4 The transition width range shown has been reduced by nearly 70%.

[0166] Refer again Figure 5 Reducing the angle of incidence from 45 degrees to 30 degrees at the central beam axis is further advantageous because it reduces the beam spot size on the dichroic filter. For example, in Figure 5 In the dichroic filters, an incident angle of 45 degrees results in a beam footprint area on the dichroic filter that is more than 1.4 times the beam cross-sectional area. However, an incident angle of 30 degrees results in a beam footprint area on the dichroic filter that is only about 1.15 times the beam cross-sectional area. Therefore, reducing the incident angle at dichroic filters 535, 540a, and 540b from 45 degrees to 30 degrees reduces the beam footprint area on the dichroic filters 535, 540a, and 540b by approximately 18%. This reduction in beam footprint area allows for the use of smaller dichroic filters.

[0167] Now for joint reference Figure 9A-B, reducing the incident angle from 45 degrees to 30 degrees also provides improved performance regarding surface distortion caused by dichroic filters in any imaging module configuration disclosed herein, as shown by the improvement in the modulation transfer function. Typically, the amount of surface distortion increases with larger area optical elements. Using a large area on a dichroic filter results in a larger amount of surface distortion, thus introducing more wavefront error into the beam. Figure 9A The effect of the folding angle on the image quality degradation caused by adding 1 peak-valley (PV) spherical power to the last mirror is shown. Figure 9B The effect of the folding angle on the image quality degradation caused by increasing the PV spherical power by 0.1 wave to the last mirror is shown. Figure 9A and Figure 9B As shown, reducing the incident angle to 30 degrees can significantly reduce the influence of surface deformation, thereby achieving performance close to the diffraction limit of the detection optics.

[0168] In some embodiments of the disclosed imaging module, the performance of the multi-channel fluorescence imaging module disclosed herein can be further improved by utilizing the polarization state of the excitation beam. Return to Reference Figure 2A , Figure 2B and Figure 6A For example, some embodiments of the multichannel fluorescence imaging module disclosed herein have an epifluorescence configuration, wherein a first dichroic filter 130 or 530 combines the optical paths of the excitation beam and the emission beam, such that both the excitation and emission beams are transmitted through the objective lenses 110, 510. As described above, the illumination source 115 may include a light source, such as a laser or other light 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, polarizing optics are included to polarize 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. In some designs, a delayer (e.g., a half-wave delayer or multiple quarter-wave delayers or delayers with other delay amounts) may be included to rotate the linear polarization.

[0169] When a linearly polarized excitation beam is incident on any dichroic filter or other planar interface, it can be p-polarized (e.g., having an electric field component parallel to the incident plane), s-polarized (e.g., having an electric field component perpendicular to the incident plane), or a combination of p-polarized and s-polarized states within the beam. The p-polarization or s-polarization state of the excitation beam can be selected and / or varied by choosing the orientation of the illumination source 115 and / or one or more of its components relative to the first dichroic filters 130, 530 and / or relative to any other surface to which the excitation beam will interact. In some embodiments where the light source outputs linearly polarized light, the light source can 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, which can rotate about its optical axis or the central axis of the beam to orient the linearly polarized light output from it. Alternatively or additionally, a retarder may be employed to rotate the linear polarization about the optical axis or the central axis of the beam. As described above, in some embodiments, such as when the light source does not output polarized light, a polarizer arranged in the optical path of the excitation beam can polarize the excitation beam. For example, in some designs, a linear polarizer is positioned in the optical path of the excitation beam. This polarizer can be rotated to provide the appropriate orientation for linear polarization, thus providing s-polarized light.

[0170] In some designs, linear polarization is rotated about the optical axis or the central axis of the beam, so that s-polarized light is incident on the dichroic reflector of the dichroic beam splitter. When s-polarized light is incident on the dichroic reflector of the dichroic beam splitter, the transition between the passband and stopband is steeper, which is the opposite of when p-polarized light is incident on the dichroic reflector of the dichroic beam splitter.

[0171] like Figure 10A and Figure 10B As shown, the p-polarization state or s-polarization state of the excitation beam can significantly affect the narrowband performance of any excitation filter (e.g., the first dichroic filter 130, 530). Figure 10A The transmission spectra of an exemplary bandpass dichroic filter between 610 nm and 670 nm are shown at incident angles of 40 degrees and 45 degrees, wherein the incident beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. Figure 10BAs shown, changing the orientation of the light source relative to the dichroic filter causes the incident beam to be s-polarized relative to the plane of the dichroic filter, resulting in a substantially steeper edge between the passband and stopband of the dichroic filter. Therefore, the illumination and imaging modules 100, 500 disclosed herein can advantageously have an illumination source 115 oriented relative to the first dichroic filters 130, 530 such that the excitation beam is s-polarized relative to the plane of the first dichroic filters 130, 530. As described above, in some embodiments, a polarizer (such as a linear polarizer) can be used to polarize the excitation beam. This polarizer can be rotated to provide an orientation of linearly polarized light corresponding to s-polarized light. Also as described above, in some embodiments, other methods of rotating linearly polarized light can be used. For example, an optical retarder (such as a half-wave retarder or various quarter-wave retarders) can be used to rotate the polarization direction. Other arrangements are also possible.

[0172] As discussed elsewhere in this article, reducing the numerical aperture (NA) of the fluorescence imaging module and / or objectives can increase the depth of field, enabling comparable imaging of both surfaces. Figure 11A-16B This demonstrates how the MTF is more similar on the first and second surfaces separated by 1 mm glass for a smaller numerical aperture compared to a larger numerical aperture.

[0173] Figure 11A and Figure 11B It is shown on the first surface ( Figure 11A ) and the second ( Figure 11B The surface has an MTF of 0.3 for NA.

[0174] Figure 12A and Figure 12B It is shown in the first ( Figure 12A ) surface and second ( Figure 12B The surface has an MTF of 0.4 for NA.

[0175] Figure 13A and Figure 13B It is shown in the first ( Figure 13A ) surface and second ( Figure 13B The surface has an MTF of 0.5 for NA.

[0176] Figure 14A and Figure 14B It is shown in the first ( Figure 14A ) surface and second ( Figure 14B The surface has an MTF of 0.6 for NA.

[0177] Figure 15A and Figure 15B It is shown in the first ( Figure 15A ) surface and second ( Figure 15BThe surface has an MTF of 0.7 for NA.

[0178] Figure 16A and Figure 16B It is shown in the first ( Figure 16A ) surface and second ( Figure 16B The surface is at an MTF of 0.8 for NA. The first and second surfaces in each of these figures correspond, for example, the top and bottom surfaces of a flow cell.

[0179] Figure 17A -B provides a graph of the calculated Strell ratio (i.e., the ratio of the peak light intensity focused or collected by the optical system to the peak light intensity focused or collected by the ideal optical system and the point source) for imaging the surface of the second flow cell through the surface of the first flow cell. Figure 17A A graph showing the Strell ratio for imaging a second flow cell surface through a first flow cell surface as a function of the intermediate fluid layer thickness (fluid channel height) for different objective and / or optical system numerical apertures is presented. As shown, the Strell ratio decreases as the distance between the first and second surfaces increases. Therefore, as the distance between the two surfaces increases, one of these surfaces will have reduced image quality. Compared to imaging systems with larger numerical apertures, for imaging systems with smaller numerical apertures, the reduction in imaging performance of the second surface is reduced as the distance between the two surfaces increases. Figure 17B A plot of the Strell ratio as a function of numerical aperture is shown for imaging a second flow cell surface through a first flow cell surface and an intermediate water layer with a thickness of 0.1 mm. The loss of imaging performance at higher numerical apertures may be attributed to increased optical aberrations introduced by the fluid used for imaging the second surface. With increasing NA, the increased optical aberrations introduced by the fluid used for imaging the second surface significantly degrade image quality. However, generally, reducing the numerical aperture of the optical system reduces the achievable resolution. The loss of image quality can be at least partially offset by providing an increased sample-plane (or object-plane) contrast-to-noise ratio, for example, by using chemical reagents for nucleic acid sequencing applications (which enhance the fluorescence emission of labeled nucleic acid clusters and / or reduce background fluorescence emission). In some cases, for example, sample carrier structures comprising hydrophilic matrix materials and / or hydrophilic coatings can be used. In some cases, such hydrophilic matrices and / or hydrophilic coatings can reduce background noise. Further discussion of sample carrier structures, hydrophilic surfaces and coatings, and methods for enhancing the contrast-to-noise ratio (e.g., for nucleic acid sequencing applications) can be found below.

[0180] In some embodiments, any one or more of the fluorescence imaging system, illumination and imaging module 100, imaging optics (e.g., optics 126), objective lens, and / or barrel lens are configured to have a reduced magnification, such as less than 10x, as will be discussed further below. This reduced magnification can adjust design constraints to allow for the implementation of other design parameters. For example, any one or more of the fluorescence microscope, illumination and imaging module 100, imaging optics (e.g., optics 126), objective lens, or barrel lens can also be configured to give the fluorescence imaging module a large field of view (FOV), such as at least 3.0 mm or greater (e.g., in terms of diameter, width, height, or longest dimension), as will be discussed further below. Any one or more of the fluorescence imaging system, illumination and imaging module 100, imaging optics (e.g., optics 126), objective lens, and / or barrel lens can be configured to provide such a field of view to the fluorescence microscope, such that the FOV has an aberration of less than, for example, 0.1 wavelengths over at least 80% of the field of view. Similarly, any one or more of the fluorescence imaging system, illumination and imaging module 100, imaging optics (e.g., optics 126), objective lens and / or barrel lens can be configured such that the fluorescence imaging module has this FOV and is diffractively limited, or is diffractively limited at this FOV.

[0181] As described above, in various embodiments, the disclosed optical system provides a large field of view (FOV). In some embodiments, the increased FOV is partially facilitated by using a larger image sensor or photodetector array. The photodetector array may, for example, have an effective area with a diagonal of at least 15 mm or greater, as will be discussed further below. As described above, in some embodiments, the disclosed optical imaging system provides a reduced magnification, for example, less than 10x, which can facilitate a large FOV design. Despite the reduced magnification, the optical resolution of the imaging module may still be sufficient because a detector array with a small pixel size or spacing can be used. The pixel size and / or spacing may, for example, be about 5 μm or smaller, as will be discussed in more detail below. In some embodiments, the pixel size is less than twice the optical resolution provided by the optical imaging system (e.g., objectives and barrel lenses) to satisfy the Nyquist theorem. Therefore, the pixel size and / or spacing of the image sensor can allow the spatial sampling frequency of the imaging module to be at least twice the optical resolution of the imaging module. For example, the spatial sampling frequency of the photodetector array can be at least 2, at least 2.5, at least 3, at least 4, or at least 5 times the optical resolution of the fluorescence imaging module (e.g., the illumination and imaging module, the objective lens and tube lens, the objective lens and optics 126 in the detection channel, the sample carrier structure or platform (configured to support the sample carrier platform) and the imaging optics between the photodetector array), or any spatial sampling frequency within any of these values.

[0182] Although this article discusses a wide range of features for fluorescence imaging modules, any features and designs described herein can be applied to other types of optical imaging systems, including but not limited to bright-field and dark-field imaging, and can be applied to luminescent or phosphorescent imaging.

[0183] Dual-wavelength excitation / four-channel imaging system: Figure 18A dual-excitation wavelength / four-channel imaging system for two-sided imaging applications is illustrated, comprising an objective and barrel lens combination scanning in a direction perpendicular to the optical axis to provide large-area imaging, for example, by tiling multiple images to create a composite image with a total field of view (FOV) much larger than that of each individual image. The system includes two excitation sources, such as lasers or laser diodes, operating at different wavelengths, and an autofocusing laser. The two excitation beams and the autofocusing laser beam are combined using a series of mirrors and / or dichroic reflectors and then delivered through the objective to either the upper or lower inner surface of the flow cell. Fluorescence emitted by a labeled oligonucleotide (or other biomolecule) tethered to one of the flow cell surfaces is collected by the objective, transmitted through the barrel lens, and guided by a series of intermediate dichroic reflectors to one of four imaging sensors according to the wavelength of the emitted light. The autofocusing laser reflected from the flow cell surface is collected by the objective, transmitted through the barrel lens, and guided by a series of intermediate dichroic reflectors to the autofocusing sensor. The system allows the objective / tube lens combination to maintain accurate focus when scanning in a direction perpendicular to the objective 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 focus adjustment mechanism mounted on the microscope turret to reduce or minimize the size of the reflected spot on the autofocus image sensor). The combination of dual-wavelength excitation and four-channel (i.e., four-wavelength) imaging capability provides high-throughput imaging of both the upper (near) and lower (far) inner surfaces of the flow cell.

[0184] Multiplexed optical readout head:

[0185] In some cases, miniaturized versions of any imaging module described herein can be assembled to create a multiplexed readhead that can be horizontally translated relative to a sample surface (e.g., the inner surface of a flow cell) in one or more directions to simultaneously image multiple portions of the surface. A non-limiting example of a multiplexed readhead is recently described in U.S. Patent Application Publication No. 2020 / 0139375A1.

[0186] For example, in some cases, a miniaturized imaging module may include a "microfluorometer" comprising an illumination or excitation source (e.g., an LED or laser diode) (or the tip of an optical fiber connected to an external light source), one or more lenses for collimating or focusing the illumination or excitation light, one or more dichroic reflectors, one or more filters, one or more mirrors, beam splitters, prisms, apertures, etc., one or more objectives, one or more custom-designed barrel lenses (for achieving double-sided imaging with minimal focus adjustment, as described elsewhere herein), one or more image sensors, or any combination thereof, as described elsewhere herein. In some cases, the miniaturized imaging module (e.g., a "microfluorometer") may also include an autofocus mechanism, a microprocessor, power and data transmission connectors, an opaque housing, etc. The resulting miniaturized imaging module may therefore include an integrated imaging package or unit with a small form factor. In some cases, 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 cases, 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 cases, one or more individual miniaturized imaging modules within a multiplexed readhead may include an autofocus mechanism.

[0187] In some cases, the multiplexed readhead described herein may comprise an assembly 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 cases, the optical design specifications and performance attributes (e.g., numerical aperture, field of view, depth of field, image resolution, etc.) of the individual miniaturized imaging modules or microfluorometers may be identical, as described elsewhere herein for other versions of the disclosed imaging modules. In some cases, multiple individual miniaturized imaging modules may be arranged in a linear arrangement comprising 1, 2, 3, 4 rows and / or columns or more than 4 rows and / or columns. In some cases, multiple individual miniaturized imaging modules may be arranged, for example, in a hexagonal close-packed arrangement. In some cases, multiple individual miniaturized imaging modules may be arranged in a circular or spiral arrangement, a randomly distributed arrangement, or any other arrangement known to those skilled in the art.

[0188] Figure 43A -B provides a non-limiting schematic diagram of a multiplexed read head as disclosed herein. Figure 43A A side view of a multiplexed readhead is shown, in which two rows of individual microfluorimeters (viewed from the end) with common optical design specifications (e.g., numerical aperture, field of view, working distance, etc.) are configured to image a common surface (e.g., the first inner surface of the flow cell). Figure 43B A top view of the same multiplexed readhead is shown, illustrating the overlapping imaging paths acquired by the individual microfluorescence meters of the multiplexed readhead when it is translated relative to the flow cell (and vice versa). In some cases, such as Figure 43B As shown, the fields of view of each microfluorescence meter can overlap. In some cases, they may not overlap. In some cases, multiplexed readheads can be designed to align with and image predetermined features (e.g., individual fluid channels) within the flow cell.

[0189] Figure 44A -B provides a non-limiting schematic of a multiplexed readhead in which a first subset of multiple individual miniaturized imaging modules is configured to image a first sample plane (e.g., the first inner surface of a flow cell), and a second subset of multiple individual miniaturized imaging modules is configured to simultaneously image a second sample plane (e.g., the second inner surface of a flow cell). Figure 44A A side view of a multiplexed readhead is shown, wherein a first subset of the individual microfluorometers is configured to image, for example, the first inner surface or upper inner surface of a flow cell, and a second subset is configured to image a second surface (e.g., the second inner surface or lower inner surface of a flow cell). Figure 44B It shows Figure 44A A top view of the multiplexed readhead shows the imaging paths acquired by the individual microfluorometers of the multiplexed readhead. Similarly, in some cases, the fields of view of the individual microfluorometers in a given subset may overlap. In some cases, they may not overlap. In some cases, the multiplexed readhead can be designed such that the miniaturized imaging modules of the first and second subsets are aligned with and imaged against predetermined features (e.g., individual fluid channels) within the flow cell.

[0190] Objectives and / or tube lenses for improved or optimized imaging with thicker coverslips: Existing design practices include the design of objectives and / or the use of commonly available off-the-shelf microscope objectives to optimize image quality when acquiring images through thin (e.g., <200 μm thick) microscope coverslips. When used for imaging on both sides of a fluid channel or flow cell, the additional height of the gap between the two surfaces (i.e., the height of the fluid channel; typically approximately 50 μm to 200 μm) introduces optical aberrations in images captured on the non-optimal side of the fluid channel, resulting in reduced optical resolution. This is primarily because the additional gap height is significant compared to the optimal coverslip thickness (typical fluid channel or gap height is 50–200 μm, relative to a coverslip thickness <200 μm). Another common design practice is to use additional “compensating” lenses in the optical path when imaging on the non-optimal side of the fluid channel or flow cell. The “compensation” lens and the mechanism required to move it into or out of the optical path (so that both sides of the flow cell can be imaged) further increase the system complexity and imaging system downtime, and may reduce image quality due to vibration, etc.

[0191] In this disclosure, the imaging system is designed to be compatible with flow cell consumables that include thicker coverslips or flow cell walls (thickness ≥ 700 μm). Objective design can be improved or optimized for coverslips equal to the actual coverslip thickness plus half the effective gap thickness (e.g., 700 μm + 1 / 2 * fluid channel (gap) height). This design significantly reduces the impact of the gap height on the image quality of both surfaces of the fluid channel and balances the optical quality of the images on both surfaces because the gap height is relatively small relative to the total thickness of the coverslip, thus reducing its impact on optical quality.

[0192] Other advantages of using a thicker coverslip include improved control over thickness tolerances during manufacturing and a reduced likelihood of deformation due to thermal and installation-induced stresses. Thickness errors and deformation of the coverslip can adversely affect the imaging quality of both the top and bottom surfaces of the flow cell.

[0193] To further improve the imaging quality of dual surfaces for sequencing applications, our optical system design focuses on improving or optimizing MTF in the medium to high spatial frequency range, which is best suited for imaging and resolving small spots or clusters (e.g., by improving or optimizing objective and / or barrel lens design).

[0194] Improved or optimized barrel-lens designs for use in conjunction with commercially available off-the-shelf objectives: For low-cost sequencer designs, commercially available off-the-shelf objectives are preferred due to their relatively low cost. However, as mentioned above, low-cost off-the-shelf objectives are primarily optimized for use with thin coverslips approximately 170 μm thick. In some cases, the disclosed optical system can utilize a barrel-lens design that compensates for thicker flow cell coverslips while enabling high image quality for both inner surfaces of the flow cell in dual-surface imaging applications. In some cases, the barrel-lens designs disclosed herein enable high-quality imaging of both inner surfaces of the flow cell without moving one or more optical elements or assemblies of the barrel-lens along the optical path, and without moving one or more optical elements or assemblies of the barrel-lens into or out of the optical path, without requiring the optical compensator to be moved into or out of the optical path.

[0195] Figure 19 Optical ray tracing plots are provided for a low-light objective design that has been improved or optimized to image surfaces on opposite sides of a 0.17 mm thick coverslip. The modulation transfer function plot of this objective (e.g.) is also provided. Figure 20 (As shown) This indicates the near-diffraction-limited imaging performance when used with a coverslip designed for a thickness of 0.17 mm.

[0196] Figure 21 Provided for imaging the surfaces of opposite sides of a 0.3 mm thick coverslip, Figure 19 The figure shows a graph of the modulation transfer function (MTF) of the same objective lens as a function of spatial frequency. The relatively small deviation of the MTF values ​​in the spatial frequency range of approximately 100 lines / mm to approximately 800 lines / mm (or period / mm) indicates that the image quality obtained is still reasonable, even when using a 0.3 mm thick coverslip.

[0197] Figure 22 This is provided for imaging surfaces separated from the 0.3 mm thick coverslip by a 0.1 mm thick aqueous fluid layer (i.e., under the conditions encountered in double-sided imaging of a flow cell when imaging distant surfaces). Figure 19 The graph shows the modulation transfer function as a function of spatial frequency for the same objective lens. Figure 22 As can be seen from the figure, in the spatial frequency range of approximately 50 lp / mm to approximately 900 lp / mm, the deviation of the MTF curve from the ideal diffraction-limited condition indicates a decrease in imaging performance.

[0198] Figure 23 and Figure 24 Provided when using Figure 19The objective lens shown is used for imaging through a 1.0 mm thick coverslip, and when the upper and lower inner surfaces are separated by a 0.1 mm thick aqueous fluid layer, the upper (or near) inner surface of the flow cell ( Figure 23 ) and lower (or distal) inner surface ( Figure 24 The graph shows the modulation transfer function of the two surfaces as a function of spatial frequency. It can be seen that the imaging performance of both surfaces is greatly reduced.

[0199] Figure 25 Provides ray tracing diagrams for lens barrel design, if compared with... Figure 19 When used in combination with the objectives shown, the tube lens provides improved double-sided imaging through a 1 mm thick coverslip. An optical design 700, including compound lenses (lens elements 702, 703, 704, 705, 706, 707, 708, 709, and 710) and tube lenses (lens elements 711, 712, 713, and 714), is improved or optimized for use with a flow cell (which includes a thick coverslip (or wall), for example, with a thickness greater than 700 μm, and a fluid channel thickness of at least 50 μm), and transmits an image of the inner surface from the flow cell 701 to an image sensor 715, where optical image quality is significantly improved and the CNR is higher.

[0200] In some cases, a tube lens (or tube lens assembly) may include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more optical lens elements, wherein the number of optical lens elements, the surface geometry of each element, and their placement order in the assembly are improved or optimized to correct optical aberrations caused by the thick walls of the flow cell, and in some cases, allow the use of commercially available off-the-shelf objectives while still maintaining high-quality double-sided imaging capabilities.

[0201] In some cases, such as Figure 25 As shown, the lens assembly may sequentially include a first asymmetric convex-convex lens 711, a second convex-flat lens 712, a third asymmetric concave-concave lens 713, and a fourth asymmetric convex-concave lens 714.

[0202] Figure 26 and Figure 27 Provides calibration when using objectives (for 0.17mm coverslips) and Figure 25 The illustrated lens assembly, when imaging through a 1.0 mm thick coverslip and when the upper and lower inner surfaces are separated by a 0.1 mm thick aqueous fluid layer, shows the upper (or near) inner surface of the flow cell. Figure 26 ) and lower (or distal) inner surface ( Figure 27The graph shows the modulation transfer function as a function of spatial frequency. It can be seen that the obtained imaging performance is almost exactly what is expected for diffraction-limited optical designs.

[0203] Figure 28 A ray-tracing diagram (left) is provided for a barrel lens design used in this disclosure, which has been improved or optimized to provide high-quality double-sided imaging performance. Since the barrel lens is no longer infinitely corrected, a suitably designed zero lens (right) can be combined with the barrel lens to compensate for barrel lenses that are not infinitely corrected for manufacturing and testing purposes.

[0204] Adaptation or optimization of tube lenses specific to imaging channels: In imaging system design, it is possible to improve or optimize both the objective lens and the tube lens within the same wavelength region for all imaging channels. Typically, the same objective lens is shared by all imaging channels (e.g., see [link to related documentation]). Figure 18 Furthermore, each imaging channel either uses the same lens barrel or has a lens barrel that shares the same design.

[0205] In some cases, the imaging systems disclosed herein may also include barrel lenses for each imaging channel, wherein the barrel lenses have been independently improved or optimized for a specific imaging channel to improve image quality, for example, to reduce or minimize distortion and field curvature, and to improve the depth of field (DOF) performance of each channel. Since the wavelength range (or bandpass) of each specific imaging channel is much narrower than the combined wavelength range of all channels, the wavelength- or channel-specific adaptation or optimization of the barrel lenses used in the disclosed systems results in significant improvements in image quality and performance. This channel-specific adaptation or optimization leads to improved image quality on the top and bottom surfaces of the flow cell in two-sided imaging applications.

[0206] Double-sided imaging in the absence of fluid in the flow cell: To achieve optimal imaging performance for both the top and bottom inner surfaces of the flow cell, motion-actuated compensators are typically required to correct optical aberrations caused by the fluid in the flow cell (usually comprising a fluid layer thickness of approximately 50–200 μm). In some of the disclosed optical system designs, the top inner surface of the flow cell can be imaged in the presence of fluid. Once the sequencing chemical cycle is complete, the fluid can be removed from the flow cell to image the bottom inner surface. Therefore, in some cases, image quality of the bottom surface can be maintained even without the use of a compensator.

[0207] Electro-optic phase plates are used to compensate for optical aberrations and / or vibrations: In some cases, combining an electro-optic phase plate (or other corrective lens) with an objective lens to eliminate optical aberrations caused by the presence of fluid can improve the quality of bifacial images without removing the fluid from the flow cell. In some cases, electro-optic phase plates (or lenses) can be used to eliminate the effects of vibrations caused by the mechanical movement of motion-actuated compensators, and can provide faster image acquisition and sequencing cycle times for genome sequencing applications.

[0208] Improved contrast-to-noise ratio (CNR), field of view (FOV), spectral separation, and timing design to increase or maximize information transfer and throughput: Another approach to increasing or maximizing 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 specific FOV. For typical large-NA optical imaging systems, this typically involves obtaining an area of ​​approximately 1 mm². 2 The image of the field of view, in which the currently disclosed imaging system design specifies a large FOV objective with a long working distance to achieve a 2mm image. 2 Or image a larger area.

[0209] In some cases, the disclosed imaging system is designed for use in combination with proprietary low-binding matrix surface and DNA amplification methods, which reduce fluorescence background caused by various confounding signals, including but not limited to nonspecific adsorption of fluorescent dyes to the matrix surface, nonspecific nucleic acid amplification products (e.g., nucleic acid amplification products appearing on the matrix surface in regions between spots or features corresponding to amplified clusters of nucleic acid molecules (i.e., specifically amplified clones), nonspecific nucleic acid amplification products that may appear in the amplified clones, the phasing, and the pre-phasing nucleic acid strands, etc.). Combining the low-binding matrix surface and DNA amplification methods with the disclosed optical imaging system (which reduces fluorescence background) can significantly reduce the time required for imaging each field of view (FOV).

[0210] The currently disclosed system design can further reduce the required imaging time by improving or optimizing the imaging sequence, wherein fluorescence images of multiple channels are acquired simultaneously or in overlapping time sequences, and wherein the spectral separation of the fluorescence signals is designed to reduce crosstalk between fluorescence detection channels and between excitation light and fluorescence signals.

[0211] The currently disclosed system design can further reduce the required imaging time by improving or optimizing the scanning motion sequence. 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 focal position is determined), and then the objective lens is moved along the Z direction to the determined focal position before image acquisition. A series of fluorescence images are acquired by cyclically traversing a series of target FOV positions. From the perspective of information transfer duty cycle, information is transferred only during the fluorescence image acquisition portion of that cycle. In the currently disclosed imaging system design, a single-step motion in which all axes (XYZ) are simultaneously repositioned is performed, and the autofocus step is used to check for focal position errors. Additional Z motion commands are issued only when the focal position error (the difference between the focal plane position and the sample plane position) exceeds a certain limit (e.g., a specified error threshold). Combined with high-speed XY motion, this method increases the system's duty cycle, thereby increasing the imaging throughput per unit time.

[0212] Furthermore, by matching the designed optical collection efficiency, modulation transfer function, and image sensor performance characteristics with the expected fluorescence photon flux and dye efficiency (related to the dye extinction coefficient and fluorescence quantum yield) of the input excitation photon flux, while taking into account the background signal and system noise characteristics, the time required to acquire high-quality (high contrast-to-noise ratio (CNR) images) can be reduced or minimized.

[0213] Effective image acquisition combined with improved or optimized stage step size and settling time results in fast imaging time (i.e., the total time required for each field of view) and higher throughput imaging system performance.

[0214] Along with a large FOV and a fast image acquisition duty cycle, the disclosed design may also include specifications for image plane flatness, color focusing performance between fluorescence detection channels, sensor flatness, image distortion, and focus quality.

[0215] Color focusing performance can be further improved by aligning the image sensors with different fluorescence detection channels, thereby overlapping the optimal focal planes of each detection channel. The design goal is to ensure that more than 90% of the field of view is acquired relative to the optimal focal plane of each channel within ±100 nm (or less), thus increasing or maximizing the transmission of the intensity signal at a single point. In some cases, the disclosed design also ensures that 99% of the field of view is acquired relative to the optimal focal plane of each channel within ±150 nm (or less), and ensures that more of the image can be acquired within ±200 nm (or less) of the entire field of view relative to the optimal focal plane of each imaging channel.

[0216] Illumination Path Design: Another factor for improving signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and / or increasing throughput is increasing the illumination power density of the sample. In some cases, the disclosed imaging system may include an illumination path design that utilizes a high-power laser or laser diode coupled to a liquid light guide. The liquid light guide eliminates the inherent spotting of coherent light sources such as lasers and laser diodes. Furthermore, the coupling optics are designed for an entry aperture with a bottom-filled liquid light guide. The bottom-filling of the liquid light guide entry aperture reduces the effective numerical aperture of the illumination beam entering the objective, thereby improving the light transmission efficiency through the objective to the sample plane. Through this design innovation, illumination power density can be up to three times that of conventional designs at a large field of view (FOV).

[0217] In some cases, by utilizing the angle dependence of s-polarization and p-polarization, the polarization orientation of the illumination beam can be adjusted to reduce the amount of backscattered and back-reflected illumination light reaching the imaging sensor.

[0218] Structured illumination systems: In some cases, the disclosed imaging modules and systems may include structured illumination optics designs to increase the effective spatial resolution of the imaging system, thereby enabling higher surface densities of cloned amplified target nucleic acid sequences (clusters) on the flow cell surface to improve sequencing throughput. Structured illumination microscopy (SIM) utilizes spatially structured (i.e., periodic) light patterns to illuminate the sample plane and relies on the generation of interference patterns called moiré fringes. For example, multiple images are acquired under slightly different illumination conditions by moving and / or rotating the structured illumination pattern to generate moiré fringes. Mathematical deconvolution of the generated interference signals can reconstruct a super-resolution image with up to approximately two times the spatial resolution improvement compared to using diffraction-limited imaging optics [Lutz (2011), “Biological Imaging by Superresolution Light Microscopy”, Comprehensive Biotechnology (Second) version), Volume 1, pp. 579-589, Elsevier; Feiner-Gracia, et al. (2018), "15-Advanced OpticalMicroscopy Techniques for the Investigation of Cell-NanoparticleInteractions", Smart Nanoparticles for Biomedicine: Micro and Nano TechnologiesPages 219-236, Elsevier; Nylk, et al. (2019), “Light-Sheet Fluorescence Microscopy With Structured Light”. Neurophotonics and Biomedical Spectroscopy [Pages 477-501, Elsevier]. Examples of structured illumination microscope imaging systems are recently described in Hong’s U.S. Patent Application Publication No. 2020 / 0218052.

[0219] Figure 41 A non-limiting schematic diagram of an imaging system 4100 is provided, which includes a structured illumination optics design with branches disclosed herein. A first branch (or arm) of the illumination optical path of 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 in a first orientation relative to the optical axis, a rotating window 4140A, and a lens 4150A. A second branch of the illumination optical path of 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 in a second orientation relative to the optical axis, a rotating window 4140B, and a lens 4150B. Diffraction gratings 4130A and 4130B can project light fringe patterns onto a sample plane.

[0220] In some cases, 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, an LED, laser, or laser diode, the output of which is subsequently collimated by corresponding collimating lenses 4120A and 4120B. In some cases, light sources 4110A and 4110B may output light of the same wavelength. In some cases, light sources 4110A and 4110B may output light of different wavelengths. Both light sources 4110A and 4110B may be configured to output light of any wavelength and / or wavelength range described elsewhere herein. During imaging, light sources 4110A and 4110B may be turned on or off using, for example, a high-speed shutter (not shown) located in the optical path or by pulses applied to the light sources at a predetermined frequency.

[0221] exist Figure 41In the example shown, the first illumination arm of system 4100 includes a fixed vertical grating 4130A for projecting a grating pattern (e.g., a vertical fringe pattern) onto a sample plane (e.g., the first inner surface 4188 of flow cell 4187) in a first orientation, and the second illumination arm includes a fixed horizontal grating 4130B for projecting a grating pattern (e.g., a horizontal fringe pattern) onto the sample plane 4188 in a second orientation. Advantageously, in this non-limiting example, the diffraction grating of imaging system 4100 does not require mechanical rotation or translation during imaging, which can provide improved imaging speed, system reliability, and system repeatability. In some cases, diffraction gratings 4130A and / or 4130B can be rotated about their respective optical axes, thereby making the angle between the fringe patterns projected onto the sample plane adjustable.

[0222] like Figure 41 As shown, in some cases, diffraction gratings 4130A and 4130B can be transmission diffraction gratings, comprising multiple diffraction elements (e.g., parallel slits or grooves) formed in a glass matrix or other suitable surface. In some cases, the grating can be implemented as a phase grating, providing a periodic variation in the refractive index of the grating material. In some cases, the grooves or feature spacing can be selected to diffract light at a suitable angle and / or tuned to the minimum resolvable feature size of the imaging sample for operation of the imaging system 4100. In other cases, the diffraction grating can be a reflection diffraction grating.

[0223] exist Figure 41 In the example shown, the orientation of the vertical and horizontal light fringe patterns is offset by approximately 90 degrees. In other cases, other orientations of the diffraction gratings can be used to produce an offset of approximately 90 degrees. For example, the diffraction gratings can be oriented such that they project light fringe patterns offset by ±45 degrees from the x-axis or y-axis of the sample plane (e.g., the surface of the first inner flow cell) 4188. This also applies when the sample carrier surface (e.g., the inner surface 4188 of the flow cell 4187) includes regularly patterned features arranged on a rectangular grating. Figure 41 The configuration of the imaging system 4100 shown can be particularly advantageous because it is possible to improve image resolution using structured illumination methods by using only two vertical grating orientations (e.g., a vertical grating orientation and a horizontal grating orientation).

[0224] In the example of system 4100, diffraction gratings 4130A and 4130B can be configured to diffract the input illumination beam into a series of intensity maxima due to constructive interference according to the following relationship:

[0225] m = order = d sin(θ) / λ

[0226] Where d = the distance between slits or grooves in the diffraction grating, θ = the incident angle of the illumination light relative to the normal to the surface of the diffraction grating, λ = the wavelength of the illumination light, and m = an integer value corresponding to the maximum intensity of the diffracted light, such as m = 0, ±1, ±2, etc. In some cases, a specific order of the diffracted illumination light, for example, the first order (m = ±1), can be projected onto the sample plane, for example, the inner flow cell surface 4188. In some cases, for example, a vertical grating 4130A can diffract the collimated beam into a first-order diffracted beam (±1 order), which is focused onto the sample plane with a first orientation, and a horizontal grating 4130B can diffract the collimated beam into a first-order diffracted beam, which is focused onto the sample plane with a second orientation. In some cases, using a beam-blocking element (not shown) (e.g., an order filter) that can be inserted into the optical path after the diffraction grating, the zero-order beam and / or all other higher-order beams (e.g., m = ±2 or higher) can be blocked, i.e., filtered out from the illumination pattern projected onto the sample plane 4188.

[0227] In the example of 4100, each branch of the structured illumination system includes optical phase modulators or phase shifters 4140A and 4140B to phase-shift each transmitted or reflected diffracted beam by diffraction gratings 4130A and 4130B. During structured imaging, the optical phase of each diffracted beam can be shifted by a fraction (e.g., 1 / 2, 1 / 2, 1 / 4, etc.) of the spacing (X) of each fringe of the structured pattern. Figure 41 In the example, phase modulators 4140A and 4140B can be implemented as rotating optical phase plates, for example, actuated by a rotary actuator or other actuator mechanism, to rotate and modulate the optical path length of each diffracted beam. For example, optical phase plate 4140A can be rotated about a vertical axis to move the image projected onto sample plane 4188 by vertical grating 4130A left and right, and optical phase plate 4140B can be rotated about a horizontal axis to move the image projected onto sample plane 4188 by horizontal grating 4130B in the vertical direction.

[0228] In other embodiments, other types of phase modulators that alter the optical path length of the diffracted light can be used (e.g., optical wedges mounted on a linear translation stage, etc.). Additionally, although optical phase modulators 4140A and 4140B are illustrated as being positioned after diffraction gratings 4130A and 4130B, in other embodiments they may be positioned at other locations in the illumination optical path. In some cases, a single optical phase modulator can operate in two different directions to produce different light fringe patterns, or a single movement can be used to adjust the position of a single optical phase modulator to simultaneously adjust the optical path lengths of both arms of the illumination optical path.

[0229] exist Figure 41In the example shown, optical component 4160 can be used to combine light from two illumination paths. Optical component 4160 may include, for example, a partially silvered mirror, a dichroic mirror (depending on the wavelength of the light output from light sources 4110A and 4110B), a mirror with a patterned or patterned reflective coating containing apertures that allows light from both arms of the illumination system to be combined in a lossless or near-lossless manner (e.g., without significant optical power loss except for a small amount of absorption by the reflective coating), a polarizing beam splitter (when light sources 4110A and 4110B are configured to produce polarized light), etc. Optical component 4160 can be positioned such that the desired diffraction order of light reflected or transmitted by each diffraction grating is spatially resolved, and unwanted orders of light are blocked. In some cases, optical component 4160 can allow first-order light output from the first illumination path to pass through and reflect first-order light output from the second illumination path. In some cases, the structured illumination pattern on the sample surface 4188 can 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 turning on and off optical shutters in the light paths of the light sources. In other cases, the structured illumination pattern can be switched by using optical switches to change the illumination path used to illuminate the sample plane.

[0230] Refer again Figure 41 Lens 4170, a semi-reflective mirror or dichroic mirror 4180, and objective lens 4185 can be used to focus the structured illumination light onto sample surface 4188 (e.g., the first inner surface of flow cell 4187). The light emitted, reflected, or scattered by sample surface 4188 is then collected by objective lens 4185, transmitted through mirror 4180, and imaged by image sensor or camera 4195. As described, mirror 4180 can be a dichroic mirror used to reflect the structured illumination light received from each branch of the illumination path into objective lens 4185 for projection onto sample surface 4188, and to allow light emitted from sample surface 4188 (e.g., fluorescence, which emits light at a wavelength different from the excitation light) to pass through for imaging onto image sensor 4195.

[0231] In some cases, system 4100 may optionally include a custom-designed barrel lens 4190, as described elsewhere herein, such that the focal point of the imaging system can be moved from the first inner surface 4188 of the flow cell 4187 to the second inner surface 4189 to achieve bi-surface imaging with minimal adjustment. In some cases, lens 4170 may include a custom-designed barrel lens, as described elsewhere herein, such that the focal point of the illumination path can be moved from the first inner surface 4188 of the flow cell 4187 to the second inner surface 4189 to achieve bi-surface imaging with minimal adjustment. In some cases, lens 4170 may be implemented as hinged along the optical axis to adjust the focal point of the structured illumination pattern on the sample plane. In some cases, system 4100 may include an autofocus mechanism (not shown) to adjust the focal point of the illumination light and / or the image at the plane of image sensor 4195. In some cases, since there is no polarizer in the optical path, therefore Figure 41 The system 4100 shown can provide high optical efficiency. Depending on the numerical aperture of the objective lens 4185, using unpolarized light may or may not have a significant impact on the contrast of the illumination pattern.

[0232] For simplicity, some optical components of the imaging system 4100 may have been removed from... Figure 41 As omitted in the foregoing discussion. Although system 4100 is shown as a single-channel detection system in this non-limiting example, it can also be implemented as a multi-channel detection system in other cases (e.g., using two different image sensors and appropriate optics and light sources emitting at two different wavelengths). Furthermore, although the illumination optical path of system 4100 is illustrated as including two branches in this non-limiting example, it can be implemented in some cases including, for example, three, four, or more than four branches, each of which includes a diffraction grating that is fixed or adjustable in relative orientation to each other.

[0233] In some cases, alternative illumination path optics designs can be used to create structured illumination. For example, in some cases, a single large rotating optical phase modulator can be placed after optical assembly 4160 and used instead of optical phase modulators 4140A and 4140B to modulate the phase of the two diffracted beams output by vertical diffraction grating 4130A and horizontal diffraction grating 4130B. In some cases, instead of being parallel to the optical axis relative to one of the diffraction gratings, the rotation axis of the single rotating optical compensator can be offset by 45 degrees (or another angle) from the optical axis of each vertical and horizontal diffraction grating to allow phase shift along the two illumination directions. In some cases, the single rotating optical phase modulator can be replaced by, for example, a wedge-shaped optical assembly that rotates about the nominal beam axis.

[0234] In an alternative illumination path design, diffraction gratings 4130A and 4130B can be mounted on their respective linear motion platforms so that they can be translated to change the optical path length (and thus the phase) of the light reflected or transmitted by the diffraction gratings 4130A and 4130B. The motion axis of the linear motion stage can be perpendicular to or otherwise offset from the orientation of their respective diffraction gratings to provide translation of the fringe pattern of the diffraction gratings along the sample plane 4188. Suitable translation stages may include, for example, crossed roller bearing stages, linear motors, high-precision linear encoders, and / or other linear actuator technologies to provide precise linear translation of the diffraction gratings.

[0235] Figure 42 Non-limiting examples of workflows for acquiring and processing images using structured illumination to enhance the spatial resolution of an imaging system are provided. In some cases, this can be performed... Figure 42 The workflow shown is for imaging the entire sample plane (e.g., imaging the inner surface of the flow cell by image tiling) or imaging a single region of a larger sample plane. Figure 41 The vertical 4130A and horizontal 4130B diffraction gratings of the system 4100 shown can be used to project illumination light fringe patterns onto sample planes with different known orientations and / or different known phase shifts. For example, the imaging system 4100 can use the vertical grating 4130A and the horizontal grating 4130B to generate horizontal and vertical illumination patterns, respectively, while the optical phase modulators 4140A and 4140B can be set to three different positions to produce the three phase shifts shown for each orientation.

[0236] During operation, a first illumination condition (e.g., a specific orientation and phase-shift setting of the diffraction grating) can be used to project a grating fringe pattern onto a sample plane (e.g., the surface of a flow cell). After capturing an image using the first illumination condition, one or more additional images can be acquired using one or more phase-shift illumination patterns (e.g., 1, 2, 3, 4, 5, 6, or more than 6 additional images acquired using 1, 2, 3, 4, 5, 6, or more than 6 phase-shift illumination patterns). If the imaging system includes a second branch of the illumination path, the image acquisition process can be repeated using a second illumination condition as a starting point (e.g., a second specific orientation and phase-shift setting of the diffraction grating). In some cases, at least 5 different phase-shift fringe patterns can be used to acquire images for at least three different orientations of the diffraction grating (e.g., spaced 60 degrees apart from each other). If no more images are acquired using different orientations of the diffraction grating or phase-shift illumination fringe patterns, an image reconstruction algorithm can be used to process the acquired images and produce a reconstructed super-resolution image. In some cases, at least 1, 2, 3, 4, 5, 6 or more different phase-shifting fringe patterns are used in each orientation to acquire images for at least 1, 2, 3, 4, 5, 6 or more different orientations of the diffraction grating.

[0237] A potential drawback of acquiring multiple images for reconstructing a single super-resolution image is the time required to adjust the orientation and / or relative phase shift of the projected light fringe pattern, the exposure time required to acquire each image, and downstream image processing. Therefore, an optical design that minimizes the time required to change the orientation and relative phase of the diffraction grating, and an efficient image reconstruction algorithm, are preferred. In some cases, fewer images may be needed to reconstruct a super-resolution image of, for example, 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.

[0238] Refer again Figure 42 For example, when tiling an image to create a higher resolution image of the entire flow cell surface, the above cycle can be repeated for different regions of a given flow cell surface. In some cases, such as when imaging a second flow cell surface, the above cycle can be repeated after adjusting the focus of the imaging system.

[0239] Other super-resolution imaging techniques: In some cases, the disclosed imaging system may include the use of alternative super-resolution imaging techniques, such as photoluminescent localization microscopy (PALM), fluorescence photoluminescent localization microscopy (FPALM), and / or stochastic optical reconstruction microscopy (STORM). [See, for example, Lutz et al. (2011), “Biological Imaging by Superresolution Light Microscopy”, Comprehensive Biotechnology (Second Edition) Volume 1, pp. 579-589 (Elsevier), which is based on statistically fitting a Gaussian distribution function to the intensity distribution observed in the image of the point spread function (PSF) of a single molecule. The Gaussian distribution function is then used to define the position of the molecule in the sample plane with much higher precision than allowed by the classical resolution limit. The same method can be used to image small, dispersed subsets of fluorescently labeled molecules (e.g., clonal amplification clusters of target nucleic acid sequences tethered to a low-nonspecific binding surface on a sample carrier or on the inner surface of a flow cell).

[0240] The spatial accuracy or resolution obtained using these methods depends on the number of photons collected from the molecule before photobleaching and the level of background noise [Lutz et al. (2011), ibid.]. If the background noise is negligible and at least 10,000 photons can be collected per molecule, a positional accuracy of 1–2 nm is demonstrated. In some cases, such as using affinity sequencing methods described elsewhere in this document, polymer-nucleotide conjugates containing multiple fluorescent markers (to ensure high photon counts) (e.g., each conjugate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers), optionally combined with low nonspecific binding surfaces disclosed elsewhere in this document (to ensure very low background signal), can facilitate the use of these super-resolution imaging techniques for gene testing and sequencing applications. Spatial accuracy or resolution decreases with decreasing numbers of photons collected; however, even with only a moderate number of photons collected, a positional accuracy or resolution of 20 nm is possible. In some cases, lateral spatial resolution improvements of 10-fold or higher can be achieved. In some cases, image resolutions superior to 500nm, 400nm, 300nm, 200nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm, or 10nm can be achieved.

[0241] The second fundamental principle of this type of imaging is to image a small number of spatially separated fluorescent molecules within a sample at any given time.

[0242] In some cases, the ability to control the fluorescence emission of small, dispersed subsets of fluorescent molecules in a sample plane is crucial for advancing super-resolution imaging. In the cases of fluorescence photodynamic localization microscopy (FPALM) and photodynamic localization microscopy (PALM), for example, using photoactivated green fluorescent protein (PA-GFP) as a marker allows for the controlled induction of fluorescent subsets in the sample using short pulses of 405 nm light to convert PA-GFP from a dark, non-fluorescent state to a 488 nm excitable fluorescent state, thereby producing spatially separated subsets of fluorescent molecules that can be imaged [Lutz et al. (2011), ibid.]. In the case of stochastic optical reconstruction microscopy (STORM), for example, the light conversion properties of cyanine dyes to Cy5–Cy3 can be used in a similar manner to enable the random induction of Cy5 fluorescence from small subsets of molecules in the sample (e.g., small subsets of molecules spatially separated by at least a few resolution units) at any given time. In some cases, such as when combined with affinity sequencing methods described elsewhere herein, polymer-nucleotide conjugates may contain a photoactivated green fluorescent protein (PA-GFP) or a subdomain thereof or a portion thereof. In some cases, polymer-nucleotide conjugates may contain a mixture of conjugates, wherein a first portion is labeled with, for example, a Cy3 label and a second portion is labeled with, for example, a Cy5 label. In some cases, polymer-nucleotide conjugates may contain a mixture of, for example, Cy3 and Cy5 labels within the same conjugate.

[0243] Super-resolution images are reconstructed from the sum of Gaussian fittings of all molecules or features (e.g., labeled nucleic acid clusters) imaged in the temporal stack of acquired images [Lutz et al. (2011), ibid.], where the intensity corresponds to the positional uncertainty of the location of each molecule or subset of molecules. The unique feature of this dataset is its ability to render images with varying localization precision or resolution. In some cases, imaging modules incorporating total internal reflection fluorescence (TIRF) optical imaging designs can be advantageous in implementing these super-resolution imaging techniques because the evanescent wave used to excite fluorescence is confined within an axial dimension of less than 200 nm from the sample carrier or flow cell surface, thus suppressing background fluorescence signals. In some cases, the imaging system may include objectives with higher numerical apertures than those used in other imaging module designs disclosed herein. Using objectives with higher numerical apertures can facilitate evanescent wave excitation and efficient photon capture from fluorescent probes. In some cases, wide-field-of-view imaging using a single-photon-sensitive EM-CCD camera or other types of image sensors can enable the simultaneous imaging of many molecules or subsets of molecules (e.g., nucleic acid sequence clusters) per frame, thereby increasing image acquisition throughput.

[0244] In some cases, the data acquisition time required to acquire sufficient images to achieve adequate feature definition and resolution can be reduced by improving the sensitivity and speed of the imaging system as follows: increasing the signal by using the affinity sequencing reagents and low nonspecific binding surfaces disclosed herein, while reducing or eliminating background, and using improved image reconstruction algorithms.

[0245] Image quality assessment: For any implementation of the optical imaging design disclosed herein, imaging performance or image quality can be evaluated using any of a variety of performance metrics known to those skilled in the art. Examples include, but are not limited to, measurements of 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.

[0246] In some cases, the disclosed optical designs for double-sided imaging (e.g., the disclosed objective lens design, lens barrel design, combination of electro-optic plates with objectives, etc., individually or in combination) can significantly improve the image quality of the upper (near) inner surface and the lower (far) inner surface of the flow cell, such that for any of the imaging performance indicators listed above (whether individually or in combination), the difference in imaging performance indicators for imaging the upper inner surface and the lower inner surface 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%.

[0247] In some cases, the disclosed optical designs for duplex imaging (e.g., including the disclosed barrel lens designs, combining an electro-optic plate with an objective lens, etc.) can significantly improve image quality, such that for any of the imaging performance metrics listed above (whether individually or in combination), compared to conventional systems (which include, for example, objectives, motion-actuated compensators (which move out of or into the optical path when imaging the near or far inner surface of the flow cell), and an image sensor), the image quality performance metrics for duplex imaging provide an improvement 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% in terms of the imaging performance metrics for duplex imaging. In some cases, a fluorescence imaging system including one or more of the disclosed barrel lens designs provides at least equal or greater improvement in imaging performance metrics for duplex imaging compared to a conventional system including objectives, motion-actuated compensators, and an image sensor. In some cases, fluorescence imaging systems, including one or more of the disclosed tube lens designs, provide at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% improvement in imaging performance for double-sided imaging compared to conventional systems that include objectives, motion-actuated compensators, and image sensors.

[0248] Imaging module specifications:

[0249] Excitation wavelength: In any disclosed optical imaging module design, the light source of the disclosed imaging module can produce visible light, such as green and / or red light. In some cases, the light source, alone or in combination with one or more optical components (e.g., excitation optical filters and / or dichroic beam splitters), can produce excitation light of about 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 can have any value within this range, such as about 620 nm.

[0250] Excitation bandwidth: In any disclosed optical imaging module design, the light source, alone or in combination with one or more optical components (e.g., excitation optical filters and / or dichroic beam splitters), can generate light at a specified excitation wavelength within a bandwidth of ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or greater. Those skilled in the art will recognize that the excitation bandwidth can have any value within this range, for example, approximately ±18 nm.

[0251] Light source power output: In any disclosed optical imaging module design, the power output of the light source and / or the excitation beam (including composite excitation beams) obtained therefrom can range from about 0.5W to about 5.0W, or greater (as will be discussed in more detail below). In some cases, the power output of the light source and / or the excitation beam obtained therefrom can be at least 0.5W, at least 0.6W, at least 0.7W, at least 0.8W, at least 1W, at least 1.1W, at least 1.2W, at least 1.3W, at least 1.4W, at least 1.5W, at least 1.6W, at least 1.8W, at least 2.0W, at least 2.2W, at least 2.4W, at least 2.6W, at least 2.8W, at least 3.0W, at least 3.5W, at least 4.0W, at least 4.5W, or at least 5.0W. In some embodiments, the power of the output of the light source and / or the excitation beam (including a composite excitation beam) obtained therefrom can be up to 5.0 W, up to 4.5 W, up to 4.0 W, up to 3.5 W, up to 3.0 W, up to 2.8 W, up to 2.6 W, up to 2.4 W, up to 2.2 W, up to 2.0 W, up to 1.8 W, up to 1.6 W, up to 1.5 W, up to 1.4 W, up to 1.3 W, up to 1.2 W, up to 1.1 W, up to 1 W, up to 0.8 W, up to 0.7 W, up to 0.6 W, or up to 0.5 W. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the power of the output of the light source and / or the excitation beam (including a composite excitation beam) obtained therefrom can range from about 0.8 W to about 2.4 W. Those skilled in the art will recognize that the power of the output of the light source and / or the excitation beam (including a composite excitation beam) obtained therefrom can have any value within that range, for example, about 1.28 W.

[0252] Light source output power and CNR: In some embodiments of the disclosed optical imaging module design, the output power of the light source and / or the power of one or more excitation beams (including composite excitation beams) obtained therefrom are sufficient, in combination with appropriate samples, to provide 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 greater, or any CNR within any range formed by any of these values, in the image acquired by the illumination and imaging module.

[0253] Fluorescent emission bands: In some cases, the disclosed fluorescence optical imaging module can be configured to detect fluorescence emission produced by any of a variety of fluorophores known to those skilled in the art. Examples of suitable fluorescent dyes for applications such as genotyping and nucleic acid sequencing (e.g., by conjugating nucleotides, oligonucleotides, or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, anthocyanins, and their derivatives, including anthocyanin derivatives such as anthocyanin dye 3 (Cy3), anthocyanin dye 5 (Cy5), and anthocyanin dye 7 (Cy7).

[0254] Fluorescence emission wavelength: In any disclosed optical imaging module design, the detection or imaging channel of the disclosed optical system 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. Those skilled in the art will recognize that the emission wavelength can have any value within this range, for example, approximately 825 nm.

[0255] Fluorescence emission bandwidth: In any disclosed optical imaging module design, the detection channel or imaging channel may include one or more optical components, such as emission optical filters and / or dichroic beam splitters, configured to collect light of a specified emission wavelength within a bandwidth of ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or greater. Those skilled in the art will recognize that the excitation bandwidth can have any value within this range, for example, approximately ±18 nm.

[0256] Numerical aperture: In some cases, in any disclosed optical system design, the numerical aperture of the objective lens and / or optical imaging module (e.g., including objective lenses and / or barrel lenses) may range from about 0.1 to about 1.4. In some cases, 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 cases, 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 lower and upper limits described in this paragraph can be combined to form a range included in this disclosure; for example, in some cases, the range of numerical aperture can be from about 0.1 to about 0.6. Those skilled in the art will recognize that numerical aperture can have any value within this range, such as about 0.55.

[0257] Optical resolution: In some cases, depending on the numerical aperture of the objective lens and / or optical system (e.g., including objective lenses and / or barrel lenses), the minimum resolvable spot (or feature) spacing distance at the sample plane, achievable by any disclosed optical system design, can range from about 0.5 μm to about 2 μm. In some cases, the minimum resolvable spot spacing distance at the sample plane can 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 cases, the minimum resolvable spot spacing 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 lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the minimum resolvable spot spacing distance may be in the range of about 0.8 μm to about 1.6 μm. Those skilled in the art will recognize that the minimum resolvable spot spacing distance may have any value within this range, for example, about 0.95 μm.

[0258] Optical resolution of the first and second surfaces at different depths: In some cases, in any optical module or system disclosed herein, the use of the novel objective and / or barrel lens designs disclosed herein can impart comparable optical resolution to the first and second surfaces (e.g., the upper and lower inner surfaces of the flow cell) with or without the need for refocusing between acquiring images of the first and second surfaces. In some cases, the optical resolution of the images of the first and second surfaces thus obtained can have 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, or 1% of each other, or any value within this range.

[0259] Magnification: In some cases, the magnification of the objective lens and / or barrel lens, and / or optical system (e.g., including the objective lens and / or barrel lens) in any of the disclosed optical configurations can range from about 2x to about 20x. In some cases, the magnification of the optical system can 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 cases, the magnification of the optical system can be at most 20x, at most 15x, at most 10x, at most 9x, at most 8x, at most 7x, at most 6x, at most 5x, at most 4x, at most 3x, or at most 2x. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the magnification of the optical system can range from about 3x to about 10x. Those skilled in the art will recognize that the magnification of the optical system can have any value within this range, for example, about 7.5x.

[0260] Objective lens focal length: In some embodiments of the disclosed optical designs, the focal length of the objective lens can be in the range of 20 mm to 40 mm. In some cases, the focal length of the objective lens can be at least 20 mm, at least 25 mm, at least 30 mm, at least 35 mm, or at least 40 mm. In some cases, the focal length of the objective lens can be at most 40 mm, at most 35 mm, at most 30 mm, at most 25 mm, or at most 20 mm. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the focal length of the objective lens can be in the range of 25 mm to 35 mm. Those skilled in the art will recognize that the focal length of the objective lens can have any value within the range of values ​​specified above, for example, about 37 mm.

[0261] Objective lens working distance: In some embodiments of the disclosed optical design, the working distance of the objective lens can be in the range of approximately 100 μm to 30 mm. In some cases, the working distance can 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 cases, the working distance can 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 lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the working distance of the objective lens can be in the range of 500 μm to 2 mm. Those skilled in the art will recognize that the working distance of the objective lens can have any value within the range specified above, for example, about 1.25 mm.

[0262] For objectives optimized for imaging with thick coverslips: In some examples of the disclosed optical designs, the objective design can be improved or optimized for coverslips of varying flow cell thicknesses. For example, in some cases, the objective can be designed to have optimal optical performance for coverslips with thicknesses from about 200 μm to about 1,000 μm. In other cases, the objective can be designed to have optimal performance for coverslips with thicknesses of 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. In some cases, objectives can be designed to have optimal performance for coverslips with thicknesses of 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. Any lower and upper limits described in this paragraph can be combined to form ranges included in this disclosure; for example, in some cases, objectives can be designed to have optimal optical performance for coverslips with thicknesses of about 300 μm to about 900 μm. Those skilled in the art will recognize that objectives can be designed to have optimal optical performance for coverslips that can have any value within this range, for example, about 725 μm.

[0263] Depth of field and depth of focus: In some cases, the depth of field and / or depth of focus of any disclosed imaging module (e.g., including objectives and / or barrel lenses) may be designed to range from about 10 μm to about 800 μm, or greater. In some cases, 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, or at least 800 μm, or greater. In some cases, the depth of field and / or depth of focus is 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 lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the range of depth of field and / or depth of focus can be 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 can have any value within the range specified above, for example, about 132 μm.

[0264] Field of View (FOV): In some embodiments, the FOV of any disclosed imaging module design (e.g., provided by a combination of objective lenses and detection channel optics, such as barrel lenses) can range from, for example, about 1 mm to about 5 mm (e.g., in terms of diameter, width, length, or longest dimension). In some cases, the FOV can 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 terms of diameter, width, length, or longest dimension). In some cases, the FOV can be at most 5.0 mm, at most 4.5 mm, at most 4.0 mm, at most 3.5 mm, at most 3.0 mm, at most 2.5 mm, at most 2.0 mm, at most 1.5 mm, or at most 1.0 mm (e.g., in terms of diameter, width, length, or longest dimension). Any lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure. For example, in some cases, the range of FOV may be from about 1.5 mm to about 3.5 mm (e.g., in terms of diameter, width, length, or longest dimension). Those skilled in the art will recognize that FOV may have any value within the range specified above, for example, about 3.2 mm (e.g., in terms of diameter, width, length, or longest dimension).

[0265] Field of view (FOV) area: In some examples of the disclosed optical system designs, the field of view area can be approximately 2 mm. 2 Approximately 5mm 2 Within a certain range. In some cases, the area of ​​the field of view can be at least 2 mm. 2 At least 3mm 2 At least 4mm 2 Or at least 5mm 2 In some cases, the field of view area can be up to 5 mm. 2 At most 4mm 2 At most 3mm 2 or at most 2mm 2 Any lower and upper limits described in this paragraph can be combined to form a range included in this disclosure; for example, in some cases, the area of ​​the field of view can be approximately 3 mm. 2 Approximately 4mm 2 Within this range. Those skilled in the art will recognize that the area of ​​the field of view can have any value within this range, for example, 2.75 mm. 2 .

[0266] Optimization of the MTF of the objective lens and / or the tube lens: In some cases, the objective lens and / or at least one tube lens in the disclosed imaging modules and systems are designed to optimize the modulation transfer function in the medium to high spatial frequency range. For example, in some cases, the objective lens and / or at least one tube lens in the disclosed imaging modules and systems are designed to optimize the modulation transfer function in the sample plane in the following spatial frequency ranges: 500 cycles / mm to 900 cycles / mm, 700 cycles / mm to 1100 cycles / mm, 800 cycles / mm to 1200 cycles / mm, or 600 cycles / mm to 1000 cycles / mm.

[0267] Optical Aberrations and Diffraction-Limited Imaging Performance: In some embodiments of any optical imaging module design disclosed herein, the objective lens and / or barrel lens may be configured to provide the imaging module with the field of view as described above, such that the FOV has an aberration of less than 0.15 wavelengths at at least 60%, 70%, 80%, 90%, or 95% of the field of view. In some embodiments, the objective lens and / or barrel lens may be configured to provide the imaging module with the field of view as described above, such that the FOV has an aberration of less than 0.1 wavelengths at at least 60%, 70%, 80%, 90%, or 95% of the field of view. In some embodiments, the objective lens and / or barrel lens may be configured to provide the imaging module with the field of view as described above, such that the FOV has an aberration of less than 0.075 wavelengths at at least 60%, 70%, 80%, 90%, or 95% of the field of view. In some embodiments, the objective lens and / or barrel lens may be configured to provide the imaging module with the field of view as described above, such that the FOV is diffractively limited at at least 60%, 70%, 80%, 90%, or 95% of the field of view.

[0268] Angle of incidence of the light beam on the dichroic reflector, beam splitter, and beam combiner: In some examples of the disclosed optical designs, the angle of incidence of the light beam on the dichroic reflector, beam splitter, or beam combiner can range from about 20 degrees to about 45 degrees. In some cases, the angle of incidence can 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 cases, the angle of incidence can be at most 45 degrees, at most 40 degrees, at most 35 degrees, at most 30 degrees, at most 25 degrees, or at most 20 degrees. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the angle of incidence can range from about 25 degrees to about 40 degrees. Those skilled in the art will recognize that the angle of incidence can have any value within the range specified above, for example, about 43 degrees.

[0269] Image sensor (photodetector array) size: In some cases, the disclosed optical system may include an image sensor having an effective area whose diagonal ranges from about 10 mm to about 30 mm or greater. In some cases, the diagonal of the effective area of ​​the image sensor is 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 cases, the diagonal of the effective area of ​​the image sensor is at most 30 mm, at most 28 mm, at most 26 mm, at most 24 mm, at most 22 mm, at most 20 mm, at most 18 mm, at most 16 mm, at most 14 mm, at most 12 mm, or at most 10 mm. Any lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the image sensor may have an effective area with a diagonal range of about 12 mm to about 24 mm. Those skilled in the art will recognize that one or more image sensors may have an effective area with a diagonal having any value within the range of values ​​specified above (e.g., about 28.5 mm).

[0270] Image sensor pixel size and pitch: In some cases, the pixel size and / or pitch selected for the image sensor used in the disclosed optical system design may range from about 1 μm to about 10 μm in at least one dimension. In some cases, 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 cases, 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 lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the pixel size and / or pitch may range from about 3 μm to about 9 μm. Those skilled in the art will recognize that pixel size and / or spacing can have any value within that range, for example, about 1.4 μm.

[0271] Oversampling: In some examples of the disclosed optical designs, a spatial oversampling scheme is used, wherein the spatial sampling frequency is at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 times the optical resolution X (lp / mm).

[0272] Maximum translation stage speed: In some examples of the disclosed optical imaging modules, the maximum translation stage speed on any axis can range from about 1 mm / s to about 5 mm / s. In some cases, the maximum translation stage speed can 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 cases, the maximum translation stage speed can be at most 5 mm / s, at most 4 mm / s, at most 3 mm / s, at most 2 mm / s, or at most 1 mm / s. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the maximum translation stage speed can range from about 2 mm / s to about 4 mm / s. Those skilled in the art will recognize that the maximum translation stage speed can have any value within this range, for example, about 2.6 mm / s.

[0273] Maximum translation stage acceleration: In some examples of the disclosed optical imaging modules, the maximum acceleration on any one axis of motion can be approximately 2 mm / s². 2 Approximately 10 mm / s 2 Within a certain range. In some cases, the maximum acceleration can be at least 2 mm / s². 2 At least 3mm / s 2 At least 4mm / s 2 At least 5mm / s 2 At least 6mm / s 2 At least 7mm / s 2 At least 8mm / s 2 At least 9mm / s 2 Or at least 10 mm / s 2 In some cases, the maximum acceleration can be up to 10 mm / s². 2 At most 9mm / s 2 At most 8mm / s 2 At most 7mm / s 2 At most 6mm / s 2 At most 5mm / s 2 At most 4mm / s 2 At most 3mm / s 2 or at most 2mm / s 2 Any lower and upper limits described in this paragraph can be combined to form ranges included in this disclosure; for example, in some cases, the maximum acceleration can be approximately 2 mm / s². 2 Approximately 8 mm / s 2 Within this range. Those skilled in the art will recognize that the maximum acceleration can have any value within this range, for example, approximately 3.7 mm / s². 2 .

[0274] Translation stage positioning repeatability: In some examples of the disclosed optical imaging modules, the repeatability of positioning for any one axis can range from about 0.1 μm to about 2 μm. In some cases, the positioning repeatability can 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 cases, the repeatability of the positioning can 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 lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the repeatability of the positioning can be in the range of about 0.3 μm to about 1.2 μm. Those skilled in the art will recognize that the repeatability of the positioning can have any value within this range, for example, about 0.47 μm.

[0275] FOV repositioning time: In some examples of the disclosed optical imaging modules, the longest time required to reposition the sample plane (field of view) relative to the optics or vice versa can range from about 0.1 seconds to about 0.5 seconds. In some cases, the longest repositioning time (i.e., the stage step size and settling time) can 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 cases, the longest repositioning time can be at most 0.5 seconds, at most 0.4 seconds, at most 0.3 seconds, at most 0.2 seconds, or at most 0.1 seconds. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the longest repositioning time can range from about 0.2 seconds to about 0.4 seconds. Those skilled in the art will recognize that the longest repositioning time can have any value within this range, for example, about 0.45 seconds.

[0276] Error threshold for autofocus correction: In some examples of the disclosed optical imaging modules, the specified error threshold used to trigger autofocus correction can be in the range of about 50 nm to about 200 nm. In some cases, the error threshold can 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 cases, the error threshold can 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 lower and upper limits described in this paragraph can be combined to form a range included in this disclosure; for example, in some cases, the error threshold can be in the range of about 75 nm to about 150 nm. Those skilled in the art will recognize that the error threshold can have any value within this range, for example, about 105 nm.

[0277] Image acquisition time: In some cases of the disclosed optical imaging modules, the image acquisition time can range from about 0.001 seconds to about 1 second. In some cases, the image acquisition time can be at least 0.001 seconds, at least 0.01 seconds, at least 0.1 seconds, or at least 1 second. In some cases, the image acquisition time can be at most 1 second, at most 0.1 seconds, at most 0.01 seconds, or at most 0.001 seconds. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the image acquisition time can range from about 0.01 seconds to about 0.1 seconds. Those skilled in the art will recognize that the image acquisition time can have any value within this range, for example, about 0.250 seconds.

[0278] Imaging time per FOV: In some cases, the imaging time per FOV may range from about 0.5 seconds to about 3 seconds. In some cases, the imaging time per FOV 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. In some cases, the imaging time per FOV may be at most 3 seconds, at most 2.5 seconds, at most 2 seconds, at most 1.5 seconds, at most 1 second, or at most 0.5 seconds. Any lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some cases, the imaging time may range from about 1 second to about 2.5 seconds. Those skilled in the art will recognize that the imaging time may have any value within this range, for example, about 1.85 seconds.

[0279] Field flatness: In some cases, images are acquired at 80%, 90%, 95%, 98%, 99%, or 100% of the field of view relative to the optimal focal plane of each fluorescence (or other imaging mode) detection channel at ±200nm, ±175nm, ±150nm, ±125nm, ±100nm, ±75nm, or ±50nm.

[0280] Systems and system components for genomics and other applications: As described above, in some embodiments, the disclosed optical imaging module can be used as a module, component, sub-assembly, or subsystem of a larger system configured to perform applications such as genomics (e.g., gene testing and / or nucleic acid sequencing applications) or other chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. Figure 39 Non-limiting examples of block diagrams for sequencing systems such as those disclosed herein are provided. In addition to the one, two, three, four, or more than four imaging modules disclosed herein (each imaging module may include one or more illumination paths and / or one or more detection paths (e.g., one or more detection channels configured to image fluorescence emission within a specific 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 cassettes, fluid systems and fluid flow control modules, reagent kits, temperature control modules, fluid dispensing robots, cassette and / or microplate handling (pick-and-place) robots, opaque housings and / or environmental control chambers, one or more processors or computers, data storage modules, data communication modules (e.g., Bluetooth, WiFi, intranet, or Internet communication 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.

[0281] Translation stages: In some embodiments of the imaging and analysis systems (e.g., nucleic acid sequencing systems) disclosed herein, the system may include one or more (e.g., one, two, three, four, or more than four) high-precision XY (or in some cases XYZ) translation stages for repositioning one or more sample carrier structures (e.g., one or more flow cells) relative to one or more imaging modules, thereby tiling one or more images (each image corresponding to the field of view of an imaging module) to reconstruct a composite image of the entire flow cell surface. In some embodiments of the imaging systems and genome analysis systems (e.g., nucleic acid sequencing systems) disclosed herein, the system may include one or more (e.g., one, two, three, four, 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 carrier structures (e.g., flow cells), thereby tiling one or more images (each image corresponding to the field of view of an imaging module) to reconstruct one or more composite images of the entire flow cell surface.

[0282] Suitable translation stages are available from many vendors, such as Parker Hannifin. Precision translation stage systems typically comprise a combination of multiple components, including but not limited to linear actuators, optical encoders, servo and / or stepper motors, and motor controllers or drive units. For the systems and methods disclosed herein, high precision and repeatability of platform movement are required to ensure accurate and reproducible positioning and imaging of, for example, fluorescence signals during repetitive steps of reagent delivery and optical detection.

[0283] Therefore, the systems disclosed herein may include a specified translation stage configured to position a sample carrier structure (or vice versa) relative to illumination and / or imaging optics with a certain precision. In one aspect of this disclosure, the precision of one or more translation stages is between about 0.1 μm and about 10 μm. In other aspects, 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. Those skilled in the art will understand that, in some cases, the positioning accuracy of the translation stage can fall within any range defined by any two of these values ​​(e.g., about 0.5 μm to about 1.5 μm). In some cases, the positioning accuracy of the translation stage can have any value within the range of values ​​included in this section, for example, about 0.12 μm.

[0284] Flow cell, microfluidic device, and cartridge: The flow cell devices and cartridges disclosed herein can be used as components of systems designed for a variety of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. Typically, 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 one or more of the microfluidic devices and cartridges described herein. Further description of the disclosed flow cell devices and cartridges can be found in PCT patent application publication WO 2020 / 118255, the entire contents of which are incorporated herein by reference.

[0285] In some cases, the systems disclosed herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 individual capillary flow cell devices, multiple capillary flow cell devices, capillary flow cell boxes, and / or microfluidic devices and boxes. In some cases, the individual capillary flow cell devices, multiple capillary flow cell devices, and / or microfluidic devices and boxes may be fixed components of the disclosed system. In some cases, the individual capillary flow cell devices, multiple capillary flow cell devices, and / or microfluidic devices and boxes may be removable, replaceable components of the disclosed system. In some cases, the individual capillary flow cell devices, multiple capillary flow cell devices, and / or microfluidic devices and boxes may be disposable or consumable components of the disclosed system.

[0286] In some embodiments, the disclosed single capillary flow cell device (or single capillary flow cell box) includes a single capillary, such as a glass or fused silica capillary, whose inner cavity forms a fluid flow path through which a reagent or solution can flow, and whose inner surface can form a sample carrier structure to which the target sample is bound or tethered. In some embodiments, the multi-capillary flow cell device (or multi-capillary flow cell box) disclosed herein may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more capillaries configured to perform an analytical technique that also includes imaging as a detection method.

[0287] In some cases, one or more capillaries may be packaged within a rack to form an easy-to-operate box, incorporating an adapter or connector for external fluid connections, and may optionally include additional integrated functional components, such as reagent reservoirs, waste reservoirs, valves (e.g., microvalves), pumps (e.g., micropumps), etc., or any combination thereof.

[0288] Figure 29The illustration shows a non-limiting example of a single glass capillary flow cell device comprising two fluid adapters (one fixed to each end of a one-piece glass capillary) designed to mate with a standard OD fluid tube to provide convenient, interchangeable fluid connections to an external fluid control system. The fluid adapters can be attached to the capillary using any of a variety of techniques known to those skilled in the art, including but not limited to press fitting, bond bonding, solvent bonding, laser welding, or any combination thereof.

[0289] Typically, the capillary tubes used in the disclosed capillary flow cell devices and capillary flow cell boxes will have at least one internally axially aligned fluid flow channel (or "cavity") extending along the entire length of the capillary tube. In some cases, the capillary tube may have two, three, four, five, or more than five internally axially aligned fluid flow channels (or "cavities").

[0290] The specified cross-sectional geometries for suitable capillaries (or their lumens) are consistent with the disclosure herein, including but not limited to circular, elliptical, square, rectangular, triangular, rounded square, rounded rectangular, or rounded triangular cross-sectional geometries. In some cases, capillaries (or their lumens) may have any specified cross-sectional size or set of sizes. For example, in some cases, the maximum cross-sectional size of the capillary lumen (e.g., the diameter if the lumen is circular, or the diagonal if the lumen is square or rectangular) may be in the range of about 10 μm to about 10 mm. In some cases, the maximum cross-sectional size of the capillary lumen 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 aspects, the maximum cross-sectional size of the capillary lumen can be up to 10 mm, up to 9 mm, up to 8 mm, up to 7 mm, up to 6 mm, up to 5 mm, up to 4 mm, up to 3 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, up to 100 μm, up to 75 μm, up to 50 μm, up to 25 μm, or up to 10 μm. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the maximum cross-sectional size of the capillary lumen can be in the range of about 100 μm to about 500 μm. Those skilled in the art will recognize that the maximum cross-sectional size of the capillary lumen can have any value within this range, for example, about 124 μm.

[0291] In some cases, for example, where the inner cavity of one or more capillaries in a flow cell device or box has a square or rectangular cross-section, the distance between the first inner surface (e.g., the top surface or upper surface) and the second inner surface (e.g., the bottom surface or lower surface) (which defines the gap height or thickness of the fluid flow channel) can range from about 10 μm to about 500 μm. In some cases, 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 cases, the gap height can be up to 500 μm, up to 475 μm, up to 450 μm, up to 425 μm, up to 400 μm, up to 375 μm, up to 350 μm, up to 325 μm, up to 300 μm, up to 275 μm, up to 250 μm, up to 225 μm, up to 200 μm, up to 175 μm, up to 150 μm, up to 125 μm, up to 100 μm, up to 90 μm, up to 80 μm, up to 70 μm, up to 60 μm, up to 50 μm, up to 40 μm, up to 30 μm, up to 20 μm, or up to 10 μm. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the gap height can range from about 40 μm to about 125 μm. Those skilled in the art will recognize that the gap height can have any value within the range of values ​​in this paragraph, for example, about 122 μm.

[0292] In some cases, the length of one or more capillaries used to manufacture the disclosed capillary flow cell device or flow cell box can range from about 5 mm to about 5 cm or greater. In some cases, the length of one or more capillaries 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, or at least 5 cm. In some cases, the length of one or more capillaries can be at most 5 cm, at most 4.5 cm, at most 4 cm, at most 3.5 cm, at most 3 cm, at most 2.5 cm, at most 2 cm, at most 1.5 cm, at most 1 cm, or at most 5 mm. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the length of one or more capillaries can 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 can have any value within this range, for example, about 1.85 cm. In some cases, the device or cassette may include multiple capillaries of two or more identical lengths. In other cases, the device or cassette may include multiple capillaries of two or more different lengths.

[0293] The capillary tubes used to construct the disclosed capillary flow cell apparatus or capillary flow cell box can be made of 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 polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI), and perfluoroelastomer (FFKM) as more chemically inert alternatives, or any combination thereof. In terms of cost and chemical compatibility, PEI falls between polycarbonate and PEEK. FFKM is also known as Kalrez.

[0294] One or more materials used to manufacture capillaries are typically optically transparent to facilitate use with spectral or imaging-based detection techniques. In some cases, the entire capillary will be optically transparent. Alternatively, in some cases, only a portion of the capillary (e.g., an optically transparent "window") will be optically transparent.

[0295] The capillaries used to construct the disclosed capillary flow cell apparatus and capillary flow cell box can be manufactured using any of a variety of techniques known to those skilled in the art, wherein the choice of manufacturing technique generally depends on the choice of material, and vice versa. Examples of suitable capillary manufacturing techniques include, but are not limited to, extrusion, stretching, precision computer numerical control (CNC) machining and boring, laser ablation, etc.

[0296] In some embodiments, the capillaries used in the disclosed capillary flow cell apparatus and cartridges may be off-the-shelf commercial products. Examples of commercial suppliers of precision capillaries include Accu-Glass (St. Louis, MO; precision glass capillaries), Polymicro Technologies (Phoenix, AZ; precision glass and fused silica capillaries), Friedrich & Dimmock, Inc. (Millville, NJ; custom precision glass capillaries), and Drummond Scientific (Broomall, PA; OEM glass and plastic capillaries).

[0297] The capillary fluid adapters attached to the capillary flow cell apparatus and cartridge disclosed herein, as well as other components of the capillary flow cell apparatus or cartridge, can be manufactured using any of a variety of suitable techniques (e.g., extrusion molding, injection molding, compression molding, precision CNC machining, etc.) and materials (e.g., glass, fused silica, ceramics, metals, polydimethylsiloxane, polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HOPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), etc.), wherein the choice of manufacturing technique generally depends on the choice of materials used, and vice versa.

[0298] Figure 30Non-limiting examples of capillary flow cell cassettes are provided, comprising two glass capillaries, fluid adapters (two for each capillary in this example), and a cassette base that mates with the capillaries and / or fluid adapters, thereby holding the capillaries in a fixed orientation relative to the cassette. In some cases, the fluid adapters may be integrated with the cassette base. In some cases, the cassette may include additional adapters that mate with the capillaries and / or capillary fluid adapters. As described elsewhere herein, in some cases, the cassette may include additional functional components. In some cases, the capillaries are permanently mounted in the cassette. In some cases, the cassette base is designed to allow one or more capillaries of the flow cell cassette to be interchangeably removed and replaced. For example, in some cases, the cassette base may include a hinged “flip-top” configuration that allows it to be opened to allow removal and replacement of one or more capillaries. In some cases, the cassette base is configured to be mounted on, for example, a stage of a fluorescence microscope or within a cassette holder of a fluorescence imaging module or instrument system of this disclosure.

[0299] In some cases, the disclosed flow cell devices may include microfluidic devices (or “microfluidic chips”) and cartridges, wherein the microfluidic devices are fabricated by forming fluid channels in one or more layers of suitable material and include one or more fluid channels (e.g., “analysis” channels) configured to perform analytical techniques, which also include imaging as a detection method. In some embodiments, 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 fluid channels (e.g., “analysis” fluid channels) configured to perform analytical techniques, which also include imaging as a detection method. In some cases, the disclosed microfluidic devices may also include additional fluid channels (e.g., for reagent dilution or mixing), reagent reservoirs, waste reservoirs, adapters for external fluid connections, etc., to provide integrated “lab-on-a-chip” functionality.

[0300] Non-limiting examples of microfluidic flow cell cassettes include: a chip having two or more parallel glass channels formed on the chip, a fluid adapter coupled to the chip, and a cassette base mated to the chip and / or the fluid adapter to position the chip relative to the cassette in a fixed orientation. In some cases, the fluid adapter may be integrated with the cassette base. In some cases, the cassette may include additional adapters mated to the chip and / or the fluid adapter. In some cases, the chip is permanently mounted in the cassette. In some cases, the cassette base is designed to allow one or more chips in the flow cell cassette to be interchangeably removed and replaced. In some cases, the cassette base may include a hinged “flip-top” configuration that allows it to be opened to remove and replace one or more chips. In some cases, the cassette base is configured to be mounted within a stage, for example, in a microscope system or a cassette holder in an imaging system. In these non-limiting examples, even though only one chip is described, it should be understood that more than one chip may be used in a microfluidic flow cell cassette. The flow cell cassettes of this disclosure may include a single microfluidic chip or multiple microfluidic chips. In some cases, the flow cell cassette of this disclosure may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more microfluidic chips. Packaging one or more microfluidic devices within the cassette can facilitate ease of operation and proper positioning of the devices in an optical imaging system.

[0301] The disclosed microfluidic devices and fluid channels within the housing can have a variety of cross-sectional geometries, including but not limited to circular, elliptical, square, rectangular, triangular, rounded square, rounded rectangular, or rounded triangular cross-sectional geometries. In some cases, the fluid channel can have any specified cross-sectional size or group of sizes. For example, in some cases, the height (e.g., gap height), width, or maximum cross-sectional size (e.g., diagonal if the fluid channel has a square, rounded square, rectangular, or rounded rectangular cross-section) of the fluid channel can range from about 10 μm to about 10 mm. In some aspects, the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel 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 aspects, the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel can be up to 10 mm, up to 9 mm, up to 8 mm, up to 7 mm, up to 6 mm, up to 5 mm, up to 4 mm, up to 3 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, up to 100 μm, up to 75 μm, up to 50 μm, up to 25 μm, or up to 10 μm. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel can range from about 20 μm to about 200 μm. Those skilled in the art will recognize that the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel can have any value within that range, for example, about 122 μm.

[0302] In some cases, the length of the fluid channel in the disclosed microfluidic device and cartridge can range from about 5 mm to about 10 cm or more. In some cases, the length of the 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 cases, the length of the fluid channel can be at most 10 cm, at most 9 cm, at most 8 cm, at most 7 cm, at most 6 cm, at most 5 cm, at most 4.5 cm, at most 4 cm, at most 3.5 cm, at most 3 cm, at most 2.5 cm, at most 2 cm, at most 1.5 cm, at most 1 cm, or at most 5 mm. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the length of the fluid channel can range from about 1.5 cm to about 2.5 cm. Those skilled in the art will recognize that the length of the fluid channels can have any value within this range, for example, about 1.35 cm. In some cases, a microfluidic device or cartridge may include multiple fluid channels of the same length. In other cases, a microfluidic device or cartridge may include multiple fluid channels of different lengths.

[0303] The disclosed microfluidic devices will include at least one layer of material having one or more fluid channels formed therein. In some cases, the microfluidic chip may include two layers bonded together to form one or more fluid channels. In some cases, the microfluidic chip may include three or more layers bonded together to form one or more fluid channels. In some cases, the microfluidic channel may have an open top. In some cases, the microfluidic channel may be fabricated within a layer (e.g., the top surface of a bottom layer) and may be sealed by bonding the top surface of the bottom layer to the bottom surface of a top layer of material. In some cases, the microfluidic channel may be fabricated within a layer, for example as a patterned channel whose depth extends through the entire thickness of the layer, and then sandwiched between and bonded to two unpatterned layers to seal the fluid channel. In some cases, the microfluidic channel is fabricated by removing a sacrificial layer from the surface of a substrate. This method does not require etching away the bulk substrate (e.g., glass or silicon wafer). Instead, the fluid channel is located on the surface of the substrate. In some cases, the microfluidic channel may be fabricated within or on the surface of a substrate and then sealed by depositing a conformal film or layer on the surface of the substrate to form a subsurface or buried fluid channel in the chip.

[0304] Microfluidic chips can be fabricated using a combination of microfabrication processes. Because the devices are microfabricated, the matrix material is typically selected based on its compatibility with known microfabrication techniques, such as photolithography, wet chemical etching, laser ablation, laser irradiation, air abrasion, injection molding, embossing, and others. The matrix material is also typically selected to be compatible with the entire range of conditions that the microfluidic device may be exposed to, including extreme pH, temperature, salt concentration, and the application of electromagnetic fields (e.g., light) or electric fields.

[0305] The disclosed microfluidic chip can be made of 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.), quartz glass (quartz), silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HOPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI), and perfluoroelastomers (FFKM) (as a more chemically inert alternative), or any combination thereof. In some preferred embodiments, the matrix material may include silica-based matrices such as borosilicate glass and quartz, as well as other suitable materials.

[0306] The disclosed microfluidic device can be fabricated using any of a variety of techniques known to those skilled in the art, wherein the choice of fabrication technique generally depends on the choice of materials used, and vice versa. Microfluidic channels on a chip can be constructed using techniques suitable for forming microstructures or micropatterns on a substrate surface. In some cases, fluid channels are formed by laser irradiation. In some cases, microfluidic channels are formed by focused femtosecond laser irradiation. In some cases, microfluidic channels are formed by photolithography and etching, including but not limited to chemical etching, plasma etching, or deep reactive ion etching. In some cases, laser etching is used to form microfluidic channels. In some cases, direct-write lithography is used to form microfluidic channels. Examples of direct-write lithography include electron beam direct writing and focused ion beam polishing.

[0307] In another preferred embodiment, the matrix material may include polymeric materials, such as plastics (e.g., polymethyl methacrylate (PMMA), polycarbonate, polytetrafluoroethylene (PTFE)). TMPolymer matrices such as polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), and polysulfone can be readily patterned or microfabricated using microfabrication techniques such as those described above. In some cases, microfluidic chips can be made from polymeric materials, for example, from microfabricated master discs, using well-known molding techniques (e.g., injection molding, embossing, die-cutting, or by polymerizing polymer precursor materials in a mold (see, for example, U.S. Patent No. 5,512,131)). In some cases, such polymeric matrix materials are preferred because they are easy to manufacture, inexpensive, and disposable, and because of their general inertness to most extreme reactive conditions. Like flow cell devices made from other materials (e.g., glass), flow cell devices made from these polymeric materials may include treated surfaces (e.g., derivatized or coated surfaces) to enhance their utility in microfluidic systems, as discussed in more detail below.

[0308] Typically, the microfabrication techniques described above are used to fabricate fluid channels and / or fluid chambers of a microfluidic device as microscale channels (e.g., grooves, notches, etc.) into the upper surface of a first substrate. The first substrate includes a top side and a bottom side having a first flat surface. In microfluidic devices prepared according to the methods described herein, multiple fluid channels (e.g., grooves and / or notches) are formed on the first flat surface. In some cases, the fluid channels (e.g., grooves and / or notches) formed in the first flat surface (before bonding to the second substrate) have a bottom wall and side walls, with the top remaining open. In some cases, the fluid channels (e.g., grooves and / or notches) formed in the first flat surface (before bonding to the second substrate) have a bottom wall and side walls, with the top remaining closed. In some cases, the fluid channels (e.g., grooves and / or notches) formed in the first flat surface (before bonding to the second substrate) have only side walls, without a top or bottom surface (i.e., the fluid channel spans the entire thickness of the first substrate).

[0309] Fluid channels and chambers can be sealed by positioning a first flat surface of the first substrate in contact with and bonding with a flat surface of the second substrate to form channels and / or chambers (e.g., interiors) of the device at the junction of these two components. In some cases, after bonding the first substrate to the second substrate, the structure can be further positioned to contact and bond to a third substrate. In some cases, the third substrate can be positioned to contact the side of the first substrate that is not in contact with the second substrate. In some cases, the first substrate is placed between the second and third substrates. In some cases, the second and third substrates can cover and / or seal grooves, notches, or orifices formed in the first substrate to form channels and / or chambers (e.g., interiors) of the device at the junction of these components.

[0310] The device may have openings oriented such that they are in fluid communication with at least one fluid channel and / or fluid chamber formed within the device, thereby forming a fluid inlet and / or fluid outlet. In some cases, the openings are formed on a first substrate. In some cases, the openings are formed on a first substrate and a second substrate. In some cases, the openings are formed on a first substrate, a second substrate, and a third substrate. In some cases, the openings are located on the top side of the device. In some cases, the openings are located on the bottom side of the device. In some cases, the openings are located at a first end and / or a second end of the device, and the channel extends in a direction from the first end to the second end.

[0311] Those skilled in the art generally understand the conditions under which substrates can be bonded together, and that such bonding is typically achieved by any of a variety of methods, the choice of which can vary depending on the properties of the substrate material used. For example, thermal bonding of substrates can be applied to many substrate materials, including, for example, glass- or silica-based substrates and some polymer-based substrates. Such thermal bonding techniques typically involve mating the substrate surfaces to be bonded under elevated temperatures and, in some cases, applied external pressure. The precise temperature and pressure used will generally vary depending on the properties of the substrate material used.

[0312] For example, for silica-based matrix materials, i.e., glass (borosilicate glass, Pyrex glass) TM Borosilicate glass, fused silica, and other substrates are typically thermally bonded 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 about 550°C, while borosilicate glass is typically thermally bonded at 800°C or close to 800°C. On the other hand, quartz substrates are typically thermally bonded at 1200°C or close to 1200°C. These bonding temperatures are usually achieved by placing the substrates to be bonded in a high-temperature annealing furnace.

[0313] On the other hand, thermally bonded polymer matrices typically utilize lower temperatures and / or pressures than silica-based matrices to prevent excessive melting and / or deformation of the matrix, such as flattening of the device's interior (i.e., fluid channels or chambers). Typically, such elevated temperatures for bonding polymer matrices range from about 80°C to about 200°C, depending on the polymer material used, and are preferably between about 90°C and about 150°C. Because the temperatures required for bonding polymer matrices are significantly lower, this bonding can often be performed without the need for high-temperature ovens used for bonding silica-based matrices. As described in more detail below, this allows for the incorporation of a heat source into a single, integrated bonding system.

[0314] Binders can also be used to bond matrices together according to well-known methods, which typically involve applying a layer of binder between the matrices to be bonded and pressing them together until the binder cures. Various binders can be used according to these methods, including, for example, commercially available UV-curable binders. According to the invention, alternative methods can also be used to bond the matrices together, including, for example, acoustic or ultrasonic welding and / or solvent welding of polymer components.

[0315] Typically, multiple microfluidic chips or devices are fabricated simultaneously using, for example, “wafer-level” manufacturing. For instance, a polymer matrix can be imprinted or molded into large, separable wafers, which are then paired and bonded together. Individual devices or bonded matrices can then be separated from the larger wafers by dicing or dicing. Similarly, for silica-based matrices, individual devices can be fabricated from larger matrix wafers or plates, allowing for higher manufacturing throughput. Specifically, multiple fluid channel structures can be fabricated on a first matrix wafer or plate, then covered and bonded to a second matrix wafer or plate, and optionally further covered and bonded to a third matrix wafer or plate. The individual devices are then separated from the larger matrix using known methods such as sawing, dicing, and breaking.

[0316] As described above, a top or second substrate covers a bottom or first substrate to seal various channels and chambers. During the bonding process according to the method of this disclosure, the bonding of the first and second substrates can be performed using vacuum and / or pressure to maintain optimal contact between the two substrate surfaces. Specifically, optimal contact between the bottom and top substrates can be maintained, for example, by aligning a flat surface of the bottom substrate with a flat surface of the top substrate and by applying a vacuum through holes provided through the top substrate. Typically, a vacuum is applied to the holes in the top substrate by placing the top substrate on a vacuum chuck, which typically includes a mounting stage or surface with an integrated vacuum source. In the case of silica-based substrates, the bonding substrates are subjected to high temperatures to produce an initial bond, allowing the bonded substrates to then be transferred to an annealing furnace without any offset relative to each other.

[0317] Alternative bonding systems for use with the apparatus described herein include, for example, a binder dispensing system for applying a binder layer between two flat surfaces of the substrate. This can be accomplished by applying the binder layer prior to the mating substrates, or by placing a quantity of binder at one edge of an adjacent substrate and allowing the wicking action of the two mating substrates to pull the binder across the space between the two substrates.

[0318] In some cases, the entire bonding system may include an automated system for placing the top and bottom substrates on a mounting surface and aligning them for subsequent bonding. Typically, such systems include translation systems for moving the mounting surface or one or more top and bottom substrates relative to each other. For example, a robotic system may be used to sequentially lift, translate, and place each of the top and bottom substrates onto a mounting table and within an alignment structure. After the bonding process, such systems may also remove the finished products from the mounting surface and transfer these paired substrates to subsequent operations, such as separation or dicing operations, annealing furnaces for silica-based substrates, etc., before placing other substrates on them for bonding.

[0319] In some cases, the fabrication of microfluidic chips involves stacking or laminating two or more layers of substrate (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 otherwise fabricating features into the surface of a first layer. A second layer is then laminated or bonded to the surface of the first layer to seal these features and provide fluid elements of the device, such as fluid channels.

[0320] As described above, in some cases, one or more capillary flow cell devices or microfluidic chips can be mounted in a housing to form a capillary flow cell housing or microfluidic housing. In some cases, the capillary flow cell housing or microfluidic housing may further include additional components integrated with the housing to provide enhanced performance for a specific application. Examples of additional components that can be integrated into the housing include, but are not limited to, adapters or connectors for fluid connection to other components of the system, fluid flow control components (e.g., microvalves, micropumps, mixing manifolds, etc.), temperature control components (e.g., resistance heating elements, metal plates used as heat sources or radiators, piezoelectric (Peltier) devices 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 microlight sources that can collectively facilitate spectral measurements and / or imaging of one or more capillary or fluid channels).

[0321] Fluid adapters, housings, and other housing assemblies can be connected to capillaries, capillary flow cell devices, microfluidic chips (or fluid channels within chips) using any of a variety of techniques known to those skilled in the art, including but not limited to press-fitting, binder bonding, solvent bonding, laser welding, and any combination thereof. In some cases, the inlets and / or outlets of the microfluidic channels in the microfluidic chip are holes on the top surface of the chip, and the fluid adapter can be attached to or coupled to the inlets and / or outlets of the microfluidic channels within the chip. In some cases, the housing may include additional adapters (i.e., in addition to the fluid adapter) that mate with the chip and / or the fluid adapter and help position the chip within the housing. These adapters can be constructed using the same manufacturing techniques and materials outlined above for the fluid adapters.

[0322] The housing (or "casing") can be made of metallic and / or polymeric materials, such as aluminum, anodized aluminum, polycarbonate (PC), acrylic (PMMA), or Ultem (PEI), while other materials are consistent with this disclosure. The housing can be manufactured using CNC machining and / or molding techniques and is designed such that one, two, or more capillary or microfluidic chips are constrained by the housing in a fixed orientation to create one or more independent flow channels. The capillary or chip can be mounted in the housing using, for example, a press-fit design or by mating with a compressible adapter made of silicone or a fluorinated elastomer. In some cases, two or more components of the housing (e.g., an upper and lower half) are assembled using, for example, screws, clips, pliers, or other fasteners, such that the two halves are separable. In some cases, two or more components of the housing are assembled using, for example, adhesives, solvent bonding, or laser welding, such that the two or more components are permanently attached.

[0323] Flow cell surface coating: In some cases, one or more inner surfaces of the capillary lumen or microfluidic channel in the disclosed flow cell device may be coated using any of a variety of surface modification techniques or polymer coatings known to those skilled in the art. In some cases, the coating may be formulated to increase or maximize the number of available binding sites (e.g., tethered oligonucleotide adaptor / primer sequences) on one or more inner surfaces to increase or maximize the foreground signal, such as the fluorescence signal generated by hybridization of a labeled nucleic acid molecule with a tethered oligonucleotide adaptor / primer sequence. In some cases, the coating may be formulated to reduce or minimize nonspecific binding of the fluorophore to 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 the background signal, such as background fluorescence generated by nonspecific binding of labeled biomolecules or autofluorescence of the sample carrier structure. In some cases, the combination of increased foreground signal and reduced background signal that can be achieved by using the disclosed coating may thus provide an improved signal-to-noise ratio (SNR) in spectroscopic measurements or an improved contrast-to-noise ratio (CNR) in imaging methods.

[0324] As will be discussed in more detail below, the disclosed hydrophilic polymer-coated flow cell device (optionally used in combination with an improved hybridization and / or amplification protocol) produces a solid-phase bioassay reaction exhibiting: (i) negligible nonspecific binding of proteins and other reaction components (thus reducing or minimizing matrix background), (ii) negligible nonspecific nucleic acid amplification products, and (iii) providing a tunable nucleic acid amplification reaction. Although this document is primarily described in the context of nucleic acid hybridization, amplification, and sequencing assays, those skilled in the art will understand that the disclosed low-binding vector can be used in any of a variety of other bioassay formats, including but not limited to sandwich immunoassays, enzyme-linked immunosorbent assays (ELISA), etc.

[0325] In a preferred aspect, one or more coating materials may be applied to the surface of the internal flow cell device, wherein the number of layers and / or the material composition of each layer are selected to modulate one or more surface properties of the internal flow cell device surface, as described in U.S. Patent Application No. 16 / 363,842. Examples of modifiable surface properties include, but are not limited to, surface hydrophilicity / hydrophobicity, total coating thickness, surface density of chemically reactive functional groups, surface density of grafted linker molecules or oligonucleotide adaptors / primers, etc. In some preferred applications, one or more surface properties of the capillary or channel lumen are modulated to, for example, (i) provide very low nonspecific binding of proteins, oligonucleotides, fluorophores, and other molecular components for chemical or biological analytical applications, including solid-phase nucleic acid amplification and / or sequencing applications, (ii) provide improved solid-phase nucleic acid hybridization specificity and efficiency, and (iii) provide improved solid-phase nucleic acid amplification rate, specificity, and efficiency.

[0326] 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) can be used to generate one or more chemically modified layers on the surface of an internal flow cell device. The choice of components used may vary to alter one or more properties of the carrier surface, such as the surface density of functional groups and / or tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the carrier surface, or the three three-dimensional properties of the carrier surface (i.e., “thickness”).

[0327] The attachment chemistry used to graft the first chemically modified layer onto the inner surface of the flow cell (capillary or channel) typically depends on both the material used to fabricate the flow cell device and the chemical properties of the layer. In some cases, the first layer may be covalently attached to the surface of the inner flow cell device. In others, the first layer may be non-covalently linked to the surface, for example, through non-covalent interactions such as electrostatic interactions, hydrogen bonds, or van der Waals interactions between the surface of the first layer and the molecular components, such as adsorption. In either case, the substrate surface can be treated prior to the attachment or deposition of the first layer. Any of a variety of surface treatment techniques known to those skilled in the art can be used to clean or treat the carrier surface. For example, glass or silicon surfaces can be acid-washed using a Piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)) and / or cleaned using oxygen plasma treatment methods.

[0328] Silane chemistry constitutes a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach additional reactive functional groups (e.g., amine or carboxyl groups), which can then be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths, such as C6, Cl2, C18 hydrocarbons, or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) onto the surface. Examples of suitable silanes that can be used to generate any of the disclosed low-binding carrier surfaces include, but are not limited to, (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), and any of a variety of PEG-silanes (e.g., having molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., having free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, etc.

[0329] Examples of preferred polymers that can be used to generate one or more layers of low-nonspecific binding material on any of the disclosed carrier surfaces include, but are not limited to, polyethylene glycol (PEG), streptavidin, polyacrylamide, polyesters, dextran, polylysine and polylysine copolymers, or any combination thereof, of various molecular weights and branching structures. Examples of conjugation chemistry that can be used to graft one or more layers of material (e.g., polymer layers) onto a carrier surface and / or to crosslink the layers to each other include, but are not limited to, biotin-streptavidin interactions (or variations thereof), His-tagged – Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxides, azides, hydrazides, alkynes, isocyanates, and silanes.

[0330] In some cases, the number of polymer or other chemical layers on the surface of the internal flow cell device can range from 1 to about 10 or greater. In some cases, the number of layers is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some cases, the number of layers can be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any lower and upper limits described in this paragraph can be combined to form the ranges included in this disclosure; for example, in some cases, the number of layers can range from about 2 to about 4. In some cases, one or more layers can all contain the same material. In some cases, each layer can contain a different material. In some cases, multiple layers can contain multiple materials.

[0331] One or more layers of a multilayer surface may contain branched polymers or may be linear. Examples of suitable branched polymers include, but are not limited to: branched PEG, branched polyvinyl alcohol (branched PVA), branched poly(vinylpyridine), branched poly(vinylpyrrolidone) (branched PVP), branched poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched poly(2-hydroxyethyl methacrylate) (branched PHEMA), branched poly(ethylene glycol) monomethyl methacrylate (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine, branched polyglucoside, and dextran.

[0332] In some cases, the branched polymer used to generate one or more layers of any multilayer surface disclosed herein may include at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, or at least 40 branches. Molecules typically exhibit a power of 2 number of branches, such as 2, 4, 8, 16, 32, 64, or 128 branches.

[0333] In some cases, after depositing one or more layers, such as polymer layers, the resulting functional end groups away from the surface may include, but are not limited to, biotin, methoxy ethers, carboxylates, amines, NHS esters, maleimides, and bissilanes.

[0334] The molecular weight of the linear, branched, or multibranched polymer used to form one or more layers of any multilayer surface disclosed herein may be at least 500 Daltons, at least 1,000 Daltons, at least 1,500 Daltons, at least 2,000 Daltons, at least 2,500 Daltons, at least 3,000 Daltons, at least 3,500 Daltons, at least 4,000 Daltons, at least 4,500 Daltons, at least 5,000 Daltons, at least 7,500 Daltons, at least 10,000 Daltons, at least 12,500 Daltons, at least 15,000 Daltons, at least 17,500 Daltons, at least 20,000 Daltons, at least 25,000 Daltons, at least 30,000 Daltons, at least 35,000 Daltons, at least 40,000 Daltons, at least 45,000 Daltons, or at least 50,000 Daltons. In some cases, the molecular weight of the linear, branched, or multibranched polymer used to form one or more layers of any multilayer surface disclosed herein may be up to 50,000 Daltons, up to 45,000 Daltons, up to 40,000 Daltons, up to 35,000 Daltons, up to 30,000 Daltons, up to 25,000 Daltons, up to 20,000 Daltons, up to 17,500 Daltons, up to 15, The molecular weights of the linear, branched, or multibranched polymers used to form any one or more layers of any multilayer surface disclosed herein may range from about 1,500 Daltons to about 20,000 Daltons. The lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure. For example, in some cases, the molecular weight of the linear, branched, or multibranched polymer used to form any one or more layers of any multilayer surface disclosed herein may be in the range of about 1,500 Daltons to about 20,000 Daltons. Those skilled in the art will recognize that the molecular weight of the linear, branched, or multibranched polymer used to produce one or more layers of any of the multilayer surfaces disclosed herein can have any value within that range, for example, about 1,260 Daltons.

[0335] In some cases, two or more layers may be covalently coupled to each other or internally cross-linked to improve the stability of the resulting surface. In some cases, for example, where at least one layer of the multilayer surface comprises a branched polymer, the number of covalent bonds between the branched polymer molecules of the deposited layer and the molecules of the previous layer can range from about one covalent bond per molecule to about 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, or more covalent bonds per molecule. In some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can be up to 32, up to 30, up to 28, up to 26, up to 24, up to 22, up to 20, up to 18, up to 16, up to 14, up to 12, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1. Any lower and upper limits described in this paragraph can be combined to form the ranges covered by this disclosure; for example, in some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can range from about 4 to about 16. Those skilled in the art will recognize that the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer can have any value within this range, for example, about 11 in some cases, or an average of about 4.6 in others.

[0336] Any reactive functional groups remaining after the material layer is coupled to the surface of the internal flow cell device can be selectively blocked by coupling small inert molecules using high-yield coupling chemistry. For example, in the case of attaching a new material layer to a previous layer using amine coupling chemistry, any residual amine groups can then be acetylated or deactivated by coupling with small amino acids (e.g., glycine).

[0337] To scale the surface density of binding sites, such as oligonucleotide adaptor / primer surface density, and to add dimensionality to hydrophilic or amphoteric surfaces, matrices incorporating multilayer coatings of PEG and other hydrophilic polymers have been developed. The adaptor / primer loading density on a surface can be significantly increased by using hydrophilic and amphoteric surface layering methods (including, but not limited to, the polymer / copolymer materials described below). Conventional PEG coating methods use monolayer primer deposition, which has been tested and reported for single-molecule sequencing applications, but does not produce high copy numbers in nucleic acid amplification applications. As described herein, “layering” can be accomplished using any compatible polymer or monomer subunit using conventional crosslinking methods, allowing for the sequential construction of surfaces comprising two or more highly crosslinked layers. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyesters, dextran, polylysine, and copolymers of polylysine and PEG. In some cases, different layers can be cross-linked through various conjugation reactions, including but not limited to biotin-streptavidin binding, azide-alkyne click reactions, amine-NHS ester reactions, thiol-maleimide reactions, and ionic interactions between positively charged and negatively charged polymers. In some cases, materials with high connector / primer densities can be constructed in solution and then layered onto a surface through multiple steps.

[0338] Exemplary PEG multilayers include PEG (8-arm, 16-arm, 8-arm) on PEG-amine-APTES. Similar concentrations were observed for 3-layer multi-arm PEG (8-arm, 16-arm, 8-arm) and (8-arm, 64-arm, 8-arm) on PEG-amine-APTES exposed to 8 μM primers, as well as for 3-layer multi-arm PEG (8-arm, 8-arm, 8-arm) using star-shaped PEG-amines instead of 16-arm and 64-arm PEG. PEG multilayers with comparable first, second, and third PEG layers were also considered.

[0339] In some cases, the resulting surface density of binding sites on the surface of the internal flow cell device, for example, the surface density of oligonucleotide adaptors / primers, ...

Claims

1. A method of sequencing a nucleic acid molecule, comprising: a) providing a flow cell comprising (i) a first plurality of primered target nucleic acid sequences tethered to a first surface, (ii) a second plurality of primered target nucleic acid sequences tethered to a second surface, wherein the second surface is axially displaced from the first surface; b) contacting the first plurality of primered target nucleic acid sequences and the second plurality of primered target nucleic acid sequences with one or more polymer-nucleotide conjugates under conditions sufficient to transiently couple (1) at least one subset of the first plurality of primered target nucleic acid sequences and the second plurality of primered target nucleic acid sequences and (2) at least one subset of a plurality of nucleotide moieties of the one or more polymer-nucleotide conjugates, each polymer-nucleotide conjugate comprising (i) a plurality of nucleotide moieties comprising a plurality of detectable labels, and (ii) a polymer core conjugated to the plurality of nucleotide moieties; and c) imaging the first surface and the second surface to detect a plurality of signals from a subset of the plurality of detectable labels in the subset of the plurality of nucleotide moieties, wherein the detecting comprises: i) illuminating the first surface and the second surface with a first light provided by a structured illumination system under a first set of illumination conditions to project a first plurality of fringes oriented in a particular direction on the first surface and the second surface; ii) capturing a first plurality of phase images of the first surface and the second surface, wherein the position of the first plurality of fringes moves across the surface during capturing of the first plurality of phase images; iii) illuminating the first surface and the second surface with a second light provided by the structured illumination system under a second set of illumination conditions to project a second plurality of fringes on the first surface and the second surface, wherein the second plurality of fringes is angularly offset from the first plurality of fringes on the surface; and iv) capturing a second plurality of phase images of the first surface and the second surface illuminated by the second plurality of fringes, wherein the position of the second plurality of fringes moves across the surface during capturing of the second plurality of fringes; and d) performing a primer extension reaction to incorporate nucleotides comprising a base complementary to a nucleotide of each primered target nucleic acid sequence of the first plurality of primered target nucleic acid sequences and the second plurality of primered target nucleic acid sequences.

2. The method of claim 1, wherein a nucleotide moiety of the plurality of nucleotide moieties is conjugated to a polymer core to form a conjugated polymer-nucleotide.

3. The method of claim 1, wherein the first plurality of fringes and the second plurality of fringes are produced by a diffraction grating.

4. The method of claim 3, wherein the diffraction grating comprises a horizontal grating, a vertical grating, or any combination thereof.

5. The method of claim 4, wherein the first surface is illuminated by the vertical grating and the second surface is illuminated by the horizontal grating.

6. The method of claim 3, wherein the diffraction grating comprises a transmissive phase diffraction grating.

7. The method of claim 1, wherein the structured illumination system comprises a light source.

8. The method of claim 1, wherein the second plurality of fringes is offset by 90 degrees from the first plurality of fringes.

9. The method of claim 1, wherein the structured illumination system comprises an optical phase modulator or a phase shifter.

10. The method of claim 9, wherein the optical phase modulator or the phase shifter offsets the first plurality of fringes and the second plurality of fringes by 1 / 2 or 1 / 4 of a pitch of the first plurality of fringes and the second plurality of fringes.

11. The method of claim 9 or 10, wherein the optical phase modulator comprises a rotating optical phase plate driven by a rotating actuator.

12. The method of claim 1, wherein the structured illumination system comprises one or more of: (i) a collimating optical element; (ii) a polarizer; and (iii) a diffraction grating.

13. The method of claim 1, wherein the structured illumination system comprises a partially silvered mirror that combines the first light and the second light.

14. The method of claim 1, wherein the first plurality of phase images or the second plurality of phase images comprises at least 1 angular orientation of the first plurality of fringes or the second plurality of fringes.

15. The method of claim 1, wherein the first plurality of phase images or the second plurality of phase images further comprises at least 1 phase shift of the first plurality of fringes or the second plurality of fringes.

16. The method of claim 1, wherein the structured illumination system comprises an optical arm comprising a light emitter that emits light and a diffraction grating that diffracts the light emitted by the light emitter to project the first plurality of fringes oriented in a particular direction on the first surface.

17. The method of claim 1, wherein the structured illumination system comprises a plurality of beam splitter slides comprising a plurality of beam splitters mounted on a linear translation stage such that each of the plurality of beam splitters has a fixed orientation relative to an optical axis of the structured illumination system, and wherein the first set of illumination conditions corresponds to a first position of the linear translation stage and the second set of illumination conditions corresponds to a second position of the linear translation stage.

18. The method of claim 17, wherein the plurality of beam splitters comprises a plurality of diffraction gratings.

19. The method of claim 18, wherein the plurality of diffraction gratings comprises two diffraction gratings.

20. The method of claim 1, wherein the structured illumination system comprises a fixed two-dimensional diffraction grating used in combination with a spatial filter wheel to project a one-dimensional diffraction pattern to the surface, and wherein the first set of illumination conditions corresponds to a first position of the spatial filter wheel and the second set of illumination conditions corresponds to a second position of the spatial filter wheel.

21. The method of claim 1, wherein the structured illumination system comprises a second optical arm, the second optical arm comprising: a second light emitter to emit light; and a second diffraction grating to diffract light emitted by the second light emitter to project a second plurality of fringes angularly offset from the first plurality of fringes on the surface.

22. The method of claim 1, wherein the surface comprises a plurality of features regularly patterned in a rectangular array or a hexagonal array.

23. The method of claim 1, wherein the coupling in (b) comprises forming transient binding complexes between nucleotide moieties and nucleotides of the primed target nucleic acid sequence, wherein the transient binding complexes lack a polymerase.

24. The method of claim 1, wherein: (i) the structured illumination system comprises: (1) a light source; (2) an optical phase modulator that rotates an optical phase plate driven by a rotational actuator; (3) a phase shifter that shifts the first plurality of fringes and the second plurality of fringes by 1 / 2 or 1 / 4 of a pitch of the first plurality of fringes and the second plurality of fringes; (4) a collimating optical element; (5) a polarizer; (6) a diffraction grating that generates the first plurality of fringes and the second plurality of fringes, wherein the diffraction grating comprises a horizontal grating, a vertical grating, a transmissive phase diffraction grating, or any combination thereof, and wherein the second plurality of fringes is offset by 90 degrees from the first plurality of fringes; and (7) a partially silvered mirror that combines light of the first plurality of fringes and light of the second plurality of fringes; and (ii) the first plurality of phase images or the second plurality of phase images comprises: (1) at least 1 angular orientation of the first plurality of fringes or the second plurality of fringes; and (2) at least 1 phase shift of the first plurality of fringes or the second plurality of fringes.

Images