High-performance fluorescence imaging module for genomic testing assays

Abstract

Fluorescence imaging system designs are described that provide a larger 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, thereby enabling higher throughput image acquisition and analysis for genomics and other imaging applications.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of January 15, 2021, application number 202180003305.6, and invention name “High-performance fluorescence imaging module for genomic testing and determination” (the application date of the corresponding PCT application is January 15, 2021, and the application number is PCT / US2021 / 013696).

[0002] Cross-references

[0003] This application claims the benefit 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 hereby incorporated by reference in their entireties. Background Art

[0004] In a typical fluorescence-based genomic test assay, for example, genotyping or nucleic acid sequencing (using real-time, cyclic or step-by-step reaction schemes), an excitation light source is used to excite a dye molecule connected to a nucleic acid molecule tethered to a substrate, generating a fluorescent photon signal at one or more spatially positioned positions on the substrate, and subsequently imaging the fluorescence onto an image sensor through an optical system. An analytical method is then used to analyze the image, find the position of the labeled molecule (or clonal amplification cluster of molecules) on the substrate, and quantify the fluorescent photon signal according to wavelength and spatial coordinates, which can then be associated with the degree of a specific chemical reaction (e.g., hybridization event or base addition event) occurring at a specified position on the substrate. Image-based methods provide large-scale parallel and multiplexing capabilities, which help reduce the cost and availability of such technologies. However, due to, for example, the over-dense accumulation of labeled molecules (or clonal amplification clusters of molecules) within a small area on the substrate surface or due to detection errors caused by a low contrast-to-noise ratio (CNR) in the image, errors may occur when attributing the fluorescent signal to the correct molecule (or clonal amplification cluster of molecules). Summary of the Invention

[0005] Disclosed herein is 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 tube lens disposed in an 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 and wherein the at least one tube lens is configured to correct imaging performance so 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 wall thickness of the flow cell 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, the images of the first inner surface and the second inner surface are acquired without moving an 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) greater than 0.3. In some embodiments, the imaging system has a numerical aperture (NA) greater than 1.5 mm. 2 field of view (FOV). In some embodiments, the optical resolution of the image of the first inner surface and the second inner surface is diffraction limited over the entire field of view (FOV). In some embodiments, at least one barrel lens comprises, in sequence, an asymmetric convex-convex lens, a convex-flat lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the imaging system comprises two or more barrel lenses designed to provide optimal imaging performance for the first inner surface and the second inner surface at two or more fluorescence wavelengths. In some embodiments, the imaging system further comprises a focusing mechanism configured to refocus the optical system between acquiring images of the first inner surface and the second inner surface. In some embodiments, the imaging system is configured to image two or more fields of view on at least one of the first inner surface or the second inner surface. In some embodiments, the first inner surface and the second inner surface of the flow cell are coated with a hydrophilic coating, and wherein the hydrophilic coating further comprises a nucleic acid colony at a density of >10,000 nucleic acid colonies / mm 2In some embodiments, the imaging system comprises 1, 2, 3 or 4 imaging channels, which are configured to detect nucleic acid communities that have been labeled with 1, 2, 3 or 4 different detectable markers on at least one of the two different surfaces. In some embodiments, the imaging system is used to monitor affinity sequencing, nucleotide base pairing sequencing, nucleotide binding sequencing or nucleotide incorporation reaction sequencing on at least one of the first and second inner surfaces, and detects the nucleotide bases bound or incorporated. 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 the sample, wherein determining the genotype of the sample includes preparing a nucleic acid molecule for sequencing extracted from the sample and then sequencing the nucleic acid molecule. In some embodiments, the at least one image sensor includes pixels whose pixel size is selected so 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 the 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 the at least one tube lens, the 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] Also disclosed herein is a method for sequencing a nucleic acid molecule, the method comprising: a) imaging a first surface and an axially displaced second surface using an optical system 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 range of greater than 1.0 mm. 2 and b) detecting a fluorescently labeled composition comprising a nucleic acid molecule or a complement thereof disposed on the first surface or the axially displaced second surface to determine the identity of the nucleotides in the nucleic acid molecule.

[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 comprises 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 a hydrophilic coating having a density of >10,000 nucleic acid colonies / mm 2In some embodiments, when the nucleic acid community is labeled with cyanine dye 3 (Cy3), the surface images of the two surfaces acquired using the optical system exhibit a contrast-to-noise ratio (CNR) of at least 5, the optical system comprising a dichroic mirror and a bandpass filter set optimized for Cy3 emission, and acquiring images under non-signal saturation conditions while immersing the surface in 25mM ACES pH 7.4 buffer. In some embodiments, the optical system comprises 1, 2, 3, or 4 imaging channels configured to detect nucleic acid communities that have been labeled with 1, 2, 3, or 4 different detectable markers disposed on at least one of the first surface and the axially displaced second surface. In some embodiments, at least one image sensor comprises pixels having a pixel size selected so 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 tube lens positioned between the objective lens and the at least one image sensor, wherein the at least one tube lens is configured to correct for an imaging performance metric for imaging a first inner surface of the flow cell and a second inner surface of the flow cell. In some embodiments, the wall thickness of the flow cell 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 at least one tube lens comprises, in sequence, an asymmetric convex-convex lens, a convex-plano lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the combination of the objective lens and the tube lens is configured to optimize the modulation transfer function over a medium to high spatial frequency range. In some embodiments, the imaging performance metric comprises a measurement of the modulation transfer function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast-to-noise ratio (CNR), or any combination thereof. In some embodiments, the optical resolution of the image of the first surface and the axially displaced second surface is diffraction limited across the field of view (FOV). In some embodiments, sequencing of the nucleic acid molecules further comprises performing affinity sequencing, nucleotide base pairing sequencing, nucleotide binding sequencing, or nucleotide incorporation reaction sequencing on at least one of the first surface and the axially displaced second surface, and detecting the bound or incorporated nucleotide bases. In some embodiments, the method further comprises determining the genotype of the sample, wherein determining the genotype of the sample comprises preparing the nucleic acid molecules for sequencing and then sequencing the nucleic acid molecules.

[0009] Disclosed herein is an imaging system configured to image two different axially displaced surfaces, the imaging system comprising an objective lens and at least one image sensor, wherein the imaging system has a numerical aperture (NA) less than 0.6 and a diameter greater than 1.0 mm. 2 The imaging system is capable of acquiring images of two different axially displaced surfaces with substantially the same optical resolution without moving an optical compensator into the optical path between the objective lens and the at least one image sensor.

[0010] In some embodiments, the imaging system has a numerical aperture greater than 0.3. In some embodiments, the imaging system further comprises a focusing mechanism for refocusing the optical system between acquiring images of two different axially displaced surfaces. In some embodiments, the imaging system is configured to image two or more fields of view on at least one of the two different axially displaced surfaces. In some embodiments, the two different axially displaced surfaces comprise two surfaces of a flow cell. In some embodiments, the two different surfaces of the flow cell are coated with a hydrophilic coating, and wherein the hydrophilic coating further comprises a nucleic acid colony at a density of >10,000 nucleic acid colonies / mm 2 In some embodiments, the imaging system includes 1, 2, 3 or 4 imaging channels, which are configured to detect nucleic acid communities that have been labeled with 1, 2, 3 or 4 different detectable markers and are arranged on at least one of the two different surfaces. In some embodiments, at least one image sensor includes pixels, and the pixel size of the pixels is selected so 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 barrel lens positioned between the objective lens and the at least one image sensor, and wherein the at least one barrel lens is configured to correct the imaging performance index for imaging the first inner surface of the flow cell and the second inner surface of the flow cell. In some embodiments, the wall thickness of the flow cell 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 barrel lenses, which are designed to provide optimal imaging performance under two or more fluorescence wavelengths. In some embodiments, the optical resolution of the image of the two different axially shifted surfaces is diffraction limited over the entire field of view (FOV).

[0011] Disclosed herein is a method for sequencing nucleic acid molecules, the method comprising: a) imaging a first surface and an axially displaced second surface using an 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 resolution greater than 1.0 mm;2 a) processing images of the first surface and the axially displaced second surface to correct for optical aberrations so that the images of the first surface and the axially displaced second surface have substantially the same optical resolution; and c) detecting a fluorescently labeled composition comprising a nucleic acid molecule or its complement disposed on the first surface or the axially displaced second surface to determine the identity of the nucleotides in the nucleic acid molecule.

[0012] In some embodiments, images of the first surface and the axially displaced second surface are acquired without moving an optical 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 refocusing the optical system alone. In some embodiments, the method further comprises 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 a nucleic acid colony at >10,000 nucleic acid colonies / mm 2In some embodiments, when the nucleic acid community is labeled with cyanine dye 3 (Cy3), the surface images of the two surfaces acquired using the optical system exhibit a contrast-to-noise ratio (CNR) of at least 5, the optical system comprising a dichroic mirror and a bandpass filter set optimized for Cy3 emission, and acquiring images under non-signal saturation conditions while immersing the surface in 25mM ACES pH 7.4 buffer. In some embodiments, the optical system comprises 1, 2, 3, or 4 imaging channels configured to detect nucleic acid communities that have been labeled with 1, 2, 3, or 4 different detectable markers disposed on at least one of the first surface and the axially displaced second surface. In some embodiments, at least one image sensor comprises pixels having a pixel size selected so 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 tube lens positioned between the objective lens and the at least one image sensor, wherein the at least one tube lens is configured to correct for an imaging performance metric for imaging a first inner surface of the flow cell and a second inner surface of the flow cell. In some embodiments, the wall thickness of the flow cell 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 at least one tube lens comprises, in sequence, an asymmetric convex-convex lens, a convex-plano lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens. In some embodiments, the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the combination of the objective lens and the tube lens is configured to optimize the modulation transfer function over a medium to high spatial frequency range. In some embodiments, the imaging performance metric comprises a measurement of the modulation transfer function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast-to-noise ratio (CNR), or any combination thereof. In some embodiments, the optical resolution of the image of the first surface and the axially displaced second surface is diffraction limited across the field of view (FOV). In some embodiments, sequencing of the nucleic acid molecules further comprises performing affinity sequencing, nucleotide binding sequencing, or nucleotide incorporation reaction sequencing on at least one of the first surface and the axially displaced second surface, and detecting the bound or incorporated nucleotide bases. In some embodiments, the method further comprises determining the genotype of the sample, wherein determining the genotype of the sample comprises preparing the nucleic acid molecules for sequencing and then sequencing the nucleic acid molecules.

[0013] Disclosed herein is a system for sequencing nucleic acid molecules, the system comprising: a) an optical system comprising an objective lens and at least one image sensor, wherein the optical system has a numerical aperture (NA) less than 0.6 and a resolution greater than 1.0 mm; 2 and a second axially displaced surface; b) a processor programmed to: i) process the images of the first surface and the axially displaced second surface to correct for optical aberrations so that the images of the first surface and the axially displaced second surface have substantially the same optical resolution; and ii) detect a fluorescently labeled composition comprising a nucleic acid molecule or its complement disposed on the first surface or the axially displaced second surface to determine the identity of the nucleotides in the nucleic acid molecule.

[0014] In some embodiments, the images of the first surface and the axially displaced second surface are acquired without moving an optical compensator into the optical path between the objective lens and the at least one image sensor. In some embodiments, the images of the first surface and the axially displaced second surface are acquired by refocusing the optical system alone. 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 wherein the hydrophilic coating further comprises a nucleic acid colony at a density of >10,000 nucleic acid colonies / mm 2 In some embodiments, the optical system includes 1, 2, 3 or 4 imaging channels, which are configured to detect nucleic acid communities that have been labeled with 1, 2, 3 or 4 different detectable markers on at least one of the first surface or the axially displaced second surface. In some embodiments, at least one image sensor includes pixels, and the pixel size of the pixels is selected so 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 the at least one image sensor, and wherein the at least one barrel lens is configured to correct the imaging performance index for imaging the first inner surface of the flow cell and the second inner surface of the flow cell. In some embodiments, the wall thickness of the flow cell 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, which are designed to provide optimal imaging performance under two or more fluorescence wavelengths.

[0015] The fluorescence imaging system disclosed herein comprises: 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 from a specified field of view of a sample plane after the sample plane is exposed to the excitation light, wherein the numerical aperture of the objective lens is at least 0.3, wherein the working distance of the objective lens is at least 700 μm, and wherein the area of ​​the field of view is at least 2 mm 2 ; and c) at least one image sensor, wherein the fluorescence collected by the objective lens is imaged onto the image sensor, and wherein the pixel size of the image sensor is selected so 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 area of ​​the field of view is at least 2.5 mm 2 In some embodiments, the area of ​​the field of view is at least 3 mm 2. In 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 comprises an XYZ displacement stage, such that the system is configured to acquire a series of two or more fluorescence images in an automated manner, wherein each image of the series is acquired for a different field of view. In some embodiments, the position of the sample plane is adjusted simultaneously in the X, Y, and Z directions to match the position of the objective lens focal plane between the 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 comprises an autofocus mechanism, which is configured to adjust the focal plane position before acquiring images of different fields of view if the error signal indicates that the difference between the position 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 comprises three or more image sensors, and wherein the system is configured to image the fluorescence of each of the three or more wavelength ranges onto a different image sensor. In some embodiments, the difference between the focal plane of each of the three or more image sensors and the position of the sample plane is less than 100 nm. In some embodiments, the difference between the focal plane of each of the three or more image sensors and the position of the sample plane is less than 50 nm. In some embodiments, the total time required to reposition the sample plane, adjust focus if necessary, and acquire an image is less than 0.4 seconds per field of view. In some embodiments, the total time required to reposition the sample plane, adjust focus if necessary, and acquire an image is less than 0.3 seconds per field of view.

[0017] Also disclosed herein is a fluorescence imaging system for dual-surface imaging of a flow cell, comprising: a) an objective lens configured to collect fluorescence generated within a specified field of view of a sample plane within the flow cell; 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 an imaging performance metric of the combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an inner surface of the flow cell, wherein the wall thickness of the flow cell is at least 700 μm, and the gap between the upper inner surface and the lower inner surface is at least 50 μm; wherein the imaging performance metric 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 an 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 corrected to compensate for a coverslip thickness of 0.17 mm (or flow cell wall thickness). In some embodiments, the fluorescence imaging system also includes an electro-optical phase plate located near the objective lens and between the objective lens and the tube lens, wherein the electro-optical phase plate provides correction for optical aberrations caused by the gap between the upper inner surface and the lower inner surface of the fluid-filled flow cell. In some embodiments, at least one tube lens is a composite lens comprising three or more optical components. In some embodiments, at least one tube lens is a composite lens comprising four optical components. In some embodiments, the four optical components include, in sequence, a first asymmetric convex-convex lens, a second convex-flat 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 a different fluorescence wavelength. 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 a different fluorescence wavelength. In some embodiments, the system includes a single objective lens, four tube lenses, and four image sensors, and each of the four tube lenses is designed to provide optimal imaging performance at a different fluorescence wavelength. In some embodiments, the design of the objective lens or at least one of the tube lenses is configured to optimize the modulation transfer function over a medium to high spatial frequency range. In some embodiments, the imaging performance metric includes a measurement of the modulation transfer function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof. In some embodiments, the difference between the imaging performance metrics for imaging the upper and lower inner surfaces of the flow cell is less than 10%.In some embodiments, the difference between imaging performance metrics for imaging the upper and lower interior surfaces of the flow cell is less than 5%. In some embodiments, the use of at least one tube lens provides at least an equivalent or greater improvement in imaging performance metrics for dual-sided imaging compared to a conventional system comprising an objective lens, a motion-activated compensator, and an image sensor. In some embodiments, the use of at least one tube lens provides at least a 10% improvement in imaging performance metrics for dual-sided imaging compared to a conventional system comprising an objective lens, a motion-activated compensator, and an image sensor.

[0019] Disclosed herein is an illumination system for imaging-based solid-phase genotyping and sequencing applications, comprising: a) a light source; b) a liquid light guide configured to collect light emitted by the light source and deliver the light to a designated illumination field on a carrier surface containing a tethered biomacromolecule.

[0020] In some embodiments, the lighting system further comprises a focusing lens. In some embodiments, the designated lighting area has a focal length of at least 2 mm. 2 In some embodiments, for an imaging system for acquiring an image of a support surface, the light delivered to the designated illumination field has a uniform intensity across the designated field of view. In some embodiments, the designated field of view has an area of ​​at least 2 mm. 2 In some embodiments, the light delivered to the designated illumination field has uniform intensity across the designated field of view when the coefficient of variation (CV) of the light intensity is less than 10%. In some embodiments, the light delivered to the designated illumination field has uniform intensity across the designated field of view when the coefficient of variation (CV) of the light intensity is less than 5%. In some embodiments, the light delivered to the designated illumination field has a speckle contrast value of less than 0.1. In some embodiments, the light delivered to the designated illumination field has a speckle contrast value of less than 0.05.

[0021] Incorporated by Reference

[0022] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term in this document and that in an incorporated reference, the term in this document controls. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 1A-1BSchematically depicted are non-limiting examples of imaging dual-surface support structures for presenting sample sites for imaging by the imaging systems disclosed herein. Figure 1A : Illustration of imaging the front and back inner surfaces of the flow cell. Figure 1B : Illustration of imaging the anterior and posterior external surfaces of the stroma.

[0025] Figure 2A-2B A non-limiting example of a multi-channel fluorescence imaging module is shown, which includes a dichroic beam splitter for transmitting an excitation beam to a sample and receiving and redirecting the resulting fluorescence emission by reflection to four detection channels, wherein the four detection channels are configured to detect fluorescence emissions of four different corresponding wavelengths or bands. Figure 2A : Top isometric view. Figure 2B : Bottom isometric view.

[0026] Figure 3A-Figure 3B Shown Figure 2A and Figure 2B The multi-channel fluorescence imaging module includes an optical path within the multi-channel fluorescence imaging module, which includes a dichroic beam splitter, which is used to transmit an excitation light beam to the sample and receive and redirect the generated fluorescence emission to four detection channels by reflection. 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 is a graph showing the relationship between dichroic filter performance and incident beam angle.

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

[0029] Figure 6A-6B An example configuration of a dichroic filter and detection channels of 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 light beam (e.g., the center angle) and the reflective surface of the dichroic filter is less than 45. Figure 6A : Schematic diagram of a multi-channel fluorescence imaging module including four detection channels. Figure 6B : Detailed view showing the angle of incidence (AOI) of a light beam on a dichroic mirror.

[0030] Figure 7 A diagram is provided which shows the corresponding Figure 6A and 6B Improved dichroic filter performance of the imaging module configuration shown in FIG.

[0031] Figure 8A diagram is provided which shows the corresponding Figure 6A and 6B Improved dichroic filter performance of the imaging module configuration shown in FIG.

[0032] Figure 9A-9B A diagram is provided showing the Figure 6A and 6B The imaging module configuration results in reduced surface deformation. Figure 9A The effect of the fold angle on the image degradation caused by adding 1 wave PV spherical power to the last mirror is shown. Figure 9B The effect of the fold angle on the image degradation caused by adding 0.1 waves of PV spherical power to the last mirror is shown.

[0033] Figures 10A-10B Graphs are provided showing improved excitation filter performance (eg, a steeper transition between the passband and surrounding stopbands) due to using s-polarization of the excitation beam. Figure 10A : Transmission spectra of an exemplary bandpass dichroic filter at incident angles of 40 and 45 degrees, where the incident light beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. Figure 10B : Changing the orientation of the light source relative to the dichroic filter so that the incident beam is s-polarized relative to the plane of the dichroic filter results 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 disclosed herein having a numerical aperture (NA) of 0.3 is shown. Figure 11A : First surface. Figure 11B : The second surface.

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

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

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

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

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

[0040] Figures 17A-17B A plot of the calculated Strehl ratio for imaging a second flow cell surface through a first flow cell surface is provided. Figure 17A : Plot of the Strehl ratio for imaging the second flow cell surface through the first flow cell surface as a function of the intermediate fluid layer thickness (fluid channel height) for different objectives and / or optical system numerical apertures. Figure 17B : Plot of Strehl ratio as a function of numerical aperture for imaging the second flow cell surface through the first flow cell surface and an 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 of the present disclosure is provided.

[0042] Figure 19 Light ray trace diagrams are provided for an objective designed to image surfaces on opposite sides of a 0.17 mm thick coverslip.

[0043] Figure 20 Provided when used to image surfaces on opposite sides of a 0.17 mm thick coverslip, Figure 19 A diagram of the modulation transfer function of an objective lens as it varies with spatial frequency is shown in FIG.

[0044] Figure 21 Provided when used to image the surface opposite the 0.3 mm thick coverslip, Figure 19 A diagram of the modulation transfer function of an objective lens as it varies with spatial frequency is shown in FIG.

[0045] Figure 22 When used to image a surface separated from a surface on the opposite side of a 0.3 mm thick cover glass by a 0.1 mm thick aqueous fluid layer, Figure 19 A diagram of the modulation transfer function of an objective lens as it varies with spatial frequency is shown in FIG.

[0046] Figure 23 Provided when used to image surfaces on opposite sides of a 1.0 mm thick coverslip, Figure 19A diagram of the modulation transfer function of an objective lens as it varies with spatial frequency is shown in FIG.

[0047] Figure 24 When used to image a surface separated from a surface on the opposite side of a 1.0 mm thick coverslip by a 0.1 mm thick aqueous fluid layer, Figure 19 A diagram of the modulation transfer function of an objective lens as it varies with spatial frequency is shown in FIG.

[0048] Figure 25 Provides ray tracing diagrams for tube lens design. Figure 19 Used in conjunction with the objective shown, the tube lens provides improved two-sided imaging through a 1 mm thick coverslip.

[0049] Figure 26 Provided when used to image surfaces on opposite sides of a 1.0 mm thick coverslip, Figure 25 Graph of the modulation transfer function of the objective lens and tube lens combination as a function of spatial frequency is shown in FIG.

[0050] Figure 27 When used to image a surface separated from a surface on the opposite side of a 1.0 mm thick coverslip by a 0.1 mm thick aqueous fluid layer, Figure 25 Graph of the modulation transfer function of the objective lens and tube lens combination as a function of spatial frequency is shown in FIG.

[0051] Figure 28 Ray trace plots are provided for the disclosed tube lens design (left), which has been optimized to provide high-quality dual-sided imaging performance. Since the tube lens is no longer infinity-corrected, an appropriately designed null lens (right) can be used in combination with the tube lens to compensate for the non-infinity-corrected tube lens for manufacturing and inspection purposes.

[0052] Figure 29 A non-limiting example of a single capillary flow cell with two fluidic adapters is shown.

[0053] Figure 30 A non-limiting example of a flow cell cartridge is shown, which includes a base, a fluidic adapter, and optionally other components, and is designed to accommodate two capillaries.

[0054] Figure 31 A non-limiting example of a system is illustrated that includes a single capillary flow cell connected to various fluid flow control components, where the single capillary is compatible with mounting on a microscope stage or in a custom imaging instrument for use in various imaging applications.

[0055] Figure 32A non-limiting example of a system is illustrated that includes a capillary flow cell cartridge with integrated diaphragm valves to reduce or minimize dead volume and conserve certain critical reagents.

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

[0057] Figure 34 One non-limiting example of controlling the temperature of a capillary flow cell by using a metal plate placed in contact with the flow cell cartridge is illustrated.

[0058] Figure 35 One non-limiting method for temperature control of a capillary flow cell including a non-contact thermal control mechanism is illustrated.

[0059] Figures 36A-36C A non-limiting example of flow cell device fabrication is illustrated. Figure 36A The preparation 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 preparation of a three-piece glass flow cell is shown.

[0060] Figures 37A-37C Non-limiting examples of glass flow cell designs are illustrated. Figure 37A A one-piece glass flow cell design is shown. Figure 37B A two-piece glass flow cell design is shown. Figure 37C A three-piece glass flow cell design is shown.

[0061] Figure 38 Illustration of visualization of cluster expansion in the capillary lumen.

[0062] Figure 39 A non-limiting example of a block diagram of the sequencing system disclosed herein is provided.

[0063] Figure 40 Non-limiting examples of flow diagrams of 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 A non-limiting example of a flow chart for acquiring and processing structured illumination images of a flow cell surface as disclosed herein is provided.

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

[0067] Figures 44A-44B A non-limiting schematic diagram of a multiplexing read head as disclosed herein is provided. Figure 44A : Side view of a multiplexed read head, wherein a first subset of a plurality of individual microfluorometers is configured to image a first surface (e.g., a first interior surface) of a flow cell, and a second subset of a plurality of individual microfluorometers is configured to image a second surface (e.g., a second interior surface) of the flow cell. Figure 44B : Figure 44A Figure 2. Top view of a multiplexing readhead showing the imaging paths acquired by the individual microfluorometers of the multiplexing readhead. DETAILED DESCRIPTION

[0068] There is a need for fluorescence imaging methods and systems that provide increased optical resolution and improved image quality for genomics applications, resulting in corresponding improvements in genomic testing accuracy. Disclosed herein are 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, a higher spatial sampling frequency, faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., images of different fields of view), improved imaging system duty cycle, and higher throughput image acquisition and analysis.

[0069] In some cases, such as for dual-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 channels with a height or thickness of 50-200 μm)), improvements in imaging performance can be achieved using novel objective lens designs that correct for optical aberrations introduced by imaging surfaces on the opposite side of the thick coverslip and / or fluid channels from the objective lens.

[0070] In some cases, such as for double-sided (flow cell) imaging applications (including the use of thick flow cell walls (e.g., wall (or cover glass) thickness >700 μm and fluid channels (e.g., fluid channels with a height or thickness of 50-200 μm)), improvements in imaging performance can be achieved even when using commercially available off-the-shelf objectives by using a novel tube lens design (unlike the tube lens in conventional microscopes that simply forms an image at the intermediate image plane) that, in combination with the objective, corrects for optical aberrations caused by the thick flow cell walls and / or the intermediate fluid layer.

[0071] In some cases, such as for multi-channel (e.g., two-color or four-color) imaging applications, improvements in imaging performance can be achieved by using multiple tube lenses, one for each imaging channel, where each tube lens design has been optimized for a specific wavelength range used in that imaging channel.

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

[0073] Various multi-channel fluorescence imaging module designs are disclosed that can include illumination and imaging light paths that include folded light paths (e.g., including one or more beam splitters or combiners, such as dichroic beam splitters or combiners) that direct an excitation beam to an objective and direct emission light transmitted through the objective to multiple detection channels. Some particularly advantageous features of the fluorescence imaging modules described herein include specifying dichroic filter incident angles that result in steeper and / or more uniform transitions between the passband and stopband wavelength regions of the dichroic filter. Such filters can be included within folded optics and can include dichroic beam splitters or combiners. Additional advantageous features of the disclosed imaging optics designs can include the position and orientation of one or more excitation light sources and one or more detection light paths relative to the objective and the dichroic filter that receives the excitation beam. The excitation beam can also be linearly polarized, and the linear polarization can be oriented such that s-polarized light is 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 emission beam due to surface deformations of the dichroic filter. The fluorescence imaging modules described herein may or may not include any of these features and may or may not include any of these advantages.

[0074] Also described herein is a device and system for analyzing a large number of different nucleic acid sequences by imaging an array of fixed nucleic acid molecules or amplified nucleic acid clusters, such as those formed on the flow cell surface. Devices and systems described herein can also be used for, for example, sequencing the genomes of the comparison, tracking gene expression, performing microRNA sequence analysis, epigenomics, aptamer and phage display library characterization, and performing other sequencing applications. Devices and systems disclosed herein include various combinations of optics, machinery, fluid, heat, electricity, and computing devices / aspects. The advantages imparted by the disclosed flow cell devices, boxes, and systems include, but are not limited to: (i) reducing the manufacturing complexity and cost of the device and system, (ii) significantly reducing 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 (e.g., syringe pumps and diaphragm valves, etc.), and (v) flexible system throughput.

[0075] Disclosed herein are capillary flow cell devices and capillary flow cell cartridges constructed from readily available, disposable single-lumen (e.g., single fluid flow channel) or multi-lumen capillaries, which may further include a fluid adapter, a cartridge base, one or more integrated fluid flow control components, or any combination thereof. Also disclosed herein are capillary flow cell-based systems, which may include one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell cartridges (or microfluidic cartridges), a fluid flow controller module, a temperature control module, an imaging module, or any combination thereof.

[0076] Design features of some disclosed capillary flow cell devices, cartridges, and systems include, but are not limited to, (i) an integrated flow channel construction, (ii) sealed, reliable, and repeatable switching between reagent streams, 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) a replaceable single fluid flow channel device or capillary flow cell cartridge comprising multiple interchangeable flow channels to provide flexible system throughput, and (iv) compatibility with multiple detection methods (e.g., fluorescence imaging).

[0077] Although the disclosed capillary flow cell devices and systems, capillary flow cell cartridges, capillary flow cell-based systems, microfluidic devices and cartridges, and microfluidic chip-based systems are primarily described 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 article 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 modes, for example, brightfield imaging, darkfield 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 belongs.

[0079] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference to "or" herein is intended to cover "and / or" unless stated otherwise.

[0080] As used herein, the term "about" refers to a number plus or minus 10% of the number. Use of the term "about" in the context of a range means the range is minus 10% of its minimum and plus 10% of its maximum.

[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 including, for example, a fluid module, a temperature control module, a displacement platform, automated fluid dispensing and / or microplate handling, a processor or computer, instrument control software, data analysis and display software, and the like.

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

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

[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. Although the excitation wavelength is typically specified as a single wavelength, such as 620 nm, it will be understood by those skilled in the art that this specification refers to a wavelength range or excitation filter bandpass centered around the specified wavelength. For example, in some cases, light at a specified excitation wavelength includes light at a specified wavelength of ±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 with light of an appropriate wavelength. Although the emission wavelength is typically specified as a single wavelength, such as 670 nm, it will be understood by those skilled in the art that this specification refers to a wavelength range or emission filter bandpass centered around a specified wavelength. In some cases, the light of a specified emission wavelength includes light of a specified wavelength ± 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 be consistent with the maximum emission peak of the fluorescent indicator.

[0086] As used herein, fluorescence is "specific" if it originates from a fluorophore that is annealed or otherwise tethered to a surface, such as a fluorescently labeled nucleic acid sequence having a region that is reverse complementary to and anneals to a corresponding segment of an oligonucleotide adaptor on the surface. This fluorescence is in contrast to fluorescence from fluorophores that are not tethered to the surface by such an annealing process, or in some cases, background fluorescence from the surface.

[0087] As used herein, "nucleic acid" (also referred to as "nucleic acid molecule," "polynucleotide," "oligonucleotide," ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is a linear polymer of nucleotides linked by covalent internucleoside bonds, or variants or functional fragments thereof. In natural examples of nucleic acids, the internucleoside bond is typically a phosphodiester bond. However, other examples optionally include other internucleoside bonds, such as phosphorothioate bonds, and may or may not include a phosphate group. 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 between PNA and DNA or RNA, and may also include other types of nucleic acid modifications.

[0088] As used herein, "nucleotide" refers to a nucleotide, nucleoside, or an analog 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 analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, and the like.

[0089] Fluorescence imaging as an information pipeline: A useful summary of the role played by fluorescence imaging systems in typical genomic assays, including nucleic acid sequencing applications, is that of an information pipeline, where a photon signal enters one end of the pipeline, such as the objective lens used for imaging, and location-specific information about the fluorescence signal emerges at the other end, such as at the location of an image sensor. As more information is transmitted through this pipeline, it is inevitable that some content will be lost in transit and never recovered. An example of this is when there are too many labeled molecules (or clonally expanded clusters of molecules) in a small area on a substrate surface to be clearly resolved in an image; at the location of the image sensor, it is difficult to distinguish between photon signals generated by adjacent molecular clusters, increasing the probability of attributing a signal to the wrong cluster and resulting in false detection.

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

[0091] 1) Matching the density of physical features 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. Mismatching these parameters can lead to information loss or, in some cases, even erroneous information. For example, spatial aliasing can 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 to the overall image quality of the optical imaging system and the quality of focus over the entire field of view.

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

[0094] 4) Maximize the separation of spectral content to reduce crosstalk between fluorescence imaging channels.

[0095] 5) Effective synchronization of image acquisition steps by repositioning the sample or optics between image captures of different fields of view to minimize downtime of the imaging system (or maximize duty cycle) and thereby maximize the overall throughput of the image capture process.

[0096] This disclosure describes a systematic approach to addressing each of the design elements outlined above and creating a component-level description of the imaging system.

[0097] Improved optical resolution and image quality to improve or maximize information transfer and throughput: A non-limiting design exercise can be to start with the optical resolution required to distinguish two adjacent features specified in terms of the number of line pairs X / mm (lp / mm) and convert this into a corresponding numerical aperture (NA). The numerical aperture requirement can then be used to evaluate the resulting impact on the 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. In addition, wavefront errors can negatively impact MTF, necessitating the use of true system MTF rather than diffraction-limited optics predictions to improve or optimize optical system designs. Note that while design practice may primarily consider the MTF of the objective lens, as used herein, MTF refers to the total system MTF (including the complete optical path from the coverslip to the image sensor).

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

[0100] In a typical design, the value of C may need to be at least 4 so that a simple thresholding method can be used to avoid misclassification of the fluorescence signal. Assuming a Gaussian distribution of fluorescence signal intensity around the mean, at C 图像 When the MTF is >4, the expected error in correctly classifying the fluorescence signal (e.g., as 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 CNRs (C ) greater than 12 (or even higher) can be achieved for clusters of clonally amplified, labeled oligonucleotide molecules tethered to a substrate surface when measured for sparse fields (i.e., when the surface density of clusters or spots is low), where the MTF value is close to 100%. 样品 ) value. Assuming the sample plane CNR value C 样品 >12, and the target classification error rate is <0.1% (therefore, C 图像 >4), then in some embodiments, the minimum value of M(1 / d) can be determined to be M(1 / d)=4 / 12-33%. Therefore, a modulation transfer function threshold of at least 33% can be used to preserve the information content of the transmitted image.

[0101] Design practice can relate the minimum separation distance d of two features or points to the optical resolution requirement (expressed as X (lp / mm) as described above), d = (1 mm) / X, that is, d is the minimum separation distance between two features or points that can be fully resolved by the optical system. In some designs disclosed herein, where the goal of the design analysis is 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, the value of d (the minimum resolvable point separation distance at the sample plane) is reduced, thereby enabling 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 in Xlp / 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 result in aliasing because the optical sensor cannot adequately sample the higher frequency information resolved by the optical system.

[0103] In some designs disclosed herein, an oversampling scheme based on the relationship S = B*Y (where B ≥ 2 and Y is the true optical system MTF limit) can be used to further improve the information transfer capability of the imaging system. As described 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, so the modulation is zero at Y (lp / mm). Therefore, in the disclosed designs, Y (lp / mm) can be advantageously significantly greater than X. For values ​​of B ≥ 2, the disclosed designs are oversampled for the sample object frequency X, i.e., S ≥ B*Y > 2*X.

[0104] The above relationship can be used to determine the system magnification and provides an upper limit for the image sensor pixel size. The image sensor pixel size is chosen to match the optical quality of the system and the spatial sampling frequency required to reduce aliasing. A lower limit for the image sensor pixel size can be determined based on the photon flux, as the relative noise contribution increases with smaller pixels.

[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 an increased depth of field. This increased depth of field can achieve dual-surface imaging, where two surfaces of different depths can be imaged simultaneously with or without refocusing. As described above, reducing the NA can reduce the optical resolution. In some embodiments, a higher excitation beam power, such as 1W or higher, can be used to generate a strong signal. Intrinsic high-contrast samples (i.e., sample surfaces comprising a strong foreground signal and a significantly reduced background signal) can also be used to facilitate the acquisition of high contrast-to-noise ratio (CNR) images, such as having a CNR value>20, which provides improved signal discrimination for base detection in nucleic acid sequencing applications, etc. In some optical system designs disclosed herein, a sample carrier structure (e.g., a flow cell with a hydrophilic surface) is used to reduce background noise.

[0106] In various embodiments, disclosed optical system provides a large field of view (FOV). For example, a FOV greater than 2mm or 3mm can be provided for some optical imaging systems including, for example, an objective lens and a tube lens. In some cases, the optical imaging system provides a reduced magnification, for example, a magnification less than 10x. In some embodiments, this reduced magnification can promote a large FOV design. Although the magnification is reduced, owing to the detector array with a small pixel size or spacing can be used, the optical resolution of such a system is still sufficient. In some embodiments, an image sensor having a pixel size smaller than twice the optical resolution provided by the optical imaging system (for example, an objective lens and a tube lens) can be used to meet the Nyquist theorem.

[0107] Other designs are also possible. In some optical designs, configured to provide dual-surface imaging, where two surfaces at different depths can be imaged simultaneously, the optical imaging system (e.g., an objective lens and / or a tube lens) is configured to reduce optical aberrations so as to image more of the two surfaces (e.g., two planes) at the two corresponding depths than at other locations (e.g., other planes) at other depths. Additionally, the optical imaging system can be configured to reduce aberrations so as to image the two surfaces (e.g., two planes) at the two corresponding depths through a transmissive layer (e.g., a glass layer (e.g., a cover glass)) 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] Multi-channel fluorescence imaging module and system: In some cases, the imaging module or system disclosed herein may include a fluorescence imaging module or system. In some cases, the fluorescence imaging system disclosed herein may include a single fluorescence excitation light source (for providing excitation light within a single wavelength or a single excitation wavelength range) and a light path configured to deliver the excitation light to a sample (e.g., a fluorescently labeled nucleic acid molecule or a cluster thereof arranged on a substrate surface). In some cases, the fluorescence imaging system disclosed herein may include a single fluorescence emission imaging and detection channel, such as a light path, which is configured to collect the fluorescence emitted by the sample and deliver an image of the sample (e.g., an image of a substrate surface on which fluorescently labeled nucleic acid molecules or a cluster thereof are arranged) to an image sensor or other light detection device. In some cases, the fluorescence imaging system may include two, three, four or more fluorescence excitation light sources and / or light paths, which are configured to deliver excitation light at two, three, four or more excitation wavelengths (or within two, three, four or more 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, which are 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 an image of the sample (e.g., an image of a substrate surface having fluorescently labeled nucleic acid molecules or clusters thereof disposed thereon) to two, three, four, or more than four image sensors or other photodetection devices.

[0109] Dual-surface imaging: In some cases, imaging systems disclosed herein, including fluorescence imaging systems, can be configured to obtain high-resolution images of a single sample carrier structure or matrix surface. In some cases, imaging systems disclosed herein, including fluorescence imaging systems, can be configured to obtain 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 (e.g., nucleic acid hybridization, amplification, and / or sequencing reactions) occurring on two or more surfaces of a flow cell as various reagents flow through the flow cell or flow around the flow cell matrix. Figure 1A and Figure 1B A schematic diagram of this dual-surface support structure is provided. Figure 1AShown is a double-surface carrier structure, such as a flow cell, which includes an internal flow channel through which an analyte or reagent can flow. The flow channel can be formed between the first and second layers, the top and bottom layers and / or the front layer and the back layer, such as between the first plate and the second plate, the top plate and the bottom plate and / or the front plate and the back plate shown. One or more plates may include a glass plate, such as a cover glass, etc. In some embodiments, the layer includes borosilicate glass, quartz or plastic. The inner surfaces of these top and bottom layers provide the walls of the flow channel, which help to limit the flow of the analyte or reagent through the flow channel of the flow cell. In some designs, these inner surfaces are flat. Similarly, the top and bottom layers can be flat. In some designs, at least one other layer (not shown) is arranged between the top and bottom layers. This other layer can cut out one or more channels therein, which help to define one or more flow channels and control the flow of the analyte or reagent in the flow channel. Additional discussion of sample carrier structures (such as flow cells) can be found below.

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

[0111] Figure 1B Another double-surface carrier structure with two surfaces is shown, and described two surfaces comprise the sample site of fluorescing that will be imaged.The sample carrier structure comprises the matrix with first outer surface and second outer surface, top outer surface and bottom outer surface and / or front outer surface and back outer surface.In some designs, these outer surfaces are flat.In various embodiments, analyte or reagent flow through these first outer surface and second outer surface. Figure 1B A plurality of fluorescent sample sites on the first and second outer surfaces, the top and bottom outer surfaces, and / or the front and back outer surfaces of the sample carrier structure are schematically shown. In some embodiments, a reaction can occur at these sites to bind the sample so that fluorescence is emitted from these sites (note that Figure 1B are schematic and not drawn to scale; for example, the size and spacing of the fluorescing 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 fluorescing sample sites on a first surface and a second surface that are at different distances from the objective lens. In some designs, only one of the first or second surfaces is focused on at a time. Thus, in such a design, one of these surfaces is imaged at a first time, and the other surface is imaged at a second time. The focus of the fluorescence imaging module can be changed after imaging one of these surfaces so that the other surface can be imaged with comparable optical resolution, since the images of the two surfaces are not in focus at the same time. In some designs, an optical compensation element 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 a fluorescence imaging configuration, 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 at the same time, i.e., simultaneously. For example, the fluorescence imaging module can have a depth of field large enough to include both surfaces. In some cases, this increased depth of field can be provided by, for example, reducing the numerical aperture of the objective lens (or microscope objective), as will be discussed in more detail below.

[0113] like Figure 1A and Figure 1B As shown, the imaging optical device (e.g., an objective lens) can be positioned at a suitable distance (e.g., a distance corresponding to the working distance) from the first surface and the second surface to form a focused image of the first surface and the second surface on the image sensor of the detection channel. Figure 1A and Figure 1B As shown in the example, the first surface can be between the objective lens and the second surface. For example, as shown, the objective lens is arranged above both the first surface and the second surface, and the first surface is arranged above the second surface. The first surface and the second surface are, for example, at different depths. The first surface and the second surface are at different distances from any one or more of the fluorescence imaging module, the illumination and imaging module, the imaging optical device, or the objective lens. The first surface and the second surface are separated from each other, with the first surface spaced above the second surface. In the example shown, the first surface and the second surface are planes and separated from each other along a direction perpendicular to the first and second planes. Similarly, in the example shown, 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. Similarly, the spacing between the first surface and the second surface can 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. Therefore, the two surfaces can be separated from each other by a distance in the longitudinal (Z) direction, which can 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 can, for example, correspond to a flow channel within a flow cell.

[0114] In various designs, the objective lens (possibly in combination with another optical component, such as a tube lens) has a depth of field and / or depth of focus that is at least as great as the longitudinal separation (along the Z direction) between the first surface and the second surface. Thus, the objective lens, alone or in combination with another optical component, can simultaneously form in-focus images of both the first surface and the second surface on the image sensor of one or more detection channels, wherein these images have comparable optical resolutions. In some embodiments, the imaging module may or may not need to refocus to capture images of both the first surface and the second surface with comparable optical resolutions. In some embodiments, the compensating optics do not need to be moved into or out of the optical path of the imaging module to form in-focus images of the first surface and the second surface. Similarly, in some embodiments, one or more optical elements (e.g., lens elements) in the imaging module (e.g., the objective lens and / or the tube lens) do not need to be moved longitudinally, for example, along the first optical path and / or the second optical path (e.g., along the optical axis of the imaging optics) to form an in-focus image of the first surface compared to the position of the one or more optical elements used to form an in-focus image of the second surface. However, in some embodiments, the imaging module includes an autofocus system that is configured to focus the first surface and the second surface simultaneously. In various embodiments, the sample is focused to fully resolve sample sites that are closely spaced together in lateral directions (e.g., X and Y directions). Thus, in various embodiments, in at least one detection channel, no optical elements enter the optical path between the sample carrier structure (e.g., between the translation stage 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 first surface of the sample carrier structure and the fluorescent sample sites on the second surface of the sample carrier structure. Similarly, in various embodiments, no optical compensation is used to form 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 is different from the optical compensation used to form a focused image of the fluorescent sample sites on the second surface of the sample carrier structure on the image sensor or photodetector array. Additionally, in certain embodiments, no optical elements in the optical path between the sample carrier structure (e.g., between a translation stage supporting the sample carrier structure) and an image sensor in at least one detection channel are adjusted differently to form a focused image of fluorescent sample sites on a first surface of the sample carrier structure as compared to forming a focused image of fluorescent sample sites on a second surface of the sample carrier structure.Similarly, in some various embodiments, no optical elements in the optical path between the sample carrier structure (e.g., between the translation stage supporting the sample carrier structure) and the image sensor in at least one detection channel are moved by a different amount or in a different direction to form an in-focus image of the fluorescing sample sites on the first surface of the sample carrier structure on the image sensor as compared to forming an in-focus image of the fluorescing sample sites on the second surface of the sample carrier structure on the image sensor. Any combination of features is possible. For example, in some embodiments, an in-focus image 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 the at least one image sensor and without moving one or more optical elements of the imaging system (e.g., an objective lens and / or a tube lens) along the optical path (e.g., an optical axis) therebetween. For example, an in-focus image of the upper and lower inner surfaces of the flow cell can be obtained without moving one or more optical elements of the tube lens into or out of the optical path or without moving one or more optical elements of the tube lens along the optical path (e.g., an optical axis) therebetween.

[0115] Any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens or tube lens can be designed to reduce or minimize optical aberrations at two positions (e.g., two planes corresponding to two surfaces on a flow cell or other sample carrier structure, such as where a sample site that fluoresces) is located. Any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens or tube lens can be designed to reduce or minimize optical aberrations at a selected position or plane relative to other positions or planes, such as at the first surface and second surface of a sample site that fluoresces on a dual-surface flow cell. For example, any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens or tube lens can be designed to reduce or minimize optical aberrations at two depths or planes at different distances from the object lens compared to aberrations associated with other depths or planes at other distances from the object lens. For example, the optical aberrations for imaging the first and second surfaces can be smaller than elsewhere in an area ranging from about 1 mm to about 10 mm from the object lens. Additionally, in some cases, any one or more of the fluorescence imaging module, illumination optical path, imaging optical path, objective lens, or tube lens can be configured to compensate for optical aberrations caused by transmission of emitted light through one or more portions of the sample support 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.) Such a layer (e.g., a coverslip or the wall of a flow cell) can comprise, for example, glass, quartz, plastic, or other transparent material having a refractive index that introduces optical aberrations.

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

[0117] Those skilled in the art will appreciate that, in some cases, disclosed imaging module or system can be an independent optical system designed for imaging sample or substrate surface. In some cases, they can include one or more processors or computers. In some cases, they can include one or more software packages providing instrument control functions and / or image processing functions. In some cases, except optical components, such as light source (for example, solid-state laser, dye laser, diode laser, arc lamp, tungsten halogen lamp etc.), lens, prism, reflector, dichroic reflector, beam splitter, optical filter, bandpass filter, light guide, optical fiber, aperture and image sensor (for example, complementary metal oxide semiconductor (CMOS) image sensor and camera, charge coupled device (CCD) image sensor and camera etc.), they can also include mechanical and / or optical mechanical components, such as XY translation stage, XYZ translation stage, piezoelectric focusing mechanism, electro-optical phase plate etc. In some cases, they can serve as module, assembly, subassembly or subsystem of the larger system designed for, for example, genomics application (for example, gene testing and / or nucleic acid sequencing application). For example, in some cases, they can function as modules, components, sub-components, or subsystems of larger systems that also include light-tight and / or other environmentally controlled enclosures, temperature control modules, flow cells and cartridges, fluid control modules, fluid dispensing robots, cartridge 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 associated software), display modules, etc., or any combination thereof. These additional components of larger systems (e.g., systems designed for genomics applications) are discussed in more detail below.

[0118] Figure 2A and Figure 2B A non-limiting example of an illumination and imaging module 100 for multi-channel fluorescence imaging is shown. The illumination and imaging module 100 includes an objective lens 110, an illumination source 115, a plurality of detection channels 120, and a first dichroic filter 130, which may include a dichroic reflector or a beam splitter. In some designs, an autofocus system may be included, which may include an autofocus laser 102, for example, that projects a light spot whose size is monitored to determine when the imaging system is in focus. Some or all components of the illumination and imaging module 100 may be coupled to a substrate 105.

[0119] The illumination or light source 115 may include any suitable light source configured to produce light of at least the desired excitation wavelength (discussed in more detail below). The light source may be a broadband light source that emits light within one or more excitation wavelength ranges (or frequency bands). The light source may be a narrowband light source that emits light within one or more narrower wavelength ranges. In some cases, the light source may produce a single isolated wavelength (or line) corresponding to the desired excitation wavelength, or multiple isolated wavelengths (or lines). In some cases, the line may have some very narrow bandwidth. Example light sources that may be suitable for the illumination source 115 include, but are not limited to, an incandescent filament, a xenon arc lamp, a mercury vapor lamp, a light emitting diode, a laser source (e.g., a laser diode), or a solid-state laser or other type of light source. 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 so 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 further include one or more additional optical components, such as lenses, filters, optical fibers, or any other suitable transmissive or reflective optical devices, to output an excitation light beam having suitable characteristics to the first dichroic filter 130. For example, a beam shaping optical device may be included to, for example, receive light from a light emitter in the light source and generate a light beam and / or provide desired beam characteristics. Such an optical device may, for example, include a collimating lens configured to reduce divergence of light 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, the light produced by the different light sources can be directed to overlap and form a concentrated excitation beam. The composite excitation beam can be composed of the excitation beams from each light source. The composite excitation beam will have greater optical power than the individual beams that overlap to form the composite beam. For example, in some embodiments that include 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 separate 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 that together form a composite beam having an optical power that is 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 the composite excitation beam) obtained therefrom can range from about 0.5 W to about 5.0 W, or greater (as will be discussed in more detail below).

[0123] Reference again Figure 2A and Figure 2B , the first dichroic filter 130 is arranged relative to the 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, which is 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, the first dichroic filter can be configured to transmit light from the 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 comprising fluorescently labeled nucleic acid molecules or their complementary sequences, relative to the illumination and imaging module 100. Thus, a first light 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 the sample is arranged or to which the sample is bound. In some cases, the sample can be arranged in or bound to different localized areas or sites on at least one surface of the sample carrier structure.

[0125] In some cases, the support structure may include two surfaces that are at different distances from the objective lens 110 (i.e., at different positions or depths along the optical axis of the objective lens 110), and the sample is disposed on the two surfaces. As described below, for example, the flow cell may include a fluid channel formed at least in part by first and second (e.g., upper and lower) inner surfaces, and the sample may be disposed at a localized location on the first inner surface, the second inner surface, or both inner surfaces. The first and second surfaces may be separated by an area corresponding to the fluid channel through which the solution flows and, therefore, are at different distances or depths relative to the objective lens 110 of the illumination and imaging module 100.

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

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

[0128] In some embodiments, object lens 110 can comprise a microscope objective lens, such as an off-the-shelf object lens. In some embodiments, object lens 110 can comprise a customized object lens. The example of customized object lens and / or customized object lens-tube lens combination is described below and in U.S. Provisional Application No. 62 / 962,723 (the entire contents of which are incorporated herein by reference) filed on January 17, 2020. Object lens 110 can be designed to reduce or minimize the optical aberration at two positions, such as at two planes corresponding to the two surfaces of flow cell or other sample carrier structures. Object lens 110 can be designed to reduce the optical aberration at selected position or plane (such as, the first surface and the second surface of double-surface flow cell) relative to other positions or planes in the light path. For example, object lens 110 can be designed to reduce the optical aberration at two depths or planes at different distances from the object lens compared to the optical aberrations related to other depths or planes at other distances from the object lens. For example, in some cases, the optical aberrations of the first and second surfaces of the imaging flow cell can be smaller than the aberrations exhibited elsewhere in an area spanning 1 to 10 mm from the front surface of the objective lens. Additionally, the custom objective lens 110 can, in some cases, be configured to compensate for optical aberrations caused by transmission of fluorescent emission light through one or more portions of a sample carrier structure, such as a layer comprising one or more flow cell surfaces on which a sample is disposed, or a layer comprising a solution filling a fluid channel of the flow cell. These layers can comprise, for example, glass, quartz, plastic, or other transparent materials having a refractive index that can introduce optical aberrations.

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

[0130] In some embodiments, the objective lens 110 can have a numerical aperture (NA) of 0.6 or less (as discussed in more detail below). Such a numerical aperture can provide an 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 a distance apart, such as the distance separating the first and second surfaces of a dual-surface flow cell.

[0131] As described above, the flow cell can include, for example, a first layer and a second layer, each including 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 can be configured to provide a sufficiently large depth of field and / or depth of focus to image the first inner surface and the second inner surface of the flow cell sequentially (by refocusing the imaging module between imaging 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 can be at least equal to or greater than the distance separating the first surface and the second surface of the flow cell to be imaged (e.g., the first inner surface and the second inner surface of the flow cell). In some cases, the first surface and the second surface, such as the first inner surface and the second inner surface of a dual-surface flow cell or other sample carrier structure, can be separated by a distance in the range of about 10 μm to about 700 μm or more (as discussed in more detail below). Thus, in some cases, the depth of field and / or depth of focus can be in the range of about 10 μm to about 700 μm, or greater (as discussed in more detail below).

[0132] In some designs, compensating optics (e.g., an "optical compensator" or "compensator") can be moved into or out of an optical path in an imaging module, e.g., through which light collected by objective 110 is passed to an image sensor, to enable the imaging module to image a first surface and a second surface of a dual-surface flow cell. The imaging module can be configured to, for example, image 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 from 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 having a high numerical aperture (NA) value (e.g., for NA values ​​of 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 compensating optical device (e.g., an optical compensator or compensator) includes a refractive optical element (e.g., a lens), a plate of optically transparent material (e.g., glass), a plate of optically transparent material (e.g., glass), or a quarter-wave plate or a half-wave plate in the case of a polarized beam. Other configurations can be used to enable the first surface and the second surface to be imaged 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, the objective lens 110 is configured to provide a sufficiently large depth of focus and / or depth of field to enable the first surface and the second surface to be imaged with comparable optical resolution without requiring such compensating optics to be moved into and out of an optical path in the imaging module, such as an optical path between the objective lens and an image sensor or photodetector array. Similarly, in various designs, the objective lens 110 is configured to provide a sufficiently large depth of focus and / or depth of field to enable the first surface and the second surface to be imaged with comparable optical resolution without requiring movement of optics, such as without translating one or more lenses or other optical components along an optical path in the imaging module, such as an optical path between the objective lens and an image sensor or photodetector array. Examples of such objective lenses are described in more detail below.

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

[0135] In some embodiments, objective 110 can be configured to provide a field of view as described above to the fluorescence imaging module such that the FOV has diffraction-limited performance, e.g., less than 0.15 waves of aberration 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 110 can be configured to provide a field of view as described above to the fluorescence imaging module such that the FOV has diffraction-limited performance, e.g., a Strehl 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] Reference again Figure 2A and 2B A first dichroic beam splitter or beam combiner is positioned in a first optical path between the light source and the sample to illuminate the sample with one or more excitation beams. The first dichroic beam splitter or beam combiner is also positioned in one or more second optical paths from the sample to different optical channels for detecting fluorescence emissions. Thus, the first dichroic filter 130 couples a first optical path of the excitation beam emitted by the illumination source 115 and a second optical path of emission light emitted by the sample into respective optical channels, where the light is directed to corresponding image sensors or photodetector arrays for capturing images of the sample.

[0138] In various embodiments, the first dichroic filter 130 (e.g., a first dichroic reflector or 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 the desired one or more excitation wavelengths). For example, the first dichroic beam splitter 130 includes a reflective surface having a dichroic reflector having a spectral transmittance response, i.e., configured to transmit light having at least some wavelengths output by the light source that form a portion of the excitation beam. The spectral transmittance response can be configured to not transmit (e.g., instead reflect) light of one or more other wavelengths, such as light of one or more other fluorescence emission wavelengths. In some embodiments, the spectral transmittance response can also be configured to not transmit (e.g., instead reflect) light of one or more other wavelengths output by the light source. Thus, the first dichroic filter 130 can be used to select which wavelength or wavelengths of light output by the light source reaches the sample. In contrast, 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 the desired fluorescence emission from the sample, and may reflect light having one or more wavelengths output from the light source that is not intended to reach the sample. Thus, in some embodiments, the dichroic reflector has a spectral transmittance that includes one or more passbands for transmitting light to be incident on the sample, and one or more stopbands that reflect light outside the passbands, for example, light at one or more emission wavelengths, and possibly one or more wavelengths output by the light source that is not intended to reach the sample. Similarly, in some embodiments, the dichroic reflector has a spectral reflectivity that includes one or more spectral regions configured to reflect one or more emission wavelengths and possibly one or more wavelengths output by the light source that is not intended to reach the sample, and includes one or more regions that transmit light outside of 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-wave stack) configured to provide an appropriate spectral transmission and reflectance profile. Figure 2A and Figure 2B Also shown is a dichroic filter 105, which may comprise, for example, a dichroic beam splitter or beam combiner, which may be used to direct the autofocus laser 102 through the objective and to the sample carrier structure.

[0139] although Figure 2A and Figure 2BThe imaging module 100 shown in and described above is configured such that the excitation beam is transmitted by the first dichroic filter 130 to the objective 110, but in some designs, the illumination source 115 can be arranged relative to the first dichroic filter 130 and / or the first dichroic filter is configured (e.g., oriented) such that the excitation beam is reflected by the first dichroic filter 130 to the objective 110. Similarly, in some such designs, the first dichroic filter 130 is configured to transmit fluorescent emissions from the sample and may transmit light having one or more wavelengths output from the light source that is not intended to reach the sample. As will be discussed below, designs that transmit rather than reflect fluorescent emissions can potentially reduce wavefront errors in the detected emissions and / or may have other advantages. In either case, in various embodiments, the first dichroic reflector 130 is arranged in the second optical path to receive fluorescent emissions from the sample, at least some of which continues to the detection channel 120.

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

[0141] Figure 3A and 3B It shows Figure 2A and 2BA ray tracing diagram of the optical path of the illumination and imaging module 100. Figure 3A Corresponds to a top view of the lighting and imaging module 100 . Figure 3B 120. 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 equally implemented in a system including more or less than four detection channels 120. For example, the multi-channel system disclosed herein can be implemented with as few as one detection channel 120, or as many as two detection channels 120, three detection channels 120, four detection channels 120, five detection channels 120, six detection channels 120, seven detection channels 120, eight detection channels 120, or more than eight detection channels 120 without departing from the spirit or scope of the present disclosure.

[0142] Figure 3A and 3B The non-limiting example of an imaging module 100 shown in FIG includes four detection channels 120, a first dichroic filter 130 (which reflects an emission light beam 150), a second dichroic filter (e.g., a dichroic beamsplitter) 135 (which separates the light beam 150 into a transmitted portion and a reflected portion), and two channel-specific dichroic filters (e.g., dichroic beamsplitters) 140 (which further separate the transmitted and reflected portions of the light beam 150 between the respective detection channels 120). The dichroic reflective surfaces in the dichroic beamsplitters 135 and 140 used to separate the light beam 150 between the detection channels are shown arranged at 45 degrees relative to the central beam axis of the light beam 150 or the optical axis of the imaging module. However, as described below, angles less than 45 degrees may be employed and may provide advantages such as a steeper transition from the passband to the stopband.

[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 optical devices 126, such as lenses (e.g., one or more tube lenses, each including one or more lens elements), which are arranged to focus a portion of the emitted light entering the detection channel 120 on a focal plane that coincides with the plane of the photodetector array 124. The optical device 126 (e.g., tube lens) combined with the objective lens 110 is 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 is bound to a surface on a flow cell or other sample carrier structure. Thus, such an image of the sample can include multiple fluorescence emission points or regions within the spatial range of the sample carrier structure where the sample is emitting fluorescence. The objective lens 110, together with the optical device 126 (e.g., tube lens), can provide a field of view (FOV) that includes a portion of the sample or the entire sample. Similarly, the photodetector arrays 124 of different detection channels 120 can be configured to capture images of the full field of view (FOV) provided by the objective lens and the tube lens or a portion thereof. In some embodiments, the photodetector arrays 124 of some or all detection channels 120 can detect emitted light emitted by a sample disposed on a sample carrier structure, such as a surface or a portion thereof, and record electronic data representing an image thereof. In some embodiments, the photodetector arrays 124 of some or all detection channels 120 can detect features in the emitted light emitted by the sample without having to capture and / or store 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 the optical device 126 and / or 122 (e.g., an element of the tube lens). In some embodiments, the FOV of the disclosed imaging module (e.g., provided by the combination of the objective lens 110 and the optical device 126 and / or 122) can be, for example, in the range of between about 1 mm and 5 mm (e.g., in terms of diameter, width, length, or longest dimension), as described below. The FOV can be selected, for example, to provide a balance between magnification and resolution of the imaging module and / or based on one or more characteristics of the image sensor and / or objective lens. For example, a relatively small FOV can be provided in conjunction with a smaller and faster imaging sensor to achieve high throughput.

[0144] Reference again Figure 3A and 3BIn some embodiments, the optical device 126 (e.g., a tube lens) in a detection channel can be configured to reduce optical aberrations in an image acquired using the optical device 126 in combination with the objective lens 110. In some embodiments including multiple detection channels for imaging at different emission wavelengths, the optical devices 126 (e.g., tube lenses) for different detection channels have different designs to reduce aberrations for the respective emission wavelengths that the particular channel is configured to image. In some embodiments, the optical device 126 (e.g., a tube lens) can be configured to reduce aberrations when imaging a particular surface (e.g., a plane, an object plane, etc.) on a sample carrier structure including a fluorescent sample disposed thereon, compared to other locations (e.g., other planes in object space). Similarly, in some embodiments, the optical device 126 (e.g., a tube lens) can be configured to reduce aberrations 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 a fluorescent sample site disposed thereon, compared to other locations (e.g., other planes in object space). In some embodiments, the optical device 126 (e.g., tube lens) in the detection channel can be designed to reduce the aberration at two depths or planes at different distances from the objective lens compared to the aberration associated with other depths or planes at other distances from the objective lens. For example, compared to other places in the region of about 1mm to about 10mm from the objective lens, the optical aberration for imaging the first surface and the second surface can be smaller. In addition, in some embodiments, the custom optical device 126 (e.g., tube lens) in the detection channel can be configured to compensate for the aberration caused by the transmission of one or more parts of the sample carrier structure (such as a layer comprising one of the surfaces on which the sample is arranged and possible solution adjacent to and in contact with the surface on which the sample is arranged) of the emitted light. The layer comprising one of the surfaces on which the sample is arranged can comprise, for example, glass, quartz, plastic or other transparent materials with a refractive index and introduce optical aberration. For example, in some embodiments, the custom optical device 126 (e.g., tube lens) in the detection channel can be configured to compensate for the optical aberration caused by the sample carrier structure (e.g., coverslip or flow cell wall) or other sample carrier structure components and possible solution adjacent to and in contact with the surface on which the sample is arranged.

[0145] In some embodiments, the optical device 126 (e.g., a tube lens) in the detection channel is configured to have a reduced magnification. The optical device 126 (e.g., a tube lens) in the detection channel can be configured to, for example, cause the fluorescence imaging module to have a magnification of less than, for example, 10 times, as will be discussed further below. This reduced magnification can change the design constraints so that other design parameters can be achieved. For example, the optical device 126 (e.g., a tube lens) can also be configured to cause the fluorescence imaging module to have a large field of view (FOV) of, for example, at least 1.0 mm or larger (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 tube lens) can be configured to provide a field of view as described above to the fluorescence imaging module 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] Reference again Figure 3A and 3B In various embodiments, the sample is located at or near the focal position 112 of the objective lens 110. Figure 2A and 2B As described, a light source, such as a laser source, provides an excitation beam to the sample to induce fluorescence. An objective lens 110 collects at least a portion of the fluorescence emission as emitted light. The objective lens 110 transmits the emitted light toward a first dichroic filter 130, which reflects a portion or all of the emitted light as a light beam 150. The light beam 150 is incident on a second dichroic filter 135 and reaches a different detection channel, each of which includes an optical device 126 that forms an image of the sample (e.g., a plurality of fluorescent sample sites on the surface of the 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.), wherein the two surfaces contain sample sites that emit fluorescent emissions. The two surfaces can be separated from each other by a certain distance in the longitudinal (Z) direction along the central axis of the excitation beam and / or the direction of the optical axis of the objective lens. The separation can, for example, correspond to a flow channel within the flow cell. The analyte or reagent can flow through the flow channel and contact the first inner surface and the second inner surface of the flow cell, thereby allowing it to contact the binding composition so that fluorescent emissions are emitted from multiple sites on the first inner surface and the second inner surface. The imaging optical device (e.g., objective lens 110) can 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, the objective lens 110 (possibly in combination with the optical device 126) can have a depth of field and / or depth of focus that is at least as large as the longitudinal spacing between the first surface and the second surface. Thus, the objective lens 110 (for each detection channel) and the optical device 126 can simultaneously form images of both the first flow cell surface and the second flow cell surface on the photodetector array 124, and these images of the first surface and the second surface are in focus and have comparable optical resolution (or can be focused by only a minor refocusing of the object to obtain images of the first surface and the second surface with comparable optical resolution). In various embodiments, the compensating optical device does not need to be moved into or out of the optical path of the imaging module (e.g., into or out of the first optical path and / or the second optical path) to form focused images of the first surface and the second surface with comparable optical resolution. Similarly, in various embodiments, one or more optical elements (e.g., lens elements) in the imaging module (e.g., the objective lens 110 or the optical device 126) do not need to be moved, for example, longitudinally along the first optical path and / or the second optical path to form a focused image of the first surface, compared to the position of the one or more optical elements when forming a focused image of the second surface. In some embodiments, the imaging module includes an autofocus system configured to quickly and sequentially refocus the imaging module on the first surface and / or the second surface such that the image has a comparable optical resolution. In some embodiments, the objective lens 110 and / or the optical device 126 are configured to bring both the first flow cell surface and the second flow cell surface into focus simultaneously with a comparable optical resolution without requiring the optical compensator to be moved into or out of the first optical path and / or the second optical path, and without requiring the one or more lens elements (e.g., the objective lens 110 and / or the optical device 126 (e.g., a tube lens)) to be longitudinally moved along the first optical path and / or the second optical path.In some embodiments, images of the first surface and / or the second surface acquired sequentially (e.g., by refocusing between surfaces) or simultaneously (e.g., without refocusing between surfaces) using the novel objective lens and / or tube 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 surface and the second surface have comparable optical resolution. In various embodiments, the sample plane is sufficiently in focus to resolve sample sites on the first flow cell surface and / or the second flow cell surface, the sample sites being closely spaced in the lateral directions (e.g., in the X and Y directions).

[0149] As described above, the dichroic filter may include an interference filter that uses optical coatings having 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 spectrum) of the dichroic filter implemented in the multi-channel fluorescence imaging module may depend at least in part on the incident angle or incident angle range of the excitation beam and / or emission beam incident on the dichroic filter. The dichroic filter relative to the detection light path (e.g., Figure 3A and Figure 3B Such an effect may be particularly pronounced if the dichroic filters 135 and 140 are used.

[0150] Figure 4 is a graph showing the relationship between dichroic filter performance and angle of incidence (AOI). Specifically, Figure 4 The graph shows the effect of the angle of incidence on the transition width or spectral span of a dichroic filter, which corresponds to the wavelength range over which the spectral response (e.g., transmission spectrum and / or reflection spectrum) transitions between the passband and stopband regions of the dichroic filter. Figure 4 The transmission edge (or reflection edge) of the spectral range with a small Δλ value in the diagram of φ(λ) corresponds to a steeper transition between the passband and stopband regions or between the transmission and reflection regions (or vice versa, between the reflection and transmission regions), while the spectral range with a relatively large spectral span (e.g., Figure 4 The transmission edge (or reflection edge) for large Δ_λ values ​​in the graphs of FIG. 3 corresponds to a less steep transition between the passband and stopband regions. In various embodiments, a steeper transition between the passband and stopband regions is generally desirable. Additionally, it may be desirable to have an increased uniformity or relatively uniform transition width across all or most of the field of view and / or beam area.

[0151] Accordingly, a fluorescence imaging module in which the dichroic mirror is arranged at a 45 degree angle relative to 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 tube lens) can have a transition width of about 50 nm for an exemplary dichroic filter, as shown in FIG. Figure 4Because the emission beam is not collimated and has a certain degree of divergence, the fluorescence imaging module can have an incidence angle range of about 5 degrees between opposite sides of the beam. Figure 4 As shown, different portions of the emission beam can be incident on the dichroic filters of each channel at various angles of incidence, ranging from 40 to 50 degrees. A relatively wide range of angles of incidence corresponds to a transition width range of approximately 40 nm to approximately 62 nm. This relatively wide range of angles of incidence thus increases the transition width of the dichroic filters in the imaging module. Therefore, the performance of a multi-channel fluorescence imaging module can be improved by providing smaller angles of incidence across the entire beam, resulting in steeper transmission edges and better differentiation of different fluorescence emission bands.

[0152] Figure 5 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 may be desirable. For example, a smaller beam footprint allows the use of smaller dichroic filters to separate the light beam into different wavelength ranges. The use of smaller dichroic filters in turn reduces manufacturing costs and increases the ease of manufacturing suitable flat dichroic filters. Figure 5 As shown, any incident angle greater than 0 degrees (normal to the surface of the dichroic filter) results in an elliptical beam footprint that is larger than the beam's cross-sectional area. An incident angle of 45 degrees results in a larger beam footprint at the dichroic reflector that is greater than 1.4 times the beam's cross-sectional area at zero degrees of incidence.

[0153] Figure 6A and Figure 6B A non-limiting example configuration of a dichroic filter and detection channels in a multi-channel fluorescence imaging module is schematically illustrated, wherein the dichroic mirror is arranged at an angle of less than 45 degrees relative to 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 tube lens). Figure 6A An imaging module 500 is depicted that includes a plurality of detection channels 520a, 520b, 520c, 520d. Figure 6B Yes Figure 6A A detailed view of the portion of imaging module 500 shown within circle 5B. As will be described in more detail, Figure 6A and Figure 6B The configuration shown in FIG. 1 includes many aspects that can result in significant improvements to conventional multi-channel fluorescence imaging module designs. However, in some cases, the fluorescence imaging modules and systems of the present disclosure can be used with respect to Figure 6A and Figure 6B One or a subset of the features described is implemented.

[0154] Figure 6A The imaging module 500 depicted in FIG includes an objective lens 510 and four detection channels 520a, 520b, 520c, and 520d arranged to receive and / or image emission light transmitted by the objective lens 510. A first dichroic filter 530 is provided to connect the excitation and detection light paths. Figure 2A and Figure 2B as well as Figure 3A and Figure 3B In contrast to the design shown in , 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 transmit the fluorescent emission from the sample to the detection channels 520a, 520b, 520c, and 520d. A second dichroic filter 535 splits the emission light 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 emission light. The dichroic filter 540a transmits at least a portion of the first portion 550a of the emission light and reflects portion 550c to the third detection channel 520c. The dichroic filter 540b transmits at least a portion of the second portion 550b of the emission light and reflects portion 550d to the fourth detection channel 520d. Although imaging module 500 is depicted as having four detection channels, in various embodiments, imaging module 500 can include more or fewer detection channels and have a correspondingly greater 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 imaging module 500 can be implemented with similar advantageous effects in a simplified imaging module that includes only two detection channels 520a, 520b and omits the additional dichroic filters 540a, 540b. In some embodiments, only one detection channel can be included. Alternatively, three or more detection channels can be employed.

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

[0156] The objective lens 510 is arranged to receive emission light emitted from the sample by the fluorescent light. In particular, the first dichroic filter 530 is arranged to receive emission light collected and transmitted by the objective lens 510. As discussed above and in Figure 6A As shown in , in some designs, an illumination source such as a laser source is arranged (e.g., Figure 2A and Figure 2B An illumination source 115 (e.g., an optical source 115) is provided to provide an excitation beam incident on a first dichroic filter 530, such that the first dichroic filter 530 reflects the excitation beam into the same objective lens 510 that transmits the emission light, for example, in an epi-fluorescence configuration. In some other designs, the illumination source can be directed 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 in FIG, the detection optics (e.g., including the detection channels 520a, 520b, 520c, 520d and any optical components along the optical path between the objective lens 510 and the detection channels 520a, 520b, 520c, 520d, such as the dichroic filters 535, 540a, 540b) can be arranged in a transmission path of the first dichroic filter 530, rather than in a reflection path of the first dichroic filter 530. In one exemplary embodiment, the objective lens 510 and the detection optics are arranged so that the objective lens 510 transmits the emission light beam 550 directly toward the second dichroic filter 535. The presence of the first dichroic filter 530 along the path of the emission light beam 550 may slightly degrade the wavefront quality of the emission light (e.g., by imparting some wavefront error to the beam 550). However, the wavefront error introduced by the light 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 light beam reflected from the dichroic reflective surface of the dichroic beam splitter. Therefore, by placing the detection optical device along the transmitted beam path of the first dichroic filter 530 rather than 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 refer to Figure 6AAs shown, dichroic filters 535, 540a, and 540b are provided in the detection optics of imaging module 500 to split a beam 550 of emitted light between detection channels 520a, 520b, 520c, and 520d. For example, dichroic filters 535, 540a, and 540b split the beam 550 based on wavelength, such that a first wavelength or wavelength band of emitted light can be received by the first detection channel 520a, a second wavelength or wavelength band of emitted light can be received by the second detection channel 520b, a third wavelength or wavelength band of emitted light can be received by the third detection channel 520c, and a fourth wavelength or wavelength band of emitted light can be received by the fourth detection channel 520d. In some embodiments, the detection channels can receive multiple separate 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 arranged at an incident angle of less than 45 degrees relative to the central beam axis of the incident light beam. Figure 6B As shown, different light beams 550, 550a, 550b have respective central beam axes 552, 552a, 552b. In various embodiments, the central beam axes 552, 552a, 552b are at the center of the cross-section of the light beam orthogonal to the propagation direction of the light beam. These central beam axes 552, 552a, 552b can correspond to the optical axis of the objective lens and / or the optical axis of the optical device in the separate channel, such as the optical axis of the corresponding tube lens. The additional rays 554, 554a, 554b of each light beam 550, 550a, 550b are at the center of the cross-section of the light beam. Figure 6B , to indicate the diameter of each beam 550, 550a, 550b. The beam diameter may be defined, for example, as the full width at half maximum diameter, D4σ (i.e., 4 times σ, where σ is the standard deviation of the horizontal or vertical edge distribution of the beam, respectively), or the second moment width, or any other suitable definition of beam diameter.

[0160] The central beam axis 552 of the emitted light beam 550 can be used as a reference point to define the angle of incidence of the light beam 550 on the second dichroic filter 535. Thus, the "angle of incidence" (AOI) of the light beam 550 can be the angle between the central beam axis 552 of the incident light beam 550 and a line N perpendicular to the surface (e.g., a dichroic reflective surface) on which the light beam is incident. When the emitted light beam 550 is incident on the dichroic reflective surface of the second dichroic filter 535 at the angle of incidence AOI, the second dichroic filter 535 transmits a first portion 550a of the emitted light (e.g., a portion having wavelengths 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 wavelengths 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 alternatively or additionally be used.

[0161] exist Figure 6A and Figure 6B , the second dichroic filter 535 is arranged so that the central beam axis 552 of the light beam 550 is incident at an angle of incidence of 30 degrees. Similarly, the additional dichroic filters 540a, 540b are arranged so that the central beam axes 552a, 552b of the first and second portions 550a, 550b of the light 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 angle of incidence can be in the range of about 20 degrees to about 45 degrees, as will be discussed further below. Moreover, the angle of incidence on each of the dichroic filters 535, 540a, 540b does not necessarily have to be the same. In some embodiments, some or all of the dichroic filters 535, 540a, 540b can be arranged so that their incident light beams 550, 550a, 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 tube lens)) and the dichroic reflective surface in the respective dichroic beam splitter. The same angle of incidence range and values ​​apply when the optical axis is used to specify the AOI.

[0162] The emitted light beams 550, 550a, 550b in the fluorescence imaging module system are typically diverging beams. As described above, the emitted light beams can have a sufficiently large beam divergence such that the beam region within the beam diameter is incident on the dichroic filter at an angle of incidence that differs by up to 5 degrees or more relative to the central beam axis and / or optical axis of the optical device. In some designs, the objective lens 510 can be configured, for example, to have an f-number or numerical aperture that is selected to produce a smaller beam diameter for a given field of view of the microscope. In one example, the f-number or numerical aperture of the objective lens 510 can be selected so that the entire diameter of the light beam 550, 550a, 550b is incident on the dichroic filter 535, 540a, 540b at an angle of incidence that is, for example, within 1 degree, 1.5 degrees, 2 degrees, 2.5 degrees, 3 degrees, 3.5 degrees, 4 degrees, 4.5 degrees, or 5 degrees of the angle of incidence of the central beam axis 552, 552a, 552b.

[0163] In some embodiments, the focal length of an objective lens suitable for producing such a narrow beam diameter can be longer than those typically employed in fluorescence microscopy 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 having a focal length of 36 mm can produce a beam 550 characterized by sufficiently small divergence that light across the entire diameter of the beam 550 is incident on the second dichroic filter 535 at an angle within 2.5 degrees of the angle of incidence of the central beam axis.

[0164] Figure 7 and Figure 8 Provided to show that due to Figure 6A and 6B Graph of improved dichroic filter performance with aspects of the imaging module configuration (or any imaging module configuration disclosed herein). Figure 7 The diagram in is similar to Figure 4 , and illustrates the effect of the angle of incidence on the transition width (e.g., the spectral span of the transmission edge) of a dichroic filter. Figure 7 An example is shown in which the dichroic filters (eg, dichroic filters 535, 540a, and 540b) and the dichroic reflective surfaces therein are oriented so that their incident light beams have an angle of incidence of 30 degrees rather than 45 degrees. Figure 7The figure shows how a reduced angle of incidence significantly improves the steepness and uniformity of the transition width across the entire beam diameter. For example, while a 45-degree angle of incidence at the central beam axis results in a transition width range of approximately 40 nm to approximately 62 nm, a 30-degree angle of incidence 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. Furthermore, the variation in transition width across the entire beam diameter decreases by nearly 40%, from a range of 22 nm to a range of 14 nm, indicating a more uniform transition steepness across the entire beam area.

[0165] Figure 8 It is shown that additional advantages can be achieved 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 of FIG. 5 , the objective lens 510 has a focal length of 36 mm and has an appropriate numerical aperture (e.g., less than 5) to reduce the range of the incident angle within the beam 550 from 30 degrees ± 5 degrees to 30 degrees ± 2.5 degrees. With this design, the range of the transition width can be reduced to about 19 nm to about 26 nm. Figure 7 When compared with the improved system, although the average transition width is basically the same (for example, the spectral span is about 23nm), the transition width variation across the entire beam diameter is further reduced to the 7nm range, indicating that the transition width is significantly improved compared to the original system. Figure 4 The transition width range shown is reduced by nearly 70%.

[0166] Reference 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. Figure 5 In Figure 5, an incident angle of 45 degrees results in a beam footprint area on the dichroic filter that is greater 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 approximately 1.15 times the beam cross-sectional area. Thus, reducing the incident angle at the dichroic filters 535, 540a, 540b from 45 degrees to 30 degrees results in an approximately 18% reduction in the beam footprint area on the dichroic filters 535, 540a, 540b. This reduction in beam footprint area allows the use of smaller dichroic filters.

[0167] Now common reference Figure 9A-B, reducing the angle of incidence from 45 degrees to 30 degrees can also provide improved performance with respect to surface deformation caused by the dichroic filter in any of the imaging module configurations disclosed herein, as demonstrated by an improvement in the modulation transfer function. Generally, the amount of surface deformation increases with larger area optical elements. If a larger area is used on the dichroic filter, a greater amount of surface deformation is encountered, thereby introducing more wavefront error into the beam. Figure 9A The effect of the fold angle on the image degradation caused by adding 1 peak-to-valley (PV) spherical power to the last mirror is shown. Figure 9B The effect of the fold angle on the image quality degradation caused by adding 0.1 wave PV spherical power to the last mirror is shown. Figure 9A and Figure 9B As shown in , reducing the incident angle to 30 degrees can significantly reduce the effects of surface deformation, achieving near-diffraction-limited performance of the detection optics.

[0168] In some embodiments of the disclosed imaging module, the polarization state of the excitation beam can be used to further improve the performance of the multi-channel fluorescence imaging module disclosed herein. Figure 2A 、 Figure 2B and Figure 6A For example, some embodiments of the multi-channel fluorescence imaging module disclosed herein have an epi-fluorescence configuration, wherein the first dichroic filter 130 or 530 merges the optical paths of the excitation beam and the emission beam so that both the excitation light and the emission light are transmitted through the objective lens 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 that forms the excitation beam. In some designs, the light source includes a linearly polarized light source, and the excitation beam may be linearly polarized. In some designs, polarization optics are included to polarize the light and / or rotate the polarization of the light. For example, a polarizer (such as a linear polarizer) may be included in the optical path of the excitation beam to polarize the excitation beam. In some designs, a retarder (e.g., a half-wave retarder or multiple quarter-wave retarders or a retarder 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 plane of incidence), s-polarized (e.g., having an electric field component perpendicular to the plane of incidence), or can have a combination of p- and s-polarization states within the beam. The p- or s-polarization state of the excitation beam can be selected and / or altered by selecting the orientation of the illumination source 115 and / or one or more of its components relative to the first dichroic filter 130, 530 and / or relative to any other surface with 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 can include an emitter, such as a solid-state laser or laser diode, that can be rotated about its optical axis or the central axis of the beam to orient the linearly polarized light output therefrom. Alternatively or additionally, a retarder can 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 disposed in the optical path of the excitation beam can polarize the excitation beam. For example, in some designs, a linear polarizer is placed in the optical path of the excitation beam. This polarizer can be rotated to provide the proper orientation of the linear polarization to provide s-polarized light.

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

[0171] like Figure 10A and Figure 10B As shown in , using 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 angles of incidence of 40 and 45 degrees, where the incident light beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. Figure 10BAs shown in , changing the orientation of the light source relative to the dichroic filter so that the incident light beam is s-polarized relative to the plane of the dichroic filter results in a substantially steeper edge between the passband and the 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 filter 130, 530 so that the excitation light beam is s-polarized relative to the plane of the first dichroic filter 130, 530. As described above, in some embodiments, a polarizer (such as a linear polarizer) can be used to polarize the excitation light beam. The 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 a variety of quarter-wave retarders) can be used to rotate the polarization direction. Other arrangements are also possible.

[0172] As discussed elsewhere herein, reducing the numerical aperture (NA) of the fluorescence imaging module and / or objective lens can increase the depth of field to enable comparable imaging of both surfaces. Figures 11A-16B It shows how for smaller numerical apertures the MTFs are more similar on the first and second surfaces separated by 1 mm of glass than for larger numerical apertures.

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

[0174] Figure 12A and Figure 12B Shown in the first ( Figure 12A ) surface and the second ( Figure 12B ) surface for MTF of NA 0.4.

[0175] Figure 13A and Figure 13B Shown in the first ( Figure 13A ) surface and the second ( Figure 13B ) surface for MTF of NA 0.5.

[0176] Figure 14A and Figure 14B Shown in the first ( Figure 14A ) surface and the second ( Figure 14B ) surface for MTF of NA 0.6.

[0177] Figure 15A and Figure 15B Shown in the first ( Figure 15A ) surface and the second ( Figure 15B) surface for MTF of NA 0.7.

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

[0179] Figure 17A -B provides a graph of the calculated Strehl Ratio (ie, the ratio of the peak light intensity focused or collected by the optical system to the peak light intensity focused or collected by an ideal optical system and a point light source) for imaging the second flow cell surface through the first flow cell surface. Figure 17A Shown for different objective lenses and / or optical system numerical aperture, as the figure of the Strehl ratio that is used for imaging the second flow cell surface by the first flow cell surface of the function of intermediate fluid layer thickness (fluid channel height).As shown in the figure, Strehl ratio reduces along with the interval between the first surface and the second surface increases.Therefore, along with the interval between the two surfaces increases, one of these surfaces will have the image quality that reduces.Compared with the imaging system with larger numerical aperture, for the imaging system with less numerical aperture, along with the increase of the interval distance between the two surfaces, the reduction of the second surface imaging performance is reduced. Figure 17B Shown is a graph of the Strehl ratio as a function of numerical aperture for imaging the second flow cell surface through the 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 the increase in optical aberrations caused by the fluid used for second surface imaging. As NA increases, the increased optical aberrations introduced by the fluid used for second surface imaging can greatly reduce image quality. However, generally, reducing the numerical aperture of the optical system can reduce the achievable resolution. By providing an increased sample plane (or object plane) contrast-to-noise ratio, for example, by using chemical reagents used for nucleic acid sequencing applications (which enhance the fluorescence emission of labeled nucleic acid clusters and / or reduce background fluorescence emission), the loss of image quality can be at least partially offset. In some cases, for example, a sample carrier structure including a hydrophilic matrix material and / or a hydrophilic coating can be used. In some cases, such hydrophilic matrix and / or hydrophilic coating can reduce background noise. Other discussions of sample carrier structures, hydrophilic surfaces and coatings and methods for enhancing contrast-to-noise ratios (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, and / or tube lens can be configured to have a reduced magnification, such as a magnification of less than 10 times, as discussed further below. This reduced magnification can adjust design constraints so that other design parameters can be achieved. For example, any one or more of the fluorescence microscope, illumination and imaging module 100, imaging optics (e.g., optics 126), objective, or tube lens can also be configured to provide the fluorescence imaging module with a large field of view (FOV), such as a field of view of at least 3.0 mm or greater (e.g., in diameter, width, height, or longest dimension), as discussed further below. Any one or more of the fluorescence imaging system, illumination and imaging module 100, imaging optics (e.g., optics 126), objective, and / or tube lens can be configured to provide the fluorescence microscope with a field of view such that the FOV has an aberration of less than, for example, 0.1 wave 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 tube lens can be configured such that the fluorescence imaging module has such a FOV and is diffraction limited, or is diffraction 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, achieving an increased FOV is facilitated in part by using a larger image sensor or photodetector array. The photodetector array can, for example, have an active area having a diagonal of at least 15 mm or greater, as discussed further below. As described above, in some embodiments, the disclosed optical imaging system provides reduced magnification, for example, less than 10x magnification, which can facilitate large FOV designs. Despite the reduced magnification, the optical resolution of the imaging module can still be sufficient because a detector array with a small pixel size or pitch can be used. The pixel size and / or pitch can, for example, be approximately 5 μm or less, as 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., the objective lens and tube lens) to satisfy the Nyquist theorem. Therefore, the pixel size and / or pitch of the image sensor can enable 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 times, at least 2.5 times, at least 3 times, at least 4 times, or at least 5 times the optical resolution of the fluorescence imaging module (e.g., the illumination and imaging module, the objective and tube lens, the objective and optical device 126 in the detection channel, the imaging optical device between the sample carrier structure or platform (configured to support the sample carrier platform) and the photodetector array), or any spatial sampling frequency within a range between any of these values.

[0182] Although broad features are discussed herein with respect to fluorescence imaging modules, any of the features and designs described herein can be applied to other types of optical imaging systems, including but not limited to brightfield and darkfield imaging, and can be applied to luminescence or phosphorescence imaging.

[0183] Dual-wavelength excitation / four-channel imaging system: Figure 18A dual-excitation wavelength / four-channel imaging system for double-sided imaging applications is shown, which includes an objective lens and a tube lens combination that scans in a direction perpendicular to the optical axis to provide large-area imaging, for example, by tiling multiple images to create a composite image whose total field of view (FOV) is much larger than each individual image. The system includes two excitation light sources operating at different wavelengths, such as lasers or laser diodes, and an autofocus laser. The two excitation beams and the autofocus laser beam are combined using a series of mirrors and / or dichroic reflectors and then delivered to the upper or lower inner surface of the flow cell through the objective lens. Fluorescence emitted by labeled oligonucleotides (or other biomolecules) tethered to one of the flow cell surfaces is collected by the objective lens, transmitted through the tube lens, and directed to one of four imaging sensors via a series of intermediate dichroic reflectors depending on the wavelength of the emitted light. The autofocus laser reflected from the flow cell surface is collected by the objective lens, transmitted through the tube lens, and directed to the autofocus sensor via a series of intermediate dichroic reflectors. The system allows the objective / tube lens combination to maintain accurate focus while 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 using a precision linear actuator, a translation stage, or a focus adjustment mechanism mounted on the microscope turret to reduce or minimize the reflected spot size 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 pickup head:

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

[0186] For example, in some cases, a miniaturized imaging module may include a "microfluorometer" that includes an illumination or excitation light 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 objective lenses, one or more custom tube lenses (for achieving two-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, a miniaturized imaging module (e.g., a "microfluorometer") may also include an autofocus mechanism, a microprocessor, power and data transmission connectors, a light-tight housing, etc. The resulting miniaturized imaging module may thus include an integrated imaging package or unit having a small form factor. In some cases, the shortest dimension (e.g., width or diameter) of the miniaturized imaging module can 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 can 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 multiplexing readhead can include an autofocus mechanism.

[0187] In some cases, a multiplexing readhead as described herein can include 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 one another. In some cases, the optical design specifications and performance attributes (e.g., numerical aperture, field of view, depth of field, image resolution, etc.) of each miniaturized imaging module or microfluorometer can be identical, as described elsewhere herein for other versions of the disclosed imaging modules. In some cases, the plurality of individual miniaturized imaging modules can be arranged in a linear arrangement comprising 1, 2, 3, 4, or more than 4 rows and / or columns. In some cases, the plurality of individual miniaturized imaging modules can be arranged in, for example, a hexagonal close-packed arrangement. In some cases, the plurality of individual miniaturized imaging modules can 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 multiplexing read head as disclosed herein. Figure 43A A side view of a multiplexed readhead is shown in which two rows of individual microfluorometers (as viewed from the end) having common optical design specifications (e.g., numerical aperture, field of view, working distance, etc.) are configured to image a common surface (e.g., a first interior surface of a flow cell). Figure 43B A top view of the same multiplexing readhead is shown showing overlapping imaging paths acquired by each microfluorometer of the multiplexing readhead as the multiplexing readhead is translated relative to the flow cell (and vice versa). Figure 43B As shown, the fields of view of the various microfluorometers can overlap. In some cases, they may not overlap. In some cases, the multiplexed read head can be designed to align with and image predetermined features (e.g., individual fluidic channels) within the flow cell.

[0189] Figure 44A -B provides a non-limiting schematic diagram of a multiplexed read head, wherein a first subset of a plurality of individual miniaturized imaging modules is configured to image a first sample plane (e.g., a first interior surface of a flow cell), and a second subset of a plurality of individual miniaturized imaging modules is configured to simultaneously image a second sample plane (e.g., a second interior surface of a flow cell). Figure 44A A side view of a multiplexed read head is shown, wherein a first subset of individual microfluorometers is configured to image, for example, a first or upper interior surface of a flow cell, and a second subset is configured to image a second surface (e.g., a second or lower interior surface of the flow cell). Figure 44B Shown Figure 44A Figure 1 is a top view of a multiplexing readhead illustrating the imaging paths acquired by each microfluorometer of the multiplexing readhead. Similarly, in some cases, the fields of view of each microfluorometer in a given subset may overlap. In some cases, they may not overlap. In some cases, the multiplexing readhead can be designed so that each miniaturized imaging module of the first and second subsets aligns with and images predetermined features (e.g., individual fluidic channels) within the flow cell.

[0190] Improved or optimized objectives and / or tube lenses for 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 to image 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 on the order of 50 μm to 200 μm) can introduce optical aberrations in images captured for 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 heights are 50-200 μm, relative to coverslip thicknesses of <200 μm). Another common design practice is to use an additional "compensation" lens in the optical path when imaging is to be performed on the non-optimal side of a fluid channel or flow cell. This "compensation" lens and the mechanisms required to move it in or out of the optical path (so that both sides of the flow cell can be imaged) further increase system complexity and imaging system downtime, and may degrade image quality due to vibration, etc.

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

[0192] Other advantages of using thicker coverslips include improved control of thickness tolerance errors during the manufacturing process and reduced potential for coverslip deformation due to thermal and mounting-induced stresses. Thickness errors and deformation of the coverslip can adversely affect imaging quality on both the top and bottom surfaces of the flow cell.

[0193] To further improve dual-surface imaging quality for sequencing applications, our optical system designs focus on improving or optimizing the MTF (e.g., by improving or optimizing the objective and / or tube lens design) in the medium to high spatial frequency range that is most suitable for imaging and resolving small spots or clusters.

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

[0195] Figure 19 Optical ray trace 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. Figure 20 ) represents nearly diffraction-limited imaging performance when used with a coverslip designed for 0.17 mm thickness.

[0196] Figure 21 Provided when used to image the surface opposite the 0.3 mm thick coverslip, Figure 19 Figure 2 shows a plot of the modulation transfer function of the same objective as a function of spatial frequency. The relatively small deviations in the MTF values ​​over the spatial frequency range from approximately 100 lines / mm to approximately 800 lines / mm (or cycles / mm) indicate that reasonable image quality is achieved even when using a 0.3 mm thick cover glass.

[0197] Figure 22 When used to image a surface separated from a surface on the opposite side of a 0.3 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid (i.e., under conditions such as those encountered in dual-sided imaging of a flow cell when imaging a far surface), Figure 19 A plot of the modulation transfer function of the same objective lens as shown in . Figure 22 As can be seen in the figure, in the spatial frequency range of about 50 lp / mm to about 900 lp / mm, the deviation of the MTF curve from the ideal diffraction-limited case indicates a degradation in imaging performance.

[0198] Figure 23 and Figure 24 Provided when using Figure 19The upper (or near) inner surface of the flow cell ( Figure 23 ) and the lower (or distal) inner surface ( Figure 24 ). It can be seen that the imaging performance of both surfaces is greatly reduced.

[0199] Figure 25 Provides ray tracing diagrams for tube lens design. Figure 19 The optical design 700 comprising the composite objective lens (lens elements 702, 703, 704, 705, 706, 707, 708, 709, and 710) and the tube lens (lens elements 711, 712, 713, and 714) is improved or optimized for use with a flow cell (which includes a thick cover glass (or wall), e.g., greater than 700 μm thick, and a fluid channel thickness of at least 50 μm) and transmits an image of the interior surface from the flow cell 701 to the image sensor 715, wherein the optical image quality is significantly improved and the CNR is higher.

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

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

[0202] Figure 26 and Figure 27 Provides the following when using objectives (corrected for 0.17 mm coverslips) and Figure 25 The upper (or near) inner surface of the flow cell ( Figure 26 ) and the lower (or distal) inner surface ( Figure 27) as a function of spatial frequency. It can be seen that the imaging performance achieved is almost as expected for a diffraction-limited optical design.

[0203] Figure 28 Ray trace plots (left) are provided for the disclosed tube lens design, which has been improved or optimized to provide high-quality dual-sided imaging performance. Since the tube lens is no longer infinity-corrected, an appropriately designed null lens (right) can be used in combination with the tube lens to compensate for the non-infinity-corrected tube lens for manufacturing and testing purposes.

[0204] Adaptation or optimization of imaging channel-specific tube lenses: In imaging system design, it is possible to improve or optimize both the objective lens and the tube lens in the same wavelength region for all imaging channels. Typically, the same objective lens is shared by all imaging channels (see, for example, Figure 18 ), and each imaging channel either uses the same tube lens or has tube lenses that share the same design.

[0205] In some cases, the imaging systems disclosed herein may also include a tube lens for each imaging channel, wherein the tube lens has 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 depth of field (DOF) performance for each channel. Because 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 tube lens used in the disclosed system results in significant improvements in imaging quality and performance. This channel-specific adaptation or optimization results in improved image quality of both the top and bottom surfaces of the flow cell in dual-surface imaging applications.

[0206] Dual-surface imaging without fluid in the flow cell: To achieve optimal imaging performance for both the top and bottom inner surfaces of a flow cell, a motion-activated compensator is typically required to correct for optical aberrations caused by the fluid in the flow cell (typically comprising a fluid layer thickness of approximately 50-200 μm). In some instances of the disclosed optical system designs, the top inner surface of the flow cell can be imaged with fluid in the flow cell. Once the sequencing chemistry cycle is complete, the fluid can be extracted from the flow cell to image the bottom inner surface. Thus, in some instances, image quality of the bottom surface can be maintained even without the use of a compensator.

[0207] Compensating for optical aberrations and / or vibrations using an electro-optic phase plate: In some cases, by combining an electro-optic phase plate (or other corrective lens) with an objective lens to eliminate optical aberrations due to the presence of fluid, dual-surface image quality can be improved without removing the fluid from the flow cell. In some cases, an electro-optic phase plate (or lens) can be used to eliminate the effects of vibrations caused by the mechanical motion of a motion-actuated compensator and can provide faster image acquisition times 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 increase or maximize information transfer in imaging systems designed for genomics applications is to increase the size of the field of view (FOV) and reduce the time required to image a particular FOV. For a typical large NA optical imaging system, it is usually necessary to obtain an area of ​​approximately 1 mm 2 In the currently disclosed imaging system design, a large FOV objective lens with a long working distance is specified to achieve an image of a 2mm field of view. 2 or larger areas for imaging.

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

[0210] The currently disclosed system design can further reduce the required imaging time through imaging sequence improvement or optimization, 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 (in which the optimal focus position is determined), and then the objective lens is moved in the Z direction to the determined focus position, and then the image is acquired. A series of fluorescence images are acquired by cycling through a series of target FOV positions. From the perspective of information transmission duty cycle, information is only transmitted during the fluorescence image acquisition portion of the cycle. In the currently disclosed imaging system design, a single-step motion is performed in which all axes (XYZ) are repositioned simultaneously, and the autofocus step is used to check for focus position error. Additional Z motion commands are only issued if the focus 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 approach increases the duty cycle of the system, thereby improving the imaging throughput per unit time.

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

[0213] The combination of efficient image acquisition with improved or optimized translation stage step size and settling time results in fast imaging time (ie, the total time required per field of view) and higher throughput imaging system performance.

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

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

[0216] Illumination Optical Path Design: Another factor for improving signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and / or increasing throughput is to increase the illumination power density at the sample. In some cases, the disclosed imaging system can 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 speckle inherent in coherent light sources (e.g., lasers and laser diodes). Additionally, the coupling optics are designed to underfill the entrance aperture of the liquid light guide. The underfill of the liquid light guide into the aperture reduces the effective numerical aperture of the illumination beam entering the objective, thereby improving the efficiency of light transmission through the objective to the sample plane. With this design innovation, the illumination power density can be tripled over conventional designs over a large field of view (FOV).

[0217] In some cases, by exploiting the angular-dependent discrimination of s- and p-polarizations, the illumination beam polarization can be oriented to reduce the amount of backscattered and backreflected illumination light that reaches the imaging sensor.

[0218] Structured Illumination Systems: In some cases, the disclosed imaging modules and systems may include structured illumination optical designs to increase the effective spatial resolution of the imaging system, thereby enabling the use of higher surface densities of clonally amplified target nucleic acid sequences (clusters) on the flow cell surface to increase 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 known as moiré fringes. For example, multiple images are acquired under slightly different illumination conditions by shifting and / or rotating the structured illumination pattern to generate moiré fringes. Mathematical deconvolution of the generated interference signal allows the reconstruction of super-resolution images with up to approximately a factor of two improvement in spatial resolution over that achieved 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 Technologies, pp. 219-236, Elsevier; Nylk, et al. (2019), “Light-Sheet Fluorescence Microscopy With Structured Light”, Neurophotonics and Biomedical Spectroscopy , pp. 477-501, Elsevier]. An example of a structured illumination microscopy imaging system was recently described in U.S. Patent Application Publication No. 2020 / 0218052 to Hong.

[0219] Figure 41 A non-limiting schematic diagram of an imaging system 4100 is provided that includes a branched structured illumination optical design 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 light emitted by 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 light emitted by 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 a light fringe pattern onto a sample plane.

[0220] In some cases, light sources 4110A and 4110B can 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 can include optical fibers coupled to, for example, LEDs, lasers, or laser diodes, which output light beams that are then collimated by corresponding collimating lenses 4120A and 4120B. In some cases, light sources 4110A and 4110B can output light of the same wavelength. In some cases, light sources 4110A and 4110B can output light of different wavelengths. Both light sources 4110A and 4110B can be configured to output light of any wavelength and / or wavelength range described elsewhere herein. During imaging, light sources 4110A and 4110B can be turned on or off using, for example, a high-speed shutter (not shown) located in the optical path or by pulsing the light source at a predetermined frequency.

[0221] exist Figure 41In the illustrated example, a first illumination arm of system 4100 includes a fixed vertical grating 4130A for projecting a grating pattern (e.g., a vertical light stripe pattern) onto a sample plane (e.g., a first inner surface 4188 of a flow cell 4187) in a first orientation, and a second illumination arm includes a fixed horizontal grating 4130B for projecting a grating pattern (e.g., a horizontal light stripe pattern) in a second orientation onto sample plane 4188. Advantageously, in this non-limiting example, the diffraction gratings of imaging system 4100 do not need to be mechanically rotated or translated 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 to allow for adjustable angles between the light stripe patterns projected onto the sample plane.

[0222] like Figure 41 As shown, in some cases, diffraction gratings 4130A and 4130B can be transmissive diffraction gratings that include a plurality of diffraction elements (e.g., parallel slits or grooves) formed in a glass substrate or other suitable surface. In some cases, the gratings can be implemented as phase gratings that provide a periodic variation in the refractive index of the grating material. In some cases, the groove or feature spacing can be selected to diffract light at an appropriate angle and / or to adjust to the minimum resolvable feature size of the imaged sample for operation of imaging system 4100. In other cases, the diffraction gratings can be reflective diffraction gratings.

[0223] exist Figure 41 In the example shown, the orientations of the vertical and horizontal light fringe patterns are offset by about 90 degrees. In other cases, other orientations of the diffraction gratings can be used to produce an offset of about 90 degrees. For example, the diffraction gratings can be oriented so that they project light fringe patterns that are offset by ±45 degrees from the x-axis or y-axis of the sample plane (e.g., the first inner flow cell surface) 4188. In the case where the sample carrier surface (e.g., the inner surface 4188 of the flow cell 4187) includes regularly patterned features arranged on a rectangular grid, Figure 41 The illustrated configuration of imaging system 4100 may be particularly advantageous because improved image resolution using structured illumination methods may be achieved using only two perpendicular grating orientations (eg, 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 an 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 the slits or grooves in the diffraction grating, θ = the angle of incidence of the illumination light relative to the normal to the diffraction grating surface, λ = the wavelength of the illumination light, and m = an integer value corresponding to the maximum intensity of the diffracted light, e.g., m = 0, ±1, ±2, etc. In some cases, a particular order of diffracted illumination light, e.g., first order (m = ±1) light, can be projected onto the sample plane, e.g., the inner flow cell surface 4188. In some cases, for example, the vertical grating 4130A can diffract a collimated beam into first order diffracted beams (±1 order) that are focused onto the sample plane in a first orientation, and the horizontal grating 4130B can diffract the collimated beam into first order diffracted beams that are focused onto the sample plane in a second orientation. In some cases, the zeroth 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 using, for example, a beam blocking element (not shown) (e.g., an order filter) that can be inserted into the optical path after the diffraction grating.

[0227] In the example of 4100, each branch of the structured lighting system includes an optical phase modulator or phase shifter 4140A and 4140B to phase shift the diffracted light transmitted or reflected by each of the diffraction gratings 4130A and 4130B. During structured imaging, the optical phase of each diffracted light beam can be shifted by a certain fraction (e.g., 1 / 2, 1 / 2, 1 / 4, etc.) of the pitch (X) of each fringe of the structured pattern. Figure 41 In the example of FIG. 4 , phase modulators 4140A and 4140B can be implemented as rotating optical phase plates 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 shift the image projected by vertical grating 4130A on sample plane 4188 left and right, and optical phase plate 4140B can be rotated about a horizontal axis to shift the image projected by horizontal grating 4130B on sample plane 4188 in the vertical direction.

[0228] In other embodiments, other types of phase modulators that change the optical path length of the diffracted light can be used (e.g., an optical wedge mounted on a linear translation stage, etc.). Furthermore, while optical phase modulators 4140A and 4140B are shown as being placed after diffraction gratings 4130A and 4130B, in other embodiments, they can be placed elsewhere in the illumination light path. In some cases, a single optical phase modulator can be operated in two different directions to produce different light fringe patterns, or a single motion can be used to adjust the position of a single optical phase modulator to simultaneously adjust the optical path length of both arms of the illumination light path.

[0229] exist Figure 41In the example shown, optical assembly 4160 can be used to combine light from the two illumination light paths. Optical assembly 4160 can include, for example, a partially silvered mirror, a dichroic mirror (depending on the wavelength of light output by light sources 4110A and 4110B), a mirror containing a pattern of holes or a patterned reflective coating that allows light from the two arms of the illumination system to be combined in a lossless or nearly lossless manner (e.g., without significant optical power loss other than a small amount of absorption by the reflective coating), a polarizing beam splitter (in the case where light sources 4110A and 4110B are configured to generate polarized light), and the like. Optical assembly 4160 can be positioned to spatially resolve desired diffraction orders of light reflected or transmitted by each diffraction grating while blocking undesired orders. In some cases, optical assembly 4160 can pass first-order light output by the first illumination light path and reflect first-order light output by the second illumination light path. In some cases, the structured illumination pattern on 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 opening and closing a shutter in the light path of the light source. In other cases, the structured illumination pattern can be switched using an optical switch to change the illumination light path used to illuminate the sample plane.

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

[0231] In some cases, the system 4100 can optionally include a custom tube lens 4190, as described elsewhere herein, such that the focus of the imaging system can be moved from the first inner surface 4188 to the second inner surface 4189 of the flow cell 4187 to enable dual surface imaging with minimal adjustments. In some cases, the lens 4170 can include a custom tube lens, as described elsewhere herein, such that the focus of the illumination light path can be moved from the first inner surface 4188 to the second inner surface 4189 of the flow cell 4187 to enable dual surface imaging with minimal adjustments. In some cases, the lens 4170 can be implemented to be articulated along the optical axis to adjust the focus of the structured illumination pattern at the sample plane. In some cases, the system 4100 can include an autofocus mechanism (not shown) to adjust the focus of the illumination light and / or the focus of the image at the plane of the image sensor 4195. In some cases, because there is no polarizer in the light path, Figure 41 The illustrated system 4100 can provide high optical efficiency. Depending on the numerical aperture of the objective lens 4185, using unpolarized light may or may not significantly affect the contrast of the illumination pattern.

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

[0233] In some cases, alternative illumination path optical 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 in place 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 of 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 of the vertical and horizontal diffraction gratings to allow phase shifting along both 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 another alternative illumination optical path design, the diffraction gratings 4130A and 4130B can be mounted on separate linear motion stages so that they can be translated to change the optical path length (and thus the phase) of 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 its respective diffraction grating to provide translation of the fringe pattern of the diffraction grating along the sample plane 4188. Suitable translation stages can 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, Figure 42 The workflow shown can be used to image the entire sample plane (e.g., imaging the inner surface of a flow cell via image tiling), or to image a single region of a larger sample plane. Figure 41 The vertical 4130A and horizontal 4130B diffraction gratings of the illustrated system 4100 can be used to project illumination light stripe patterns onto a sample plane having 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 grating light fringe pattern can be projected onto a sample plane (e.g., a flow cell surface) using a first illumination condition (e.g., a particular orientation and phase shift setting of a diffraction grating). After capturing an image using the first illumination condition, one or more additional images can be acquired using one or more phase-shifted 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-shifted illumination patterns). If the imaging system includes a second branch of the illumination light path, the image acquisition process can be repeated using the second illumination condition as a starting point (e.g., a second particular orientation and phase shift setting of the diffraction grating), and the image acquisition process can be repeated. In some cases, images can be acquired using at least five different phase-shifted light fringe patterns for at least three different orientations of the diffraction grating (e.g., spaced 60 degrees relative to each other). If no more images are acquired using different orientations of the diffraction grating or phase-shifted illumination light fringe pattern, an image reconstruction algorithm can be used to process the acquired images and generate a reconstructed super-resolution image. In some cases, images are acquired for at least 1, 2, 3, 4, 5, 6, or more than 6 different orientations of the diffraction grating using at least 1, 2, 3, 4, 5, 6, or more than 6 different phase-shifted light fringe patterns at each orientation.

[0237] A potential disadvantage 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 diffraction grating orientation and relative phase, as well as an efficient image reconstruction algorithm, is preferred. In some cases, fewer images may be required to reconstruct a super-resolution image of, for example, a flow cell surface comprising discrete fluorescently labeled clusters of amplified target nucleic acid sequences tethered to a low nonspecific binding surface as described elsewhere herein, than would typically be required to reconstruct a higher resolution image of a conventional sample (e.g., a stained tissue sample).

[0238] Reference again Figure 42 The above cycle can be repeated for different regions of a given flow cell surface, for example, where the image is tiled to create a higher resolution image of the entire flow cell surface. In some cases, the above cycle can be repeated after adjusting the focus of the imaging system, for example, if a second flow cell surface is to be imaged.

[0239] Other Super-resolution Imaging Techniques: In some cases, the disclosed imaging systems may include the use of alternative super-resolution imaging techniques, such as photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), and / or stochastic optical reconstruction microscopy (STORM). [See, e.g., Lutz, et al. (2011), “Biological Imaging by Superresolution Light Microscopy”, Comprehensive Biotechnology (Second Edition), Volume 1, pages 579-589, Elsevier), which is based on statistical curve fitting of the intensity distribution observed in the image of the point spread function (PSF) of a single molecule to a Gaussian distribution function. The Gaussian distribution function is then used to define the position of the molecule in the sample plane with much higher precision than that 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 low nonspecific binding surfaces on a sample carrier or 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 it is photobleached and the level of background noise [Lutz, et al. (2011), supra]. 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, for example, using affinity sequencing methods described elsewhere herein, polymer-nucleotide conjugates (e.g., each conjugate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 markers) containing multiple fluorescent markers (to ensure high photon counts), optionally combined with low nonspecific binding surfaces disclosed elsewhere herein (to ensure very low background signals), can facilitate the use of these super-resolution imaging techniques for genetic testing and sequencing applications. Spatial accuracy or resolution decreases as the number of photons collected decreases, but even when only a moderate number of photons are collected, a positioning accuracy or resolution of 20 nm is possible. In some cases, a 10-fold or higher increase in lateral spatial resolution can be achieved. In some cases, image resolutions better than 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm, or 10 nm can be achieved.

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

[0242] In some cases, the ability to control the fluorescence emission of a small, dispersed subset of fluorescent molecules in the sample plane is key to facilitating super-resolution imaging. In the case of fluorescence photoactivated localization microscopy (FPALM) and photoactivated localization microscopy (PALM), for example, the use of photoactivatable green fluorescent protein (PA-GFP) as a marker can allow controlled induction of a fluorescent subset in the sample using a short pulse of 405 nm light to convert PA-GFP from a dark, non-fluorescent state to a 488 nm excitable fluorescent state, thereby generating a spatially separated subset of fluorescent molecules that can be imaged [Lutz, et al. (2011), supra]. In the case of stochastic optical reconstruction microscopy (STORM), for example, the photoconversion properties of the cyanine dye pair Cy5–Cy3 can be used in a similar manner to enable the random induction of Cy5 fluorescence from a small subset of molecules in the sample (e.g., a small subset of molecules that are spatially separated by at least a plurality of resolution units) at any given time. In some cases, for example, when combined with affinity sequencing methods described elsewhere herein, the polymer-nucleotide conjugate can comprise a photoactivatable green fluorescent protein (PA-GFP) or a subdomain thereof or a portion thereof. In some cases, the polymer-nucleotide conjugate can comprise 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, the polymer-nucleotide conjugate can comprise a mixture of, for example, Cy3 and Cy5 labels within the same conjugate.

[0243] A super-resolution image is reconstructed from the sum of Gaussian fits of all molecules or features (e.g., labeled nucleic acid clusters) imaged in a time stack of acquired images [Lutz, et al. (2011), supra], where the intensity corresponds to the positional uncertainty of the position of each molecule or subset of molecules. Unique to this dataset is the ability to render images with varying localization accuracy or resolution. In some cases, imaging modules that include total internal reflection fluorescence (TIRF) optical imaging designs can be advantageous in implementing the use of these super-resolution imaging techniques because the evanescent wave used to excite fluorescence is confined to an axial dimension less than 200 nm from the sample carrier or flow cell surface, thereby suppressing background fluorescence signals. In some cases, the imaging system can include an objective lens with a higher numerical aperture than used in other imaging module designs disclosed herein. The use of a higher numerical aperture objective lens can facilitate evanescent wave excitation and efficient capture of photons from fluorescent probes. In some cases, wide-field imaging using a single-photon-sensitive EM-CCD camera or other type of image sensor can enable simultaneous imaging of many molecules or subsets of molecules (e.g., nucleic acid sequence clusters) per frame, thereby increasing the throughput of image acquisition.

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

[0245] Evaluating Image Quality: For any embodiment 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, measurement of the modulation transfer function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof.

[0246] In some cases, the disclosed optical designs for dual-sided imaging (e.g., the disclosed objective lens designs, tube lens designs, use of an electro-optical phase plate in combination with an objective lens, 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 (either individually or in combination), the difference between the 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 dual-sided imaging (e.g., including the disclosed tube lens designs, use of an electro-optical phase plate in combination 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), the image quality performance metric for dual-sided imaging provides 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% over the imaging performance metric for dual-sided imaging compared to a conventional system (which includes, for example, an objective lens, a motion-actuated compensator (which moves out of or into the optical path when imaging the near or far inner surface of the flow cell), and an image sensor). In some cases, a fluorescence imaging system including one or more of the disclosed tube lens designs provides at least equal or more improved imaging performance metrics for dual-sided imaging compared to a conventional system including an objective lens, a motion-actuated compensator, and an image sensor. In some cases, a fluorescence imaging system including one or more of the disclosed tube lens designs provides at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% improvement in imaging performance metrics for dual-sided imaging compared to a conventional system including an objective lens, a motion-actuated compensator, and an image sensor.

[0248] Imaging module specifications:

[0249] Excitation Light Wavelength: In any disclosed optical imaging module design, the light source of the disclosed imaging module can generate visible light, such as green light 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 generate excitation light of approximately 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. One skilled in the art will recognize that the excitation wavelength can have any value within this range, such as approximately 620 nm.

[0250] Excitation Light 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 beamsplitters), can produce 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, such as approximately ±18 nm.

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

[0252] Light Source Output Power and CNR: In some embodiments of the disclosed optical imaging module designs, the output power of the light source and / or the power of one or more excitation beams (including a composite excitation beam) obtained therefrom is sufficient, in combination with an appropriate sample, 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 in an image acquired by the illumination and imaging module, or any CNR within any range formed by any of these values.

[0253] Fluorescence emission bands: In some cases, the disclosed fluorescence optical imaging modules 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 use in, for example, genotyping and nucleic acid sequencing applications (e.g., by conjugation to nucleotides, oligonucleotides, or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, cyanine, and derivatives thereof, including the cyanine derivatives cyanine dye 3 (Cy3), cyanine dye 5 (Cy5), cyanine dye 7 (Cy7), and the like.

[0254] Fluorescence Emission Wavelength: In any disclosed optical imaging module design, the detection channel or imaging channel of the disclosed optical system can include one or more optical components, such as emission optical filters and / or dichroic beam splitters, configured to collect emission light at approximately 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. One skilled in the art will recognize that the emission wavelength 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 can include one or more optical components, such as emission optical filters and / or dichroic beamsplitters, 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 the objective lens and / or tube lens) can be in the range of about 0.1 to about 1.4. In some cases, the numerical aperture can 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 can 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 values ​​described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases, the range of numerical aperture can be about 0.1 to about 0.6. Those skilled in the art will recognize that the numerical aperture can have any value within this range, for example, about 0.55.

[0257] Optical Resolution: In some cases, depending on the numerical aperture of the objective and / or the optical system (e.g., including the objective and / or the tube lens), the minimum resolvable spot (or feature) separation 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 (or feature) separation 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 separation distance may be at most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most 0.7 μm, at most 0.6 μm, or at most 0.5 μm. Any lower and upper limits described in this paragraph may be combined to form a range included in the present disclosure. For example, in some cases, the minimum resolvable spot separation 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 separation 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 lens and / or tube lens designs disclosed herein can provide comparable optical resolution of the first and second surfaces (e.g., the upper and lower inner surfaces of a flow cell) with or without 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 acquired can be 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 tube lens and / or the optical system (e.g., including the objective lens and / or tube lens) in any disclosed optical configuration can be in the range of about 2 times to about 20 times. In some cases, the optical system magnification can be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, or at least 20 times. In some cases, the optical system magnification can be at most 20 times, at most 15 times, at most 10 times, at most 9 times, at most 8 times, at most 7 times, at most 6 times, at most 5 times, at most 4 times, at most 3 times, or at most 2 times. Any lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases, the optical system magnification can be in the range of about 3 times to about 10 times. Those skilled in the art will recognize that the optical system magnification can have any value within this range, for example, about 7.5 times.

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

[0261] Objective Working Distance: In some embodiments of the disclosed optical designs, the working distance of the objective can be in the range of about 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 at most 30 mm, at most 25 mm, at most 20 mm, at most 15 mm, at most 10 mm, at most 8 mm, at most 6 mm, at most 4 mm, at most 2 mm, at most 1 mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm. Any lower and upper values ​​described in this paragraph can be combined to form a range included in the present 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 of values ​​specified above, for example, about 1.25 mm.

[0262] Objectives optimized for imaging through 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 a thickness of about 200 μm to about 1,000 μm. In some cases, the objective can be designed to have optimal performance for coverslips with a thickness 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, the objective lens can be designed to have optimal performance for a coverslip having a thickness of at most 1,000 μm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, or at most 200 μm. Any of the lower and upper values ​​described in this paragraph can be combined to form a range encompassed by the present disclosure. For example, in some cases, the objective lens can be designed to have optimal optical performance for a coverslip having a thickness of about 300 μm to about 900 μm. One skilled in the art will recognize that the objective lens can be designed to have optimal optical performance for a coverslip having 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 the objective and / or tube lens) design can range from about 10 μm to about 800 μm, or greater. In some cases, the depth of field and / or depth of focus can 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 values ​​described in this paragraph can be combined to form ranges included in the present disclosure, for example, in some cases, the depth of field and / or depth of focus can range from about 100 μm to about 175 μm. Those skilled in the art will recognize that the depth of field and / or depth of focus can have any value within the range of values ​​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 an objective lens and detection channel optics (e.g., a tube lens)) can be, for example, in the range of about 1 mm to about 5 mm (e.g., in 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 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 diameter, width, length, or longest dimension). Any lower and upper values ​​described in this paragraph can be combined to form ranges encompassed by the present disclosure, e.g., in some cases, the FOV can range from about 1.5 mm to about 3.5 mm (e.g., in diameter, width, length, or longest dimension). One skilled in the art will recognize that the FOV can have any value within the range of values ​​specified above, e.g., about 3.2 mm (e.g., in diameter, width, length, or longest dimension).

[0265] Field of View (FOV) Area: In some examples of the disclosed optical system designs, the area of ​​the FOV can be around 2 mm 2 to about 5mm 2 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 can be up to 5 mm 2 , up to 4mm 2 , up to 3mm 2 or up to 2mm 2 Any lower and upper values ​​described in this paragraph can be combined to form a range included in the present disclosure. For example, in some cases, the area of ​​the field of view can be around 3 mm. 2 to about 4mm 2 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 Objective and / or Tube Lens MTF: In some cases, the design of the objective and / or at least one tube lens in the disclosed imaging modules and systems is configured to optimize the modulation transfer function in a medium to high spatial frequency range. For example, in some cases, the design of the objective and / or at least one tube lens in the disclosed imaging modules and systems is configured 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 of the optical imaging module designs disclosed herein, the objective lens and / or the tube lens can be configured to provide a field of view as described above to the imaging module, such that the FOV has an aberration of less than 0.15 waves over at least 60%, 70%, 80%, 90%, or 95% of the field of view. In some embodiments, the objective lens and / or the tube lens can be configured to provide a field of view as described above to the imaging module, such that the FOV has an aberration of less than 0.1 waves over at least 60%, 70%, 80%, 90%, or 95% of the field of view. In some embodiments, the objective lens and / or the tube lens can be configured to provide a field of view as described above to the imaging module, such that the FOV has an aberration of less than 0.075 waves over at least 60%, 70%, 80%, 90%, or 95% of the field of view. In some embodiments, the objective lens and / or tube lens can be configured to provide a field of view as described above to the imaging module such that the FOV is diffraction limited over at least 60%, 70%, 80%, 90% or 95% of the field of view.

[0268] Angle of incidence of light beams on dichroic reflectors, beam splitters, and beam combiners: In some examples of the disclosed optical designs, the angle of incidence of the light beams on the dichroic reflectors, beam splitters, or beam combiners 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 of the lower and upper values ​​described in this paragraph can be combined to form ranges included in the present disclosure, for example, in some cases, the angle of incidence can be in the range of 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 of values ​​specified above, for example, about 43 degrees.

[0269] Image sensor (photodetector array) size: In some cases, the disclosed optical system can include an image sensor having an active area with a diagonal in the range of about 10 mm to about 30 mm or more. In some cases, the image sensor has an active area that 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 diagonal. In some cases, the image sensor has an active area that 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 diagonal. Any of the lower and upper values ​​described in this paragraph can be combined to form ranges included in the present disclosure, for example, in some cases, an image sensor can have an active area that is in the range of about 12 mm to about 24 mm diagonal. Those skilled in the art will recognize that one or more image sensors may have an active area with a diagonal having any value within the range of values ​​specified above (eg, approximately 28.5 mm).

[0270] Image Sensor Pixel Size and Pitch: In some cases, the pixel size and / or pitch selected for an image sensor used in the disclosed optical system designs can be in the range of about 1 μm to about 10 μm in at least one dimension. In some cases, the pixel size and / or pitch can 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 can 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 values ​​described in this paragraph can be combined to form ranges included in the present disclosure, for example, in some cases, the pixel size and / or pitch can be in the range of about 3 μm to about 9 μm. Those skilled in the art will recognize that the pixel size and / or pitch can have any value within this range, for example, approximately 1.4 μm.

[0271] Oversampling: In some examples of the disclosed optical designs, a spatial oversampling scheme is utilized, where 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 stage speed: In some examples of the disclosed optical imaging modules, the maximum stage speed in any one axis can be in the range of about 1 mm / s to about 5 mm / s. In some cases, the maximum 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 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 of the lower and upper values ​​described in this paragraph can be combined to form ranges included in the present disclosure, for example, in some cases, the maximum stage speed can be in the range of about 2 mm / s to about 4 mm / s. Those skilled in the art will recognize that the maximum 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 in any one axis of motion can be about 2 mm / s 2 to about 10 mm / s 2 In some cases, the maximum acceleration can be at least 2 mm / s 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 may be up to 10 mm / s 2 , up to 9mm / s 2 , up to 8mm / s 2 , up to 7mm / s 2 , up to 6mm / s 2 , up to 5mm / s 2 , up to 4mm / s 2 , up to 3mm / s 2 , or at most 2mm / s 2 Any lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure. For example, in some cases, the maximum acceleration can be about 2 mm / s. 2 to about 8mm / s 2 Those skilled in the art will recognize that the maximum acceleration can have any value within this range, for example, approximately 3.7 mm / s 2 .

[0274] Translation Stage Positioning Repeatability: In some examples of the disclosed optical imaging modules, the repeatability of positioning for any one axis can be in the range of about 0.1 μm to about 2 μm. In some cases, the repeatability of positioning 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 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 limit and upper limit described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases, the repeatability of 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 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 maximum time required to reposition the sample plane (field of view) relative to the optics, or vice versa, can be in the range of about 0.1 seconds to about 0.5 seconds. In some cases, the maximum repositioning time (i.e., scanning stage step size and stabilization 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 maximum 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 of the lower and upper values ​​described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases, the maximum repositioning time can be in the range of about 0.2 seconds to about 0.4 seconds. One skilled in the art will recognize that the maximum 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, a specified error threshold for triggering 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 values ​​described in this paragraph can be combined to form a range included in the present 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 be in the range of 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 values ​​described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases, the image acquisition time can be in the range of 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 field of view can be in the range of about 0.5 seconds to about 3 seconds. In some cases, the imaging time per FOV can 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 can 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 of the lower and upper values ​​described in this paragraph can be combined to form ranges included in the present disclosure, for example, in some cases, the imaging time can be in the range of about 1 second to about 2.5 seconds. One skilled in the art will recognize that the imaging time can have any value within this range, for example, about 1.85 seconds.

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

[0280] Systems and system components for genomics and other applications: As described above, in some embodiments, the disclosed optical imaging modules can be used as a module, component, subassembly, or subsystem of a larger system configured to perform, for example, genomics applications (e.g., genetic 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 one, two, three, four, or more than four imaging modules disclosed herein (each of which may include one or more illumination light paths and / or one or more detection light paths (e.g., one or more detection channels configured to image fluorescence emissions 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, a flow cell or cartridge, a fluid system and fluid flow control module, a reagent cartridge, a temperature control module, a fluid dispensing robot, a cartridge and / or microplate handling (pick and place) robot, a light-tight housing and / or environmental control chamber, one or more processors or computers, a data storage module, a data communication module (e.g., Bluetooth, WiFi, intranet or Internet communication hardware and associated software), a display module, 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 Stage: In some embodiments of the imaging and analysis systems (e.g., nucleic acid sequencing systems) disclosed herein, the systems 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, e.g., to tile 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 genomic analysis systems (e.g., nucleic acid sequencing systems) disclosed herein, the systems 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), e.g., to tile 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 commercially available from a number of suppliers, such as Parker Hannifin. Precision translation stage systems typically include a combination of 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 stage movement are required to ensure accurate and reproducible positioning and imaging of, for example, fluorescent signals during repeated steps of interleaved reagent delivery and optical detection.

[0283] Thus, the systems disclosed herein can include specifying the precision with which a translation stage is configured to position a sample carrier structure relative to illumination and / or imaging optics (or vice versa). In one aspect of the 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, about 0.1 μm or less. Those skilled in the art will appreciate that, in some cases, the positioning accuracy of the translation stage can fall within any range bounded 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 segment, for example, about 0.12 μm.

[0284] Flow cells, microfluidic devices, and cartridges: The flow cell devices and flow cell 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 can include one or more 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. Additional descriptions 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 single capillary flow cell devices, multiple capillary flow cell devices, capillary flow cell boxes and / or microfluidic devices and boxes. In some cases, a single capillary flow cell device, multiple capillary flow cell devices and / or microfluidic devices and boxes can be fixed components of the disclosed systems. In some cases, a single capillary flow cell device, multiple capillary flow cell devices and / or microfluidic devices and boxes can be removable, replaceable components of the disclosed systems. In some cases, a single capillary flow cell device, multiple capillary flow cell devices and / or microfluidic devices and boxes can be disposable or consumable components of the disclosed systems.

[0286] In some embodiments, a single capillary flow cell device (or single capillary flow cell cartridge) disclosed herein includes a single capillary, e.g., a glass or fused silica capillary, whose lumen 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 a sample of interest is bound or tethered. In some embodiments, a multi-capillary capillary flow cell device (or multi-capillary flow cell cartridge) disclosed herein can 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 can be packaged within a rack to form a box that is easy to handle, incorporates adapters or connectors for making external fluid connections, and can optionally include additional integrated functional parts such as reagent reservoirs, waste reservoirs, valves (e.g., microvalves), pumps (e.g., micropumps), etc., or any combination thereof.

[0288] Figure 29A non-limiting example of a single glass capillary flow cell device is shown, which includes two fluid adapters (one fixed to each end of a one-piece glass capillary) designed to mate with standard OD fluidic tubing to provide a convenient, interchangeable fluid connection to an external fluid control system. The fluidic adapter 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-fit, adhesive bonding, solvent bonding, laser welding, etc., or any combination thereof.

[0289] Typically, the capillaries used in the disclosed capillary flow cell devices and capillary flow cell cartridges will have at least one internal axially aligned fluid flow channel (or "lumen") extending the entire length of the capillary. In some cases, the capillary may have two, three, four, five, or more than five internal axially aligned fluid flow channels (or "lumens").

[0290] The cross-sectional geometries of a plurality of appointments for suitable capillary (or its inner chamber) are consistent with the disclosure herein, including but not limited to circular, oval, square, rectangular, triangle, rounded square, rounded rectangle or rounded triangle cross-sectional geometries. In some cases, the capillary (or its inner chamber) can have any specified cross-sectional dimensions or size groups. For example, in some cases, the maximum cross-sectional dimension of the capillary lumen (for example, if the inner chamber is circular, it is a diameter, or if the inner chamber is square or rectangular, it is a diagonal) can be in the range of about 10 μm to about 10 mm. In some cases, the maximum cross-sectional dimension 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 respects, the maximum cross-sectional dimension of capillary lumen can be 10mm at the most, 9mm at the most, 8mm at the most, 7mm at the most, 6mm at the most, 5mm at the most, 4mm at the most, 3mm at the most, 2mm at the most, 1mm at the most, 900 μm at the most, 800 μm at the most, 700 μm at the most, 600 μm at the most, 500 μm at the most, 400 μm at the most, 300 μm at the most, 200 μm at the most, 100 μm at the most, 75 μm at the most, 50 μm at the most, 25 μm at the most or 10 μm at the most.Any lower limit value and the upper limit value described in this paragraph can be combined to form the scope included by the present disclosure, for example, in some cases, the maximum cross-sectional dimension of capillary lumen can be in the scope of approximately 100 μm to approximately 500 μm.It will be appreciated by those skilled in the art that the maximum cross-sectional dimension of capillary lumen can have any numerical value in this scope, for example, approximately 124 μm.

[0291] In some cases, for example, where the lumen of one or more capillaries in a flow cell device or cartridge has a square or rectangular cross-section, the distance between a first interior surface (e.g., a top or upper surface) and a second interior surface (e.g., a bottom 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 can 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 at most 500 μm, at most 475 μm, at most 450 μm, at most 425 μm, at most 400 μm, at most 375 μm, at most 350 μm, at most 325 μm, at most 300 μm, at most 275 μm, at most 250 μm, at most 225 μm, at most 200 μm, at most 175 μm, at most 150 μm, at most 125 μm, at most 100 μm, at most 90 μm, at most 80 μm, at most 70 μm, at most 60 μm, at most 50 μm, at most 40 μm, at most 30 μm, at most 20 μm or at most 10 μm. Any lower and upper values ​​described in this paragraph can be combined to form ranges included in the present disclosure, for example, in some cases, the gap height can be in the range of 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 for making disclosed capillary flow cell device or flow cell box can be in the range of about 5mm to about 5cm or larger.In some cases, the length of one or more capillaries can be less than 5mm, at least 5mm, at least 1cm, at least 1.5cm, at least 2cm, at least 2.5cm, at least 3cm, at least 3.5cm, at least 4cm, at least 4.5cm or at least 5cm.In some cases, the length of one or more capillaries can be at most 5cm, at most 4.5cm, at most 4cm, at most 3.5cm, at most 3cm, at most 2.5cm, at most 2cm, at most 1.5cm, at most 1cm or at most 5mm.Any lower limit and upper limit value described in this paragraph can be combined to form the scope included in the present disclosure, for example, in some cases, the length of one or more capillaries can be in the range of about 1.5cm to about 2.5cm.It will be appreciated by those skilled in the art that the length of one or more capillaries can have any numerical value within the scope, for example, about 1.85cm. In some cases, a device or cartridge can include a plurality of two or more capillaries of the same length. In some cases, a device or cartridge can include a plurality of two or more capillaries of different lengths.

[0293] The capillaries used to construct the disclosed capillary flow cell devices or capillary flow cell cassettes 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 polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI), and perfluoroelastomer (FFKM) as a more chemically inert alternative, or any combination thereof. In terms of cost and chemical compatibility, PEI is between polycarbonate and PEEK. FFKM is also known as Kalrez.

[0294] The one or more materials used to make the capillary are typically optically transparent to facilitate use with spectroscopy-based 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 devices and capillary flow cell cartridges 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, drawing, precision computer numerical control (CNC) machining and boring, laser ablation, and the like.

[0296] In some embodiments, the capillaries used in the disclosed capillary flow cell devices and cartridges can be commercially available 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] Fluid adapters for capillaries attached to the capillary flow cell devices and cartridges disclosed herein, as well as other components of the capillary flow cell devices or cartridges, 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 quartz, ceramic, metal, polydimethylsiloxane, polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HOPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), etc.), where the choice of manufacturing technique again typically depends on the choice of material used, and vice versa.

[0298] Figure 30A non-limiting example of a capillary flow cell box is provided, which includes two glass capillaries, a fluid adapter (in this example, two for each capillary) and a box base that matches the capillaries and / or the fluid adapters so that the capillaries are maintained in a fixed orientation relative to the box. In some cases, the fluid adapter can be integrated with the box base. In some cases, the box can include another adapter that matches the capillaries and / or the capillary fluid adapters. As described elsewhere in this article, in some cases, the box can include other functional components. In some cases, the capillaries are permanently installed in the box. In some cases, the box base is designed to allow one or more capillaries of the flow cell box to be interchangeably removed and replaced. For example, in some cases, the box base can include a hinged "flip" structure that allows it to be opened so that one or more capillaries can be removed and replaced. In some cases, the box base is configured to be mounted on, for example, a stage of a fluorescence microscope or to be mounted in a box holder of a fluorescence imaging module or instrument system of the present disclosure.

[0299] In some cases, the disclosed flow cell device can include a microfluidic device (or "microfluidic chip") and a box, wherein the microfluidic device is manufactured by forming a fluid channel in one or more layers of suitable materials, and includes one or more fluid channels (e.g., "analysis" channels) that are configured to perform analytical techniques that also include imaging as a detection method. In some embodiments, the microfluidic device or box disclosed herein can 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) that are configured to perform analytical techniques that also include imaging as a detection method. In some cases, the disclosed microfluidic device can also include additional fluid channels (e.g., for dilution or mixing of reagents), reagent reservoirs, waste reservoirs, adapters for making external fluid connections, etc., to provide an integrated "chip lab" function within the device.

[0300] Non-limiting examples of microfluidic flow cell cartridges include: a chip having two or more parallel glass channels formed on the chip, a fluid adapter coupled to the chip, and a cartridge base that mates with the chip and / or fluid adapter so that the chip is placed in a fixed orientation relative to the cartridge. In some cases, the fluid adapter can be integrated with the cartridge base. In some cases, the cartridge can include an additional adapter that mates with the chip and / or fluid adapter. In some cases, the chip is permanently mounted in the cartridge. In some cases, the cartridge base is designed to allow one or more chips in the flow cell cartridge to be interchangeably removed and replaced. In some cases, the cartridge base can include a hinged "clamshell" structure that allows it to be opened so that one or more chips can be removed and replaced. In some cases, the cartridge base is configured to be mounted on, for example, a stage of a microscope system or within a cartridge holder of an imaging system. In non-limiting examples, even if only one chip is described, it should be understood that more than one chip can be used in the microfluidic flow cell cartridge. The flow cell cartridge of the present disclosure can include a single microfluidic chip or multiple microfluidic chips. In some cases, a flow cell cartridge of the present disclosure can 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 in a cartridge can facilitate easy handling and correct positioning of the device in an optical imaging system.

[0301] The fluid channels within the disclosed microfluidic devices and cartridges can have a variety of cross-sectional geometries, including, but not limited to, circular, oval, square, rectangular, triangular, rounded square, rounded rectangular, or rounded triangular cross-sectional geometries. In some cases, the fluid channels can have any specified cross-sectional dimension or set of dimensions. For example, in some cases, the height (e.g., gap height), width, or maximum cross-sectional dimension (e.g., diagonal if the fluid channel has a square, rounded square, rectangular, or rounded rectangular cross-section) of the fluid channel can be in the range of 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 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 height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel can be at most 10 mm, at most 9 mm, at most 8 mm, at most 7 mm, at most 6 mm, at most 5 mm, at most 4 mm, at most 3 mm, at most 2 mm, at most 1 mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 75 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any lower and upper values ​​described in this paragraph can be combined to form ranges included in the present disclosure, for example, in some cases, the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel can be in the range of about 20 μm to about 200 μm. Those skilled in the art will recognize that the height (eg, gap height), width, or maximum cross-sectional dimension of the fluidic channel can have any value within this range, eg, approximately 122 μm.

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

[0303] The disclosed microfluidic device 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 that are combined together to form one or more fluid channels. In some cases, the microfluidic chip may include three or more layers that are combined 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 can be manufactured in a layer (e.g., the top surface of the bottom layer) and can be sealed by bonding the top surface of the bottom layer to the bottom surface of the top layer of the material. In some cases, the microfluidic channel can be manufactured in a layer, such as a patterned channel, the depth of the patterned channel extending through the entire thickness of the layer, and then sandwiched between two non-patterned layers and bonded thereto to seal the fluid channel. In some cases, the microfluidic channel is manufactured by removing a sacrificial layer on the surface of the substrate. This method does not require the bulk substrate (e.g., glass or silicon wafer) to be etched away. Instead, the fluid channel is located on the surface of the substrate. In some cases, the microfluidic channel can be manufactured in or on the surface of the substrate, and then sealed to form a subsurface or buried fluid channel in the chip by depositing a conformal film or layer on the surface of the substrate.

[0304] The combination of microfabrication processes can be used to manufacture microfluidic chips. Because the device is microfabricated, usually the compatibility of the substrate material will be selected based on them and known microfabrication techniques (for example, photoetching, wet chemical etching, laser ablation, laser irradiation, air abrasion technology, injection molding, embossing and other technologies). Usually the substrate material is also selected to be compatible with the entire range of conditions to which it may be exposed to the microfluidic device, including the application of extreme pH, temperature, salt concentration and electromagnetic field (for example, light) or electric field.

[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), cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI) and perfluoroelastomer (FFKM) (as a chemically more inert alternative), or any combination thereof. In some preferred cases, the matrix material can include a silica-based matrix such as borosilicate glass and quartz, as well as other suitable materials.

[0306] The disclosed microfluidic device 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 materials used, and vice versa. The microfluidic channels on the chip can be constructed using techniques suitable for forming microstructures or micropatterns on the substrate surface. In some cases, the fluid channels are formed by laser irradiation. In some cases, the microfluidic channels are formed by focused femtosecond laser irradiation. In some cases, the microfluidic channels are formed by photolithography and etching, including but not limited to chemical etching, plasma etching or deep reactive ion etching. In some cases, laser etching is used to form the microfluidic channels. In some cases, direct write lithography is used to form the microfluidic channels. Examples of direct write lithography include electron beam direct writing and focused ion beam milling.

[0307] In another preferred example, the matrix material may include a polymer material, for example, a plastic (eg, polymethyl methacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON) TM), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), polysulfone, etc.). Such polymer matrices can be easily patterned or micro-machined using microfabrication techniques such as those described above. In some cases, microfluidic chips can be made of polymer materials, such as micromachined master, using well-known molding techniques (e.g., injection molding, embossing, compression molding, or by polymerizing polymer precursor materials in a mold (see, e.g., U.S. Patent No. 5,512,131). In some cases, such polymer matrix materials are preferred because they are easy to manufacture, low in cost, and have disposability, as well as their general inertness to most extreme reaction conditions. As with flow cell devices made of other materials (e.g., glass), for example, flow cell devices made of these polymeric materials can include treated surfaces (e.g., derivatized or coated surfaces) to enhance their utility in microfluidic systems, as will be discussed in more detail below.

[0308] Generally, the fluid channel and / or fluid chamber of microfluidic device are manufactured into the upper surface of the first matrix as microscale channel (for example, groove, recess etc.) using above-mentioned microfabrication technology.The first matrix includes the top side and bottom side with the first flat surface.In the microfluidic device prepared according to the method described herein, a plurality of fluid channels (for example, groove and / or recess) are formed on the first flat surface.In some cases, the fluid channel (for example, groove and / or recess) formed in the first flat surface (before being attached to the second matrix) has bottom wall and side wall, and wherein top remains open.In some cases, the fluid channel (for example, groove and / or recess) formed in the first flat surface (before being attached to the second matrix) has bottom wall and side wall, and top remains closed.In some cases, the fluid channel (for example, groove and / or recess) formed in the first flat surface (before being attached to the second matrix) only has side wall, and does not have top surface or bottom surface (that is, fluid channel spans the whole thickness of the first matrix.

[0309] In some cases, the first substrate can be placed in a groove, recess or orifice that is formed on the first substrate, so that the second substrate can be sealed. In some cases, the second substrate and the third substrate can be placed in a groove, recess or orifice that is formed on the first substrate, so that the second substrate can be sealed in a groove, recess or orifice that is formed on the first substrate, so that the second substrate can be sealed in a groove, recess or orifice that is formed on the first substrate, so that the second substrate and the third ...

[0310] This device can have opening, and this opening is oriented so that they are communicated with at least one fluid in the fluid passage and / or the fluid chamber formed in the inside of this device, thereby form fluid inlet and / or fluid outlet.In some cases, opening is formed on the first matrix.In some cases, opening is formed on the first matrix and the second matrix.In some cases, opening is formed on the first matrix, the second matrix and the 3rd matrix.In some cases, opening is positioned at the top side of device.In some cases, opening is positioned at the bottom side of device.In some cases, opening is positioned at the first end and / or the second end of device, and passage extends along the direction from the first end to the second end.

[0311] Those skilled in the art generally understand that the conditions that matrix can be combined together, and this combination of matrix is ​​carried out by any one of multiple methods usually, and its selection can change according to the character of the host material used.For example, the heat bonding of matrix can be applied to many host materials, including for example, matrix based on glass or silicon dioxide and some matrixes based on polymer.Such heat bonding technology is generally included in the temperature that rises and applies under the condition of external pressure in some cases and makes the matrix surface to be combined cooperate.Employed precise temperature and pressure will change according to the character of the materials used matrix usually.

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

[0313] On the other hand, thermal bonding of polymeric matrices will typically utilize lower temperatures and / or pressures than silica-based matrices to prevent excessive melting and / or deformation of the matrices, such as flattening of the interior of the device (i.e., fluid channels or chambers). Typically, such elevated temperatures for bonding polymeric matrices will vary 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 to bond polymeric matrices are significantly lower, such bonding can typically be performed without the high-temperature ovens used for bonding silica-based matrices. As described in more detail below, this allows the heat source to be incorporated into a single, integrated bonding system.

[0314] The adhesive can also be used to bond the substrates together according to well-known methods, which generally involve applying a layer of adhesive between the substrates to be bonded and pressing them together until the adhesive cures. According to these methods, a variety of adhesives can be used, including, for example, commercially available UV-curable adhesives. According to the present invention, alternative methods can also be used to bond the substrates together, including, for example, sonic or ultrasonic welding and / or solvent welding of polymeric components.

[0315] Typically, will use for example " wafer level " manufacturing to manufacture a plurality of described microfluidic chips or device simultaneously.For example, can polymer matrix imprint or be molded into large separable piece, then it is paired and combine together.Then can separate single device or the matrix of combination from larger sheet by cutting or dicing.Similarly, for the matrix based on silicon dioxide, can make single device by bigger matrix wafer or plate, thereby allow higher manufacturing process output.Particularly, can make a plurality of fluid channel structures on the first matrix wafer or plate, then it is covered and combined with it with the second matrix wafer or plate, and optionally, further cover and combine with it with the 3rd matrix wafer or plate.Then, use known method for example sawing, dicing and fracture etc., each device is cut up from larger matrix.

[0316] As mentioned above, top or second matrix cover on bottom or first matrix to seal various passages and chamber.In carrying out bonding process according to method for the present disclosure, first matrix and second matrix can be combined and can use vacuum and / or pressure to carry out to keep two matrix surfaces in optimal contact.Especially, can for example mate the flat surface of bottom matrix with the flat surface of top matrix and keep the optimal contact of bottom matrix and top matrix by applying vacuum through the hole that passes top matrix arrangement.Usually, carry out applying vacuum to the hole in top matrix by top matrix is ​​placed on vacuum chuck, this vacuum chuck usually comprises mounting platform or the surface with integrated vacuum source.In the situation that based on the matrix of silicon dioxide, make the matrix of combination stand high temperature to produce initial combination, thereby can then transfer the matrix of combination in annealing furnace, and relative to each other without any offset.

[0317] Alternative bonding systems for use in conjunction with the devices described herein include, for example, bonding agent dispensing systems for applying a bonding agent layer between two planar surfaces of a substrate. This can be accomplished by applying a bonding agent layer prior to mating the substrates, or by placing a quantity of bonding agent at one edge of adjacent substrates and allowing the wicking action of the two mating substrates to pull the bonding agent through the space between the two substrates.

[0318] In some cases, the entire bonding system can include an automated system for placing the top and bottom substrates on the mounting surface and aligning them for subsequent bonding. Typically, such systems include a translation system for moving the mounting surface or one or more top and bottom substrates relative to each other. For example, a robotic system can be used to sequentially lift, translate, and place each of the top and bottom substrates on a mounting table and within an alignment structure. After the bonding process, such a system can also remove the finished product 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 thereon for bonding.

[0319] In some cases, the manufacture of microfluidic chips includes stacking or laminating two or more layers of substrates (e.g., patterned and non-patterned polymer sheets) to produce the chip. For example, in a microfluidic device, the microfluidic features of the device are typically created by laser irradiation, etching, or otherwise manufacturing 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 the fluidic elements of the device, e.g., fluid channels.

[0320] As described above, in some cases, one or more capillary flow cell devices or microfluidic chips can be installed in a box base to form a capillary flow cell box or a microfluidic box. In some cases, the capillary flow cell box or the microfluidic box may further include additional components integrated with the box to provide enhanced performance for specific applications. Examples of additional components that can be integrated into the box include, but are not limited to, adapters or connectors for fluid connection with other components of the system, fluid flow control components (e.g., micro valves, micro pumps, mixing manifolds, etc.), temperature control components (e.g., resistive heating elements, metal plates used as heat sources or heat sinks, piezoelectric (Peltier) devices for heating or cooling, temperature sensors) or optical components (e.g., optical lenses, windows, filters, reflectors, prisms, optical fibers and / or light emitting diodes (LEDs) or other miniature light sources that can be used together to facilitate spectral measurement and / or imaging of one or more capillaries or fluid channels.

[0321] Fluid adapters, box bases and other box components can be connected to capillaries, capillary flow cell devices, microfluidic chips (or fluid channels within a chip) 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, etc., or any combination thereof. In some cases, the inlet and / or outlet 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 inlet and / or outlet of the microfluidic channels within the chip. In some cases, the box can include an additional adapter (i.e., in addition to the fluid adapter) that cooperates with the chip and / or the fluid adapter and helps position the chip within the box. These adapters can be constructed using the same manufacturing techniques and materials as outlined above for the fluid adapters.

[0322] The box base (or "housing") can be made of metal and / or polymer materials, such as aluminum, anodized aluminum, polycarbonate (PC), acrylic (PMMA) or Ultem (PEI), while other materials are also consistent with the present disclosure. The housing can be manufactured using CNC machining and / or molding techniques and is designed so that one, two or more capillaries or microfluidic chips are constrained by the base in a fixed orientation to produce one or more independent flow channels. The capillaries or chips can be mounted in the base using, for example, a press-fit design or by cooperating with a compressible adapter made of silicone or fluoroelastomer. In some cases, two or more components (e.g., the upper half and the lower half) of the box base are assembled using, for example, screws, clamps, pliers or other fasteners so that the two halves are separable. In some cases, two or more components of the box base are assembled using, for example, a bonding agent, solvent bonding or laser welding so that the two or more components are permanently attached.

[0323] Flow cell surface coatings: In some cases, one or more inner surfaces of the capillary lumens or microfluidic channels in the disclosed flow cell devices can 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 can be formulated to increase or maximize the number of available binding sites (e.g., tethered oligonucleotide adapter / primer sequences) on one or more inner surfaces to increase or maximize foreground signals, such as fluorescence signals generated by hybridization of labeled nucleic acid molecules with tethered oligonucleotide adapter / primer sequences. In some cases, the coating can be formulated to reduce or minimize nonspecific binding of fluorophores 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 background signals, such as background fluorescence generated by nonspecific binding of labeled biomolecules or autofluorescence of sample carrier structures. The combination of increased foreground signal and reduced background signal that can be achieved using the disclosed coatings in some cases can therefore 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 devices (optionally used in combination with improved hybridization and / or amplification protocols) produce solid-phase bioassay reactions that exhibit: (i) negligible nonspecific binding of proteins and other reaction components (thereby reducing or minimizing matrix background), (ii) negligible nonspecific nucleic acid amplification products, and (iii) provide tunable nucleic acid amplification reactions. Although primarily described herein in the context of nucleic acid hybridization, amplification, and sequencing assays, those skilled in the art will appreciate that the disclosed low-binding supports can be used in any of a variety of other bioassay formats, including but not limited to sandwich immunoassays, enzyme-linked immunosorbent assays (ELISAs), and the like.

[0325] In a preferred aspect, one or more layers of coating material can be applied to the surface of the internal flow cell device, wherein the number of layers and / or the material composition of each layer is selected to adjust one or more surface properties of the surface of the internal flow cell device, as described in U.S. Patent Application No. 16 / 363,842. Examples of surface properties that can be adjusted 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 adapters / primers, etc. In some preferred applications, one or more surface properties of the capillary or channel lumen are adjusted to, for example, (i) provide very low non-specific binding of proteins, oligonucleotides, fluorophores, and other molecular components of chemical or biological analysis 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 create one or more chemically modified layers on the surface of the internal flow cell device, where the choice of components used may be varied to alter one or more properties of the support surface, for example, the surface density of functional groups and / or tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the support surface, or the three-dimensional properties of the support surface (i.e., "thickness").

[0327] The attachment chemistry for grafting the first chemically modified layer to the inner surface of the flow cell (capillary or channel) generally depends on the material and the chemical properties of the layer for manufacturing the flow cell device. In some cases, the first layer can be covalently attached to the internal flow cell device surface. In some cases, the first layer can be non-covalently connected to the surface of the inner flow cell device by non-covalent interactions such as electrostatic interactions, hydrogen bonds or the van der Waals interactions between the surface and the molecular components of the first layer, for example, adsorbed on the surface. In either case, the substrate surface can be processed before attaching or depositing the first layer. Any one of a variety of surface treatment techniques well known to those skilled in the art can be used to clean or process the carrier surface. For example, Piranha solution (sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) mixture) can be used to pickle glass or silicon surfaces and / or use oxygen plasma treatment method to clean.

[0328] Silane chemistry constitutes a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more 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) to the surface. Examples of suitable silanes that can be used to create any of the disclosed low-binding support surfaces include, but are not limited to, (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), and any of a variety of PEG-silanes (e.g., having a molecular weight of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., having free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, and the like.

[0329] Examples of preferred polymers that can be used to create one or more layers of low nonspecific binding material on the surface of any disclosed support include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyesters, dextran, polylysine, and polylysine copolymers, or any combination thereof. Examples of conjugation chemistries that can be used to graft one or more layers of material (e.g., polymer layers) to the support surface and / or cross-link the layers to each other include, but are not limited to, biotin-streptavidin interactions (or variations thereof), His tag-Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxies, 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 be in the range of 1 to about 10 or more. 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 a range included in the present disclosure, for example, in some cases, the number of layers can be in the range of about 2 to about 4. In some cases, one or more layers can all comprise the same material. In some cases, each layer can comprise a different material. In some cases, multiple layers can comprise multiple materials.

[0331] One or more layers of the multilayer surface can comprise a branched polymer or can be linear. Examples of suitable branched polymers include, but are not limited to, branched PEG, branched polyvinyl alcohol (branched PVA), branched poly(vinyl pyridine), branched poly(vinyl pyrrolidone) (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(oligo(ethylene glycol) monomethyl ether methacrylate (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine, branched polyglucosides, and dextran.

[0332] In some cases, the branched polymers used to create one or more layers of any multilayer surface disclosed herein can 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. The 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 deposition of one or more layers, such as polymer layers, the resulting functional end groups distal to the surface may include, but are not limited to, biotin, methoxy ethers, carboxylates, amines, NHS esters, maleimides, and bis-silanes.

[0334] The molecular weight of the linear, branched, or multi-branched polymer used to form one or more layers of any multi-layer surface disclosed herein can 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 multi-branched polymer used to form one or more layers of any multi-layer surface disclosed herein can be at most 50,000 Daltons, at most 45,000 Daltons, at most 40,000 Daltons, at most 35,000 Daltons, at most 30,000 Daltons, at most 25,000 Daltons, at most 20,000 Daltons, at most 17,500 Daltons, at most 15,000 Daltons, at most 2 ... In some embodiments, the molecular weight of the polymer may be from about 1,000 daltons to about 12,500 daltons, from about 10,000 daltons to about 7,500 daltons, from about 5,000 daltons to about 4,500 daltons, from about 4,000 daltons to about 3,500 daltons, from about 3,000 daltons to about 2,500 daltons, from about 2,000 daltons to about 1,500 daltons, or from about 1,000 daltons to about 500 daltons. One skilled in the art will recognize that the molecular weight of the linear, branched, or multi-branched polymer used to create one or more layers of any multi-layered surface disclosed herein can have any value within this range, for example, about 1,260 Daltons.

[0335] In some cases, two or more layers can be covalently coupled to one another 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 be in the range of about one covalent bond per molecule and 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 than 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 most 32, at most 30, at most 28, at most 26, at most 24, at most 22, at most 20, at most 18, at most 16, at most 14, at most 12, 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 of the lower and upper values ​​described in this paragraph can be combined to form ranges encompassed by the present 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. One 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 other cases.

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

[0337] In ord...

Claims

1. A method for sequencing a nucleic acid molecule, the method comprising: a) imaging the first surface and the 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 of less than 0.6 and a diameter of greater than 1.0 mm 2 Field of view; b) processing the images of the first surface and the axially displaced second surface to correct for optical aberrations so that the images of the first surface and the axially displaced second surface have substantially the same resolution; as well as c) detecting a fluorescently labeled composition comprising the nucleic acid molecule or its complement disposed on the first surface or the axially displaced second surface to determine the identity of the nucleotides in the nucleic acid molecule.

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

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

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

5. The method of claim 1, wherein the first surface and the axially displaced second surface constitute two surfaces of a flow cell. The method of claim 5 , wherein the two surfaces of the flow cell are coated with a hydrophilic coating.

7. The method of claim 6, wherein the hydrophilic coating comprises more than 10,000 nucleic acid colonies / mm 2 The surface density of the labeled nucleic acid population is arranged thereon.

8. A method as claimed in claim 7, wherein when the nucleic acid community is labeled with cyanine dye 3, the images of the first surface and the axially displaced second surface acquired using the optical system show a contrast-to-noise ratio of at least 5, the optical system comprising a dichroic mirror and a bandpass filter set optimized for cyanine dye 3 emission, and the images are acquired under non-signal saturation conditions while immersing the first surface and the axially displaced second surface in 25mMACES pH 7.4 buffer.

9. A method as claimed in claim 1, wherein the optical system comprises 1, 2, 3 or 4 imaging channels, and the imaging channels are configured to detect nucleic acid communities arranged on at least one of the first surface and the axially displaced second surface and labeled with 1, 2, 3 or 4 different detectable markers.

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

11. The method of claim 1 , wherein the optical system further comprises at least one tube lens positioned between the objective lens and the at least one image sensor, and wherein the at least one tube lens is configured to correct an imaging performance metric for imaging the first surface and the axially displaced second surface, wherein the first surface and the axially displaced second surface are, respectively, a first inner surface of a flow cell and a second inner surface of the flow cell. 12 . The method of claim 11 , wherein the flow cell comprises a cover glass having a thickness of at least 200 μm and a gap between the first inner surface and the second inner surface of at least 50 μm.

13. The method of claim 11, wherein the at least one tube lens comprises, in sequence, an asymmetric convex-convex lens, a convex-plano lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens.

14. The method of claim 11, wherein the optical system further comprises two or more tube lenses, the two or more tube lenses including the at least one tube lens, the two or more tube lenses being designed to provide optimal imaging performance at two or more fluorescence wavelengths.

15. The method of claim 11, wherein the combination of the objective lens and the at least one tube lens is configured to optimize a modulation transfer function in a medium to high spatial frequency range.

16. The method of claim 11, wherein the imaging performance metrics include measurements of modulation transfer function at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast-to-noise ratio, or any combination thereof.

17. The method of claim 1, wherein the resolution of the images of the first surface and the axially displaced second surface is diffraction limited across a field of view.

18. The method of claim 1, further comprising sequencing the nucleic acid molecule, wherein the sequencing of the nucleic acid molecule comprises: performing a sequencing by affinity, sequencing by nucleotide binding or sequencing by nucleotide incorporation reaction on at least one of the first surface and the axially displaced second surface, and The bound or incorporated nucleotide base of the nucleic acid molecule is detected.

19. The method of claim 1, further comprising determining a genotype of a sample, wherein determining the genotype of the sample comprises preparing the nucleic acid molecules for sequencing and sequencing the nucleic acid molecules.

20. A system for sequencing a nucleic acid molecule, comprising: a) an optical system comprising an objective lens and at least one image sensor, wherein the optical system has a numerical aperture of less than 0.6 and a diameter of greater than 3.0 mm 2 a field of view and configured to acquire images of the first surface and the axially displaced second surface; as well as b) a processor programmed to: i) processing the image of the first surface and the image of the axially displaced second surface to correct for optical aberrations so that the images of the first surface and the axially displaced second surface have substantially the same resolution; and ii) detecting a fluorescently labeled composition comprising the nucleic acid molecule or its complement disposed on the first surface or the axially displaced second surface to determine the identity of the nucleotides in the nucleic acid molecule.

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

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

23. The system of claim 20, wherein the optical system has a numerical aperture greater than 0.

3.

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

25. The system of claim 24, wherein the two surfaces of the flow cell are coated with a hydrophilic coating, and wherein the hydrophilic coating further comprises a nucleic acid colony at greater than 10,000 nucleic acid colonies / mm 2 The surface density of the labeled nucleic acid population is arranged thereon.

26. A system as described in claim 20, wherein the optical system includes 1, 2, 3 or 4 imaging channels, and the imaging channels are configured to detect nucleic acid communities labeled with 1, 2, 3 or 4 different detectable markers arranged on at least one of the first surface or the axially displaced second surface.

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

28. The system of claim 20, wherein the system comprises at least one tube lens positioned between the objective lens and the at least one image sensor, and wherein the at least one tube lens is configured to correct for an imaging performance metric for imaging the first surface and the axially displaced second surface, wherein the first surface and the axially displaced second surface are, respectively, a first inner surface of a flow cell and a second inner surface of the flow cell.

29. The system of claim 28, wherein the flow cell comprises a cover glass having a thickness of at least 200 μm and a gap between the first inner surface and the second inner surface of at least 50 μm.

30. The system of claim 28, wherein the optical system comprises two or more tube lenses, including the at least one tube lens, the two or more tube lenses being designed to provide optimal imaging performance at two or more fluorescence wavelengths.

Images