Flow cell device and optical system for nucleic acid sequencing

Abstract

Described herein are fluorescence imaging system designs, flow cell devices, and methods that enable imaging of three or more axially displaced surfaces without the use of any optical compensators. The optical systems and flow cell devices herein provide higher throughput analysis at lower cost for genomics and other imaging applications.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 385,386, filed November 29, 2022, which is incorporated herein by reference in its entirety. [Background technology]

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

[0003] Described herein are flow cell devices, systems (e.g., optical systems), and methods of using them for analyzing biological polymers. In some embodiments, the biological polymer is a nucleic acid. In some embodiments, methods of using these devices and systems include sequencing of nucleic acids. The flow cell devices described herein can include multiple axially displaced fluid channels and three, four, or more axially displaced surfaces facing the channels, which advantageously allow more samples (e.g., increased sample volume and / or sample types) to be placed on a single flow cell than conventional flow cells. The optical systems and methods described herein can image two, three, four, or more surfaces of a flow cell device that are axially displaced from one another, thereby advantageously achieving increased sequencing throughput within a set system runtime. In this manner, the optical systems and methods described herein can increase the effectiveness and efficiency of sequencing analysis. The devices, systems, and methods described herein enable imaging of three or more axially displaced surfaces of a flow cell device without moving any optical compensators in, out of, or along the optical path, thereby providing a simpler, more convenient optical system that is less susceptible to vibration-induced errors. Furthermore, the devices, systems, and methods described herein advantageously allow for easy switching between imaging of conventional flow cells, e.g., with one or two surfaces, and the multi-surface flow cells described herein, which have three or more axially displaced surfaces. Such switching does not require adding or removing any optical elements, e.g., optical compensators, in or out of the optical path. The devices, systems, and methods described herein enable imaging at numerical apertures (NA) less than 0.6 with sufficient image quality for accurate sequencing analysis, enable independent imaging of multiple axially displaced surfaces, and allow the objective lens to be adjusted to change the NA to a desired value less than 0.6. The devices, systems, and methods described herein can reduce the reagent consumption required for sequencing analysis of the same amount of sample compared to existing flow cell devices and optical systems.

[0004] In one aspect, the disclosure provides an optical system for sequencing nucleic acids, the optical system comprising: an objective lens having a field of view (FOV) greater than 1.0 square millimeters (mm); an excitation energy source configured to irradiate one or more surfaces of a flow cell; and at least one image sensor configured to acquire one or more images of the one or more surfaces of the flow cell, wherein the one or more surfaces are axially displaced from one another along an optical axis of the optical system, wherein the optical system exhibits a root-mean-square (RMS) wavefront error of less than 0.09λ, where λ is a central wavelength of the excitation energy source, the RMS wavefront error is measured using a Shack-Hartmann wavefront sensor across the FOV of the objective lens with broadband illumination from the excitation energy source, and the RMS wavefront error is an average of the individual RMS wavefront error values ​​for at least three color channels.

[0005] In some embodiments, the RMS wavefront error is for a field of view (FOV) of about 1.5 mm in the x or y direction orthogonal to the axial axis. In some embodiments, the one or more surfaces include four surfaces, and the RMS wavefront error is greater for the fourth surface than for the first, second, or third of the four surfaces. In some embodiments, the RMS wavefront error of the optical system is less than the diffraction limit of the optical system. In some embodiments, the optical system has a numerical aperture (NA) of less than 0.7. In some embodiments, the optical resolution of the optical system is sufficient to resolve two objects bound to one or more surfaces of the flow cell, the distance between the two objects being at least about 500 nm. In some embodiments, the optical system is configured to acquire one or more images of one or more surfaces of the flow cell in less than 20 minutes per mm, including sequencing time. In some embodiments, the one or more images of one or more surfaces of the flow cell are from two or more different color channels. In some embodiments, the one or more images of one or more surfaces of the flow cell are from four different color channels. In some embodiments, the optical system is configured to complete an imaging cycle in less than 6 minutes. In some embodiments, the optical system further comprises a flow cell, and the one or more images of the one or more surfaces of the flow cell include optical signals emanating from samples immobilized on the one or more surfaces of the flow cell. In some embodiments, the samples include in situ samples of cells, tissues, or both. In some embodiments, the excitation energy source is configured to uniformly illuminate an area of ​​the one or more surfaces of the flow cell greater than 1 mm2, with illumination power varying by less than 10% across the illuminated area. In some embodiments, the objective lens comprises an optical aperture stop having an adjustable size configured to change the numerical aperture of the optical system. In some embodiments, the optical aperture stop is configured to change the numerical aperture of the optical system in a range of 0.4 to 0.6. In some embodiments, the one or more images of the one or more surfaces of the flow cell are acquired without moving an optical compensator into the optical path of the optical system.In some embodiments, the flow cell has a top wall thickness or a bottom wall thickness of at least 700 micrometers (μm) along an axial direction perpendicular to the image plane. In some embodiments, the flow cell has a gap of at least 50 μm in a first fluid channel or a second fluid channel of the flow cell along an axial direction perpendicular to the image plane. In some embodiments, the flow cell has an interposer of at least 500 μm along an axial direction perpendicular to the image plane between the first and second fluid channels of the flow cell. In some embodiments, the system comprises an optical system and a controller including at least one processor. In some embodiments, the at least one processor is configured to execute executable instructions for correcting optical aberrations. In some embodiments, the at least one processor is configured to execute executable instructions for generating an optical resolution approximately the same as a resolution of one or more images of one or more surfaces of the flow cell. In some embodiments, at least one processor is programmed to instruct the system to repeatedly perform a sequencing method, the sequencing method comprising: contacting a plurality of primed target nucleic acid sequences bound to one or more surfaces with a nucleotide conjugate under conditions sufficient to form a binding complex between one of the plurality of primed target nucleic acid sequences and a nucleotide moiety of the nucleotide conjugate if the nucleotide moiety is complementary to a nucleotide of the primed target nucleic acid sequence; and imaging one or more surfaces of the flow cell to detect the binding complex, thereby determining the identity of the nucleotide of the primed target nucleic acid sequence. In some embodiments, the imaging to detect the binding complex is performed in the absence of incorporation of the nucleotide moiety into the primed nucleic acid sequence. In some embodiments, each of the plurality of primed target nucleic acid sequences comprises a primer sequence comprising a reversible terminator moiety sufficient to prevent incorporation of the nucleotide moiety into the primed nucleic acid sequence during imaging.In some embodiments, the binding complex comprises a plurality of target nucleic acid sequences, two or more of the nucleotide moieties bound to the plurality of target nucleic acid sequences, and optionally two or more DNA polymerase molecules. In some embodiments, the sequencing method is incorporation sequencing. In some embodiments, the incorporation sequencing comprises real-time sequencing by synthesis (SBS). In some embodiments, the system comprises an optical system, a flow cell, and a fluidics system configured to deliver one or more reagents to the flow cell.

[0006] In one aspect, the present disclosure provides an optical system for sequencing nucleic acids, the optical system comprising: an excitation energy source configured to irradiate one or more surfaces of a flow cell, wherein the one or more surfaces are axially displaced from one another along an optical axis of the optical system; an objective lens having a field of view (FOV) of greater than 1.0 mm; and at least one image sensor configured to acquire one or more images of the one or more surfaces of the flow cell, wherein the optical resolution of the optical system is sufficient to resolve two objects immobilized on the one or more surfaces of the flow cell, and wherein the distance between the two objects is at least about 0.5 micrometers (μm) in a plane perpendicular to the optical axis of the optical system.

[0007] In some embodiments, the distance between the two objects is at least about 400 nm. In some embodiments, the optical system further comprises a numerical aperture (NA) of less than 0.7. In some embodiments, the distance between the two objects is at least about 500 nm. In some embodiments, the optical system is configured to acquire flow cell images in less than 20 minutes per mm², including sequencing time. In some embodiments, the one or more images of the one or more surfaces of the flow cell are from two or more different color channels. In some embodiments, the one or more images of the one or more surfaces of the flow cell are from four different color channels. In some embodiments, the optical system is configured to complete an imaging cycle in less than 6 minutes. In some embodiments, the optical system further comprises a flow cell, wherein the one or more images of the one or more surfaces of the flow cell include light signals emanating from a sample immobilized on the one or more surfaces of the flow cell. In some embodiments, the sample comprises an in situ sample of cells, tissue, or both. In some embodiments, the excitation energy source is configured to uniformly illuminate an area, produce an illuminated area of ​​greater than 1 mm² of the one or more surfaces of the flow cell, and have an illumination power variation of less than 10% across the illuminated area. In some embodiments, the objective lens includes an optical aperture stop having an adjustable size configured to change the NA of the optical system. In some embodiments, the optical aperture stop is configured to change the NA of the optical system in a range of 0.4 to 0.6. In some embodiments, one or more images of one or more surfaces of the flow cell are acquired without moving an optical compensator into the optical path of the optical system. In some embodiments, the flow cell has a top wall thickness or a bottom wall thickness of at least 700 micrometers (μm) along an axial direction perpendicular to the image plane. In some embodiments, the flow cell has a gap of at least 50 μm in the first fluid channel or the second fluid channel of the flow cell along an axial direction perpendicular to the image plane.In some embodiments, the flow cell has an interposer between the first and second fluid channels of the flow cell that is at least 500 μm along an axial direction perpendicular to the image plane. In some embodiments, the system includes an optical system and a controller including at least one processor. In some embodiments, the at least one processor is configured to process one or more images of one or more surfaces of the flow cell to correct for optical aberrations. In some embodiments, the at least one processor is configured to process one or more images of one or more surfaces of the flow cell to produce an optical resolution that is approximately the same as the resolution of the flow cell image. In some embodiments, at least one processor is programmed to instruct the system to repeatedly perform a sequencing method, the sequencing method comprising: contacting a plurality of primed target nucleic acid sequences bound to one or more surfaces with a nucleotide conjugate, such that if the nucleotide moiety of the nucleotide conjugate is complementary to a nucleotide of the primed target nucleic acid sequence, a binding complex is formed between the primed target nucleic acid sequence of one of the plurality of primed target nucleic acid sequences and the nucleotide moiety; and imaging one or more surfaces of the flow cell to detect the binding complex and thereby determine the identity of the nucleotide of the primed target nucleic acid sequence. In some embodiments, the imaging to detect the binding complex is performed in the absence of incorporation of the nucleotide moiety into the primed nucleic acid sequence. In some embodiments, each of the plurality of primed target nucleic acid sequences comprises a primer sequence comprising a reversible terminator moiety sufficient to prevent incorporation of the nucleotide moiety into the primed nucleic acid sequence during imaging. In some embodiments, the binding complex comprises a plurality of target nucleic acid sequences, two or more of the nucleotide moieties bound to the plurality of target nucleic acid sequences, and, optionally, two or more DNA polymerase molecules. In some embodiments, the sequencing method is incorporation sequencing. In some embodiments, incorporation sequencing comprises real-time sequencing by synthesis (SBS).In some embodiments, the system comprises an optical system, a flow cell, and a fluidics system configured to deliver one or more reagents to the flow cell.

[0008] In one aspect, the disclosure provides an optical system for sequencing nucleic acids, comprising: an excitation energy source configured to irradiate one or more surfaces of a flow cell, wherein the one or more surfaces are axially displaced from one another along an optical axis of the optical system; an objective lens having a field of view (FOV) of greater than 10 mm; and at least one image sensor configured to acquire one or more images of the one or more surfaces of the flow cell, wherein the excitation energy source irradiates the FOV of the objective lens and the at least one image sensor has an incident light flux variation of less than 15% across the FOV.

[0009] In some embodiments, the variation of the incident light flux across the FOV comprises the root mean square of the energy difference. In some embodiments, the variation of the incident light flux across the FOV comprises a ratio of the root mean square of the energy difference to the average energy level, wherein the ratio is less than 15%. In some embodiments, the optical system further comprises a numerical aperture (NA) less than 0.7. In some embodiments, the optical resolution of the optical system is sufficient to resolve two objects where the distance between the two objects is at least about 500 nm. In some embodiments, the optical system is configured to acquire a flow cell image in less than 20 minutes per mm, including sequencing time. In some embodiments, the one or more images of one or more surfaces of the flow cell are from two or more different color channels. In some embodiments, the one or more images of one or more surfaces of the flow cell are from four different color channels. In some embodiments, the optical system is configured to complete an imaging cycle in less than 6 minutes. In some embodiments, the optical system further comprises a flow cell, and the flow cell image comprises an optical signal emitted from a sample immobilized on one or more surfaces of the flow cell. In some embodiments, the sample comprises an in situ sample of cells, tissue, or both. In some embodiments, the excitation energy source is configured to uniformly illuminate an area of ​​the flow cell greater than 1 mm2, with less than a 10% variation in illumination power across the illuminated area. In some embodiments, the objective lens comprises an optical aperture stop having an adjustable size configured to vary the NA of the optical system. In some embodiments, the optical aperture stop is configured to vary the NA of the optical system between 0.4 and 0.6. In some embodiments, one or more images of one or more surfaces of the flow cell are acquired without moving an optical compensator into the optical path of the optical system. In some embodiments, the flow cell has a top wall thickness or a bottom wall thickness of at least 700 micrometers (μm) along an axial direction perpendicular to the image plane.In some embodiments, the flow cell has a gap of at least 50 μm in the first fluid channel or the second fluid channel of the flow cell along an axial direction perpendicular to the image plane. In some embodiments, the flow cell has an interposer of at least 500 μm between the first fluid channel and the second fluid channel of the flow cell along an axial direction perpendicular to the image plane. In some embodiments, the system comprises an optical system and a controller including at least one processor. In some embodiments, the at least one processor is configured to process one or more images of one or more surfaces of the flow cell to correct for optical aberrations. In some embodiments, the at least one processor is configured to process the flow cell images to produce an optical resolution approximately the same as the resolution of the one or more images of the one or more surfaces of the flow cell. In some embodiments, at least one processor is programmed to instruct the repetitive execution of a sequencing method, the sequencing method comprising: contacting a plurality of primed target nucleic acid sequences bound to one or more surfaces with a nucleotide conjugate, such that if the nucleotide moiety of the nucleotide conjugate is complementary to a nucleotide of the primed target nucleic acid sequence, a binding complex is formed between the primed target nucleic acid sequence of one of the plurality of primed target nucleic acid sequences and the nucleotide moiety; and imaging one or more surfaces of the flow cell to detect the binding complex and thereby determine the identity of the nucleotide of the primed target nucleic acid sequence. In some embodiments, the imaging to detect the binding complex is performed in the absence of incorporation of the nucleotide moiety into the primed nucleic acid sequence. In some embodiments, each of the plurality of primed target nucleic acid sequences comprises a primer sequence comprising a reversible terminator moiety sufficient to prevent incorporation of the nucleotide moiety into the primed nucleic acid sequence during imaging. In some embodiments, the binding complex comprises a plurality of target nucleic acid sequences, two or more of the nucleotide moieties bound to the plurality of target nucleic acid sequences, and, optionally, two or more DNA polymerase molecules.In some embodiments, the sequencing method is in-built sequencing. In some embodiments, the in-built sequencing comprises real-time sequencing by synthesis (SBS). In some embodiments, the system comprises an optical system, a flow cell, and a fluidics system configured to deliver one or more reagents to the flow cell.

[0010] Incorporation by Reference All publications, patents, and patent applications mentioned in this application are herein incorporated 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 specification and a term in an incorporated reference, the term in this specification shall control.

[0011] The novel features of the inventive concept are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0012] [Figure 1A] 1A-1C are schematic diagrams illustrating non-limiting examples of imaging multiple surface support structures to present sample sites for imaging by the imaging systems disclosed herein. 1D-1C are diagrams illustrating imaging of the front and back interior surfaces of a flow cell. [Figure 1B] 1A-1C are schematic diagrams illustrating non-limiting examples of imaging multiple surface support structures to present sample sites for imaging by the imaging systems disclosed herein. [Figure 2A]1 shows a top isometric view of a non-limiting example of a multi-channel fluorescence imaging module comprising a dichroic beam splitter for transmitting an excitation light beam to a sample, receiving the resulting fluorescence emission, and redirecting it by reflection to four detection channels configured to detect the fluorescence emission at four different respective wavelengths or wavelength bands. [Figure 2B] 1 shows a bottom isometric view of a non-limiting example of a multi-channel fluorescence imaging module comprising a dichroic beam splitter for transmitting an excitation light beam to a sample, receiving the resulting fluorescence emission, and redirecting it by reflection to four detection channels configured to detect the fluorescence emission at four different respective wavelengths or wavelength bands. [Figure 2C] 2A and 2B show non-limiting examples of a multi-channel fluorescence imaging module comprising a dichroic beam splitter for transmitting an excitation light beam to a sample, receiving the resulting fluorescent emissions, and redirecting by reflection to four detection channels configured to detect the fluorescent emissions at four different respective wavelengths or wavelength bands.

[0023] FIG. 2B shows a top view of the optical path within the multi-channel fluorescence imaging module of FIGS. 2A and 2B, comprising a dichroic beam splitter for transmitting an excitation light beam to a sample, receiving the resulting fluorescent emissions, and redirecting by reflection to four detection channels configured to detect the fluorescent emissions at four different respective wavelengths or wavelength bands. [Figure 2D] 2A and 2B show non-limiting examples of a multi-channel fluorescence imaging module comprising a dichroic beam splitter for transmitting an excitation light beam to a sample, receiving the resulting fluorescent emission, and redirecting by reflection to four detection channels configured to detect the fluorescent emission at four different respective wavelengths or wavelength bands.

[0023] FIG. 2B shows a side view of the optical path within the multi-channel fluorescence imaging module of FIGS. 2A and 2B comprising a dichroic beam splitter for transmitting an excitation light beam to a sample, receiving the resulting fluorescent emission, and redirecting by reflection to four detection channels configured to detect the fluorescent emission at four different respective wavelengths or wavelength bands. [Figure 3]1 is a graph showing the relationship between dichroic filter performance and beam incidence angle. [Figure 4] 1 is a graph illustrating the relationship between beam footprint size and beam incident angle onto a dichroic filter. [Figure 5A] 1A and 1B are schematic diagrams of an example of a dichroic filter and detection channel configuration for a multi-channel fluorescence imaging module, where the dichroic filter has a reflective surface that is tilted so that the angle between the incident beam (e.g., central angle) and the reflective surface of the dichroic filter is less than 45°.

[0023] FIG. 1C is a schematic diagram of a multi-channel fluorescence imaging module with four detection channels. [Figure 5B] 1 shows a schematic diagram of an example of a dichroic filter and detection channel configuration for a multi-channel fluorescence imaging module, where the dichroic filter has a reflective surface that is tilted so that the angle between the incident beam (e.g., central angle) and the reflective surface of the dichroic filter is less than 45°. A detailed diagram showing the angle of incidence (AOI) of the light beam on the dichroic reflector is shown. [Figure 6] 5A and 5B provide graphs illustrating the performance of an improved dichroic filter corresponding to the imaging module configuration shown in FIGS. 5A and 5B. [Figure 7] 5A and 5B provide graphs illustrating the performance of an improved dichroic filter corresponding to the imaging module configuration shown in FIGS. 5A and 5B. [Figure 8A] 5A and 5B provide graphs showing the reduction in surface deformation resulting from the imaging module configurations of Figures 5A and 5B. Figure 6 shows the effect of fold angle on image degradation induced by adding 1 wave of PV spherical power to the last mirror. [Figure 8B] 5A and 5B provide graphs showing the reduction in surface deformation resulting from the imaging module configurations of Figures 5A and 5B. Figure 6 illustrates the effect of fold angle on image quality degradation caused by adding 0.1 waves of PV spherical power to the final mirror. [Figure 9A]1 provides a graph showing the improvement in performance of an excitation filter (e.g., the area between the passband and the surrounding stopband) resulting from the use of s-polarized excitation beam. Figure 1 shows the transmission spectra of an exemplary bandpass dichroic filter at angles of incidence of 40 degrees and 45 degrees, where the incident beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. [Figure 9B] A graph is provided showing the improvement in excitation filter performance (e.g., the area between the passband and the surrounding stopband) resulting from using s-polarized excitation beam. 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 significantly sharpens the edge between the passband and stopband. [Figure 10A] 1 shows the modulation transfer function (MTF) of an example multi-plane imaging system disclosed herein with a numerical aperture (NA) of 0.3, first surface. [Figure 10B] 1 shows the modulation transfer function (MTF) of an example multi-plane imaging system disclosed herein with a numerical aperture (NA) of 0.3. [Figure 11A] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.4, first surface. [Figure 11B] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.4. [Figure 12A] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.5, first surface. [Figure 12B] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.5. [Figure 13A] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.6, first surface. [Figure 13B] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.6. [Figure 14A]1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.7, first surface. [Figure 14B] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.7. [Figure 15A] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.8, first surface. [Figure 15B] 1 shows the MTF of an example multi-plane imaging system disclosed herein with a NA of 0.8. [Figure 16A] 1 provides plots of the Strehl ratio calculated for imaging a second flow cell surface through a first flow cell surface. 1 is a plot of the Strehl ratio for imaging a second flow cell surface through a first flow cell surface as a function of the thickness of the intervening fluid layer (height of the fluid channel) for different objectives and / or optical system numerical apertures. [Figure 16B] 1 provides a plot of the Strehl ratio calculated for imaging a second flow cell surface through a first flow cell surface. FIG. 2 is a plot of the Strehl ratio as a function of numerical aperture for imaging a second flow cell surface through a first flow cell surface and an intervening layer of 0.1 mm thick water. [Figure 17] 1 provides a schematic diagram of the disclosed dual-wavelength excitation / four-channel emission fluorescence imaging system. [Figure 18] A ray tracing diagram of an objective lens design designed to image the opposite surface of a 0.17 mm thick cover slip is provided. [Figure 19] 19 provides a plot of the modulation transfer function of the objective shown in FIG. 18 as a function of spatial frequency when used to image the opposite surface of a 0.17 mm thick coverslip. [Figure 20] 19 provides a plot of the modulation transfer function of the objective shown in FIG. 18 as a function of spatial frequency when used to image the opposite surface of a 0.3 mm thick coverslip. [Figure 21]19 provides a plot of the modulation transfer function of the objective shown in FIG. 18 as a function of spatial frequency when used to image a surface separated from the opposite surface of a 0.3 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 22] 19 provides a plot of the modulation transfer function of the objective shown in FIG. 18 as a function of spatial frequency when used to image the opposite surface of a 1.0 mm thick coverslip. [Figure 23] 19 provides a plot of the modulation transfer function of the objective shown in FIG. 18 as a function of spatial frequency when used to image a surface separated from the opposite surface of a 1.0 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 24] We provide a ray tracing diagram of a tube lens design that provides improved multi-planar imaging through a 1 mm thick cover slip when used in combination with the objective shown in FIG. [Figure 25] 25 provides a plot of the modulation transfer function of the objective and tube lens combination shown in FIG. 24 as a function of spatial frequency when used to image the opposite surface of a 1.0 mm thick coverslip. [Figure 26] provides a plot of the modulation transfer function of the objective and tube lens combination shown in FIG. 24 as a function of spatial frequency when used to image a surface separated from the opposite surface of a 1.0 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 27] A ray tracing diagram of the disclosed tube lens design (left) is provided, which is optimized to provide high-quality multi-planar imaging performance. Because the tube lens is not infinity corrected, a properly designed null lens (right) may be used in combination with the tube lens to compensate for the non-infinity corrected tube lens for manufacturing and testing purposes. [Figure 28] 1 shows a non-limiting example of a single capillary flow cell with two fluidic adapters. [Figure 29]1 shows one non-limiting example of a flow cell cartridge designed to hold two capillaries, including a chassis, fluidic adapters, and optionally other components. [Figure 30] A non-limiting example of a system is shown that includes a single capillary flow cell connected to various fluid flow control components, where the single capillary is suitable for mounting on a microscope stage or within custom imaging equipment for use in various imaging applications. [Figure 31] One non-limiting example of a system with a capillary flow cell cartridge with an integral diaphragm valve is shown to reduce or minimize dead volume and conserve certain key reagents. [Figure 32] 1 shows a non-limiting example of a system including a capillary flow cell, a microscope setup, and a temperature control mechanism. [Figure 33] 1 shows a non-limiting example of controlling the temperature of a capillary flow cell by using a metal plate placed in contact with the flow cell cartridge. [Figure 34] 1 illustrates one non-limiting approach to temperature control of a capillary flow cell with a non-contact thermal control mechanism. [Figure 35A] 1 shows a non-limiting example of the fabrication of a flow cell device. 1 shows the preparation of a one-piece glass flow cell. [Figure 35B] 1 shows a non-limiting example of the fabrication of a flow cell device. 1 shows the preparation of a two-piece glass flow cell. [Figure 35C]

[0023] Figure 1 shows a non-limiting example of the fabrication of a flow cell device.

[0024] Figure 1 shows the preparation of a three-piece glass flow cell. [Figure 36A] 1 shows non-limiting examples of glass flow cell designs. 2 shows a one-piece glass flow cell design. [Figure 36B] 1 shows non-limiting examples of glass flow cell designs. 2 shows a two-piece glass flow cell design. [Figure 36C] 1 shows non-limiting examples of glass flow cell designs. 2 shows a three-piece glass flow cell design. [Figure 37]Visualization of cluster (e.g., polony) amplification within the capillary lumen is shown. [Figure 38] 1 provides a non-limiting example of a block diagram of a sequencing system disclosed herein. [Figure 39] 1 provides a non-limiting example flow chart of the sequencing methods disclosed herein. [Figure 40A] 1 provides non-limiting examples of kits disclosed herein. 1 shows a kit comprising a flow cell and a plurality of numbered reagent wells, according to some embodiments. [Figure 40B] 1 provides non-limiting examples of kits disclosed herein. 1 shows the decorative appearance of kits according to some embodiments. [Figure 41] 1 provides a non-limiting example of a schematic diagram of a structured illumination system disclosed herein. [Figure 42] 1 provides a non-limiting example of a flow chart for acquiring and processing structured illumination images of a flow cell surface as disclosed herein. [Figure 43A] 1 provides a non-limiting schematic diagram of a multiplexed readhead disclosed herein;FIG. 2 is a side view of a multiplexed readhead in which individual microfluometers are configured to image a common surface, e.g., the inner surface of a flow cell; [Figure 43B] 1 provides a non-limiting schematic diagram of a multiplexed readhead disclosed herein;FIG. 2 is a top view of a multiplexed readhead showing the imaging paths acquired by the individual microfluometers of the multiplexed readhead;FIG. [Figure 44A] 4 provides a non-limiting schematic diagram of a multiplexed readhead disclosed herein. A side view of a multiplexed readhead in which a first subset of a plurality of individual microfluometers 4401 is configured to image a first surface, e.g., a first inner surface of a flow cell, and a second subset of a plurality of individual microfluometers is configured to image a second surface, e.g., a second inner surface of a flow cell. [Figure 44B]44A provides a non-limiting schematic diagram of a multiplexed readhead disclosed herein.FIG. 44B is a top view of the multiplexed readhead of FIG. 44A, showing the imaging paths acquired by the individual microfluometers 4401 of the multiplexed readhead. [Figure 45] 1 shows a non-limiting example of an optical imaging system having multiple image sensors configured for transmission imaging of a flow cell based on sequential illumination by multiple light sources, each light source emitting a different color, according to some embodiments herein. A liquid sample is introduced into the flow cell on a hydrophobic pad and flows through the flow cell by a pulling force. [Figure 46] 1 provides a non-limiting schematic diagram of a method, according to some embodiments herein, utilizing an optical system for imaging the surface of a flow cell for nucleic acid sequencing. [Figure 47A] 1 is a non-limiting cross-sectional view of an optical system for imaging a surface of a flow cell according to some embodiments herein; [Figure 47B] 47A and 47B illustrate optical systems according to various embodiments described herein, and a comparison of the optical system of FIG. [Figure 48A] 47A provides a non-limiting comparison of the imaging area of ​​a flow cell and an optical system.

[00140] Figure 47B shows a non-limiting example of a flow cell with 424 individual tiles imaged by an IDEX instrument core. [Figure 48B]

[00130] Figure 4 provides a non-limiting comparison of the imaging area of ​​a flow cell and an optical system.

[00131] Figure 4 shows a non-limiting example of a flow cell with fewer than 40 individual tiles imaged by the optical system described herein (see Figures 45, 46, 47A-47B). [Figure 49A] 1 provides a non-limiting cross-sectional view of an optical system configured for multi-planar imaging of a multi-planar flow cell. The optical system shown includes a piezo-driven wedge block for fast focus. The optical system is shown configured to focus on the back inner surface of the flow cell. [Figure 49B]1 provides a non-limiting cross-sectional view of an optical system configured for multi-planar imaging of a multi-faceted flow cell. The optical system shown includes a piezo-driven wedge block for fast focus. The optical system is shown configured to focus on the front inner surface of the flow cell. [Figure 50] 1 provides a non-limiting cross-sectional view of an optical system configured to image a large-area surface, the optical system comprising multiple optical subsystems, each with an optimized FOV that overlaps with the FOV of each adjacent optical subsystem, thereby providing a large-area FOV. [Figure 51A]

[0013] Figure 1 provides a non-limiting cross-sectional view of a focus lens assembly. The focus lens assembly is configured to maintain a fixed position within an optical path (e.g., an optical axis) and allow relative movement between at least a first lens and a second lens housed within a lens housing of the focus lens assembly.

[0014] Figure 1 illustrates a focus lens assembly having a first lens and a second lens. [Figure 51B] 51A provides a non-limiting cross-sectional view of a focus lens assembly. The focus lens assembly is configured to maintain a fixed position within an optical path (e.g., an optical axis) and allow relative movement between at least a first lens and a second lens housed within a lens housing of the focus lens assembly. Compare with FIG. 51A, which shows the same focus lens assembly with relative movement of the second lens. [Figure 52] 1 provides a non-limiting cross-sectional view of an optical system configured to image a curved, large-area surface, the optical system comprising a plurality of optical subsystems, each arranged substantially orthogonal to the surface, with the FOV of each subsystem overlapping the FOV of each adjacent optical subsystem, thereby providing a system for imaging a curved, large-area surface. [Figure 53A]

[0023] Figure 1 provides a non-limiting cross-sectional view of an optical system configured to image a capillary flow cell. In this example, an optical system configured to image a large area of ​​a curved surface is rotated about the x-axis and translated along the x-axis to acquire an image of the entire inner surface of the capillary flow cell. The optical axis of the central optical subsystem is shown aligned with the z-axis. [Figure 53B] 1 provides a non-limiting cross-sectional view of an optical system configured to image a capillary flow cell. In this example, an optical system configured to image a large area of ​​a curved surface is rotated about the x-axis and translated along the x-axis to acquire an image of the entire inner surface of the capillary flow cell. The optical axis of the central optical subsystem is shown aligned with the z-axis. The optical axis of the central optical subsystem is shown rotated 90 degrees to align with the y-axis. [Figure 54A] 1 provides a non-limiting cross-sectional view of an optical system configured to image a capillary flow cell without requiring a stage to rotate the optical system about the x-axis. The optical system shown includes a piezo-driven wedge block for fast focusing. The optical system is shown configured to focus on the inner surface of the capillary flow cell closest to the light source. [Figure 54B] 1 provides a non-limiting cross-sectional view of an optical system configured to image a capillary flow cell without requiring a stage to rotate the optical system about the x-axis. The optical system shown includes a piezo-driven wedge block for fast focusing. The optical system is shown configured to focus on the inner surface of the capillary flow cell, far from the light source. [Figure 55] 1 is a bar graph showing the results of trapping assays performed by reacting various fluorescently labeled multivalent molecules with the correct corresponding DNA templates. [Figure 56] 1 is a bar graph showing the results of a trapping assay in which increasing concentrations of various fluorescently labeled multivalent molecules were reacted with the corresponding correct DNA template. [Figure 57]Figure 1 shows four graphs showing the results of a trapping assay comparing the signal intensity of fluorescently labeled multivalent molecules with nucleotide arms containing either the N3 linker, Linker 6, Linker 8, or propargyl linker. The multivalent molecules were labeled with either the CF680 or CF532 fluorophore. Two different concentrations of multivalent molecules (20 nM and 80 nM) were tested. The graphs show the trapping time in seconds (x-axis) and the P90 signal intensity (y-axis). [Figure 58] Figure 1 shows four graphs showing the results of trapping assays comparing signal intensities of fluorescently labeled multivalent molecules with nucleotide arms containing either the N3 linker, Linker 6, Linker 8, or propargyl linker. The multivalent molecules were labeled with either the AF647 or CF570 fluorophore. Two different concentrations of multivalent molecules (20 nM and 80 nM) were tested. The graphs show trapping time in seconds (x-axis) and P90 signal intensity (y-axis). [Figure 59] Three graphs show the results of a real-time imaging trapping kinetic assay comparing the signal intensity of fluorescently labeled multivalent molecules with nucleotide arms containing linkers 6 or one of 10 to 16. Three different concentrations of multivalent molecules (15, 7.5, and 2.5 nM) were tested. The graphs show trapping time in seconds (x-axis) and signal intensity (y-axis). [Figure 60] 59 is a graph showing the results of a binding kinetics study of fluorescently labeled multivalent molecules with nucleotide arms containing linkers 6 or one of 10-16. The graph shows the concentration (x-axis, nM) and rate (y-axis) of the multivalent molecules. The legend for this figure is also applicable to Figure 59. [Figure 61] FIG. 1 is a bar graph showing binding constants (K) measured for fluorescently labeled multivalent molecules having nucleotide arms containing linkers 6 or one of 10-16. [Figure 62] 1 illustrates an example of a sequencing system according to some embodiments. [Figure 63] 1 illustrates a computer system that is programmed or otherwise configured to implement the methods provided herein. [Figure 64A]1A-1C illustrate exemplary embodiments of a multi-surface sample support structure or multi-surface flow cell with multiple surfaces axially displaced from one another, according to certain embodiments herein. 1D-1C illustrate non-limiting examples of a four-surface flow cell including an interposer layer. [Figure 64B] 64A shows an exemplary embodiment of a multi-sided sample support structure or multi-sided flow cell with multiple surfaces axially displaced from one another, according to certain embodiments herein. 64B shows a non-limiting example of an exploded view of the flow cell of FIG. [Figure 64C] 1A-1C show exemplary embodiments of a multi-sided sample support structure or multi-sided flow cell with multiple surfaces axially displaced from one another, according to certain embodiments herein. 1D-1C show non-limiting examples of cross-sectional views of flow cells using a single inlet and outlet for a pair of flow channels. [Figure 64D] 1A-1C show exemplary embodiments of a multi-sided sample support structure or multi-sided flow cell with multiple surfaces axially displaced from one another, according to certain embodiments herein. 1D-1C show non-limiting examples of cross-sectional views of flow cells using a single inlet and outlet for a pair of flow channels. [Figure 64E] 1 shows an exemplary embodiment of a multi-sided sample support structure or multi-sided flow cell with multiple surfaces axially displaced from one another, according to certain embodiments herein. 1 shows a non-limiting example of a flow cell with separate inlets and outlets for a pair of flow channels. [Figure 64F] 1 shows an exemplary embodiment of a multi-sided sample support structure or multi-sided flow cell with multiple surfaces axially displaced from one another, according to certain embodiments herein. 1 shows a non-limiting example of a flow cell with a pair of inlets or outlets for a pair of flow channels. [Figure 65] 1 illustrates an example of a flow cell according to some embodiments. [Figure 66] 1 illustrates an example of a flow cell according to some embodiments. [Figure 67] 1 illustrates an example of a flow cell according to some embodiments. [Figure 68] 1 illustrates an example of a flow cell according to some embodiments. [Figure 69] 1 illustrates an example of a flow cell according to some embodiments. [Figure 70]1 illustrates an example of a flow cell according to some embodiments. [Figure 71] 1 illustrates an example of a flow cell according to some embodiments. [Figure 72] 1 illustrates an example of a flow cell according to some embodiments. [Figure 73] 1 illustrates an example of a flow cell according to some embodiments. [Figure 74] 1 illustrates an example of a flow cell according to some embodiments. [Figure 75] 1 illustrates an example of a flow cell according to some embodiments. [Figure 76] 1 illustrates an example of a flow cell according to some embodiments. [Figure 77] 1 illustrates an example of a flow cell according to some embodiments. [Figure 78] 1 illustrates an example of a flow cell according to some embodiments. [Figure 79] 1 illustrates an example of a flow cell according to some embodiments. [Figure 80] 1 illustrates an example of a flow cell according to some embodiments. [Figure 81] 1 illustrates an example of a flow cell according to some embodiments. [Figure 82] 1 illustrates an example of a flow cell according to some embodiments. [Figure 83] 1 illustrates an example of a flow cell according to some embodiments. [Figure 84] 1 illustrates an example of a flow cell according to some embodiments. [Figure 85] 1 illustrates an example of a flow cell according to some embodiments. [Figure 86] 1 illustrates an example of a flow cell according to some embodiments. [Figure 87] 1 illustrates an example of a flow cell according to some embodiments. [Figure 88] 1 illustrates an example of a flow cell according to some embodiments. [Figure 89] 1 illustrates an example of a flow cell according to some embodiments. [Figure 90]1 illustrates an example of a flow cell according to some embodiments. [Figure 91] 1 illustrates an example of a flow cell according to some embodiments. [Figure 92] 1 illustrates an example of a flow cell according to some embodiments. [Figure 93] 1 illustrates an example of a flow cell according to some embodiments. [Figure 94] 1 illustrates an example of a flow cell according to some embodiments. [Figure 95] 1 illustrates an example of a flow cell according to some embodiments. [Figure 96] 1 illustrates an example of a flow cell according to some embodiments. [Figure 97] 1 illustrates an example of a flow cell according to some embodiments. [Figure 98] 1 illustrates an example of a flow cell according to some embodiments. [Figure 99] 1 illustrates an example of a flow cell according to some embodiments. [Figure 100] 1 illustrates an example of a flow cell according to some embodiments. [Figure 101] 1 illustrates an example of a flow cell according to some embodiments. [Figure 102] 1 illustrates an example of a flow cell according to some embodiments. [Figure 103] 1 illustrates an example of a flow cell according to some embodiments. [Figure 104] 1 illustrates an example of a flow cell according to some embodiments. [Figure 105] 1 illustrates an example of a flow cell according to some embodiments. [Figure 106] 1 illustrates an example of a flow cell according to some embodiments. [Figure 107] 1 illustrates an example of a flow cell according to some embodiments. [Figure 108] 1 illustrates an example of a flow cell according to some embodiments. [Figure 109] 1 illustrates an example of a flow cell according to some embodiments. [Figure 110]1 illustrates an example of a flow cell according to some embodiments. [Figure 111] 1 illustrates an example of a flow cell according to some embodiments. [Figure 112] 1 illustrates an example of a flow cell according to some embodiments. [Figure 113] 1 illustrates an example of a flow cell according to some embodiments. [Figure 114] 1 illustrates an example of a flow cell according to some embodiments. [Figure 115] 1 illustrates an example of a flow cell according to some embodiments. [Figure 116] 1 illustrates an example of a flow cell according to some embodiments. [Figure 117] 1 illustrates an example of a flow cell according to some embodiments. [Figure 118] 1 illustrates an example of a flow cell according to some embodiments. [Figure 119] 1 illustrates an example of a flow cell according to some embodiments. [Figure 120] 1 illustrates an example of a flow cell according to some embodiments. [Figure 121] 1 illustrates an example of a flow cell according to some embodiments. [Figure 122] 1 illustrates an example of a flow cell according to some embodiments. [Figure 123] 1 illustrates an example of a flow cell according to some embodiments. [Figure 124] 1 illustrates an example of a flow cell according to some embodiments. [Figure 125] 1 illustrates an example of a flow cell according to some embodiments. [Figure 126] 1 illustrates an example of a flow cell according to some embodiments. [Figure 127] 1 illustrates an example of a flow cell according to some embodiments. [Figure 128] 1 illustrates an example of a flow cell according to some embodiments. [Figure 129] 1 illustrates an example of a flow cell according to some embodiments. [Figure 130]1 illustrates an example of a flow cell according to some embodiments. [Figure 131] 1 illustrates an example of a flow cell according to some embodiments. [Figure 132] 1 illustrates an example of a flow cell according to some embodiments. [Figure 133] 1 illustrates an example of a flow cell according to some embodiments. [Figure 134] 1 illustrates an example of a flow cell according to some embodiments. [Figure 135] 1 illustrates an example of a flow cell according to some embodiments. [Figure 136] 1 illustrates an example of a flow cell according to some embodiments. [Figure 137] 1 illustrates an example of a flow cell according to some embodiments. [Figure 138] 1 illustrates an example of a flow cell according to some embodiments. [Figure 139] 1 illustrates an example of a flow cell according to some embodiments. [Figure 140] 1 illustrates an example of a flow cell according to some embodiments. [Figure 141] 1 illustrates an example of a flow cell according to some embodiments. [Figure 142] 1 illustrates an example of a flow cell according to some embodiments. [Figure 143] 1 illustrates an example of a flow cell according to some embodiments. [Figure 144] 1 illustrates an example of a flow cell according to some embodiments. [Figure 145] 1 illustrates an example of a flow cell according to some embodiments. [Figure 146] 1 illustrates an example of a flow cell according to some embodiments. [Figure 147] 1 illustrates an example of a flow cell according to some embodiments. [Figure 148] 1 illustrates an example of a flow cell according to some embodiments. [Figure 149] 1 illustrates an example of a flow cell according to some embodiments. [Figure 150]1 illustrates an example of a flow cell according to some embodiments. [Figure 151] 1 illustrates an example of a flow cell according to some embodiments. [Figure 152] 1 illustrates an example of a flow cell according to some embodiments. [Figure 153] 1 illustrates an example of a flow cell according to some embodiments. [Fig. 154] 1 illustrates an example of a flow cell according to some embodiments. [Figure 155] 1 illustrates an example of a flow cell according to some embodiments. [Figure 156] 1 illustrates an example of a flow cell according to some embodiments. [Figure 157] 1 illustrates an example of a flow cell according to some embodiments. [Figure 158] 1 illustrates an example of a flow cell according to some embodiments. [Figure 159] 1 illustrates an example of a flow cell according to some embodiments. [Figure 160] 1 illustrates an example of a flow cell according to some embodiments. [Figure 161] 1 illustrates an example of a flow cell according to some embodiments. [Figure 162] 1 illustrates an example of a flow cell according to some embodiments. [Figure 163] 1 illustrates an example of a flow cell according to some embodiments. [Fig. 164] 1 illustrates an example of a flow cell according to some embodiments. [Figure 165] 1 illustrates an example of a flow cell according to some embodiments. [Figure 166] 1 illustrates an example of a flow cell according to some embodiments. [Figure 167] 1 illustrates an example of a flow cell according to some embodiments. [Figure 168] 1 illustrates an example of a flow cell according to some embodiments. [Figure 169] 1 illustrates an example of a flow cell according to some embodiments. [Figure 170A]10 shows an example of the RMS wavefront error for each surface of a four-sided flow cell according to some embodiments. [Figure 170B] 10 shows an example of the RMS wavefront error for each surface of a four-sided flow cell according to some embodiments. [Figure 170C] 10 shows an example of the RMS wavefront error for each surface of a four-sided flow cell according to some embodiments. [Figure 170D] 10 shows an example of the RMS wavefront error for each surface of a four-sided flow cell according to some embodiments. [Figure 171A] 10 shows an example of a comparison of runtimes for a two-sided and four-sided embodiment, according to some embodiments. 11 shows an example of runtime for two sides, according to some embodiments. [Figure 171B] 10A-10C illustrate an example of a comparison of runtimes for a two-sided and four-sided embodiment, according to some embodiments. 10B-10C illustrate an example of a runtime for a four-sided embodiment, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0013] Next-generation sequencing (NGS) analysis systems require increased throughput and flexibility. Disclosed herein are systems, kits, compositions, and methods that may provide one or more of the following advantages: higher system throughput for fluorescence imaging-based genomics applications; compatibility with conventional flow cell devices and / or optical systems; flexibility in sample analysis or comparison (e.g., larger sample volumes and / or increased sample types); improved optical resolution (including high-performance optical resolution); and improved image quality. The disclosed optical illumination and imaging system designs may provide one or more of the following advantages: improved dichroic filter performance, improved uniformity of the dichroic filter frequency response, improved excitation beam filtering, larger field of view, improved spatial resolution, modulation transfer, improved contrast-to-noise ratio, and improved image quality; higher spatial sampling frequencies; faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., different fields of view); improved imaging system duty cycle; and higher throughput of image acquisition and analysis. The disclosed flow cells can improve throughput and sample processing speed while maintaining low background noise.

[0014] system

[0009] Provided herein, in some embodiments, is a system useful for nucleic acid analysis. In some embodiments, the system comprises an imaging system. In some embodiments, the system comprises a fluid control system. In some embodiments, the system comprises a multivalent molecule or a mixture of multivalent molecules. In some embodiments, the system comprises a solid phase for performing nucleic acid analysis. In some embodiments, the solid phase is a flow cell disclosed herein. In some embodiments, the system disclosed herein comprises one or more kits disclosed herein. In some aspects, the kit can be a kit described in International Patent Application No. PCT / US2021 / 057441, the entire contents of which are incorporated herein by reference.

[0015] 1. Imaging System Disclosed herein, in some embodiments, are imaging systems useful for imaging one or more surfaces for nucleic acid analysis. In some embodiments, the imaging system includes an adjustable numerical aperture. In some examples, the imaging system does not include a compensator. In some examples, the imaging system includes a compensator. Imaging systems with a wide field of view can provide increased throughput and reduced sample imaging time.

[0016] In some embodiments, the imaging system disclosed herein is an optical system. In some embodiments, an optical system of the present disclosure is provided in FIG. 45. In some embodiments, the optical system 4500 eliminates the need for dichroic or correction optics, such as tube lenses, for multi-surface imaging of a flow cell. The multi-surfaces can be two, three, four, or more. The optical system 4500 disclosed herein can be used as a component of systems designed for various chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. As shown in FIG. 45, in some embodiments, the optical system includes multiple image sensors 4501-4504 configured to image a flow cell 4521. In some embodiments, the image sensors 4501-4504 may be CCD image sensors. In some embodiments, the image sensors 4501-4504 may be CMOS image sensors. In some embodiments, pixel shifters 4505-4508 are used to translate the object being imaged relative to the corresponding image sensors 4505-4508. In some embodiments, the optical system includes a multi-band bandpass filter 4509. In some embodiments, the multi-band bandpass filter is a multi-band fluorescence bandpass filter. In some embodiments, the multi-band bandpass filter is a three-band fluorescence bandpass filter. In some embodiments, the three-band fluorescence bandpass filter is referred to as a three-band notch filter. In some embodiments, the imaging optics 4510-4513 are disposed between the image sensors 4501-4504 and the flow cell 4521. In some embodiments, the imaging optics 4505-4508, also referred to as an imaging optics assembly, focus light emitted from the flow cell 4521 onto one of the image sensors, e.g., 4501, 4502, 4503, or 4504. In some embodiments, the optical system includes an integrated field flattening assembly. In some embodiments, the optical system includes aberration correction. In some embodiments, the optical system lacks a bandpass filter. In some embodiments, the optical system lacks a cutoff filter.In some embodiments, the optical system lacks a dichroic mirror. In some embodiments, the liquid handling system 4514 distributes the sample 4515 to the flow cell 4521. In some embodiments, the liquid handling system 4514 distributes the liquid sample to a hydrophobic pad 4516 attached to the flow cell 4521. In some embodiments, the liquid handling system 4514 is a droplet dispensing system. In some embodiments, the droplet dispensing system 4514 delivers the sample 4515 as droplets to the hydrophobic pad 4516 of the flow cell 4521. In some embodiments, the liquid sample 4515 is drawn into the interior 4517 of the flow cell 4521 by a pulling force. In some embodiments, the pulling force is initiated by a vacuum pump 4518. In some embodiments, the flow cell 4521 comprises an interior channel 4517 surrounded by a bottom plate 4519 and a top plate 4520. In some embodiments, the top plate 4520 and the bottom plate 4519 are transparent. In some embodiments, the top plate comprises a front inner surface 4528. In some embodiments, the bottom plate comprises a back inner surface 4529. In some embodiments, the sample present in the interior channel 4517 of the flow cell 4521 is illuminated by multiple light sources 4522, 4523, or 4524. In some embodiments, each of the individual light sources 4522, 4523, and 4524 emits light of a different color or spectrum 4525, 4526, and 4527. In some embodiments, the optical system 4500 includes a heater.

[0017] While in FIG. 45 , the flow cell 4521 is shown as having two interior surfaces, in other embodiments, the flow cell 4521 can comprise two or more axially displaced channels and three or more axially displaced interior surfaces. As used herein, “axially displaced” can be used to refer to the relative arrangement of surfaces. In some examples, two surfaces can be axially displaced if the normal vector extending from one surface intersects the normal vector of the other surface. In some examples, two surfaces can be axially displaced if the normal vector extending from one surface is collinear with the normal vector of the other surface. In some examples, two surfaces can be axially displaced if they are displaced along the axis of the optical path of an optical system that includes the two surfaces.

[0018] In some embodiments, a notch filter refers to a band-stop filter. In some embodiments, a notch filter refers to a band-stop filter. In some embodiments, a filter notch refers to a band-stop or stopband. In some embodiments, a filter notch refers to a band-pass or passband. In some embodiments, a multi-band notch filter refers to a multi-band band-pass filter. In some embodiments, a multi-band notch filter refers to a multi-band band-stop filter.

[0019] In some embodiments, the imaging optics 4510 of the optical system 4500 includes a demagnification of 1. In some embodiments, the optical system includes a 1 mm 2 Exceeding 2mm 2 Exceeding 4mm 2 Exceeding 10mm 2 Exceeding 20mm 2 Exceeding 36mm 2 Exceeding 40mm 2 Exceeding 60mm 2 Exceeding 80mm 2 Exceeding or 100mm 2 In some embodiments, the optical system has a field of view (FOV) of more than 2 mm. 2 Less than 4mm 2 Less than 10mm2 Less than 20mm 2 Less than 36mm 2 Less than 40mm 2 Less than 60mm 2 Less than 80mm 2 Less than or 100mm 2 In some embodiments, the optical system has a field of view (FOV) of less than 0.6. In some embodiments, the optical system has a numerical aperture (NA) of less than 0.6. In some embodiments, the NA is about 0.1 to about 0.50, about 0.20 to about 0.40, or about 0.30. In some embodiments, the NA is 0.25. In some embodiments, the NA is about 0.1, 0.15, 0.20, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or 0.60. In some embodiments, the NA is greater than 0.1. In some embodiments, multiple image sensors are configured to capture the FOV. In some embodiments, the multiple light sources comprise a first light source 4522 configured to emit a first wavelength range 4525, a second light source 4523 configured to emit a second wavelength range 4526, and a third light source 4524 configured to emit a third wavelength range 4527. In some embodiments, the first fluorophore is excited by a first wavelength range 4525 of a first light source 4522. In some embodiments, the second fluorophore is excited by a second wavelength range 4526 of a second light source 4523. In some embodiments, the third fluorophore is excited by a third wavelength range 4527 of a third light source 4524. In some embodiments, the sample comprises a plurality of biological polymers. In some embodiments, the optical system 4500 does not include a dichroic filter. In some embodiments, the optical system 4500 does not include a tube lens.

[0020] Described herein are various methods for various chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. FIG. 46 shows a schematic diagram of an imaging method 4601 for imaging a sample 4515 contained within a flow cell 4521 using the optical system 4500 shown in FIG. 45 , according to some embodiments herein. In some embodiments, the imaging method may be configured for nucleic acid sequencing. In some embodiments, the sample 4515 is contained within or flows through an internal channel 4517 of the flow cell 4521, as shown in FIG. 45 . In some embodiments, the sample includes a biological polymer. In some embodiments, the biological polymer includes units. In some embodiments, a fluorophore is complementary to a unit of the biological polymer. In some embodiments, a fluorophore is attached to a nucleotide complementary to a unit of the biological polymer. In some embodiments, two or more detectably different fluorophores are attached to a nucleotide complementary to a unit of the biological polymer. In some embodiments, the biological polymer is a nucleic acid sequence. In some embodiments, the units are nucleotides complementary to fluorophore-labeled nucleotides. In some embodiments, multiple light sources emit light that is transmitted through the sample.

[0021] Described herein are various methods for sequencing biological polymers (e.g., nucleic acid molecules). Figure 46 shows a non-limiting schematic diagram of a sequencing method and apparatus 4601 and a base calling method 4602. In some embodiments, the method includes illuminating a sample 4515 using an optical system 4500 including a first light source 4522 of a plurality of light sources, the first light source 4522 emitting a first wavelength range 4525 that excites a first fluorophore in the sample 4515, and acquiring a first image of the sample 4515, the optical system 4500 including a plurality of image sensors 4501-4504, and further including a plurality of image sensors 4501-4504, the sample 4515 being illuminated by the plurality of light sources 4522. irradiating the sample 4515 using a plurality of second light sources 4523, the second light sources 4523 emitting a second wavelength range 4526 that excites a second fluorophore in the sample 4515, and acquiring a second image of the sample 4515; irradiating the sample 4515 using a plurality of third light sources 4524, the third light sources 4524 emitting a third wavelength range 4526 that excites a third fluorophore in the sample. the first signal being extracted from a first region of interest (ROI) of the composite image; identifying the presence of a second nucleotide via a second signal emitted by a second fluorophore, the second signal being extracted from the second ROI of the composite image; identifying the presence of a third nucleotide via a third signal emitted by a third fluorophore, the third signal being extracted from the third ROI of the composite image; and identifying the presence of a fourth nucleotide via a first signal and a third signal emitted by the first fluorophore and a third fluorophore, respectively, the first signal and the third signal being extracted from the fourth ROI of the composite image.In some embodiments, the optical system 4500 further includes a flow cell 4521, the flow cell 4521 being disposed in an optical path between the plurality of image sensors 4501-4504 and the plurality of light sources 4522-4524. In some embodiments, the optical system 4500 further includes at least one pixel shifter 4505-4508. In some embodiments, the optical system 4500 further includes a multi-band bandpass filter 4509 disposed in an optical path between the plurality of image sensors 4501-4504 and the flow cell 4521. In some embodiments, the method further includes imaging optics 4510-4513 disposed in an optical path between the multi-band bandpass filter 4509 and the flow cell 4521. In some embodiments, the optical system 4500 has a demagnification of 1x. In some embodiments, the magnification of the optical system 4500 is 1x, 1.5x, 2x, 3x, 4x, 5x, 6x, 8x, or 10x. In some embodiments, the optical system is about 1 mm. 2 More than 2mm 2 or more, about 4mm 2 Above, about 10 mm 2 or more, about 20mm 2 or more, about 36mm 2 Above, about 40 mm 2 or more, about 60mm 2 or more, about 80mm 2 or more, or about 100 mm 2In some embodiments, the optical system has a field of view (FOV) of less than 0.6. In some embodiments, the optical system has a numerical aperture (NA) of about 0.6. In some embodiments, the optical system has a numerical aperture (NA) of greater than 0.6. In some embodiments, the NA is 0.25. In some embodiments, the NA is about 0.25. In some embodiments, the NA is about 0.25 or greater. In some embodiments, the NA is about 0.25 or less. In some embodiments, the NA is 0.4-0.5. In some embodiments, the NA is about 0.4-0.5. In some embodiments, the NA is about 0.4 or greater. In some embodiments, the NA is about 0.5 or less. In some embodiments, the FOV is captured by multiple image sensors 4501-4504.

[0022] In some embodiments, sequencing is described in U.S. Patent No. 10,768,173, filed September 23, 2019, which is incorporated herein by reference in its entirety. In some embodiments, a first fluorophore is associated with a first nucleotide conjugate. In some embodiments, the nucleotide conjugate is a polymer-nucleotide conjugate. In some embodiments, a multivalent molecule disclosed elsewhere herein is or comprises a nucleotide conjugate or a polymer-nucleotide conjugate disclosed herein. In some embodiments, a second fluorophore is associated with a second nucleotide conjugate. In some embodiments, a third fluorophore is associated with a nucleotide conjugate. In some embodiments, the first fluorophore and the third fluorophore are associated with a fourth nucleotide conjugate. In some embodiments, the nucleotide conjugate may comprise a polymer-nucleotide conjugate. In some embodiments, the nucleotide conjugate may comprise a particle-nucleotide conjugate.

[0023] In some embodiments, fluorophores that can function as the first fluorophore, the second fluorophore, and / or the third fluorophore include fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluoresceinthiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, and rhodamine. and rhodamine derivatives, such as TRITC, TMR, Lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, Lissamine rhodamine B sulfonyl chloride, Lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives, such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives, such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 Hydrazide, BODIPY 493 / 503 C3 Hydrazide, BODIPY FL C3 Hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanines and derivatives such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes,Examples of suitable dyes include, but are not limited to, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and their derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbenes, DEG dyes, NR dyes, near-infrared dyes, and others known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oregon), 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press, New York (1999); or Hermanson, Bioconjugate Techniques, 2nd Edition, or any derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated form and contain two indolenine, benzoindolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which is 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium, may include 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may include1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indo 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3 ,3-dimethyl-3H-indol-1-ium-5-sulfonate), and Cy7 (which may include 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for "cyanine" and the first number identifies the number of carbon atoms between the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule. In some embodiments, the reporter moieties can be FRET pairs, allowing multiple classifications to be performed under a single excitation and imaging step. As used herein, FRET can include excitation exchange (Förster) transfer or electron exchange (Dexter) transfer.

[0024] Described herein is an optical system 4700 for imaging a sample in a flow cell that does not include a focusing step.

[0025] Described herein is an optical system 4700 for imaging samples in a flow cell for analysis of biological polymers (e.g., nucleic acid sequencing). In some embodiments, the system 4700, as shown in FIGS. 47A-47B, is more compact and has higher throughput than conventional optical systems. Table 1 and FIGS. 48A-48B provide a non-limiting example comparing sequencing cycle times for a standard flow cell and optical system with the optical systems described herein. Table 1 shows the cycle and run times, and respective calculations, for a standard flow cell having 424 individual tiles (e.g., active areas (e.g., areas undergoing active illumination), regions of interest (e.g., areas undergoing active detection), etc.) shown in FIG. 48A. A comparison is shown in FIG. 48B with a flow cell having fewer than 40 individual tiles optimized for imaging on the optical systems described herein. In some embodiments, one image corresponds to the area of ​​one tile. In some embodiments, when flow cell 4521, also shown in FIG. 48B, is imaged by the optical system, each tile is exposed to three successive light pulses from three separate LED light sources, each emitting a different wavelength. In some embodiments, each different wavelength matches the excitation spectrum of a different fluorophore, as described herein. In some embodiments, the image sensor of optical system 4500 generates an image synchronously with each excitation pulse, with one image covering the entire area of ​​one tile, and each pixel of the image representing the amount of fluorescence emitted from a fluorophore. In some embodiments, two separate surfaces are imaged into one tile by optical system 4500. In some embodiments, a total of eight images with a total exposure time of 0.3 seconds are acquired by optical system 4500, 4700, which includes eight imaging modules (e.g., optical subsystems). In Table 1, the row titled "Current" and highlighted in blue indicates that the total time over 322 cycles for the standard flow cell shown in Figure 48A, when imaged with the IDEX optical system shown in Figure 47B, is 36.17 hours.In comparison, the row titled "Sleq" shows that the total time for a Sleq cell (see FIG. 48B) when imaged with the optical system 4700 shown in FIGS. 47A-B is 13.63 hours to 14.28 hours. The bottom row of Table 1 shows that the total time is 1.11 hours when only 25 cycles are performed. The reduced sequencing time demonstrates the benefit of the larger FOV enabled by the optical system 4700 described herein.

[0026] [Table 1]

[0027] Figure 48A provides a diagram of the imaging area of ​​a flow cell described herein having 424 individual tiles, and Figure 48B provides a diagram of the imaging area of ​​a flow cell described herein having fewer than 40 tiles.

[0028] Figure 47A provides a non-limiting cross-sectional view of an optical system for imaging the surface of a flow cell 4521. In some embodiments, the optical system includes an LED bank heat sink 4701, a light pipe illuminator 4702, the flow cell 4521, a section of imaging optics 4703, one or more pixel shifters 4704, and multiple image sensors 4705. As shown in Figure 47B, the optical system 4700 is smaller than comparable instruments such as an IDEX instrument core. Advantages of smaller optical instruments include, but are not limited to, reduced cabling requirements, a reduced number of available failure modes, reduced heat exchange requirements, and a reduced benchtop footprint.

[0029] Described herein, in some embodiments, is an optical system 4900 configured for multi-planar imaging of a flow cell 4905, as shown in the non-limiting schematic diagrams of FIGS. 49A-49B. The multi-planar imaging can be two, three, four, or more. The optical system 4900 disclosed herein can be used in systems designed for various chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. As shown in FIGS. 49A-49B, the optical system includes an image sensor 4912 that can be configured to image the flow cell 4905. In some embodiments, the sample flow is aligned with the x-axis, as shown in FIGS. 49A-49B. In some embodiments, there can be multiple image sensors 4912. The image sensor 4912 can be a CCD image sensor. In some embodiments, the image sensor 4912 can be a CMOS image sensor. In some embodiments, the optical system 4900 includes a pixel shifter 4911. The pixel shifter 4911 can be configured to improve image resolution. In some embodiments, a pixel shifter 4911 translates the object being imaged relative to the image sensor 4912. In some embodiments, the optical system comprises a filter 4910. In some embodiments, the filter 4910 is a multi-band filter. In some embodiments, the filter 4509 is a multi-band stop-band filter. In some embodiments, the filter 4910 is a three-band fluorescence stop-band filter. In some embodiments, a three-band fluorescence stop-band filter is referred to as a three-band notch filter. In some embodiments, the system comprises imaging optics 4909. In some embodiments, the imaging optics 4909 comprises an objective lens.

[0030] In some embodiments, filter 4910 is disposed between image sensor 4912 and flow cell 4905. In some embodiments, imaging optics 4909, also referred to as an imaging optics assembly, focuses light emitted from flow cell 4909 onto image sensor 4912. In some embodiments, optical system 4900 comprises an integrated field flattening assembly. In some embodiments, the optical system comprises an aberration correction module. In some embodiments, the optical system comprises a wedge block 4916 configured to adjust the optical path length of the optical system. In some embodiments, wedge block 4916 comprises a first wedge piece 4907, a second wedge piece 4906, or a combination thereof. In some embodiments, the system comprises a piezo drive 4908 configured to move the position of first wedge piece 4907 and second wedge piece 4906 relative to each other, thereby adjusting the optical path length of the optical system. In some embodiments, the first wedge piece 4907 can be actuated to move relative to the second wedge piece 4906 in a range of 0.1 μm to 1000 μm. In some embodiments, the first wedge piece 4907 can be actuated to move relative to the second wedge piece 4906 in a range of 100 μm to 800 μm. In some embodiments, the first wedge piece 4907 can be actuated to move relative to the second wedge piece 4906 in a range of 300 μm to 700 μm. Relative movement of the wedge pieces 4907, 4906 can be achieved by moving the first wedge piece 4907 and / or the second wedge piece 4906 relative to a stationary sample or sample stage during imaging of one or more images. In some embodiments, movement of the wedge pieces 4907, 4906 can be at least in the XY plane. In some embodiments, movement of the wedge pieces 4907, 4906 can be solely in the XY plane. In some embodiments, the flow cell 4905 is configured for multi-plane imaging (DSI). In some embodiments, the flow cell 4905 includes a front inner surface 4904, a back inner surface 4905, or a combination thereof.In some embodiments, the front inner surface 4904 and / or the back inner surface 4903 comprise the sample site 4902. In some embodiments, the optical system comprises an optical axis 4913. In some embodiments, the optical system comprises an optimal imaging volume 4915. In certain aspects, the optimal imaging volume 4915 includes a field of view (FOV), an illumination area, an acquisition area, a focal plane, a depth of focus, a region and / or a volume in which the sample site 4902 emits an acceptable level of brightness or higher, or a combination thereof. Typically, in the field of microscopy, the brightness of an object in the center of the FOV may be greatest at the center and decrease towards the corners and / or edges.

[0031] In some embodiments, the optical system lacks a bandpass filter, in some embodiments, the optical system lacks a cutoff filter, in some embodiments, the optical system lacks a dichroic mirror or dichroic filter.

[0032] 2. Wedge Block Assembly Various embodiments of optical systems that include focusing element assemblies are described herein.

[0033] In some embodiments, the focus element assembly is an autofocus element. By way of example, Figures 51A-51B provide non-limiting cross-sectional views of a focus lens assembly. The focus lens assembly is configured to maintain a fixed position within an optical path (e.g., an optical axis) and to allow relative movement between at least a first lens and a second lens housed within a lens housing of the focus lens assembly.

[0034] In some embodiments, the autofocus element is configured for initial focus. In some embodiments, the autofocus element is housed within the lens barrel. In some embodiments, the autofocus element is built into and / or integrated with the lens barrel. In some embodiments, the autofocus element is housed within the lens barrel of the lens assembly. In some embodiments, the autofocus element is configured to improve reliability and reduce the mechanical footprint of the optical system. In some embodiments, the autofocus element comprises a wedge block assembly, a piezo drive, a wedge block-piezo drive assembly, or a combination thereof.

[0035] In some embodiments, the focusing element assembly includes a first focusing element and a second focusing element. In some embodiments, the first and second focusing elements are contained within a housing that includes the first and second focusing element assemblies. In some embodiments, the first focusing element is configured to move relative to the second focusing element to focus between the first surface of the flow cell and the second surface of the flow cell, respectively. In some embodiments, the first focusing element and / or the second focusing element are lenses. In some embodiments, the first focusing element and / or the second focusing element are prisms. In some embodiments, the focusing element assembly is a wedge block assembly, and the first focusing element and the second focusing element are wedge blocks configured to move relative to each other. In some embodiments, the optical system includes a wedge block assembly 4916, as shown in FIGS. 49A-49B. In certain aspects, the wedge block assembly 4916 includes a first wedge piece 4907 and a second wedge piece 4906. In some embodiments, the wedge block assembly 4916 has an adjustable optical path length. In some embodiments, the first wedge piece 4907 is configured to move relative to the second wedge piece 4906. In some embodiments, relative movement of the first wedge piece 4907 relative to the second wedge piece 4906 changes the optical path length of the wedge block assembly 4916 due to a change in the physical thickness of the wedge block assembly 4916, as shown in FIGS. 49A-49B. In some embodiments, the wedge block assembly 4916 includes a gap separating the first wedge piece 4907 from the second wedge piece 4906. In some embodiments, this gap maintains a constant distance regardless of the relative positions of the first wedge piece 4907 and the second wedge piece 4906. In some embodiments, the first wedge piece 4907 and the second wedge piece 4906 are composed of fused silica.In some embodiments, the first wedge piece 4907 and the second wedge piece 4906 are composed of fused silica with a refractive index of 1.5. In some embodiments, the first wedge piece 4907 is coupled to the piezo drive 4908. In some embodiments, the optical system comprises a housing. In some embodiments, the wedge block assembly 4916 and the piezo drive 4908 are contained within the housing. In some embodiments, the wedge block assembly 4916 and the piezo drive 4908 comprise a wedge block-piezo drive assembly. In some embodiments, the second wedge piece 4906 of the wedge block assembly 4916 contacts the housing. In some embodiments, the second wedge piece 4906 of the wedge block assembly 4916 contacts the flow cell 4905.

[0036] In some embodiments, the position of the first wedge piece 4907 relative to the second wedge piece 4906 determines the position of the focal plane along the optical axis 4913 (e.g., the z-axis). In some embodiments, the top wedge piece 4907 is aligned with the bottom wedge piece 4906, as shown in FIG. 49A. In such embodiments, the physical distance of the wedge block assembly 4916 aligns the focal plane with the back inner surface, in this case, the sample site 4902 on the back inner surface is in focus. In some embodiments, the piezo drive 4908 moves the top wedge piece 4907 to a position relative to the bottom wedge piece 4906, as shown in FIG. 49B, which results in a greater physical thickness of the wedge block 4916 in the optical path than in the aligned state shown in FIG. 49A. In such embodiments, the focal plane is shifted to align with the front inner surface, in this case, the sample site 4902 on the front inner surface.

[0037] 3. Stage Various embodiments of an optical system including a stage are described herein. The stage may be a tilt stage. The stage may be a tip-tilt stage. The stage may be capable of rotation. The stage may be configured to translate three different axes simultaneously, all perpendicular to one another. The stage may be configured to translate three different axes simultaneously, all perpendicular to one another. The stage may be configured to translate in three different axes simultaneously and rotate about those axes, all perpendicular to one another. The stage may translate multiple optical subsystems 5001 relative to the flow cell 4905, as shown in FIG. 50. The stage may translate the flow cell 4905 relative to the multiple optical subsystems 5001, as shown in FIG. 50. The stage may translate a single optical subsystem 4914. The stage may rotate multiple optical subsystems 5001 about the x-axis of the capillary flow cell 5201, as shown in FIGS. 53A-53B. The stage can translate the multiple optical subsystems 5001 along the x-axis, which coincides with the long axis of the capillary flow cell 5201, as shown in Figures 52A-53B.

[0038] 4. Pixel Shifter Various embodiments of optical systems including a pixel shifter 4911 are described herein. In some embodiments, the pixel shifter 4911 enables sub-pixel resolution imaging. In certain aspects, the resolution of an optical system can be increased by using the pixel shifter 4911 without increasing the actual optical system resolution. In some embodiments, the pixel shifter 4911 effectively doubles the resolution of the image sensor 4912. In some embodiments, the piezoelectric actuator is configured for a predetermined lateral pixel shift that coincides with the image plane (e.g., in the x-y plane). In some embodiments, the piezoelectric actuator is configured for pixel shifting in the optical axis 4913 (e.g., the z-axis or a plane containing the z-axis). In some embodiments, the tilt stage is configured for pixel shifting in XZ, YZ, or XYZ. In some examples, the tilt stage is configured for pixel shifting in two dimensions. In some embodiments, the optical system including the pixel shifter is configured to image a 3D sample object. In some examples, the optical system including the pixel shifter is configured to image a 2D sample object.

[0039] In some embodiments, the 3D object may include a sample site 4902. In some embodiments, the sample site 4902 is an amplified nucleic acid. In some embodiments, the sample site may include one or more polonies.

[0040] The pixel shifter 4911 may utilize polarized light.

[0041] 5. Multiple Imaging Systems In some embodiments, the optical system shown in FIG. 50 comprises multiple optical subsystems 4914. In some embodiments, each optical subsystem 4914 of the multiple optical subsystems 5001 comprises an image sensor 4912, a pixel shifter 4911, a filter 4910, imaging optics 4909, a piezo drive-wedge block assembly, a light source 4901, or a combination thereof. In some embodiments, the image sensor 4912 is a cell phone-style camera. In some embodiments, the multiple optical subsystems 5001 comprise an array of optical subsystems. In some embodiments, the array of optical subsystems may be configured for multiple focal depths, multiple wavelengths, or a combination thereof. In some embodiments, each optical subsystem 4914 of the multiple optical subsystems 5001 is configured for a focal depth, and at least two of the multiple optical subsystems have different focal depths. In some embodiments, each optical subsystem of the multiple optical subsystems is configured to detect a wavelength, and the wavelengths detected by at least two of the multiple optical subsystems are different. In some embodiments, the image sensor 4912 of each optical subsystem 4914 of the multiple optical subsystems 5001 comprises an array of image sensors 4912. In some embodiments, a high-resolution, low-cost camera is configured to provide imaging with aberrations compensated by software. In some embodiments, the optical system comprises a single optical subsystem 4914, which comprises a single optimal imaging volume, as shown in FIGS. 49A-49B. In FIGS. 49A-49B, the optimal imaging volume 4915 has a limited extent along the x-axis. Certain factors can affect the width of the optimal imaging volume in the x-y plane (e.g., focal plane). The x-y plane, or focal plane, contains a cross-section of the optimal imaging volume and may be referred to as the illumination area, acquisition area, or a combination thereof. Surfaces containing sample sites 4902 that extend beyond the optimal FOV may not be optimally illuminated by the light source, optimally captured by the image sensor, optimally resolved by the optical system, or a combination thereof.Such non-optimal surface regions exhibit non-uniform brightness and non-uniform resolution, as can be observed at the edges and / or corners of the image in Figure 37, where the sample site becomes darker and less resolved from the center of the image towards the edges and / or corners. Figure 50 shows an embodiment in which the surface covered by the sample site 4902 extends beyond the optimal imaging volume 4915 of one optical subsystem 4916, and the overlapping optimal imaging volumes 4915 overlap to provide a composite optimal imaging volume.

[0042] In some embodiments, the optical system has an optimized FOV of 6 mm x 6 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm to about 9 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm to about 1 mm, about 0.5 mm to about 3 mm, about 0.5 mm to about 6 mm, about 0.5 mm to about 9 mm, about 1 mm to about 3 mm, about 1 mm to about 6 mm, about 1 mm to about 9 mm, about 3 mm to about 6 mm, about 3 mm to about 9 mm, or about 6 mm to about 9 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm, about 1 mm, about 3 mm, about 6 mm, or about 9 mm. In some embodiments, the system has an optimized FOV of at least about 0.5 mm, about 1 mm, about 3 mm, or about 6 mm. In some embodiments, the system has an optimized FOV of at most about 1 mm, about 3 mm, about 6 mm, or about 9 mm.

[0043] In some embodiments, the optical system has an optimized illumination area of ​​6 mm x 6 mm. In some embodiments, the system has an optimized illumination area of ​​about 0.5 mm to about 9 mm. In some embodiments, the system has an optimized illumination area of ​​about 0.5 mm to about 1 mm, about 0.5 mm to about 3 mm, about 0.5 mm to about 6 mm, about 0.5 mm to about 9 mm, about 1 mm to about 3 mm, about 1 mm to about 6 mm, about 1 mm to about 9 mm, about 3 mm to about 6 mm, about 3 mm to about 9 mm, or about 6 mm to about 9 mm. In some embodiments, the system has an optimized illumination area of ​​about 0.5 mm, about 1 mm, about 3 mm, about 6 mm, or about 9 mm. In some embodiments, the system has an optimized illumination area of ​​at least about 0.5 mm, about 1 mm, about 3 mm, or about 6 mm. In some embodiments, the system has an optimized illumination area of ​​at most about 1 mm, about 3 mm, about 6 mm, or about 9 mm.

[0044] In some embodiments, the optical system is configured to rapidly image the surface. In some embodiments, the optical system is configured to rapidly image the surface of a flow cell. In some embodiments, the optical system is configured to rapidly image the first surface and the second surface of a flow cell. In some embodiments, the entire active area (e.g., region of interest ROI) of surface 4903 or 4904 of flow cell 4905 is imaged in five imaging steps. In some embodiments, the active area (e.g., region of interest) of surface 4903 or 4904 is imaged in about 1 imaging step to about 10 imaging steps. In some embodiments, the active area of ​​the surface (e.g., region of interest) is imaged for about 1 to about 2 imaging steps, about 1 to about 3 imaging steps, about 1 to about 4 imaging steps, about 1 to about 5 imaging steps, about 1 to about 6 imaging steps, about 1 to about 10 imaging steps, about 2 to about 3 imaging steps, about 2 to about 4 imaging steps, about 2 to about 5 imaging steps, about 2 to about 6 imaging steps, about The surface may be imaged in between 2 imaging steps and about 10 imaging steps, between 3 imaging steps and about 4 imaging steps, between 3 imaging steps and about 5 imaging steps, between 3 imaging steps and about 6 imaging steps, between 3 imaging steps and about 10 imaging steps, between 4 imaging steps and about 5 imaging steps, between 4 imaging steps and about 6 imaging steps, between 4 imaging steps and about 10 imaging steps, between 5 imaging steps and about 6 imaging steps, between 5 imaging steps and about 10 imaging steps, or between 6 imaging steps and about 10 imaging steps. In some embodiments, an active area of ​​the surface (e.g., a region of interest) is imaged in about 1 imaging step, about 2 imaging steps, about 3 imaging steps, about 4 imaging steps, about 5 imaging steps, about 6 imaging steps, or about 10 imaging steps.In some embodiments, an active area of ​​a surface (e.g., a region of interest) is imaged in at least about 1 imaging step, about 2 imaging steps, about 3 imaging steps, about 4 imaging steps, about 5 imaging steps, or about 6 imaging steps. In some embodiments, an active area of ​​a surface (e.g., a region of interest) is imaged in at most about 2 imaging steps, about 3 imaging steps, about 4 imaging steps, about 5 imaging steps, about 6 imaging steps, or about 10 imaging steps.

[0045] In some embodiments, each imaging step includes imaging at least a portion of the active area or entire ROI. In some embodiments, each imaging step includes imaging at least overlapping portions of the ROI, and overlapping portions within the ROI may also be imaged in different imaging steps. In some embodiments, the entire ROI may be imaged in multiple imaging steps, one portion at a time in an imaging step (with or without any overlapping areas).

[0046] In some embodiments, the image acquired using the optical system described herein is a flow cell image. The flow cell image can include a FOV that covers at least a portion of the entire active area or ROI on the surface of the flow cell or another different sample support structure. The flow cell images can be registered to each other to cover the entire ROI on the surface of the flow cell.

[0047] 6. Light delivery components In some embodiments, the optical system comprises a light delivery component. In some embodiments, the light delivery component is a waveguide. In some embodiments, the light delivery component is a light pipe 4702, as shown in FIG. 47 . In some embodiments, the light delivery component is an optical fiber. In some embodiments, the light source delivers light to the flow cell via the light delivery component. In some embodiments, the light source delivers light to the flow cell via a light pipe. In some embodiments, the light delivery component is disposed between the light source 4901 and the flow cell 4905. In some embodiments, a second light delivery component is disposed between the flow cell and the image sensor.

[0048] 7. Imaging Channels The optical systems described herein can be configured to image one or more fluorophores. In some embodiments, the optical systems are configured to differentially image two, three, or more different fluorophores. In certain aspects, the optical systems comprise one or more imaging channels. In some embodiments, a first imaging channel of the one or more imaging channels is configured to image a first fluorophore of the one or more fluorophores. In some embodiments, a second imaging channel of the one or more imaging channels is configured to image a second fluorophore of the one or more fluorophores. In some embodiments, a third imaging channel of the one or more imaging channels is configured to image a third fluorophore of the one or more fluorophores. In some embodiments, the imaging channel comprises at least one of a light source 4901, a filter 4910, an image sensor 4912, or a combination thereof.

[0049] 8. Heater In some embodiments, systems and devices are described herein that include a heater. In some examples, the flow cell 4905 further includes a heater. In some embodiments, the heater is integrated with the flow cell. In some embodiments, the heater is integrated with the multi-sided imaging flow cell 4905. In some embodiments, the heater is integrated with the capillary flow cell 5201. In some embodiments, the integrated heater is a transparent heater block integrated heater. In some embodiments, the heater is an infrared (IR) heater. In some embodiments, the transparent heater conforms to the surface of the flow cell. In some embodiments, the transparent heater conforms to and completely surrounds a flow cell having a non-rectangular cross-section. In some embodiments, the transparent heater conforms to and completely surrounds a flow cell having a circular cross-section. In some embodiments, the transparent heater conforms to and completely surrounds the capillary flow cell 5201. In some embodiments, the transparent heater is transparent in all imaging channels of one or more imaging channels of the optical system.

[0050] 9. Aberration correction In some embodiments, aberration correction methods may be applied to enable imaging through air bubbles that may occur within the flow cell. In some embodiments, uneven flow cell surfaces allow for normal or off-axis illumination. In some embodiments, the optical systems described herein may include magnetic positioning of various elements. In some embodiments, the optical systems may be configured to image flow cells with rounded edges. Information regarding imaging of rounded edges or curved surfaces is described in International Patent Application No. PCT / US2022 / 037831, which is incorporated herein by reference in its entirety.

[0051] 10. Integrated Field Flattener The illumination area and / or FOV of existing fluorescence microscope imaging systems may be limited by the size of a single lens system and / or a single image sensor. The system's ability to systematically capture brightness across the FOV is sometimes referred to as the system's field uniformity. Non-uniformity in brightness and resolution across the FOV is observed, in some instances, from the center to the edge of the FOV. In some instances, illumination non-uniformity is caused by the non-uniform field curvature effect of the lens system, which are typically single-lens systems. Systems, devices, and methods designed to improve field uniformity may be referred to as field flatteners or field flattening, respectively. The optical systems described herein may include a field flattener. In some instances, the field flattener comprises multiple optical subsystems 5001 designed to overlap and cover the active area of ​​the flow cell surface. When the image of one individual optical subsystem 4914 of the multiple optical subsystems 5001 begins to become non-uniform (e.g., increased blur, loss of intensity at corners and edges), the optimal imaging volume 4915 of a second optical subsystem 4914 may overlap. In some examples, the optimal imaging volume 4915 of the first optical subsystem overlaps with the second optical subsystem and the third optical subsystem.

[0052] In some embodiments, the surface 5101 of the flow cell comprising the sample site 4902 is not flat, as shown in Figure 52. In certain aspects, each optical subsystem 4914 of the plurality of optical subsystems 5001 is positioned to match the contours of the active area of ​​the flow cell, as shown in Figure 52.

[0053] 11. Multivalent molecules The present disclosure provides multivalent molecules that function as reagents for labeling nucleotides in nucleic acid templates for nucleic acid sequencing reactions. In some embodiments, the multivalent molecule comprises a core attached to at least one nucleotide arm. In some embodiments, at least one nucleotide arm can comprise a core-binding moiety. In some embodiments, at least one nucleotide arm can comprise a spacer. In some embodiments, at least one nucleotide arm can comprise a linker. In some embodiments, at least one nucleotide arm can comprise a nucleotide unit. In some embodiments, at least one nucleotide arm can comprise a core-binding moiety, a spacer, a linker, and a nucleotide unit. In some embodiments, the core can comprise a bead, particle, or nanoparticle. In some embodiments, the core can comprise an alkyl, alkenyl, or alkynyl core, such as may be present in a branched polymer or dendrimer. In some embodiments, the core can comprise a moiety that mediates the attachment of the core to the nucleotide arm. In some embodiments, the core can be attached to multiple nucleotide arms. In some examples, the core can be attached to about 1 to about 50 nucleotide arms. In some examples, the core is attached to about 2 to about 20 nucleotide arms. In some examples, the core is attached to about 2 to about 4 nucleotide arms. In some examples, the core is attached to about 4 to about 10 nucleotide arms. In some examples, the core is attached to about 10 to about 15 nucleotide arms. In some examples, the core is attached to about 15 to about 20 nucleotide arms.

[0054] The present disclosure provides multivalent molecules comprising a core attached to at least one biotinylated nucleotide arm. In some embodiments, at least one biotinylated nucleotide arm can comprise a core-binding moiety. In some embodiments, at least one biotinylated nucleotide arm can comprise a spacer. In some embodiments, at least one biotinylated nucleotide arm can comprise a linker. In some embodiments, at least one biotinylated nucleotide arm can comprise a nucleotide unit. In some embodiments, at least one biotinylated nucleotide arm can comprise a core-binding moiety, a spacer, a linker, and a nucleotide unit. In some embodiments, the core can comprise a streptavidin- or avidin-type moiety, and the biotin unit of the biotinylated nucleotide arm can mediate the conjugation of the core to the biotinylated nucleotide arm. The streptavidin- or avidin-type core can be a tetrameric biotin-binding protein capable of binding one, two, three, or up to four biotinylated nucleotide arms.

[0055] In some embodiments, the core can comprise streptavidin or avidin proteins, as well as streptavidin- or avidin-type moieties, including any derivatives, analogs, and other non-natural forms of streptavidin or avidin that can bind to at least one biotin moiety. Streptavidin or avidin moieties can include natural or recombinant, as well as mutant and derivatized molecules. Streptavidin and avidin mutants can include any one or any combination of two or more amino acid insertions, deletions, substitutions, or truncations. Mutants can also include fusion polypeptides. Many different forms of streptavidin and avidin are commercially available.

[0056] Multivalent molecules can be configured to reduce dissociation of nucleotide arms from cores using streptavidin or avidin cores that have high affinity for the biotin moieties on biotinylated nucleotide arms. A mixture of multivalent molecules can be prepared, the mixture containing two or more subpopulations of multivalent molecules, each subpopulation containing multivalent molecules with one type of nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP). Multivalent molecules configured to have high affinity between the core and the nucleotide arms can reduce undesired dissociation of nucleotide arms from cores and exchange of nucleotide arms between different cores. Exchange of nucleotide arms during sequencing reactions can result in inaccurate base calling and reduced sequencing accuracy. In some embodiments, a multivalent molecule with improved stability (e.g., reduced dissociation of biotinylated nucleotide arms) can include dye-labeled streptavidin, where the streptavidin subunit has a Lys121Arg mutation that can exhibit reduced dissociation of the biotinylated nucleotide arms from the streptavidin core.

[0057] The streptavidin moiety can include full-length or truncated forms that have high affinity for binding to biotin. For example, the streptavidin moiety can be about 10 -14 mol / L, or approximately 10 -15 Dissociation constant (K in mol / L d) can exhibit increased stability. In some embodiments, the streptavidin moiety can include any amino acid substitution mutation at the site that can be labeled with a dye. For example, the dye-labeling site can include a lysine at position 121, which can overlap with the biotin-binding site. In some embodiments, a dye attached to streptavidin at Lys121 can block or inhibit biotin binding to the dye-labeled streptavidin. A multivalent molecule including a dye-labeled streptavidin with a lysine at position 121 can exhibit dissociation of the biotinylated nucleotide arm from the streptavidin core (e.g., SEQ ID NO: 1). A multivalent molecule with improved stability can include a dye-labeled streptavidin with a Lys121Arg mutation, which can exhibit decreased dissociation of the biotinylated nucleotide arm from the streptavidin core.

[0058] In some embodiments, the streptavidin moiety enhances binding affinity for biotin (e.g., K d About 10 -16 The biotin-binding fragment can include any amino acid substitution that improves retention of biotin at temperatures up to about 60°C, about 65°C, about 70°C, or about 80°C, or that has a combination of increased affinity for biotin binding and improved retention of biotin.

[0059] The avidin moiety can include full-length or truncated forms that have high affinity for binding to biotin. For example, the avidin moiety can be about 10 -14 mol / L, or approximately 10 -15 Dissociation constant (K in mol / L d) can be represented. In some embodiments, avidin can include substitutions of any one or any combination of eight arginine residues. Avidin can include partially deglycosylated and non-glycosylated forms. Avidin moieties can include derivatized forms, such as N-acylavidins, e.g., N-acetyl, N-phthalyl, and N-succinyl avidins, as well as commercially available products including EXTRAVIDIN, CAPTAVIDIN (in which the tyrosine residues of the four biotin-binding sites are selectively nitrated to produce an avidin that reversibly binds biotin), NEUTRAVIDIN (chemically deglycosylated and contains modified arginine residues), and NEUTRALITE AVIDIN (in which five of the eight arginine residues are substituted with neutral amino acids, two lysine residues are substituted with glutamic acid, and Asp17 is substituted with isoleucine, e.g., SEQ ID NO: 2). Amino acids having neutral nonpolar side chains include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and valine. Amino acids having neutral polar side chains include asparagine, cysteine, glutamine, serine, threonine, tryptophan, and tyrosine.

[0060] In some embodiments, the core can be labeled with a detectable reporter moiety. The core can be a homotetramer of streptavidin or avidin. Each subunit of the homotetramer can contain at least one lysine residue that can be conjugated to a fluorophore. The labeling reaction can use N-hydroxysuccinimide (NHS) ester-linked fluorophores. The maximum number of fluorophores that can be bound to a streptavidin or avidin subunit can be determined by the number of lysine residues in the subunit.

[0061] When preparing labeled streptavidin or avidin core, the labeling reaction can be optimized to achieve a predetermined degree of labeling (sometimes abbreviated as DoL). The degree of labeling can be expressed as a molar ratio in the form of label / protein. Dye-core conjugates with lower degrees of labeling exhibit weaker fluorescence intensity. Dye-core conjugates with very high degrees of labeling (e.g., DoL>6) may exhibit reduced fluorescence due to self-quenching from the conjugated fluorophore. In some embodiments, the predetermined degree of labeling of streptavidin or avidin core may depend on the dye. Fluorescent dyes include, but are not limited to, CF647, CF680, CF570, and CF532 dyes from Biotium, AF647, AF680, AF568, and AF532 from Thermo Fisher Scientific, IFluor647, IFluor680, IFlour568, and IFlour532 from AATBio, DY648P1, DY679P1, DY585, and DY530 from Dyomics, and AFDy647, IFlour680LT, AFDye568, and AFDye532 from Fluoroprobes. The predetermined degree of labeling can be about 1-10, or about 3-8, or about 3.5-7, or about 1.6-4.

[0062] Because red fluorophores are brighter (more intense) than green dyes, imaging both red- and green-labeled multivalent molecules on the same support (e.g., flow cell) can result in color bleeding. Increasing or decreasing the degree of labeling of subpopulations of multivalent molecules can improve signal balance from a mixture of labeled multivalent molecules. For example, the degree of labeling of a subpopulation of multivalent molecules labeled with a red fluorophore can be reduced compared to the degree of labeling of a subpopulation of multivalent molecules labeled with a green fluorophore. In some embodiments, the degree of labeling of a subpopulation of multivalent molecules labeled with a red fluorophore can be about 1-3, or about 2-3, or about 3-6. In some embodiments, the degree of labeling of a subpopulation of multivalent molecules labeled with a green fluorophore can be about 4-7.

[0063] Solution fluorescence measurements can be used to determine the relative brightness of labeled streptavidin or avidin cores. Alternatively, the degree of labeling can be determined by contacting clonally amplified template molecules immobilized on a flow cell with primers, polymerase, and fluorescently labeled multivalent molecules under conditions suitable for binding the multivalent molecules to the complex polymerase without incorporating nucleotide units into the primers, and using a functional assay (e.g., a flow cell trap assay) in which signal intensity can be detected.

[0064] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise the same type of nucleotide unit. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, all of which have a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0065] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise different types of nucleotide units. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and a second attached arm can have a second nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, where the first nucleotide unit and the second nucleotide unit are different.

[0066] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms, where one nucleotide arm of the multiple nucleotide arms comprises a spacer. In some embodiments, the multiple nucleotide arms can comprise the same type of spacer. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have the same spacer.

[0067] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide arms, wherein one nucleotide arm of the plurality of nucleotide arms comprises a spacer. In some embodiments, the plurality of nucleotide arms can comprise spacers of different types. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein at least a first attached arm can have a first type of spacer and a second attached arm can have a second type of spacer, wherein the first spacer unit and the second spacer unit are different.

[0068] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms, where one nucleotide arm of the multiple nucleotide arms comprises a linker. In some embodiments, the multiple nucleotide arms can comprise the same type of linker. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have the same linker. In some embodiments, the first type of linker and the second type of linker can be selected from any of the spacers described herein.

[0069] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms, wherein one nucleotide arm of the multiple nucleotide arms comprises a linker. In some embodiments, the multiple nucleotide arms can comprise different types of linkers. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, wherein at least a first attached arm can have a first type of linker and a second attached arm can have a second type of linker, wherein the first linker unit and the second linker unit are different. In some embodiments, the first type of linker and the second type of linker can be selected from any of the linkers described herein. In some embodiments, the first type of linker and the second type of linker can be selected from any of the spacers described herein.

[0070] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms, where one nucleotide arm of the multiple nucleotide arms comprises a linker and a spacer. In some embodiments, the multiple nucleotide arms can comprise the same type of spacer and linker. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have the same spacer and linker. In some embodiments, the spacer and linker can be selected from any of the spacers and linkers described herein.

[0071] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms, where one of the multiple nucleotide arms comprises a reactive group. In some embodiments, the multiple nucleotide arms can comprise the same type of reactive group. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have the same reactive group. In some embodiments, the reactive group can comprise an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0072] In some embodiments, the reactive group is present in a linker. In some embodiments, the reactive group in the linker can react with a chemical reagent. For example, reactive groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Reactive groups such as aryl and benzyl can react with HPd / C. Reactive groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). Reactive groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). Reactive groups such as urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0073] In some embodiments, the nucleotide arms can have the same type of reactive group in the linker, and the reactive group can include an azide, azido, or azidomethyl group. In some embodiments, the azide, azido, or azidomethyl group in the linker can react with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0074] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide arms, wherein one of the plurality of nucleotide arms can comprise a linker comprising a reactive group. In some embodiments, the plurality of nucleotide arms can comprise different types of reactive groups in the linker. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein at least a first attached arm can have a first type of reactive group in a first linker unit and a second attached arm can have a second type of reactive group in a second linker unit, wherein the first reactive group and the second reactive group are different.

[0075] In some embodiments, the first reactive group in the first linker unit and the second reactive group in the second linker unit can be selected from the group consisting of alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, keto groups, isocyanate groups, phosphate groups, thio groups, disulfide groups, carbonate groups, urea groups, and silyl groups, in any combination.

[0076] In some embodiments, the first reactive group and the second reactive group can react with chemical agents. For example, reactive groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Reactive groups such as aryl and benzyl can react with HPd / C. Reactive groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). Reactive groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). Reactive groups such as urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0077] In some embodiments, the nucleotide arms can have different types of reactive groups in the linker, and the reactive groups can include azide, azido, or azidomethyl groups. In some embodiments, the azide, azido, or azidomethyl groups in the linker can react with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0078] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise nucleotide units having the same type of sugar 3'OH group. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, all of which have nucleotide units having the same type of sugar 3'OH group.

[0079] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise nucleotide units having the same type of sugar 3' blocking group (e.g., chain-terminating moiety). For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, all of which can have nucleotide units with the same type of sugar 3' blocking group. In some embodiments, the sugar 3' blocking group can comprise an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the sugar 3' blocking group can comprise a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the sugar 3' blocking group can comprise an azido, azido, or azidomethyl group.

[0080] In some embodiments, sugar 3' blocking groups can be reacted with chemical reagents. For example, sugar 3' blocking groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Sugar 3' blocking groups such as aryl and benzyl can react with HPd / C. Sugar 3' blocking groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). Sugar 3' blocking groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). Sugar 3' blocking groups such as urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0081] In some embodiments, sugar 3' blocking groups (e.g., azide, azido, and azidomethyl) can be reacted with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0082] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise nucleotide units with different sugar 3' blocking groups. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first nucleotide unit with a first 3' blocking group and a second attached arm can have a second nucleotide unit with a second 3' blocking group, where the first and second 3' blocking groups are different.

[0083] In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl groups, in any combination. In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, or 3'-O-benzyl groups, in any combination. In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of azide, azido, or azidomethyl groups, in any combination.

[0084] In some embodiments, the first 3' blocking group and the second 3' blocking group can react with chemical agents. For example, 3' blocking groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). 3' blocking groups such as aryl and benzyl can react with HPd / C. 3' blocking groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). 3' blocking groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). 3' blocking groups such as urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0085] In some embodiments, the first 3' blocking group and the second 3' blocking group (e.g., azide, azido, and azidomethyl) can be reacted with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0086] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise nucleotide units having a first sugar 3'OH blocking group. In some embodiments, the multiple nucleotide arms can comprise nucleotide units having a second 3'OH blocking group. In some examples, the first 3'OH blocking group and the second 3'OH blocking group can be different. For example, the multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where (a) at least a first arm can comprise a first nucleotide unit having a sugar moiety that includes a 3'OH group, (b) at least a second arm can comprise a second nucleotide unit having a first 3'OH blocking group, and (c) at least a third arm can comprise a third nucleotide unit having a second blocking group, wherein the first 3' blocking group and the second 3' blocking group are different from each other.

[0087] In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl groups, in any combination. In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, or 3'-O-benzyl groups, in any combination. In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of azide, azido, or azidomethyl groups, in any combination.

[0088] In some embodiments, the first 3' blocking group and the second 3' blocking group can react with chemical agents. For example, 3' blocking groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). 3' blocking groups such as aryl and benzyl can react with HPd / C. 3' blocking groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). 3' blocking groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). 3' blocking groups such as urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0089] In some embodiments, the first 3' blocking group and the second 3' blocking group (e.g., azide, azido, and azidomethyl) can be reacted with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0090] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can have a core. In some embodiments, the core can be labeled with at least one detectable reporter moiety to form a labeled core. In some embodiments, a labeled core attached to two or more nucleotide arms can comprise a labeled multivalent molecule. In some embodiments, a streptavidin or avidin core can be labeled with one to six or more reporter moieties. In some embodiments, the reporter moiety can comprise a fluorophore.

[0091] A mixture of multivalent molecules having different units in the nucleotide arms can be used to achieve differentiation between different multivalent molecules. In some embodiments, the core of a first multivalent molecule can be labeled with a reporter moiety to distinguish it from a second, labeled (or unlabeled) multivalent molecule. For example, the units in the nucleotide arms of a labeled first multivalent molecule can be different from the units in the nucleotide arms of a labeled second multivalent molecule. Any unit in the first multivalent molecule (e.g., a spacer, linker, reactive group, nucleotide base, sugar 3'OH, 3' blocking group, or a combination thereof) can be different from the corresponding unit in the second multivalent molecule, and the first and second reporter moieties correspond to differentiation units. In some embodiments, the first and second reporter moieties can be spectrally distinguishable from each other.

[0092] In some embodiments, the core of a first multivalent molecule can be labeled with a first reporter moiety corresponding to a base in the attached nucleotide arm (e.g., dATP, dGTP, dCTP, dTTP, or dUTP), and the core of a second multivalent molecule can be labeled with a second reporter moiety corresponding to a base in the attached nucleotide arm (e.g., dATP, dGTP, dCTP, dTTP, or dUTP), where the base in the first multivalent molecule is different from the base in the second multivalent molecule. In some embodiments, the first reporter moiety and the second reporter moiety are spectrally distinguishable from each other. In some embodiments, detection of the first reporter moiety indicates a binding event, incorporation event, or a combination of a binding event and an incorporation event of the first multivalent molecule having the first base, and detection of the second reporter moiety indicates a binding event, incorporation event, or a combination of a binding event and an incorporation event of the second multivalent molecule having the second base. The binding event can be binding of the multivalent molecule to a multiplex polymerase. The incorporation event may be the incorporation of a nucleotide unit onto the 3' end of an extendible primer in a multiplex polymerase, where the nucleotide unit is part of a multivalent molecule.

[0093] 12. Mixtures of multivalent molecules The present disclosure provides separate batches (subpopulations) of labeled multivalent molecules. In some embodiments, separate batches of labeled multivalent molecules can be prepared with different reporter moieties for each batch. In some embodiments, the different reporter moieties can correspond to specific bases within the nucleotide arms. A particular batch can be distinguished from other batches based on the reporter moieties attached to the core. Two, three, four, five, or more separate batches (subpopulations) can be mixed together to form a plurality of labeled multivalent molecules comprising two or more subpopulations of spectrally distinguishable multivalent molecules. In some embodiments, at least one batch of multivalent molecules in the mixture can be unlabeled (e.g., dark multivalent molecules).

[0094] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, which can include a mixture of at least two subpopulations of multivalent molecules labeled with different reporter moieties. In some embodiments, at least a first subpopulation of multivalent molecules can be labeled with a first reporter moiety corresponding to a first nucleotide unit on a nucleotide arm. In some embodiments, at least a second subpopulation of multivalent molecules can be labeled with a second reporter moiety corresponding to a second nucleotide unit on a nucleotide arm. In some examples, the first reporter moiety and the second reporter moiety can be different from each other. In some embodiments, the plurality of multivalent molecules can further include at least a third subpopulation of multivalent molecules labeled with a third reporter moiety, where the first reporter moiety, the second reporter moiety, and the third reporter moiety can be different from each other. In some embodiments, the plurality of multivalent molecules can further include at least a fourth subpopulation of multivalent molecules labeled with a fourth reporter moiety, where the first reporter moiety, the second reporter moiety, the third reporter moiety, and the fourth reporter moiety can be different from one another. In some embodiments, additional subpopulations of labeled multivalent molecules (e.g., a fifth, sixth, seventh, eighth, ninth, tenth, or more) can be added to the mixture. In some embodiments, the reporter moiety can be a fluorophore. In some embodiments, the multivalent molecules of the first subpopulation can be labeled with a first fluorophore, and the second fluorophore of the multivalent molecules can be labeled with a second fluorophore. In some examples, the first fluorophore and the second fluorophore can be different.

[0095] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, which can include a mixture of at least two subpopulations of multivalent molecules labeled with different reporter moieties. In some embodiments, at least a first subpopulation of multivalent molecules can be labeled with a first reporter moiety corresponding to a first nucleotide unit on a nucleotide arm. In some embodiments, at least a second subpopulation of multivalent molecules can be unlabeled (e.g., dark multivalent molecules).

[0096] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, including a mixture of at least three subpopulations of multivalent molecules labeled with different reporter moieties. In some embodiments, at least a first subpopulation of multivalent molecules can be labeled with a first reporter moiety corresponding to a first nucleotide unit on a nucleotide arm. In some embodiments, at least a second subpopulation of multivalent molecules can be labeled with a second reporter moiety corresponding to a second nucleotide unit on a nucleotide arm. In some embodiments, at least a third subpopulation of multivalent molecules can be unlabeled (e.g., dark multivalent molecules). In some embodiments, the first and second reporter moieties can be different from each other.

[0097] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, including a mixture of at least four subpopulations of multivalent molecules labeled with different reporter moieties. In some embodiments, the mixture of multivalent molecules can have at least a first subpopulation of multivalent molecules that can be labeled with a first reporter moiety corresponding to a first nucleotide unit on a nucleotide arm. In some embodiments, the mixture of multivalent molecules can have at least a second subpopulation of multivalent molecules that can be labeled with a second reporter moiety corresponding to a second nucleotide unit on a nucleotide arm. In some embodiments, the mixture of multivalent molecules can have at least a third subpopulation of multivalent molecules that are labeled with a third reporter moiety. In some embodiments, the mixture of multivalent molecules can have at least a fourth subpopulation of multivalent molecules that can be unlabeled (e.g., dark multivalent molecules). In some examples, the first reporter moiety, second reporter moiety, and third reporter moiety can be different from one another.

[0098] Embodiments include mixtures of four different types of multivalent molecules, including (1) a first subpopulation of multivalent molecules, each comprising a dATP nucleotide unit and a core labeled with a first type of fluorophore, (2) a second subpopulation of multivalent molecules, each comprising a dGTP nucleotide unit and a core labeled with a second type of fluorophore, (3) a third subpopulation of multivalent molecules, each comprising a dCTP nucleotide unit and a core labeled with a third type of fluorophore, and (4) a fourth subpopulation of multivalent molecules, each comprising a dTTP nucleotide unit and a core labeled with a fourth type of fluorophore, wherein the first fluorophore, second fluorophore, third fluorophore, and fourth fluorophore are spectrally distinguishable. In some embodiments, any one of the subpopulations of multivalent molecules can be unlabeled for use as a "dark" multivalent molecule.

[0099] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, wherein each multivalent molecule in the plurality can comprise a core attached to at least one nucleotide arm. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five nucleotide arms. In some embodiments, each multivalent molecule in the plurality can comprise a streptavidin or avidin core attached to two to five biotinylated nucleotide arms.

[0100] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, wherein each multivalent molecule in the plurality can comprise a core attached to at least one nucleotide arm having one type of nucleotide unit, including dATP, dGTP, dCTP, dTTP, or dUTP. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five nucleotide arms, wherein the nucleotide arms have one type of nucleotide unit, including dATP, dGTP, dCTP, dTTP, or dUTP. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five biotinylated nucleotide arms, wherein the biotinylated nucleotide arms have one type of nucleotide unit, including dATP, dGTP, dCTP, dTTP, or dUTP.

[0101] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, including a mixture (subpopulation) of two or more different types of multivalent molecules. In some embodiments, the plurality of multivalent molecules can have at least a first multivalent molecule among the plurality of multivalent molecules. In some examples, at least the first multivalent molecule can comprise a core attached to at least one nucleotide arm having a first type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, or dUTP. In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule. In some embodiments, the plurality of multivalent molecules can comprise at least the first multivalent molecule and at least a second multivalent molecule among the plurality of multivalent molecules. In some examples, at least the second multivalent molecule can comprise a core attached to at least one nucleotide arm having a second type of nucleotide different from the first nucleotide in the first multivalent molecule. In some embodiments, a first multivalent molecule can include a core attached to two to five biotinylated nucleotide arms, where the biotinylated arms can have a first type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, or dUTP. In some embodiments, a second multivalent molecule can include a core attached to two to five biotinylated nucleotide arms, where the biotinylated arms can have a second type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, or dUTP, where the first type of nucleotide and the second type of nucleotide are different. In some embodiments, a mixture can include two, three, four, five, or more different types of multivalent molecules with nucleotides selected in any combination from the group consisting of dATP, dGTP, dCTP, dTTP, or dUTP.

[0102] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, wherein each multivalent molecule in the plurality can comprise a core attached to at least one nucleotide arm. In some embodiments, at least one nucleotide arm attached to a core can have the same spacer. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five nucleotide arms. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five biotinylated nucleotide arms.

[0103] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, wherein each multivalent molecule in the plurality can comprise a core attached to at least one nucleotide arm. In some embodiments, at least one nucleotide arm attached to a core can have the same linker. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five nucleotide arms. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five biotinylated nucleotide arms.

[0104] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, wherein each multivalent molecule in the plurality can comprise a core attached to at least one nucleotide arm. In some embodiments, all of the nucleotide arms attached to the core can have the same spacer and linker. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five nucleotide arms. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five biotinylated nucleotide arms.

[0105] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, including a mixture (subpopulation) of two or more different types of multivalent molecules. In some embodiments, the plurality of multivalent molecules can comprise at least a first multivalent molecule of the plurality of multivalent molecules, which can comprise a core attached to at least one nucleotide arm having a spacer of a first type. In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule, which can comprise a core attached to at least one nucleotide arm having a spacer of a second type. In some embodiments, the plurality of multivalent molecules can comprise a mixture of at least the first multivalent molecule and at least the second multivalent molecule. In some examples, the second type of spacer in the second multivalent molecule can be different from the first spacer in the first multivalent molecule. In some embodiments, the first multivalent molecule can comprise a core attached to two to five biotinylated nucleotide arms, and the biotinylated arms can have a spacer of a first type. In some embodiments, the second multivalent molecule can include a core bound to two to five biotinylated nucleotide arms, and the biotinylated arms can have a second type of spacer, where the first type of spacer and the second type of spacer are different.

[0106] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, including a mixture (subpopulation) of two or more different types of multivalent molecules. In some embodiments, the plurality of multivalent molecules can comprise at least a first multivalent molecule of the plurality of multivalent molecules comprising a core attached to at least one nucleotide arm having a first type of linker. In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule comprising a core attached to at least one nucleotide arm having a second type of linker. In some embodiments, the plurality of multivalent molecules can comprise a mixture of at least the first multivalent molecule and at least the second multivalent molecule. In some examples, the second type of linker in the second multivalent molecule can be different from the first linker in the first multivalent molecule. In some embodiments, the first multivalent molecule can comprise a core attached to two to five biotinylated nucleotide arms, and the biotinylated arms can have a first type of linker. In some embodiments, the second multivalent molecule can include a core attached to two to five biotinylated nucleotide arms, and the biotinylated arms can have a second type of linker, and the first type of spacer and the second type of spacer are different.

[0107] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, wherein each multivalent molecule in the plurality can comprise a core attached to at least one nucleotide arm. In some embodiments, all of the nucleotide arms attached to the core can have the same reactive group in the linker. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five nucleotide arms. In some embodiments, each multivalent molecule can comprise a core attached to two to five biotinylated nucleotide arms. In some embodiments, the reactive group can comprise an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea group, or silyl group. In some embodiments, each multivalent molecule can comprise a reactive group capable of reacting with a chemical agent. For example, reactive groups such as alkyl, alkenyl, alkynyl, and aryl groups can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Reactive groups such as aryl and benzyl can react with HPd / C. Reactive groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups containing beta-mercaptoethanol or dithiothritol (DTT). Reactive groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). Reactive groups such as urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the reactive group can comprise an azide, azido, or azidomethyl group, hi some embodiments, the azide, azido, or azidomethyl group in the linker can react with a chemical agent.In some embodiments, the chemical agent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0108] The present disclosure provides compositions, systems, and kits comprising a plurality of multivalent molecules, including a mixture (subpopulation) of two or more different types of multivalent molecules. In some embodiments, the plurality of multivalent molecules can have at least a first multivalent molecule (first subpopulation) in the plurality of multivalent molecules. In some embodiments, at least the first subpopulation can include a core attached to at least one nucleotide arm having a first type of reactive group in the linker. In some embodiments, the plurality of multivalent molecules can have at least a second multivalent molecule (second subpopulation) including a core attached to at least one nucleotide arm having a second type of reactive group in the linker. In some examples, the first reactive group in the first type of linker in the first subpopulation is different from the second reactive group in the second type of linker in the second subpopulation. In some embodiments, the first multivalent molecule can include a core attached to two to five biotinylated nucleotide arms, and the biotinylated arms can have a first type of reactive group in the linker. In some embodiments, the second multivalent molecule can include a core attached to two to five biotinylated nucleotide arms, and the biotinylated arms can have a second type of reactive group in a linker, where the first reactive group is different from the second reactive group.

[0109] In some embodiments, the first reactive group and the second reactive group can be selected from the group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, and silyl groups, in any combination. In some embodiments, each polyvalent molecule can include a first reactive group or a second reactive group capable of reacting with a chemical agent. For example, reactive groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Reactive groups such as aryl and benzyl can react with HPd / C. Reactive groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). Reactive groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). Reactive groups such as urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the first reactive group or the second reactive group can be selected from the group consisting of azide, azido, or azidomethyl groups, in any combination. In some embodiments, the azide, azido, or azidomethyl reactive groups in the linker can react with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0110] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, wherein each multivalent molecule in the plurality can comprise a core attached to at least one nucleotide arm, and all of the nucleotide arms attached to the core can have a nucleotide unit having the same sugar 3'-OH group. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five nucleotide arms. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to two to five biotinylated nucleotide arms.

[0111] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, each of which can comprise a core attached to at least one nucleotide arm, and all of the nucleotide arms attached to the core can have nucleotide units with sugar 3'-OH groups substituted with the same 3'-blocking group. In some embodiments, each of the multivalent molecules can comprise a core attached to two to five nucleotide arms. In some embodiments, each of the multivalent molecules can comprise a core attached to two to five biotinylated nucleotide arms. In some embodiments, the sugar 3'-blocking group can comprise an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl group. In some embodiments, each of the multivalent molecules can comprise a 3'-blocking group capable of reacting with a chemical agent. For example, 3'-blocking groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). 3'-blocking groups such as aryl and benzyl can react with HPd / C. 3'-blocking groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). 3'-blocking groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). 3' blocking groups such as urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.In some embodiments, the 3'-blocking group can comprise a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the 3'-blocking group can comprise an azide, azido, or azidomethyl group. In some embodiments, the azide, azido, or azidomethyl 3'-blocking group can react with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can comprise tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0112] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, including a mixture (subpopulation) of two or more different types of multivalent molecules. In some embodiments, the plurality of multivalent molecules can comprise at least a first multivalent molecule of the plurality of multivalent molecules, which can comprise a core attached to at least one nucleotide arm having a first nucleotide unit with a first type of sugar 3'OH blocking group (chain-terminating moiety). In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule, which comprises a core attached to at least one nucleotide arm having a second nucleotide unit with a second type of sugar 3' blocking group (chain-terminating site). In some embodiments, the plurality of multivalent molecules can comprise a first multivalent molecule and a second multivalent molecule. In some examples, the first 3' blocking group can be different from the second 3' blocking group. In some embodiments, the first multivalent molecule can comprise a core attached to two to five biotinylated nucleotide arms, and the biotinylated arms can have a first type of 3' blocking group. In some embodiments, the second multivalent molecule can include a core bound to two to five biotinylated nucleotide arms, and the biotinylated arms can have a second type of 3' blocking group, wherein the first 3' blocking group is different from the second 3' blocking group.

[0113] In some embodiments, the first 3' blocking group and the second 3' blocking group can be selected from the group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, and silyl groups, in any combination. In some embodiments, each polyvalent molecule can include a first 3' blocking group or a second 3' blocking group that can react with a chemical agent. For example, 3' blocking groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). 3' blocking groups such as aryl and benzyl can react with HPd / C. 3'-blocking groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). 3'-blocking groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). 3'-blocking groups such as urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the first and second 3'-blocking groups can be selected from the group consisting of 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, and 3'-O-benzyl groups, in any combination. In some embodiments, the first 3' blocking group or the second 3' blocking group can be selected from the group consisting of azide, azido, or azidomethyl groups, in any combination. In some embodiments, the azide, azido, or azidomethyl 3' blocking group reacts with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound.In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety, hi some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0114] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, including a mixture (subpopulation) of two or more different types of multivalent molecules. In some embodiments, the plurality of multivalent molecules can comprise at least a first multivalent molecule of the plurality of multivalent molecules, which can comprise a core attached to at least one nucleotide arm having a first nucleotide unit with a sugar 3' OH group. In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule, which comprises a core attached to at least one nucleotide arm having a second nucleotide unit with a first type of sugar 3' blocking group. In some embodiments, the plurality of multivalent molecules can comprise a first multivalent molecule and a second multivalent molecule. In some embodiments, the first multivalent molecule can comprise a core attached to two to five biotinylated nucleotide arms, and the biotinylated arms can have sugar 3' OH groups. In some embodiments, the second multivalent molecule can comprise a core attached to two to five biotinylated nucleotide arms, and the biotinylated arms can have a first type of 3' blocking group.

[0115] In some embodiments, the first 3' blocking group can be selected from the group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, and silyl groups, in any combination. In some embodiments, each polyvalent molecule can include a first 3' blocking group capable of reacting with a chemical agent. For example, 3' blocking groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). 3' blocking groups such as aryl and benzyl can react with HPd / C. 3'-blocking groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). 3'-blocking groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). 3'-blocking groups such as urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the first 3'-blocking group can be selected from the group consisting of a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, and a 3'-O-benzyl group, in any combination. In some embodiments, the first 3'-blocking group can be selected from the group consisting of an azide, azido, or azidomethyl group, in any combination. In some embodiments, the azide, azido, or azidomethyl 3' blocking group can be reacted with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety.In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0116] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, including a mixture (subpopulation) of three or more different types of multivalent molecules. In some embodiments, the plurality of multivalent molecules can comprise at least a first multivalent molecule. In some embodiments, the at least the first multivalent molecule can comprise a core attached to at least one nucleotide arm having a first nucleotide unit with a sugar 3' OH group. In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule. In some embodiments, the at least the second multivalent molecule can comprise a core attached to at least one nucleotide arm having a second nucleotide unit with a first type of sugar 3' blocking group. In some embodiments, the plurality of multivalent molecules can comprise at least a third multivalent molecule. In some embodiments, the at least the third multivalent molecule can comprise a core attached to at least one nucleotide arm having a third nucleotide unit with a second type of sugar 3' blocking group. In some examples, the first 3' blocking group and the second 3' blocking group are different. In some embodiments, a first multivalent molecule can include a core bound to two to five biotinylated nucleotide arms, where the biotinylated arms can have a sugar 3' OH group. In some embodiments, a second multivalent molecule can include a core bound to two to five biotinylated nucleotide arms, where the biotinylated arms can have a first type of 3' blocking group. In some embodiments, a third multivalent molecule can include a core bound to two to five biotinylated nucleotide arms, where the biotinylated arms can have a second type of 3' blocking group.

[0117] In some embodiments, the first 3' blocking group and the second 3' blocking group can be selected from the group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, and silyl groups, in any combination. In some embodiments, each polyvalent molecule can include a first 3' blocking group or a second 3' blocking group that can react with a chemical agent. For example, 3' blocking groups such as alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). 3' blocking groups such as aryl and benzyl can react with HPd / C. 3'-blocking groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can react with phosphines or thiol groups, including beta-mercaptoethanol or dithiothritol (DTT). 3'-blocking groups such as carbonate can react with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). 3'-blocking groups such as urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the first and second 3'-blocking groups can be selected from the group consisting of 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, and 3'-O-benzyl groups, in any combination. In some embodiments, the first 3' blocking group and the second 3' blocking group can be selected from the group consisting of azide, azido, or azidomethyl groups, in any combination. In some embodiments, the azide, azido, or azidomethyl 3' blocking group can be reacted with a chemical agent. In some embodiments, the chemical agent can include a phosphine compound.In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety, hi some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0118] 13.Light source In some embodiments, the light source comprises a solid-state laser, a dye laser, a diode laser, an arc lamp, or a tungsten-halogen lamp. Any of a variety of light sources may be used to provide imaging or excitation light, including, but not limited to, a tungsten lamp, a tungsten-halogen lamp, an arc lamp, a laser, a light-emitting diode (LED), or a laser diode. In some examples, the combination of one or more light sources and additional optical components, such as lenses, filters, apertures, diaphragms, mirrors, etc., may be configured as an illumination system (or subsystem). In some embodiments, the light source 4901 shown in FIGS. 49A-53B is a solid-state light source. In some embodiments, the solid-state light source is a light-emitting diode (LED). In some embodiments, the light source 4901 is configured to emit multiple wavelengths. In some embodiments, the light source includes multiple light sources. In some embodiments, each of the multiple light sources is configured to emit light of a different wavelength. In some embodiments, the light source 4901 is configured to emit light of a first wavelength at a first time, light of a second wavelength at a second time, and light of a third wavelength at a third time. In some embodiments, light of a first wavelength is emitted at a first time, light of a second wavelength at a second time, and light of a third wavelength at a third time, sequentially. In some embodiments, the multiple light sources are configured to deliver successive colors for a timed pulse sequence. In some embodiments, the multiple optical subsystems 5001 are configured to increase the speed of detection. In some embodiments, the solid-state light source is not a laser. In some applications, the light source comprises a filter that narrows the spectrum of light emitted from the light source. In some embodiments, the light source is referred to as an excitation source. In some embodiments, the light emitted from the light source is referred to as excitation light. In some examples, a light source that produces light in the wavelength ranges of the present disclosure may be used.

[0119] 14. Fluorescence Imaging Viewed as an Information Pipeline A useful abstraction of the role that fluorescence imaging systems play in typical genomic assay technologies (including nucleic acid sequencing applications) is that of an information pipeline, where photon signals enter one end of the pipeline, e.g., the objective lens used for imaging, and location-specific information about the fluorescent signal emerges at the other end, e.g., the image sensor. As more information is pumped down this pipeline, some content inevitably becomes lost during this transfer process and cannot be recovered. An example of this is when there are too many labeled molecules (or clonally amplified molecular clusters) within a small area of ​​the substrate surface to be clearly resolved in the image, making it difficult for the image sensor to distinguish between photon signals originating from neighboring molecular clusters, thus increasing the likelihood of attributing signals to the wrong cluster and leading to detection errors. In some instances, the clusters are polonies.

[0120] 15. Optical Imaging Module Design Therefore, the goal in designing the optical imaging module is to maximize the flow of information through this detection pipeline and minimize detection errors. The inventors of this disclosure considered several design factors, including the following:

[0121] 1) Match the physical feature density on the substrate surface being imaged with the overall image quality of the optical imaging system and the pixel sampling frequency of the image sensor being used. Mismatches between these parameters can lead to loss of information, or in some cases, the generation of erroneous information; for example, spatial aliasing can occur if the pixel sampling frequency is lower than twice the optical resolution limit.

[0122] 2) Match the size of the imaged area to the overall image quality of the optical imaging system and the focus quality across the field of view.

[0123] 3) Match the optical system design light collection efficiency, modulation transfer function, and image sensor performance characteristics to the input excitation photon flux and expected fluorescence photon flux and dye efficiency (related to the dye extinction coefficient and fluorescence quantum yield), taking into account background signal and system noise characteristics.

[0124] 4) Maximize the separation of spectral components to reduce crosstalk between fluorescence imaging channels.

[0125] 5) Effective synchronization of image acquisition steps while repositioning the sample or optics between image captures of different fields of view minimizes downtime (or maximizes the duty cycle) of the imaging system, and therefore maximizes the overall throughput of the image capture process.

[0126] This disclosure addresses each of the design elements outlined above and describes a systematic method for creating component-level specifications for imaging systems.

[0127] 16. Improved optical resolution and image quality to enhance or maximize information transfer and throughput One non-limiting design practice is to start with the optical resolution required to distinguish two adjacent features, specified in number of line pairs per mm (lp / mm), X, and convert it to a corresponding numerical aperture (NA) requirement, which can then be used to evaluate the resulting effect on the modulation transfer function and image contrast.

[0128] The standard modulation transfer function (MTF) describes the spatial frequency response of image contrast (modulation) transmitted through an optical system, where image contrast decreases as a function of spatial frequency and increases with increasing NA. This function limits the contrast / modulation achievable for a given NA. Furthermore, wavefront errors can adversely affect MTF, and therefore it is desirable to improve or optimize optical system designs using the true system MTF instead of that predicted by diffraction-limited optics. Note that as used herein, MTF refers to the MTF of the entire system (including the complete optical path from the cover glass to the image sensor), although design practice may primarily consider the MTF of the objective lens.

[0129] In genomics applications, when the target to be imaged is a dense array of "spots" (either randomly distributed or patterned) on a surface, downstream analysis can determine the minimum modulation transfer value required to resolve two adjacent spots and distinguish between four possible states (e.g., on-off, on-on, off-on, and off-off). For example, assume the spots are small enough to approximate point sources. Suppose the detection task is to determine whether two adjacent spots separated by a distance d are on or off (in other words, bright or dark), and define the contrast-to-noise ratio (CNR) of the fluorescence signal arising from the spots at the sample plane (or object plane) as C. サンプル Then, under ideal conditions, the CNR of the readout signals of two adjacent spots on the image sensor plane, i.e., C 画像 is C 画像 =C サンプル *It can be roughly approximated as MTF(1 / d), which is the MTF value at spatial frequency = (1 / d).

[0130] In a typical design, C 画像 Since the value of can be at least 4, a simple threshold method can be used to avoid misclassification of the fluorescent signal. Assuming that the fluorescent signal intensity is Gaussian distributed around the mean value, C 画像>4, the expected error in correctly classifying a fluorescent signal (e.g., on or off) is less than <0.035%. The use of high CNR sequencing and surface chemistry is described in U.S. Patent Nos. 10,876,148 and 10,704,094, each of which is incorporated by reference in its entirety. In some embodiments, high CNR sequencing and surface chemistry provides sample surface CNRs (CNRs) of greater than 12 (or even higher) for clusters of clonally amplified labeled oligonucleotide molecules tethered to a substrate surface when measured in a sparse field of view (e.g., where the surface density of clusters or spots is low) with MTF values ​​approaching 100%. サンプル ) value can be achieved. サンプル >12, and the classification error rate is <0.1% (hence, C 画像 >4), in some implementations, a minimum value for M(1 / d) can be determined as M(1 / d) = 4 / 12 ~ 33%. Therefore, a modulation transfer function threshold of at least 33% may be used to preserve the information content of the transferred image.

[0131] In design practice, the minimum separation distance d between two features or spots can be related to the optical resolution requirement (specified in X (lp / mm) as discussed above) as d = (1 mm) / X, e.g., d is the minimum separation distance between two features or spots that can be fully resolved by the optical system. In some designs disclosed herein, if the goal of the design analysis is to increase or maximize relative information transfer, 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 the minimum resolvable spot separation distance d at the sample plane is reduced, thereby enabling the use of higher feature densities.

[0132] In design practice, the Nyquist criterion is used to determine the minimum spatial sampling frequency at the sample plane, where the spatial sampling frequency S is greater than or equal to 2*X, where X is the optical resolution of the imaging system, specified in terms of X lp / mm as previously described. If the spatial sampling frequency of the system is close to the Nyquist criterion (as is often the case), then an imaging system resolution higher than S will result in aliasing because the high frequency information resolved by the optical system cannot be adequately sampled by the image sensor.

[0133] In some of the designs disclosed herein, an oversampling scheme based on the relationship S=B*Y (where B≧2 and Y is the MTF limit of the true optical system) can be used to further improve the information transfer capability of the imaging system. As previously mentioned, X (lp / mm) corresponds to the smallest practical non-zero (>33%) modulation transfer value, but Y (lp / mm) is the optical resolution limit, so the modulation at Y (lp / mm) is zero. Thus, in the disclosed designs, Y (lp / mm) may advantageously not be significantly greater than X. For values ​​of B≧2, the disclosed designs are oversampled with respect to the sample object frequency X, e.g., S≧B*Y>2*X.

[0134] The above relationship can be used to determine the system magnification and provide an upper limit for the image sensor pixel size. The choice of image sensor pixel size is matched to the optical quality of the system, as well as the spatial sampling frequency required to reduce aliasing. A lower limit for the image sensor pixel size can be determined based on photon throughput, as the relative noise contribution increases with smaller pixels.

[0135] However, other design approaches are possible. For example, reducing the NA below 0.6 (e.g., 0.5 or less) can result in an increased depth of field. Such an increased depth of field can enable multi-plane imaging, in which two surfaces at different depths can be imaged simultaneously, with or without refocusing. As discussed above, reducing the NA can reduce optical resolution. In some implementations, the use of higher excitation beam power, e.g., 1 watt or greater, can be used to generate a strong signal. Intrinsically high-contrast samples (e.g., including sample surfaces exhibiting strong foreground signals and dramatically reduced background signals) can also be used to facilitate the acquisition of high-contrast-to-noise ratio (CNR) images, e.g., CNR values ​​greater than 20, thereby improving signal discrimination for base calling, such as in nucleic acid sequencing applications. In some optical system designs disclosed herein, a sample support structure, such as a flow cell with a hydrophilic surface, is used to reduce background noise.

[0136] In various implementations, a wide field of view (FOV) is provided by the disclosed optical systems. For example, an FOV greater than 2 or 3 mm can be provided in some optical imaging systems, e.g., with an objective lens and a tube lens. In some examples, the optical imaging systems provide reduced magnification, e.g., less than 10x. Such reduced magnification can facilitate wide FOV designs in some implementations. Despite the reduced magnification, the optical resolution of such systems can still be sufficient because detector arrays with small pixel sizes or pitches can be used. In some implementations, image sensors with pixel sizes smaller than twice the optical resolution provided by the optical imaging system (e.g., the objective lens and the tube lens) can be used to satisfy the Nyquist theorem.

[0137] Still other designs are possible. In some optical designs configured to provide multi-planar imaging capable of simultaneously imaging two surfaces at different depths, the optical imaging system (e.g., objective lens and / or tube lens) is configured to reduce optical aberrations when imaging the two surfaces (e.g., two planes) at their respective depths relative to other locations (e.g., other planes) at other depths. Furthermore, the optical imaging system may be configured to reduce aberrations for imaging the two surfaces (e.g., two planes) at their respective depths through a transparent layer (e.g., a glass layer (e.g., a cover slip)) on the sample support structure and through a solution (e.g., an aqueous solution) containing or in contact with a sample at at least one of the two surfaces.

[0138] Multi-Channel Fluorescence Imaging Modules and Systems: In some examples, the imaging modules or systems disclosed herein may include fluorescence imaging modules or systems. In some examples, the fluorescence imaging systems disclosed herein may include a single fluorescence excitation light source (for providing excitation light at a single wavelength or within a single excitation wavelength range) and an optical path configured to deliver the excitation light to a sample (e.g., fluorescently labeled nucleic acid molecules or clusters thereof disposed on a substrate surface). In some examples, the fluorescence imaging systems disclosed herein may include a single fluorescence emission imaging and detection channel, e.g., an optical path configured to collect fluorescence emitted from the sample and deliver an image of the sample (e.g., an image of the substrate surface on which the fluorescently labeled nucleic acid molecules or clusters thereof are disposed) to an image sensor or other light detection device. In some examples, the fluorescence imaging system may include two, three, four, or more fluorescence excitation light sources and / or optical paths configured to deliver excitation light at two, three, four, five, or more excitation wavelengths (or within two, three, four, or five or more excitation wavelength ranges). In some examples, the fluorescence imaging systems disclosed herein may comprise two, three, four, or five or more fluorescence emission imaging and detection channels configured to collect fluorescence emitted by a sample at two, three, four, or five or more emission wavelengths (or within two, three, four, or five or more emission wavelength ranges) and deliver images of the sample (e.g., images of a substrate surface on which fluorescently labeled nucleic acid molecules or clusters thereof are disposed) to two, three, four, or five or more image sensors or other light detection devices.

[0139] 17. Multi-planar imaging In some examples, imaging systems disclosed herein, including fluorescence imaging systems, can be configured to acquire high-resolution images of a single sample support structure or substrate surface. In some examples, imaging systems disclosed herein, including fluorescence imaging systems, can be configured to acquire high-resolution images of two or more sample support structure or substrate surfaces, for example, two, three, four, or more surfaces of a flow cell. The multiple surfaces of a sample support structure or flow cell device can be axially displaced from one another along the axial or z-direction. The multiple surfaces of a sample support structure or flow cell device can be interior surfaces facing the fluidic channel(s) disclosed herein. The fluidic channels or capillaries of a sample support structure or flow cell can be axially displaced from one another along the axial or z-direction. In one example, the two or more surfaces can be the top and bottom of a first channel and the top and bottom of a second channel axially displaced from the first channel. In this example, an interposer can be present between the first and second channels. The multiple surfaces of a sample support structure or flow cell can be curved surfaces (e.g., as described elsewhere herein). For example, the curved surface can be a rounded surface. The thickness of a portion of the flow cell (e.g., the top surface, the interposer layer, the bottom surface, etc.) can be at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 millimeters or more. The thickness of a portion of the flow cell (e.g., the top surface, the interposer layer, the bottom surface, etc.) can be at most about 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 millimeters or less.

[0140] In some examples, 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 around the flow cell substrate. Figures 1A and 1B provide schematic diagrams of a multiple-surface support structure. Figures 64A-64F provide schematic diagrams of a four-sided support structure as a flow cell.

[0141] FIG. 1A illustrates multiple surface support structures, such as flow cells, that include internal flow channels through which analytes or reagents can flow. The flow channels can be formed between first and second, top and bottom, and / or front and back layers, such as first and second, top and bottom, as shown. One or more of the layers can include glass plates, such as a cover glass. In some implementations, the layers include borosilicate glass, quartz, or plastic. The inner surfaces of these top and bottom layers provide flow channel walls that help confine the flow of analytes or reagents through the flow channel of the flow cell. In some designs, these inner surfaces are planar. Similarly, the top and bottom layers can be planar. In some designs, at least one additional layer (not shown) is disposed between the top and bottom layers. This additional layer can have one or more channels cut therein that define one or more flow channels and help control the flow of analytes or reagents within the flow channels. Further details of sample support structures, such as flow cells, can be found below.

[0142] 1A schematically illustrates multiple fluorescent sample sites on the first and second, top and bottom, and / or front and back interior surfaces of a flow cell. In some implementations, reactions can occur at these sites to bind sample such that fluorescence is emitted from these sites. (Note that FIG. 1A is a schematic diagram and is not drawn to scale; e.g., the size and spacing of the fluorescent sample sites may be smaller than shown.)

[0143] FIG. 1B illustrates another multi-surface support structure having two surfaces containing fluorescent sample sites to be imaged. The sample support structure includes a substrate having first and second, top and bottom, and / or front and back exterior surfaces. In some designs, these exterior surfaces are planar. In various implementations, analytes or reagents flow across these first and second exterior surfaces. FIG. 1B also illustrates a schematic representation of multiple fluorescent sample sites on the first and second, top and bottom, and / or front and back exterior surfaces of the sample support structure. In some implementations, reactions can occur at these sites to bind sample such that fluorescence is emitted from these sites. (Note that FIG. 1B is a schematic representation and is not drawn to scale; e.g., the size and spacing of the fluorescent sample sites may be smaller than shown.) For example, the support structures in FIGS. 64A-64E having one or more surfaces can have a sample site distribution on each surface similar to that shown in FIG. 1A or FIG. 1B.

[0144] In some examples, the fluorescence imaging modules and systems described herein can be configured to image such fluorescent sample sites on each of multiple surfaces at different distances from the objective lens. In some designs, only one of the multiple surfaces is focused at a time. Thus, in such designs, one of the surfaces is imaged at a first time point and another surface is imaged at a second time point. Because the images of the multiple surfaces are not simultaneously focused, the focus of the fluorescence imaging module can be changed after imaging one of the surfaces to image the next surface with equivalent optical resolution. In some designs, an optical compensation element can be introduced into the optical path between the sample support structure and the image sensor to image one of the surfaces. The depth of field in such fluorescence imaging configurations may not be large enough to include two or more of the multiple surfaces. In some implementations of the fluorescence imaging modules described herein, two or more surfaces may be imaged at the same time, e.g., simultaneously. For example, the fluorescence imaging module can have a depth of field large enough to include two or more surfaces. In some instances, this increased depth of field may be provided by, for example, reducing the numerical aperture of the objective lens (or microscope objective lens), as discussed in more detail below.

[0145] As shown in FIGS. 1A and 1B, the imaging optics (e.g., an objective lens) can be positioned at an appropriate distance (e.g., a distance corresponding to the working distance) from the surface to form a focused image of the surface on the image sensor of the detection channel. The first surface, e.g., 6418, in FIG. 64C can be between the objective lens and the second surface, e.g., 6419. For example, as shown in FIGS. 1A and 1B, the objective lens is positioned above multiple surfaces. The first surface is positioned above the second surface. As shown in FIG. 64C, the second surface 6419 is positioned above the third surface 6420, which is positioned above the fourth surface 6421. The multiple surfaces can be at different depths. These surfaces are at different distances from one or more of the fluorescence imaging module, the illumination and imaging module, the imaging optics, or the objective lens. The multiple surfaces are spaced apart from one another along the z-direction. The surfaces can be planar and spaced apart from one another along a direction perpendicular to the plane. In some embodiments, the objective lens has an optical axis, and the surfaces are spaced apart from one another along the optical axis. Similarly, the separation between the surfaces can correspond to the axial distance along the optical path of the excitation beam and / or the axial distance along the optical axis through the fluorescence imaging module and / or the objective lens. Thus, the surfaces may be spaced apart from one another in the axial (Z) direction along the central axis of the excitation beam and / or the optical axis of the objective lens and / or the fluorescence imaging module. In some implementations, this separation can correspond to a flow path within a flow cell, for example, between the first and second surfaces or between the third and fourth surfaces. In some implementations, this separation can correspond to an interposer substrate within a flow cell, for example, between the second and third surfaces.

[0146] In various designs, the objective lens (in some instances, combined with another optical component, e.g., a tube lens) has a depth of field and / or depth of focus at least as large as the axial separation (in the Z direction) between two adjacent surfaces of the plurality of surfaces. In some embodiments, the depth of field and / or depth of focus is at least as large as the axial separation (in the Z direction) between the first surface and the last surface (e.g., the fourth surface) along the optical path from the objective lens. Thus, the objective lens, alone or in combination with additional optical components, can simultaneously form in-focus images of at least two adjacent surfaces on the image sensors of one or more detection channels, with these images having comparable optical resolution. In some implementations, focusing the imaging module may or may not be required to capture images of the at least two adjacent surfaces with comparable optical resolution. In some implementations, forming in-focus images of the surfaces does not require moving correction optics into or out of the optical path of the imaging module. Similarly, in some implementations, one or more optical elements (e.g., lens elements) in the imaging module (e.g., objective lens and / or tube lens) do not need to be moved axially, e.g., along the first optical path and / or the second optical path (e.g., along the optical axis of the imaging optics), to form a focused image of a surface, e.g., a first surface, compared to the position of the one or more optical elements when used to form a focused image of another one of the surfaces, e.g., a second surface, a third surface, or a fourth surface. However, in some implementations, the imaging module includes an autofocus system configured to simultaneously focus at least two adjacent surfaces. In various implementations, the sample is focused to adequately resolve closely spaced sample sites in the lateral direction (e.g., the X and Y directions).Thus, in various implementations, to form focused images of fluorescent sample sites on one surface of the sample support structure and two other surfaces of the sample support structure, no optical elements are inserted into the optical path between the sample support structure (e.g., a translation stage supporting the sample support structure) and the image sensor (or photodetector array) in at least one detection channel. In various implementations, to form focused images of fluorescent sample sites on one surface of the sample support structure, e.g., the first surface, on the image sensor or photodetector array, no optical compensation is used that is not identical to the optical compensation used to form focused images of fluorescent sample sites on another surface of the sample support structure, e.g., the second surface, third surface, or fourth surface, on the image sensor or photodetector array. Furthermore, in certain implementations, optical elements in the optical path between the sample support structure (e.g., a translation stage supporting the sample support structure) and the image sensor in at least one detection channel are not adjusted differently to form a focused image of fluorescent sample sites on one surface, e.g., the first surface of the sample support structure, than to form a focused image of fluorescent sample sites on another surface, e.g., the second, third, or fourth surface, of the sample support structure. Similarly, in some various embodiments, optical elements in the optical path between the sample support structure (e.g., a translation stage supporting the sample support structure) and the image sensor in at least one detection channel are not moved a different amount or in a different direction to form a focused image of fluorescent sample sites on one surface, e.g., the first surface, of the sample support structure on the image sensor than to form a focused image of fluorescent sample sites on another surface, e.g., the second, third, or fourth surface, of the sample support structure on the image sensor. Any combination of the features herein is possible.For example, in some implementations, in-focus images of the first and second inner surfaces of the flow cell can be acquired without moving the optical compensator in or out of the optical path between the flow cell and the at least one image sensor, and without moving one or more optical elements of the imaging system (e.g., the objective lens and / or the tube lens) along the optical path (e.g., the optical axis) therebetween. For example, in-focus images of the first, second, and third inner surfaces of the flow cell can be acquired without moving one or more optical elements of the tube lens in or out of the optical path or along the optical path (e.g., the optical axis) therebetween.

[0147] Any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at multiple locations, such as planes corresponding to multiple surfaces on a flow cell or other sample support structure where the fluorescent sample site is located. Any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at selected locations or planes compared to other locations or planes, such as the surface containing the fluorescent sample site on a flow cell. For example, any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at two depths or planes at different distances from the objective lens compared to aberrations associated with other depths or planes at other distances from the objective lens. For example, optical aberrations may be smaller when imaging a surface than at other locations within a region from about 1 mm to about 10 mm from the objective lens. Additionally, any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be configured to correct for optical aberrations caused by transmission of the emitted light through one or more portions of the sample support structure, such as, in some instances, one of the surfaces on which the sample is attached, and in some instances, a layer containing a solution in contact with the sample. This layer (e.g., a cover glass or a wall of a flow cell) may comprise, for example, glass, quartz, plastic, or other transparent material that has a refractive index and introduces optical aberrations.

[0148] Thus, when imaging multiple surfaces, e.g., three or four surfaces, the imaging performance may be substantially the same. For example, the optical transfer function (OTF) and / or modulation transfer function (MTF) for imaging the multiple surfaces may be substantially the same. One or both of these transfer functions may be, for example, within 20%, 15%, 10%, 5%, 2.5%, or 1% of each other, or within any range formed by any of these values ​​at one or more specified spatial frequencies, or when averaged over a range of spatial frequencies. Thus, without moving an optical compensator in or out of the optical path between the flow cell and the at least one image sensor, and without moving one or more optical elements of the imaging system (e.g., the objective lens and / or the tube lens) along the optical path (e.g., the optical axis) therebetween, the imaging performance metrics for imaging each surface of the multiple surfaces of the flow cell may be substantially the same. For example, the imaging performance metrics for imaging the first, second, third, and fourth surfaces of the flow cell may be substantially the same without moving one or more optical elements of the tube lens into or out of the optical path, or along the optical path between them. In some embodiments, the optical path is the optical axis. Additional discussion regarding MTF is included below and in U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020, which is incorporated by reference in its entirety.

[0149] Those skilled in the art will understand that the disclosed imaging systems or modules may, in some instances, be standalone optical systems designed to image a sample or substrate surface. In some instances, they may include one or more processors or computers. In some instances, they may include one or more software packages providing instrument control and / or image processing functions. In some instances, in addition to optical components such as light sources (e.g., solid-state lasers, dye lasers, diode lasers, arc lamps, tungsten-halogen lamps, etc.), lenses, prisms, mirrors, dichroic reflectors, beam splitters, optical filters, optical bandpass filters, light guides, apertures, and image sensors (e.g., complementary metal-oxide semiconductor (CMOS) image sensors and cameras, charge-coupled device (CCD) image sensors and cameras, etc.), they may also include mechanical and / or opto-mechanical components such as XY translation stages, XYZ translation stages, piezoelectric focusing mechanisms, electro-optic phase plates, etc. In some instances, they may function as modules, components, subassemblies, or subsystems of a larger system designed, for example, for genomics applications (e.g., genetic testing and / or nucleic acid sequencing applications). For example, in some instances, they may function as modules, components, subassemblies, or subsystems of a larger system that further includes light-tight and / or other environmental control housings, temperature control modules, flow cells and cartridges, fluidic 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, such as systems designed for genomics applications, are discussed in more detail below.

[0150] 2A and 2B show a non-limiting example of an illumination and imaging module 100 for multi-channel fluorescence imaging. The illumination and imaging module 100 may include an objective lens 110, an illumination light source 115, multiple detection channels 120, and a first dichroic filter 130, which may comprise a dichroic reflector or beam splitter. Some designs may also include an autofocus system including an autofocus laser 102, for example, which projects a spot and monitors its size to determine when the imaging system is in focus. Some or all components of the illumination and imaging module 100 may be coupled to a base plate 105.

[0151] The illumination or light source 115 may include any suitable light source configured to generate light at least at 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 bands). The light source may be a narrowband light source that emits light within one or more narrower wavelength ranges. In some examples, the light source may generate a single isolated wavelength (or line) corresponding to the desired excitation wavelength, or multiple isolated wavelengths (or lines). In some examples, the lines may have several very narrow bandwidths. Examples of light sources suitable for use as the illumination light source 115 include, but are not limited to, incandescent filaments, xenon arc lamps, mercury lamps, light-emitting diodes, laser sources such as laser diodes or solid-state lasers, or other types of light sources. As discussed below, in some designs, the light source may include a polarized light source, such as a linearly polarized light source. In some implementations, the light source is oriented such that s-polarized light is incident on one or more surfaces of one or more optical components, for example, on the dichroic reflective surfaces of one or more dichroic filters.

[0152] The illumination light source 115 may further include one or more additional optical components, such as lenses, filters, optical fibers, or any other suitable transmissive or reflective optics, as needed to output an excitation light beam with appropriate characteristics toward the first dichroic filter 130. For example, beam shaping optics may be included to receive light from the light emitters in the light source and generate the beam and / or provide desired beam characteristics. Such optics may comprise, for example, a collimating lens configured to reduce divergence and / or increase collimation and / or collimate the light.

[0153] In some implementations, multiple light sources are included in the illumination and imaging module 100. In some such implementations, different light sources may generate light with different spectral characteristics, e.g., to excite different fluorescent dyes. In some implementations, light generated by different light sources may be directed to overlap and form a combined excitation light beam. This combined excitation light beam may be composed of excitation light beams from each light source. The combined excitation light beam has greater optical power than the individual beams that overlap to form the composite beam. For example, in some implementations including two light sources generating two excitation light beams, the combined excitation light beam formed from the two individual excitation light beams may have an optical power that is the sum of the optical powers of the individual beams. Similarly, in some implementations, three, four, five, or more light sources may be included, each of which may output an excitation light beam that together form a combined beam having an optical power that is the sum of the optical powers of the individual beams.

[0154] In some implementations, the light source 115 outputs a sufficient amount of light to generate a sufficiently strong fluorescent emission. A stronger fluorescent emission can increase the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the image acquired by the fluorescent imaging module. In some implementations, the output of the light source and / or the resulting excitation light beam (including the combined excitation light beam) may range in power from about 0.5 watts (W) to about 5.0 W or more (discussed in more detail below).

[0155] 2A and 2B, a first dichroic filter 130 is positioned relative to the light source to receive light from the light source. The first dichroic filter may comprise a dichroic mirror, a dichroic reflector, a dichroic beam splitter, or a dichroic beam combiner configured to transmit light in a first spectral region (or wavelength range) and reflect light having a second spectral region (or wavelength range). The first spectral region may include one or more spectral bands, for example, one or more spectral bands in the ultraviolet and blue wavelength ranges. Similarly, the second spectral region may include one or more spectral bands, for example, one or more spectral bands spanning wavelengths from green to red and infrared. Other spectral regions or wavelength ranges are possible.

[0156] In some implementations, the first dichroic filter can be configured to transmit light from the light source to a sample support structure, such as a microscope slide, capillary, flow cell, microfluidic chip, or other substrate or support structure. The sample support structure supports and positions a sample, e.g., a composition including a fluorescently labeled nucleic acid molecule or its complement, relative to the illumination and imaging module 100. Thus, the first optical path extends from the light source through the first dichroic filter to the sample. In various implementations, the sample support structure includes at least one surface on which the sample is disposed or to which the sample is bound. In some examples, the sample may be disposed or bound within different localized regions or sites on at least one surface of the sample support structure.

[0157] In some examples, the support structure may include two, three, four, or more surfaces on which samples are disposed, located at different distances from the objective lens 110 (e.g., at different axial positions or depths along the optical axis of the objective lens 110). As discussed below, for example, a flow cell may comprise a fluid channel formed at least in part by a first inner surface and a second (e.g., upper and lower) inner surface, and a 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 are separated by a region corresponding to the fluid channel through which the solution flows and may therefore be at different distances or depths relative to the objective lens 110 of the illumination and imaging module 100. The flow cell may comprise a second fluid channel formed at least in part by a third inner surface and a fourth (e.g., upper and lower) inner surface, and a sample may be disposed at a localized location on the third inner surface, the fourth inner surface, or both. The third surface and the fourth surface are separated by a region corresponding to a second fluid channel through which the solution flows, and therefore may be at different distances or depths relative to the objective lens 110 of the illumination and imaging module 100. The first fluid channel and the second fluid channel may be separated axially by an interposer substrate disposed therebetween.

[0158] An objective lens 110 may be included in the first optical path between the first dichroic filter and the sample. This objective lens may be configured, for example, with a focal length, a working distance, and / or positioned to focus light from the light source(s) onto the surface of the sample, such as a microscope slide, a capillary, a flow cell, a microfluidic chip, or other substrate or support structure. Similarly, the objective lens 110 may be configured with an appropriate focal length, a working distance, and / or positioned to collect light reflected, scattered, or emitted from the sample (e.g., fluorescent emission) and form an image of the sample (e.g., a fluorescence image).

[0159] In some implementations, the objective lens 110 may include a microscope objective lens, such as a commercially available objective lens. In some implementations, the objective lens 110 may include a custom objective lens. Examples of custom objective lenses and / or combinations of custom objective lenses with tube lenses are described below and in U.S. Patent Application No. 17 / 999,023, which are incorporated herein by reference in their entirety. The objective lens 110 may be designed to reduce or minimize optical aberrations at two, three, four, or more locations. For example, such locations may include planes corresponding to multiple surfaces of a flow cell. The objective lens 110 may be designed to reduce optical aberrations at selected locations or planes, such as the first and second surfaces of a multi-sided flow cell or the first, second, third, and fourth surfaces of a four-sided flow cell, relative to other locations or planes in the optical path. For example, the objective lens 110 can be designed to reduce optical aberrations at two, three, or four depths or planes located at different distances from the objective lens compared to optical aberrations associated with other depths or planes at other distances from the objective lens. For example, in some instances, optical aberrations when imaging the surface of a flow cell may be smaller than those exhibited elsewhere within a region extending from 1 to 10 mm from the front surface of the objective lens. Furthermore, in some instances, the custom objective lens 110 can be configured to compensate for optical aberrations caused by the transmission of fluorescent emission light through one or more portions of the sample support structure, such as a layer including one or more flow cell surfaces on which samples are disposed, or a layer including a solution filling the fluidic channels of the flow cell. These layers may include, for example, glass, quartz, plastic, or other transparent materials with refractive indices that may introduce optical aberrations.

[0160] In some implementations, the objective lens 110 may have a numerical aperture (NA) of 0.6 or greater (discussed in more detail below). Such a numerical aperture may result in reduced depth of focus and / or depth of field, improved background discrimination, and increased imaging resolution.

[0161] In some implementations, the objective lens 110 may have a numerical aperture (NA) of 0.6 or less (discussed in more detail below). Such an NA may result in an increased depth of focus and / or depth of field. Such an increased depth of focus and / or depth of field may improve the ability to image distantly separated planes, such as the first and second surfaces, the second and third surfaces, or the third and fourth surfaces. In some implementations, the objective lens 110 may have a numerical aperture (NA) of 0.5 or less, e.g., 0.4. Such an NA may provide lower optical aberrations that need to be compensated for by the optical system than higher NA values. Such an NA may provide the ability to focus and image additional image planes, such as planes at the third or fourth surfaces of the flow cell, with minimal changes to the design of an optical system configured to image one or more surfaces of the flow cell (e.g., no need to add or remove compensators in the optical path from the objective lens to the flow cell being imaged).

[0162] As discussed above, the flow cell may comprise, for example, first and second layers including first and second inner surfaces, respectively, through which analytes or reagents can flow, separated by a fluidic channel. The flow cell may also comprise third and fourth layers including third and fourth inner surfaces, respectively, through which analytes or reagents can flow, separated by a second fluidic channel. In some implementations, the objective lens 110 and / or the illumination and imaging module 100 may be configured to provide a depth of field and / or depth of focus large enough to image at least two adjacent surfaces of the flow cell, either sequentially by refocusing the imaging module while imaging the at least two surfaces, or simultaneously by ensuring a sufficiently large depth of field and / or depth of focus at comparable optical resolution. In some examples, the depth of field and / or depth of focus may be at least as large as or greater than the distance separating two adjacent surfaces of the flow cell being imaged. In some examples, two adjacent surfaces, e.g., the first and second inner surfaces of a two-sided flow cell, or the third and fourth surfaces of a four-sided flow cell, can be separated by a distance ranging from about 10 μm to about 700 μm, or more (discussed in more detail below). In some examples, the depth of field and / or depth of focus can therefore range from about 10 μm to about 700 μm, or more (discussed in more detail below).

[0163] In some designs, adaptive optics (e.g., “optical compensator” or “compensator”) can be moved in and out of an optical path within the imaging module, e.g., an optical path through which light collected by the objective lens 110 is delivered to an image sensor, so that the imaging module can image the surface of the flow cell. The imaging module can be configured to image one surface, e.g., a first surface, if, for example, a first adaptive optic is included in the optical path between the objective lens and an image sensor or photodetector array configured to capture images of the first surface. The imaging module can be configured to image another surface, e.g., a second surface, if, for example, a second adaptive optic is included in the optical path between the objective lens and an image sensor or photodetector array configured to capture images of the second surface. In such a design, the imaging module can be configured to image yet another surface, e.g., a third surface, if the first adaptive optic and the second adaptive optic are removed from or not included in the optical path between the objective lens 110 and an image sensor or photodetector array configured to capture images of the third surface. The need for an optical compensator may be more pronounced when using an objective 110 with a high numerical aperture (NA) value, e.g., at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, or even higher. In some implementations, the optical compensator (e.g., an optical compensator or compensator) includes a refractive optical element such as a lens, a plate of optically transparent material such as glass, or, in the case of polarized light beams, a quarter-wave plate or a half-wave plate. Other configurations may be used to allow surfaces to be imaged at different times. For example, one or more lenses or optical elements may be configured to translate in, out of, or along the optical path between the objective 110 and the image sensor.

[0164] In certain designs, the objective lens 110 is configured to be adjusted to change the NA of the optical system. In some embodiments, the objective lens 110 can be adjusted to adjust the NA of the optical system within a range of 0.25 to 0.6. In some embodiments, the objective lens 110 can be adjusted to adjust the NA of the optical system within a range of 0.35 to 0.55. In some embodiments, the objective lens 110 can be adjusted to adjust the NA of the optical system within a range of 0.4 to 0.5. In some embodiments, the NA of the optical system can be adjusted by changing the objective lens 110. In some embodiments, the NA of the optical system can be adjusted by simply changing the objective lens 110, without moving any optical compensators in, out of, or along the optical path from the objective lens to the sample. In some embodiments, the NA of the optical system can be adjusted by changing optical elements within the objective lens 110, without removing an existing objective lens and adding a new one.

[0165] In some embodiments, the objective lens 110 can include an aperture stop, and adjusting the size of the aperture stop can change the NA of the optical system. In some embodiments, changing the size of the aperture stop includes moving the objective lens or any other optical element in, out of, or parallel to the optical path between the objective lens and the image sensor. As a non-limiting example, changing the size of the aperture stop includes rotating an optical element or a portion of the objective lens about an axial or longitudinal axis of the objective lens. As another non-limiting example, changing the size of the aperture stop includes moving an optical element or a portion of the objective lens orthogonal to the axial or longitudinal axis of the objective lens.

[0166] In some embodiments, the NA of the optical system can be adjusted without changing the size of the aperture stop. Instead, the objective lens and / or tube lens can be redesigned from an optical system having a first NA, e.g., an NA of 0.5, to bring the NA of the optical system to a predetermined different value, e.g., 0.4. The redesign can include changing one or more characteristics of the objective lens and / or tube lens, including, but not limited to, diameter, size, magnification, length, cover thickness, working distance, and lens design that functions to correct aberrations.

[0167] In some embodiments, the NA of the optical system can be adjusted from the NA used to image a conventional single-sided or double-sided flow cell (e.g., NA 0.5 to NA 0.4) to image a multi-sided flow cell having three or more axially displaced surfaces with a predetermined image quality. In some embodiments, the NA of the optical system can remain unchanged from the NA used to image a conventional single-sided or double-sided flow cell (e.g., NA 0.4) to image a multi-sided flow cell having three or more axially displaced surfaces with a predetermined image quality.

[0168] The optical system herein allows for the objective lens to be adjusted to change the NA (e.g., between 0.5 and 0.4, or in the range of 0.4 to 0.5). Furthermore, the optical system can use the same NA, e.g., NA = 0.4, to image flow cell devices with total thicknesses within the compatible range of NA, e.g., between about 220 μm and about 360 μm. Thus, the optical system herein can provide the flexibility and compatibility to image both (1) conventional flow cells with one or two surfaces and (2) multifaceted flow cells herein (e.g., with three, four, or more axially displaced surfaces) with sufficient image quality, e.g., a CNR of at least 5, 10, 15, or 20.

[0169] However, in certain designs, objective lens 110 is configured to provide a sufficiently large depth of focus and / or depth of field so that such adaptive optics can image the surface with comparable optical resolution without moving the adaptive optics in and / or out of an optical path within the imaging module, such as the optical path between the objective lens and an image sensor or photodetector array. In various designs, objective lens 110 is configured to provide a sufficiently large depth of focus and / or depth of field so that the surface can be imaged with comparable optical resolution without moving the optics, such as translating one or more lenses or other optical components along an optical path within the imaging module, such as the optical path between the objective lens and an image sensor or photodetector array. Examples of such objective lenses are described in more detail below.

[0170] In some implementations, the objective lens (or microscope objective lens) 110 can be configured to have a reduced magnification. For example, the objective lens 110 may be configured so that the fluorescence imaging module has a magnification of less than 2x to less than 10x (discussed in more detail below). Such a reduced magnification changes design constraints, potentially allowing other design parameters to be achieved. For example, as discussed in more detail below, the objective lens 110 may be configured so that the fluorescence imaging module has a wide field of view (FOV), for example, in the range of about 1.0 mm to about 5.0 mm (e.g., diameter, width, length, or longest dimension).

[0171] In some implementations, as discussed in more detail below, the objective lens 110 may be configured to provide the fluorescence imaging module with such a field of view that the FOV has diffraction-limited performance, such as, for example, less than 0.15 waves of aberration across at least 60%, 70%, 80%, 90%, or 95% of the field of view.

[0172] In some implementations, as discussed in more detail below, the objective lens 110 may be configured to provide the fluorescence imaging module with such a field of view so that the FOV has diffraction-limited performance, such as a Strehl ratio of greater than 0.8 across at least 60%, 70%, 80%, 90%, or 95% of the field of view.

[0173] 2A and 2B, a first dichroic beam splitter or combiner is positioned in a first optical path between the light source and the sample to illuminate the sample with one or more excitation beams. This first dichroic beam splitter or combiner is also positioned in one or more second optical paths to different optical channels used to detect fluorescent emissions from the sample. Thus, a first dichroic filter 130 combines the first optical path of the excitation beam emitted by the illumination light source 115 and the second optical path of the emission light emitted by the sample specimen into various optical channels that direct light to respective image sensors or photodetector arrays to capture images of the sample.

[0174] In various implementations, 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 light source 115 only within a specified wavelength band, or in some instances, within multiple wavelength bands including a desired excitation wavelength or wavelengths. For example, the first dichroic beam splitter 130 includes a reflective surface with a dichroic reflector having a spectral transmittance response configured to transmit light having at least some of the wavelengths output by the light source that form part of the excitation beam. The spectral transmittance response can be configured to not transmit (e.g., to instead reflect) light of one or more other wavelengths, e.g., one or more other fluorescent emission wavelengths. In some implementations, the spectral transmittance response can also be configured to not transmit (e.g., to instead reflect) light of one or more other wavelengths output by the light source. Thus, the first dichroic filter 130 can be utilized to select which wavelength or wavelengths of light output by the light source reach the sample. Conversely, the dichroic reflector in the first dichroic beamsplitter 130 has a spectral reflectance response that reflects light having one or more wavelengths corresponding to the desired fluorescent emission from the sample and, in some instances, reflects light having one or more wavelengths output from the light source that are not intended to reach the sample. Thus, in some implementations, the dichroic reflector has a spectral transmittance that includes one or more passbands that transmit light incident on the sample and one or more stopbands that reflect light outside the passbands, e.g., light at one or more emission wavelengths and, in some instances, light at one or more wavelengths output by the light source that are not intended to reach the sample. Similarly, in some implementations, the dichroic reflector has a spectral reflectance that includes one or more spectral regions configured to reflect one or more emission wavelengths and, in some instances, one or more wavelengths output by the light source that are not intended to reach the sample, and one or more regions that transmit light outside of these reflection regions.The dichroic reflector included in the first dichroic filter 130 may comprise a reflective filter, such as an interference filter (e.g., a quarter wave stack) configured to provide the appropriate spectral transmission and reflection distributions. Figures 2A and 2B also show a dichroic filter 105, which may comprise, for example, a dichroic beam splitter or beam combiner, that may be used to direct the autofocus laser 102 through the objective lens and onto the sample support structure.

[0175] In some embodiments, the dichroic filter 105, 130, 530 can include one or more spectral passband(s) that increase the intensity, and therefore the SNR, of the optical signal(s) passing through the filter. Thus, the dichroic filter(s) of the present invention can have a more uniform frequency response than conventional dichroic filter(s) that have narrower spectral passbands.

[0176] 2A and 2B and discussed above is configured such that the excitation beam is transmitted to the objective lens 110 by the first dichroic filter 130, in some designs the illumination light source 115 may be positioned 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 lens 110. Similarly, in some such designs, the first dichroic filter 130 is configured to transmit fluorescent emission from the sample and, in some instances, to transmit light having one or more wavelengths output from the light source that are not intended to reach the sample. As discussed below, designs in which fluorescent emission is transmitted rather than reflected may reduce wavefront errors in the detected emission and / or may have other advantages. In either case, in various implementations, a first dichroic reflector 130 is positioned in the second optical path to receive fluorescent emission from the sample, at least a portion of which continues to the detection channel 120.

[0177] 2C and 2D illustrate the light paths within the multichannel fluorescence imaging module of FIGS. 2A and 2B. In the example shown in FIGS. 2A and 2C, the detection channel 120 is positioned to receive fluorescent emission from the sample specimen that is transmitted by the objective lens 110 and reflected by the first dichroic filter 130. As mentioned above and further described below, in some designs, the detection channel 120 may be positioned to receive a portion of the emission light that is transmitted rather than reflected by the first dichroic filter. In either case, the detection channel 120 may include optics for receiving at least a portion of the emission light. For example, the detection channel 120 may include one or more lenses, such as a tube lens, and may also include one or more image sensors or detectors, such as a photodetector array (e.g., a CCD or CMOS sensor array) for imaging or otherwise generating a signal based on the received light. The tube lens may include, for example, one or more lens elements configured to form an image of the sample on the sensor or photodetector array and capture the image. Additional description of detection channels is provided below and in U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020, which is incorporated herein by reference in its entirety. In some examples, improved optical resolution may be achieved using image sensors with relatively high sensitivity, small pixels, and high pixel counts, in combination with an appropriate sampling scheme, which may include oversampling or undersampling.

[0178] 2C and 2D are ray tracing diagrams illustrating the optical paths of the illumination and imaging module 100 of FIGS. 2A and 2B. FIG. 2C corresponds to a top view of the illumination and imaging module 100. FIG. 2D corresponds to a side view of the illumination and imaging module 100. These illustrated illumination and imaging modules 100 include four detection channels 120. However, it will be understood that the disclosed illumination and imaging modules may similarly be implemented in systems including more or fewer than four detection channels 120. For example, the multi-channel systems disclosed herein may be implemented with a minimum of one detection channel 120, or a maximum of 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.

[0179] 2C and 2D includes four detection channels 120, a first dichroic filter 130 that reflects a beam of emission light 150, a second dichroic filter (e.g., a dichroic beam splitter) 135 that splits the beam 150 into transmitted and reflected portions, and two channel-specific dichroic filters (e.g., dichroic beam splitters) 140 that further split the transmitted and reflected portions of the beam 150 among the individual detection channels 120. The dichroic reflective surfaces of the dichroic beam splitters 135 and 140 for splitting the beam 150 among the detection channels are shown as being disposed at 45 degrees with respect to the central beam axis of the beam 150 or the optical axis of the imaging module. However, as discussed below, angles less than 45 degrees may be employed and may provide advantages such as a sharper transition from the passband to the stopband.

[0180] The different detection channels 120 include imaging devices 124, which may include image sensors or photodetector arrays (e.g., CCD or CMOS detector arrays). The different detection channels 120 further include optics 126, such as lenses (e.g., one or more tube lenses, each comprising one or more lens elements), arranged to focus a portion of the emission light entering the detection channel 120 onto a focal plane coincident with the plane of the photodetector array 124. The optics 126 (e.g., tube lenses) in combination with the objective lens 110 are configured to form an image of the sample on the photodetector array 124 to capture an image of the sample, for example, an image of a surface on a flow cell or other sample support structure after the sample has bound to the surface. Such an image of the sample may therefore include multiple fluorescent spots or regions across the spatial extent of the sample support structure from which the sample fluoresces. The objective lens 110, together with the optics 126 (e.g., tube lenses), may provide a field of view (FOV) that includes a portion of the sample or the entire sample. Similarly, the photodetector arrays 124 of the different detection channels 120 can be configured to capture an image of the entire field of view (FOV) provided by the objective lens and tube lens, or a portion thereof. In some implementations, the photodetector arrays 124 of some or all of the detection channels 120 can detect emission light emitted by a sample disposed on a sample support structure, e.g., the surface of a flow cell, or a portion thereof, and record electronic data representing the image. In some implementations, the photodetector arrays 124 of some or all of the detection channels 120 can detect features of emission light emitted from a specimen without capturing and / or storing an image of the sample disposed on the flow cell surface and / or the entire field of view (FOV) provided by the objective lens and optics 126 and / or 122 (e.g., elements of a tube lens). In some implementations, the FOV of the disclosed imaging modules (e.g., provided by the combination of the objective lens 110 and optics 126 and / or 122) can range, for example, from about 1 mm to 5 mm (e.g., diameter, width, length, or longest dimension), as discussed further below.The FOV may be selected, for example, to balance the magnification and resolution of the imaging module and / or based on one or more characteristics of the image sensor and / or objective lens. For example, a relatively small FOV may be provided in combination with a smaller, faster image sensor to achieve high throughput.

[0181] 2C and 2D , in some implementations, the optics 126 within a detection channel (e.g., a tube lens) can be configured to reduce optical aberrations in images acquired using the optics 126 in combination with the objective lens 110. In some implementations with multiple detection channels for imaging at different emission wavelengths, the optics 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 implementations, the optics 126 (e.g., tube lenses) can be configured to reduce aberrations when imaging a particular surface (e.g., a plane, an object plane, etc.) on the sample support structure where the fluorescent sample site is located compared to other locations (e.g., other planes in object space). In some implementations, the optical system 126 (e.g., a tube lens) can be configured to reduce aberrations when imaging multiple surfaces (e.g., first and second planes, first and second object planes, etc.) on a sample support structure (e.g., a two-sided flow cell or a four-sided flow cell) on which fluorescent sample sites are located, compared to other locations (e.g., other planes in object space). For example, the optical system 126 (e.g., a tube lens) in the detection channel can be designed to reduce aberrations at two, three, or more depths or planes located at different distances from the objective lens, compared to aberrations associated with other depths or planes at other distances from the objective lens. For example, optical aberrations may be smaller when imaging multiple surfaces than at other locations within a region from about 1 mm to about 10 mm from the objective lens. Additionally, the custom optics 126 (e.g., a tube lens) in the detection channel can, in some implementations, be configured to correct for aberrations caused by transmission of the emitted light through one or more portions of the sample support structure, such as a layer including one of the surfaces on which the sample is disposed, as well as, in some instances, a solution adjacent to and in contact with the surface on which the sample is disposed. The layer including one of the surfaces on which the sample is disposed can include, for example, glass, quartz, plastic, or other transparent material having a refractive index that introduces optical aberrations.Custom optics 126 (e.g., a tube lens) in the detection channel may be configured, for example, in some implementations, to correct optical aberrations caused by the sample support structure, e.g., a cover glass or flow cell wall, or other sample support structure component, as well as, in some instances, a solution adjacent to and in contact with the surface on which the sample is placed.

[0182] In some implementations, the optics 126 (e.g., tube lens) in the detection channel may be configured to have a reduced magnification. For example, as discussed further below, the optics 126 (e.g., tube lens) in the detection channel may be configured such that the fluorescence imaging module has a magnification of, for example, less than 10x. Such a reduced magnification may change design constraints, thereby enabling other design parameters to be achieved. For example, as discussed further below, the optics 126 (e.g., tube lens) may be configured such that the fluorescence imaging module has a wide field of view (FOV), for example, at least 1.0 mm or more (e.g., diameter, width, length, or longest dimension).

[0183] In some implementations, as discussed further below, the optical system 126 (e.g., a tube lens) may be configured to provide the fluorescence imaging module with a field of view such that the FOV has an aberration of less than 0.15 waves across at least 60%, 70%, 80%, 90%, or 95% of the field of view.

[0184] 2C and 2D , in various implementations, the sample is located at or near the focal position 112 of the objective lens 110. As described above with reference to FIGS. 2A and 2B , a light source, such as a laser light source, provides an excitation beam to the sample to induce fluorescence. At least a portion of the fluorescent emission is collected as emission light by the objective lens 110. The objective lens 110 transmits the emission light toward a first dichroic filter 130, which reflects some or all of the emission light as beam 150 incident on a second dichroic filter 135, into different detection channels, each comprising an optical system 126 that forms an image of the sample (e.g., multiple fluorescent sample sites on the surface of a sample support structure) onto a photodetector array 124.

[0185] As discussed above, in some implementations, the sample support structure includes a flow cell, such as a flow cell having multiple surfaces (e.g., two or more inner surfaces) containing sample sites that emit fluorescent light. These surfaces may be separated from each other by a fixed distance in the longitudinal (Z) direction, along the direction of the central axis of the excitation beam and / or the optical axis of the objective lens. This separation may correspond, for example, to one or more flow paths within the flow cell. Analytes or reagents may flow through the flow path(s) and contact the surface of the flow cell, thereby contacting the binding composition such that fluorescent light is emitted from multiple sites on the surface. The imaging optics (e.g., objective lens 110) may be positioned at an appropriate distance from the sample (e.g., a distance corresponding to the working distance) to form a focused image of the sample on one or more detector arrays 124. As discussed above, in various designs, objective lens 110 (in some examples combined with optics 126) may have a depth of field and / or depth of focus at least as large as the longitudinal separation between two adjacent surfaces or between any two of the multiple surfaces. Thus, the objective lens 110 and optical system 126 (for each detection channel) can simultaneously form images of multiple surfaces on the photodetector array 124, and the images of these surfaces are in focus and have comparable optical resolution (or can be brought into focus with only slight refocusing of the object to obtain images with comparable optical resolution). In various implementations, the adaptive optics system does not need to be moved in or out of the optical path of the imaging module (e.g., in or out of the first optical path and / or the second optical path) to form in-focus images of surfaces with comparable optical resolution. Similarly, in various implementations, one or more optical elements (e.g., lens elements) in the imaging module (e.g., the objective lens 110 or the optical system 126) do not need to be moved longitudinally along the first optical path and / or the second optical path to form an in-focus image of, for example, a first surface, compared to the position of the one or more optical elements when used to form an in-focus image of a second surface.In some implementations, the imaging module includes an autofocus system configured to rapidly and sequentially refocus the imaging module on one or more of the multiple surfaces so that the images have equivalent optical resolution. In some implementations, the objective lens 110 and / or the optical system 126 are configured so that at least two of the multiple surfaces, e.g., two adjacent surfaces, are simultaneously focused with equivalent optical resolution without moving an optical compensator into or out of the first optical path and / or the second optical path and without longitudinally moving one or more lens elements (e.g., the objective lens 110 and / or the optical system 126 (e.g., a tube lens)) along the first optical path and / or the second optical path. In some implementations, images of surfaces acquired sequentially (e.g., with refocusing between surfaces) or simultaneously (e.g., without refocusing between surfaces) using the novel objective lens and / or tube lens designs disclosed herein may be further processed using appropriate image processing algorithms to increase the effective optical resolution of the images so that the images have equivalent optical resolution. In various implementations, the sample plane is in sufficient focus to resolve sample sites on the flow cell surface, and the sample sites are closely spaced laterally (e.g., in the X and Y directions).

[0186] As discussed above, dichroic filters may include interference filters that selectively transmit and reflect light of different wavelengths based on the principles of thin-film interference using optical coating layers with different refractive indices and specific thicknesses. Therefore, the spectral response (e.g., transmission spectrum and / or reflection spectrum) of a dichroic filter implemented in a multichannel fluorescence imaging module may depend, at least in part, on the angle of incidence, or range of angles of incidence, at which light in the excitation and / or emission beams strikes the dichroic filter. Such effects may be particularly pronounced with respect to dichroic filters in the detection light path (e.g., dichroic filters 135 and 140 in Figures 2C and 2D).

[0187] FIG. 3 is a graph illustrating the relationship between dichroic filter performance and beam angle of incidence (AOI). Specifically, the graph in FIG. 3 illustrates the effect of the angle of incidence on the transition width or spectral span of a dichroic filter, which corresponds to the range of wavelengths over which the spectral response (e.g., transmission spectrum and / or reflection spectrum) transitions between the passband and stopband regions of the dichroic filter. Thus, a transmission end (or reflection end) with a relatively narrow spectral span (e.g., a small delta λ value in the graph in FIG. 3 ) corresponds to a sharper transition between the passband and stopband regions, or between the transmission and reflection regions (or vice versa), whereas a transmission end (or reflection end) with a relatively wide spectral span (e.g., a large delta λ value in the graph in FIG. 3 ) corresponds to a less sharp transition between the passband and stopband regions. In various embodiments, a sharper transition between the passband and stopband regions is generally desirable. Furthermore, it may be desirable to increase the consistency or to have a relatively constant transition width across the entire or a majority of the field of view and / or beam area.

[0188] Thus, a fluorescence imaging module in which the dichroic mirror is positioned at 45 degrees relative to the central beam axis of the emission light or the optical axis of the light path (e.g., the objective lens and / or tube lens) can have a transition width of approximately 50 nm for the dichroic filter, as shown in Figure 3. Because the emission light beam is not collimated and has some divergence, the fluorescence imaging module may have a range of incidence angles of approximately 5 degrees between opposite sides of the beam. Thus, as shown in Figure 3, different portions of the emission light beam may be incident on the channel-splitting dichroic filter at various incidence angles, from 40 to 50 degrees. This relatively large range of incidence angles corresponds to a range of transition widths from approximately 40 nm to approximately 62 nm. This relatively large range of incidence angles therefore increases the transition width of the dichroic filter within the imaging module. Therefore, the performance of a multichannel fluorescence imaging module may be improved by providing a smaller incidence angle across the entire beam, thereby making the transmission edge sharper and allowing better discrimination between different fluorescence emission bands.

[0189] Figure 4 is a graph showing the relationship between beam footprint size (DBS) and the beam incidence angle (DBS angle) on a dichroic filter. In some instances, a smaller beam footprint may be desirable. For example, a smaller beam footprint allows for the use of a smaller dichroic filter to split a beam into different wavelength ranges. Using a smaller dichroic filter reduces manufacturing costs and improves the ease of fabricating a properly flat dichroic filter. As shown in Figure 4, any incidence angle greater than 0 degrees (e.g., perpendicular to the surface of the dichroic filter) results in an elliptical beam footprint with an area larger than the cross-sectional area of ​​the beam. A 45-degree incidence angle results in a large beam footprint on the dichroic reflector that is 1.4 times larger than the cross-sectional area of ​​the beam when incident at 0 degrees.

[0190] 5A and 5B schematically illustrate non-limiting example configurations of dichroic filters and detection channels in a multichannel fluorescence imaging module in which the dichroic mirrors are positioned at an angle of 45 degrees or less relative to the central beam axis of the emission light or the optical axis of the light path (e.g., the objective lens and / or tube lens). FIG. 5A illustrates an imaging module 500 including multiple detection channels 520a, 520b, 520c, and 520d. FIG. 5B is a detailed view of the portion of imaging module 500 within circle 5B shown in FIG. 5A. As described in more detail below, the configurations illustrated in FIGS. 5A and 5B include many aspects that may provide significant improvements over conventional multichannel fluorescence imaging module designs. However, in some examples, the fluorescence imaging modules and systems of the present disclosure may be implemented using one or a subset of the features described with respect to FIGS. 5A and 5B without departing from the spirit or scope of the present disclosure.

[0191] The imaging module 500 shown in Figure 5A 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 combine the excitation and detection light paths. In contrast to the designs shown in Figures 2A and 2B and 2C and 2D, the first dichroic filter 530 (e.g., a dichroic beam splitter or combiner) is configured to reflect light from the light source toward the objective lens 510 and the sample and transmit fluorescent emission from the sample into the detection channels 520a, 520b, 520c, and 520d. The second dichroic filter 535 splits the beam of emission light 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. Dichroic filter 540a transmits at least a portion of the first portion 550a of the emission light and reflects portion 550c into a third detection channel 520c. Dichroic filter 540b transmits at least a portion of the second portion 550b of the emission light and reflects portion 550d into a fourth detection channel 520d. While imaging module 500 is shown with four detection channels, in various embodiments, imaging module 500 may include more or fewer detection channels with more or fewer dichroic filters, as needed, to provide a portion of the emission light to each detection channel. For example, in some embodiments, features of imaging module 500 may be implemented with similar advantage in an imaging module that includes only two detection channels 520a, 520b and omits the additional dichroic filters 540a, 540b. In some implementations, only one detection channel may be included. Alternatively, three or more detection channels may be employed.

[0192] 5A may include some or all of the same or similar components as detection channel 120 shown in Figures 2A-3B. For example, the 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 the respective image sensors or photodetector arrays.

[0193] The objective lens 510 is positioned to receive emission light emitted by fluorescence from the specimen. In particular, the first dichroic filter 530 is positioned to receive the emission light collected and transmitted by the objective lens 510. As described above and shown in FIG. 5A , in some designs, an illumination light source such as a laser light source (e.g., illumination light source 115 in FIGS. 2A and 2B ) is positioned to provide an excitation beam that is incident on the first dichroic filter 530, which then reflects the excitation beam back to the same objective lens 510, which transmits the emission light, e.g., in an epifluorescence configuration. In some other designs, the illumination light source may be directed to the specimen by other optical components along a different optical path that do not include the same objective lens 510. In such configurations, the first dichroic filter 530 may be omitted.

[0194] Similarly, as described above and shown in FIG. 5A , the detection optics (e.g., including detection channels 520a, 520b, 520c, 520d and any optical components, such as dichroic filters 535, 540a, 540b, along the optical path between objective lens 510 and detection channels 520a, 520b, 520c, 520d) may be positioned on the transmission path of first dichroic filter 530 rather than on the reflection path of first dichroic filter 530. In one embodiment, objective lens 510 and detection optics are positioned such that objective lens 510 directly transmits beam of emission light 550 toward second dichroic filter 535. The wavefront quality of the emission light may be somewhat degraded by the presence of first dichroic filter 530 along the path of beam of emission light 550 (e.g., by imparting some wavefront error to beam 550). However, the wavefront error introduced by the beam transmitted through the dichroic reflector of the dichroic beamsplitter is typically significantly smaller (e.g., an order of magnitude smaller) than the wavefront error of the beam reflected from the dichroic reflecting surface of the dichroic beamsplitter. Thus, the wavefront quality of the emitted light and subsequent imaging quality in a multichannel fluorescence imaging module can be significantly improved by placing the detection optics along the transmitted beam path of the first dichroic filter 530 rather than along the reflected beam path.

[0195] 5A , within the detection optics of imaging module 500, dichroic filters 535, 540a, and 540b are provided to split beam 550 of emission light among detection channels 520a, 520b, 520c, and 520d. For example, dichroic filters 535, 540a, and 540b split beam 550 based on wavelength such that a first wavelength or wavelength band of emission light can be received by first detection channel 520a, a second wavelength or wavelength band of emission light can be received by second detection channel 520b, a third wavelength or wavelength band of emission light can be received by third detection channel 520c, and a fourth wavelength or wavelength band of emission light can be received by fourth detection channel 520d. In some implementations, multiple separate wavelengths or wavelength bands can be received by the detection channels.

[0196] In contrast to the multichannel fluorescence imaging module designs shown in Figures 2A and 2B and 2C and 2D, imaging module 500 has dichroic filters 535, 540a, and 540b positioned at angles of incidence within 45 degrees relative to the central beam axis of the incident beam. As shown in Figure 5B, different beams 550, 550a, and 550b have respective central beam axes 552, 552a, and 552b. In various implementations, the central beam axes 552, 552a, and 552b are at the centers of the cross sections of the beams perpendicular to the beam propagation direction. These central beam axes 552, 552a, and 552b may correspond to the optical axes of the objective lens and / or the optical axes of the optics within the individual channels, such as the optical axes of the respective tube lenses. 5B shows additional rays 554, 554a, 554b of each beam 550, 550a, 550b to illustrate the diameter of each beam 550, 550a, 550b. The beam diameter may be defined, for example, as the full width at half the maximum diameter, D4σ (e.g., four times σ, where σ is the standard deviation of the horizontal or vertical marginal distribution of the beam, respectively), or second moment width, or any other suitable definition of beam diameter.

[0197] The central beam axis 552 of the beam of emission light 550 may serve as a reference point for defining the angle of incidence of the beam 550 with respect to the second dichroic filter 535. Thus, the "angle of incidence" (AOI) of the beam 550 may be the angle between the central beam axis 552 of the incident beam 550 and a line N that is normal to the surface on which the beam is incident, e.g., a dichroic reflective surface. When the beam of emission light 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 emission light (e.g., a portion having a wavelength within the passband region of the second dichroic filter 535) and reflects a second portion 550b of the emission light (e.g., a portion having a wavelength within the stopband region of the second dichroic filter 535). The first portion 550a and the second portion 550b may be similarly described with respect to the central beam axes 552a, 552b, respectively. As mentioned above, an optical axis may alternatively or additionally be used.

[0198] In the exemplary configurations of FIGS. 5A and 5B, the second dichroic filter 535 is positioned such that the central beam axis 552 of the beam 550 is incident at a 30-degree angle of incidence. Similarly, the additional dichroic filters 540a and 540b are positioned such that the central beam axes 552a and 552b of the first and second portions 550a and 550b of the beam 550 are also incident at a 30-degree angle of incidence. However, in various implementations, these angles of incidence may be other angles less than 45 degrees. In some examples, for example, the angles of incidence may range from about 20 degrees to about 45 degrees, as described further below. Furthermore, the angles of incidence on each of the dichroic filters 535, 540a, and 540b need not necessarily be the same. In some embodiments, some or all of the dichroic filters 535, 540a, and 540b may be positioned such that their incident beams 550, 550a, and 550b have different angles of incidence. As mentioned above, the angle of incidence may be relative to the optical axis of the optics in the imaging module, such as the objective lens and / or the optics in the detection channel (e.g., tube lens), and the dichroic reflective surface in the respective dichroic beam splitter. The same ranges and values ​​for the angle of incidence apply when the optical axis is used to identify the AOI.

[0199] The emission light beams 550, 550a, 550b in a fluorescence imaging module system are typically diverging beams. As described above, the emission light beams can have such a large beam divergence that a beam region within the beam diameter is incident on a dichroic filter at an angle of incidence that differs by up to 5 degrees or more from the angle of incidence of the central beam axis and / or optical axis of the optical system. In some designs, the objective lens 510 can be configured, for example, with an f-number or numerical aperture 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 overall diameter of the beams 550, 550a, 550b is incident on the dichroic filters 535, 540a, 540b at an angle of incidence that is, for example, within 1 degree, 1.5 degrees, 2 degrees, 2.5 degrees, 3 degrees, 3.5 degrees, 4 degrees, 4.5 degrees, or 5 degrees of the angle of incidence of the central beam axis 552, 552a, 552b.

[0200] In some implementations, the focal length of an objective lens suitable for producing such a narrow beam diameter may be longer than those typically used in fluorescence microscopes or imaging systems. For example, in some implementations, the focal length of the objective lens may range from 20 mm to 40 mm, as discussed further below. In one example, an objective lens 510 with a focal length of 36 mm may produce a beam 550 characterized by a divergence small enough that light across the entire diameter of the beam 550 enters the second dichroic filter 535 at an angle within 2.5 degrees of the angle of incidence of the central beam axis.

[0201] 6 and 7 provide graphs showing improved dichroic filter performance according to embodiments of the imaging module configurations of FIGS. 5A and 5B (or any of the imaging module configurations disclosed herein). The graph in FIG. 6 is similar to the graph in FIG. 3 and illustrates the effect of incidence angle on the transition width (e.g., the spectral span of the transmission edge) of a dichroic filter. FIG. 6 shows an example in which dichroic filters (e.g., dichroic filters 535, 540a, and 540b) and the dichroic reflective surfaces therein are oriented such that the incident beam has an angle of incidence of 30 degrees instead of 45 degrees. FIG. 6 also shows how this reduced angle of incidence significantly improves the sharpness and uniformity of the transition width across the beam diameter. For example, 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, whereas 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 is reduced from approximately 51 nm to approximately 23 nm, indicating a sharper transition between the passband and stopband. Additionally, the variation in transition width across the beam diameter is reduced by approximately 40%, from a range of 22 nm to a range of 14 nm, indicating a more uniformly sharp transition across the entire beam area.

[0202] FIG. 7 illustrates an additional benefit that can be realized in any of the imaging module configurations disclosed herein by selecting an appropriate f-number or numerical aperture of the objective lens to reduce beam divergence. In some implementations, longer focal lengths are used. In the example of FIG. 7, the focal length of objective lens 510 is 36 mm, which, with an appropriate numerical aperture (e.g., less than 5), reduces the range of incident angles within beam 550 to 30°±5° to 30°±2.5°. This design can reduce the range of transition widths to approximately 19 nm to approximately 26 nm. Compared to the improved system of FIG. 6, the average transition width is substantially the same (e.g., the spectral span is approximately 23 nm), but the variation in transition width across the beam diameter is further reduced to a range of 7 nm, representing approximately a 70% reduction compared to the transition width range illustrated in FIG. 3.

[0203] Referring again to FIG. 4, reducing the angle of incidence at the central beam axis from 45 degrees to 30 degrees is even more advantageous because it reduces the beam spot size on the dichroic filters. As shown in FIG. 4, at a 45-degree angle of incidence, the beam footprint on the dichroic filters has an area greater than 1.4 times the cross-sectional area of ​​the beam. However, at a 30-degree angle of incidence, the beam footprint on the dichroic filters has an area only approximately 1.15 times the cross-sectional area of ​​the beam. Therefore, reducing the angle of incidence on dichroic filters 535, 540a, and 540b from 45 degrees to 30 degrees reduces the area of ​​the beam footprint on dichroic filters 535, 540a, and 540b by approximately 18%. This reduction in beam footprint area allows for the use of smaller dichroic filters.

[0204] Referring now also to FIGS. 8A-8B, reducing the angle of incidence from 45 degrees to 30 degrees can also improve performance with respect to surface deformations induced by dichroic filters in any of the imaging module configurations disclosed herein, as indicated by an improved modulation transfer function. Generally, the larger the area of ​​an optical element, the greater the amount of surface deformation. When a larger area dichroic filter is used, the greater the amount of surface deformation, thereby introducing more wavefront error into the beam. FIG. 8A illustrates the effect of fold angle on image degradation caused by adding 1 wave of peak-to-valley (PV) spherical power to the final mirror. FIG. 8B illustrates the effect of fold angle on image degradation caused by adding 0.1 wave of PV spherical power to the final mirror. As shown in FIGS. 8A and 8B, reducing the angle of incidence to 30 degrees significantly reduces the effect of surface deformations, achieving near-diffraction-limited performance for the detection optics.

[0205] In some implementations of the disclosed imaging modules, utilizing the polarization state of the excitation beam can further improve the performance of the multichannel fluorescence imaging modules disclosed herein. For example, referring back to FIGS. 2A, 2B, and 5A, some implementations of the multichannel fluorescence imaging modules disclosed herein have an epi-fluorescence configuration in which a first dichroic filter 130 or 530 combines the optical paths of the excitation and emission light beams, thereby transmitting both the excitation and emission light through the objective lens 110, 510. As discussed above, the illumination light source 115 can 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 can 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, can be included in the optical path of the excitation beam to polarize the excitation beam. A retarder, such as a half-wave retarder or multiple quarter-wave retarders, or a retarder with other amounts of retardance, may be included in some designs to rotate linear polarization.

[0206] When the linearly polarized excitation beam is incident on any dichroic filter or other planar interface, it may be p-polarized (e.g., with an electric field component parallel to the plane of incidence), s-polarized (e.g., with an electric field component perpendicular to the plane of incidence), or may have a combination of p- and s-polarization states within the beam. The p- or s-polarization state of the excitation beam can be selected and / or changed by selecting the orientation of the illumination source 115 and / or one or more components thereof relative to the first dichroic filter 130, 530 and / or relative to any other surface with which the excitation beam interacts. In some implementations in which the light source outputs linearly polarized light, the light source can be configured to provide s-polarized light. For example, the light source may comprise a light emitter, such as a solid-state laser or laser diode, that can be rotated about its optical axis or central beam axis to adjust the orientation of the linearly polarized light output therefrom. Alternatively, or additionally, a retarder can be used to rotate the linearly polarized light about the optical axis or central beam axis. As mentioned above, in some implementations, for example, if the light source does not output polarized light, the excitation beam can be polarized by a polarizer disposed in the path of the excitation beam. For example, in some designs, a linear polarizer is disposed in the path of the excitation beam. By rotating this polarizer, the linearly polarized light can be properly oriented to provide s-polarized light.

[0207] In some designs, the linearly polarized light can be rotated around the optical axis or central beam axis so that s-polarized light is incident on the dichroic reflector of the dichroic beamsplitter. When s-polarized light is incident on the dichroic reflector of the dichroic beamsplitter, the transition between the passband and the stopband becomes sharper, as opposed to when p-polarized light is incident on the dichroic reflector of the dichroic beamsplitter.

[0208] As shown in Figures 9A and 9B, the use of p- or s-polarization states of the excitation beam can significantly affect the narrowband performance of any excitation filter, such as the first dichroic filter 130, 530. Figure 9A shows the transmission spectrum between 610 nm and 670 nm of an exemplary passband dichroic filter at 40- and 45-degree angles of incidence, where the incident beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. As shown in Figure 9B, 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 significantly sharpens the edge between the passband and stopband of the dichroic filter. Therefore, the illumination and imaging modules 100, 500 disclosed herein may advantageously have the illumination light source 115 oriented relative to the first dichroic filter 130, 530 so that the excitation beam is s-polarized relative to the plane of the first dichroic filter 130, 530. As discussed above, in some implementations, a polarizer, such as a linear polarizer, may be used to polarize the excitation beam. This polarizer may be rotated to provide a linear polarization orientation corresponding to s-polarized light. Also, as discussed above, in some implementations, other approaches for rotating the linear polarization may be used. For example, an optical retarder, such as a half-wave retarder or multiple quarter-wave retarders, may be used to rotate the polarization direction. Other arrangements are also contemplated.

[0209] As discussed elsewhere herein, reducing the numerical aperture (NA) of the fluorescence imaging module and / or objective lens can increase the depth of field and enable comparable imaging of multiple surfaces, e.g., three, four, or more surfaces. Figures 10A-15B show how the MTF is more similar for a first surface and a second surface separated by 1 mm of glass for smaller numerical apertures than for larger numerical apertures.

[0210] 10A and 10B show the MTF at the first surface (FIG. 10A) and the second surface (FIG. 10B) when the NA is 0.3.

[0211] 11A and 11B show the MTF at the first surface (FIG. 11A) and the second surface (FIG. 11B) when the NA is 0.4.

[0212] 12A and 12B show the MTF at the first surface (FIG. 12A) and the second surface (FIG. 12B) when the NA is 0.5.

[0213] 13A and 13B show the MTF at the first surface (FIG. 13A) and the second surface (FIG. 13B) when the NA is 0.6.

[0214] 14A and 14B show the MTF at the first surface (FIG. 14A) and the second surface (FIG. 14B) when the NA is 0.7.

[0215] Figures 15A and 15B show the MTF at the first surface (Figure 15A) and the second surface (Figure 15B) when the NA is 0.8. The first and second surfaces in these figures correspond to, for example, the top and bottom surfaces of a flow cell.

[0216] 16A-16B provide plots of calculated Strehl ratios (e.g., the ratio of the peak light intensity focused or collected by an optical system to the peak light intensity focused or collected by an ideal optical system and point light source) for imaging a second flow cell surface through a first flow cell surface. FIG. 16A shows plots of the Strehl ratio for imaging a second flow cell surface through a first flow cell surface as a function of the thickness of the intervening fluid layer (height of the fluid channel) for different objective and / or optical system numerical apertures. In some embodiments, the Strehl ratio decreases as the separation between two adjacent surfaces, e.g., the first and second surfaces, increases. Thus, one of the surfaces may experience a decrease in image quality as the separation between the two surfaces increases. The decrease in surface imaging performance as the separation distance between two adjacent surfaces increases is reduced in imaging systems with smaller numerical apertures, e.g., NAs of 0.5 or 0.4, compared to imaging systems with larger numerical apertures. Figure 16B shows a plot of the Strehl ratio as a function of numerical aperture for imaging a first flow cell surface and a second flow cell surface through an intervening 0.1 mm layer of water. The loss in imaging performance at higher numerical apertures can be attributed to the increase in optical aberrations introduced by the fluid for imaging the second surface. As the numerical aperture increases, the image quality can be significantly degraded due to the increase in optical aberrations introduced by the fluid for imaging the second, third, or fourth surfaces. However, generally, reducing the numerical aperture of an optical system reduces the achievable resolution. This loss in image quality can be at least partially offset by increasing the contrast-to-noise ratio at the sample plane (or object plane), for example, by using chemicals for nucleic acid sequencing applications that enhance the fluorescence emission of labeled nucleic acid clusters and / or reduce background fluorescence emission. In some examples, a sample support structure including, for example, a hydrophilic substrate material and / or a hydrophilic coating can be employed. In some examples, such a hydrophilic substrate and / or hydrophilic coating can reduce background noise.Further discussion of sample support structures, hydrophilic surfaces and coatings, and methods for enhancing contrast-to-noise ratios for, for example, nucleic acid sequencing applications can be found below.

[0217] In some implementations, 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 may be configured to have a reduced magnification, such as a magnification of less than 10x, as discussed further below. Such reduced magnification may accommodate design constraints to achieve other design parameters. For example, any one or more of the fluorescence microscope, illumination and imaging module 100, imaging optics (e.g., optics 126), objective lens, or tube lens may also be configured to provide the fluorescence imaging module with a wide field of view (FOV), such as a field of view (e.g., diameter, width, height, or longest dimension) of at least 3.0 mm or greater, 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 lens, and / or tube lens may be configured to provide the fluorescence microscope with a field of view such that the FOV has an aberration of less than 0.1 waves, for example, across 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., optical system 126), objective lens, and / or tube lens may be configured such that the fluorescence imaging module has such an FOV and is diffraction limited, or is diffraction limited over such an FOV.

[0218] As discussed above, in various implementations, a wide field of view (FOV) is provided by the disclosed optical system. In some implementations, obtaining an increased FOV is facilitated, in part, by the use of a larger image sensor or photodetector array. For example, the photodetector array may have an active area with a diagonal of at least 15 mm or more, as discussed further below. As discussed above, in some implementations, the disclosed optical imaging system provides a reduced magnification, e.g., less than 10x, which may facilitate wide FOV designs. Despite the reduced magnification, the optical resolution of the imaging module may still be sufficient because a detector array with a small pixel size or pitch may be used. The pixel size and / or pitch may be, for example, about 5 μm or less, as discussed in further detail below. In some implementations, the pixel size is less than twice the optical resolution provided by the optical imaging system (e.g., the objective lens and tube lens) to satisfy the Nyquist theorem. Thus, the pixel dimensions and / or pitch of the image sensor(s) may be such that the spatial sampling frequency of the imaging module is at least two times the optical resolution of the imaging module. For example, the spatial sampling frequency of the photodetector array may be at least two times, at least 2.5 times, at least three times, at least four times, or at least five times the optical resolution of the fluorescence imaging module (e.g., the illumination and imaging module, the objective lens and tube lens, the objective lens and optics 126 in the detection channel, the sample support structure configured to support the sample support stage or imaging optics between the stage and the photodetector array), or any spatial sampling frequency in a range between any of these values.

[0219] Although broad features are described herein with respect to fluorescence imaging modules, any of the features and designs discussed herein may be applied to other types of optical imaging systems, including, but not limited to, bright-field and dark-field imaging, and may be applied to luminescence or phosphorescence imaging.

[0220] 18. Dual-wavelength excitation / four-channel imaging system Figure 17 shows a dual-excitation wavelength / four-channel imaging system for two- or four-surface imaging applications, including a combination of an objective lens and a tube lens that is scanned perpendicular to the optical axis to provide large-area imaging, for example, by tiling several images to create a composite image with a total field of view (FOV) much wider than each of the individual images. The system includes two excitation light sources, e.g., lasers or laser diodes, operating at different wavelengths and an autofocus laser. The two excitation light beams and the autofocus laser light beam are combined using a series of mirrors and / or dichroic reflectors and delivered to the flow cell surface through the objective lens. Fluorescence emitted by a labeled oligonucleotide (or other biomolecule) tethered to one of the flow cell surfaces is collected by the objective lens, transmitted through the tube lens, and directed by a series of intermediate dichroic reflectors to one of four image sensors depending on the wavelength of the emitted light. Autofocus laser light reflected from the flow cell surface is collected by the objective lens, transmitted through a tube lens, and directed to the autofocus sensor by a series of intermediate dichroic reflectors. This system allows precise focus to be maintained while the objective / tube lens combination is scanned in a direction perpendicular to the objective's optical axis (e.g., using a high-precision linear actuator, translation stage, or a focus adjustment mechanism mounted on the microscope turret to adjust the relative distance between the flow cell surface and the objective lens, thereby reducing or minimizing the reflected light spot size on the autofocus image sensor). Dual-wavelength excitation, used in combination with four-channel (e.g., four-wavelength) imaging capability, provides high-throughput imaging of multiple surfaces of the flow cell.

[0221] 19. Super-resolution optical system The super-resolution imaging techniques described herein can be used to image very small sample site features that exist at high surface densities, such as nucleic acid polonies (e.g., spots containing amplified target nucleic acids). In some embodiments, the stochastic light switching techniques described herein can be used to improve image resolution. Alternatively, the structured illumination techniques described herein can be used to improve image resolution in optical systems. In some examples, the super-resolution imaging techniques can include structured illumination.

[0222] In some instances, improved imaging performance for multi-surface (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or cover glass) thickness greater than 700 μm) and fluidic channels (e.g., fluidic channel height or thickness between 50 and 200 μm) can be achieved by using novel objective lens designs that correct for optical aberrations introduced by the thick cover glass and / or the imaging surface of the fluidic channel opposite the objective lens.

[0223] In some instances, improved imaging performance for multi-surface (flow cell) imaging applications, including those involving the use of thick flow cell walls (e.g., wall (or cover glass) thickness greater than 700 µm) and fluidic channels (e.g., fluidic channel height or thickness between 50 and 200 µm), can be achieved using novel tube lens designs, even when using commercially available, off-the-shelf objective lenses, unlike conventional microscope tube lenses that only form an image at an intermediate image plane and, in combination with the objective lens, correct for optical aberrations caused by the thick flow cell walls and / or intervening fluid layers.

[0224] In some examples, for example, improved imaging performance for multi-channel (e.g., two-color or four-color) imaging applications may be achieved by using multiple tube lenses (one for each imaging channel), with each tube lens design optimized for the particular wavelength range used in that imaging channel.

[0225] In some examples, for example, improved imaging performance for multi-surface (flow cell) imaging applications can be achieved by using an electro-optic phase plate in combination with an objective lens to compensate for optical aberrations caused by a fluid layer separating the upper (near) and lower (far) inner surfaces of the flow cell. In some examples, 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.

[0226] Various multichannel fluorescence imaging module designs have been disclosed that may include illumination and imaging optical paths that include a folding optical path (e.g., including one or more beam splitters or beam combiners, such as dichroic beam splitters or combiners) that direct an excitation light beam to an objective lens and direct emission light transmitted through the objective lens to multiple detection channels. Some particularly advantageous features of the fluorescence imaging modules described herein include specifying a dichroic filter angle of incidence to create a sharper and / or more uniform transition between the wavelength ranges of the dichroic filter's passband and stopband. Such filters may be included within the folding optical system and may include a dichroic beam splitter or combiner. Additional advantageous features of the disclosed imaging optical system designs include the position and orientation of one or more excitation light sources and one or more detection optical paths relative to the objective lens and the dichroic filter that receives the excitation beam. The excitation beam may be linearly polarized, and the orientation of the linear polarization may be such that s-polarized light is incident on the dichroic reflective surface of the dichroic filter. Such features may improve filtering of the excitation beam and / or reduce wavefront errors introduced into the emission light beam by surface deformations of the dichroic filter. The fluorescence imaging modules described herein may or may not include any of these features and may or may not include any of these advantages.

[0227] Also described herein are devices and systems configured to analyze multiple different nucleic acid sequences, for example, by imaging arrays of immobilized nucleic acid molecules or amplified nucleic acid clusters formed on a flow cell surface. The devices and systems described herein may also be useful, for example, for performing sequencing for comparative genomics, tracking gene expression, performing microRNA sequence analysis, epigenomics, characterizing aptamer and phage display libraries, and performing other sequencing applications. The devices and systems disclosed herein include various combinations of optical, mechanical, fluidic, thermal, electrical, and computing devices / aspects. Advantages offered by the disclosed flow cell devices, cartridges, and systems include, but are not limited to, (i) reduced device and system manufacturing complexity and cost, (ii) significantly reduced consumable costs (e.g., compared to currently available nucleic acid sequencing systems), (iii) compatibility with common flow cell surface functionalization methods, (iv) flexible flow control when combined with microfluidic components such as syringe pumps and diaphragm valves, and (v) flexible system throughput.

[0228] Disclosed herein are capillary flow cell devices and cartridges constructed from prefabricated, disposable, single-lumen (e.g., single fluid flow path) or multi-lumen capillaries, which may also include a fluidic adapter, a cartridge chassis, one or more integrated fluid flow control components, or any combination thereof. Also disclosed herein are capillary flow cell-based systems that 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 control module, a temperature control module, an imaging module, or any combination thereof.

[0229] Design features of some of the disclosed capillary flow cell devices, cartridges, and systems include, but are not limited to: (i) a single flow path architecture; (ii) sealed, reliable, and repeatable switching between reagent streams that can be implemented with a simple load / unload mechanism to ensure the fluid interface between the system and capillary is sealed, thereby facilitating capillary replacement and system reuse and allowing precise control of reaction conditions such as reagent concentration, pH, and temperature; (iii) interchangeable single fluid flow path devices or capillary flow cell cartridges with multiple flow paths that can be used interchangeably to provide flexible system throughput; and (iv) compatibility with various detection methods, such as fluorescence imaging.

[0230] 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 described primarily in the context of their use for nucleic acid sequencing applications, various aspects of the disclosed devices and systems may be applied not only to nucleic acid sequencing but also to any other type of chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. It is understood that various aspects of the disclosed methods, devices, and systems can be evaluated individually, collectively, or in combination with one another. While discussed primarily in the context of fluorescence imaging (including, for example, fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, etc.), those skilled in the art will appreciate that many of the disclosed optical design approaches and features are also applicable to other imaging modes, such as bright-field imaging, dark-field imaging, and phase-contrast imaging.

[0231] (a) Multiplexed optical readhead In some examples, miniaturized versions of any of the imaging modules described herein can be assembled to create a multiplexed readhead that can be translated horizontally in one or more directions relative to a sample surface, e.g., the inner surface of a flow cell, to simultaneously image multiple portions of the surface. A non-limiting example of a multiplexed readhead was recently described in U.S. Patent Application No. 16 / 735,001, which is incorporated herein by reference in its entirety.

[0232] For example, in some examples, the compact imaging module may include a "microfluorometer" including an illumination or excitation light source, such as an LED or laser diode (or the tip of an optical fiber connected to an external light source), one or more lenses for collimating or focusing the illumination or excitation light, one or more dichroic reflectors, one or more optical filters, one or more mirrors, beam splitters, prisms, apertures, etc., one or more objective lenses, one or more custom tube lenses that enable multiple surface imaging with minimal focus adjustment as described elsewhere herein, one or more image sensors as described elsewhere herein, or any combination thereof. In some examples, the compact imaging module (e.g., a "microfluorometer") may further include an autofocus mechanism, a microprocessor, power and data transfer connectors, a light-tight housing, etc. Thus, the resulting compact imaging module may comprise an integrated imaging package or unit having a compact form factor. In some examples, the smallest dimension (e.g. width or diameter) of the miniature imaging module may be less than 5 cm, less than 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, less than 1.2 cm, less than 1 cm, less than 0.8 cm, or less than 0.6 cm. In some examples, the longest dimension (e.g. height or length) of the miniature imaging module may be less than 16 cm, less than 14 cm, less than 12 cm, less than 10 cm, less than 9 cm, less than 8 cm, less than 7 cm, less than 5 cm, less than 5 cm, less than 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, less than 1.2 cm, or less than 1 cm. In some examples, one or more individual miniature imaging modules in a multiplexed readhead may be equipped with an autofocus mechanism.

[0233] In some examples, the multiplexed readheads described herein may include an assembly of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 or more miniature imaging modules or microfluorometers held in fixed positions relative to one another. In some examples, the optical design specifications and performance characteristics, such as numerical aperture, field of view, depth of field, image resolution, etc., of the individual miniature imaging modules or microfluorometers may be the same as those described elsewhere herein for other versions of the disclosed imaging modules. In some examples, the multiple individual miniature imaging modules may be arranged in a linear arrangement including 1, 2, 3, 4, 5, or more rows and / or columns. In some examples, the multiple individual miniature imaging modules may be arranged in a close-packed arrangement, for example, a hexagonal arrangement. In some examples, the multiple individual miniature imaging modules may be arranged in a circular or spiral arrangement, a randomly distributed arrangement, or any other arrangement known to those skilled in the art.

[0234] 43A-43B provide non-limiting schematic diagrams of a multiplexed readhead disclosed herein. FIG. 43A shows a side view of a multiplexed readhead in which two rows of individual microfluorometers (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., the first inner surface of a flow cell. FIG. 43B shows a top view of the same multiplexed readhead, illustrating the overlapping imaging paths acquired by the individual microfluorometers of the multiplexed readhead as the readhead is translated relative to the flow cell (or vice versa). In some examples, the individual fields of view of the individual microfluorometers may overlap, as shown in FIG. 43B. In some examples, they may not overlap. In some examples, the multiplexed readhead may be designed to align and image predetermined features within the flow cell, e.g., individual fluidic channels.

[0235] 44A-44B are non-limiting schematic diagrams of a multiplexed readhead in which a first subset of a plurality of individual miniature imaging modules is configured to image a first sample surface, e.g., a first inner surface of a flow cell, and a second subset of a plurality of individual miniature imaging modules is configured to simultaneously or sequentially image a second sample surface, e.g., a second inner surface of the flow cell. FIG. 44A shows a side view of a multiplexed readhead in which a first subset of individual microfluorometers is configured to image, e.g., a first inner or upper inner surface of a flow cell, and a second subset is configured to image a second surface, e.g., a second inner or lower inner surface of a flow cell. FIG. 44B shows a top view of the multiplexed readhead of FIG. 44A, illustrating the imaging paths acquired by the individual microfluorometers of the multiplexed readhead. Also, in some instances, the individual fields of view of the individual microfluorometers within a given subset may overlap. In some instances, th...

Claims

1. 1. An optical system for sequencing nucleic acids, comprising: 1.0 square millimeter (mm ２ an objective lens having a field of view (FOV) greater than an excitation energy source configured to irradiate one or more surfaces of the flow cell; at least one image sensor configured to acquire one or more images of the one or more surfaces of the flow cell, the one or more surfaces being axially displaced from one another along an optical axis of the optical system; The optical system exhibits a root-mean-square (RMS) wavefront error of less than 0.09λ, where λ is a central wavelength of the excitation energy source, the RMS wavefront error being measured using a Shack-Hartmann wavefront sensor across the FOV of the objective lens using broadband illumination from the excitation energy source, and the RMS wavefront error being an average of individual RMS wavefront error values ​​for at least three color channels.

2. 10. The optical system of claim 1, wherein the RMS wavefront error is for a field of view (FOV) of about 1.5 mm in an x- or y-direction orthogonal to an axial axis.

3. 2. The optical system of claim 1, wherein the one or more surfaces include four surfaces, and the RMS wavefront error is greater for a fourth surface than for a first, second, or third surface of the four surfaces.

4. The optical system of claim 1 , wherein the RMS wavefront error of the optical system is less than the diffraction limit of the optical system.

5. The optical system of claim 1 , wherein the optical system has a numerical aperture (NA) of less than 0.

7.

6. 10. The optical system of claim 1, wherein the optical resolution of the optical system is sufficient to resolve two objects bound to the one or more surfaces of the flow cell, the distance between the two objects being at least about 500 nm.

7. The optical system operates over a 1 mm ２ 10. The optical system of claim 1, configured to acquire the one or more images of the one or more surfaces of the flow cell in less than 20 minutes per acquisition.

8. The optical system of claim 1 , wherein the one or more images of the one or more surfaces of the flow cell are from two or more different color channels.

9. The optical system of claim 8 , wherein the one or more images of the one or more surfaces of the flow cell are from four different color channels.

10. The optical system of claim 1 , wherein the optical system is configured to complete an imaging cycle in less than six minutes.

11. 10. The optical system of claim 1, further comprising a flow cell, wherein the one or more images of the one or more surfaces of the flow cell include optical signals emitted from samples immobilized on the one or more surfaces of the flow cell.

12. The optical system of claim 1 , wherein the sample comprises an in situ sample of cells, tissue, or both.

13. The excitation energy source is configured to cover less than 1 mm of the one or more surfaces of the flow cell. ２ 10. The optical system of claim 1, configured to uniformly illuminate an area greater than 100 nm and to have illumination power vary by less than 10% across the illuminated area.

14. The optical system of claim 1 , wherein the objective lens comprises an optical aperture stop having an adjustable size configured to change the NA of the optical system.

15. 15. The optical system of claim 14, wherein an optical aperture stop is configured to vary the NA of the optical system in the range of 0.4 to 0.

6.

16. The optical system of claim 1 , wherein the one or more images of the one or more surfaces of the flow cell are acquired without moving an optical compensator into the optical path of the optical system.

17. The optical system of claim 1 , wherein the flow cell has a top or bottom wall thickness of at least 700 micrometers (μm) along an axis perpendicular to an image plane.

18. The optical system of claim 1 , wherein the flow cell has a gap of at least 50 μm along an axial direction perpendicular to the image plane in the first fluid channel or the second fluid channel of the flow cell.

19. 10. The optical system of claim 1, wherein the flow cell has an interposer between a first fluid channel and a second fluid channel of the flow cell that is at least 500 μm along an axial direction perpendicular to the image plane.

20. The optical system of claim 1; a controller comprising at least one processor.

21. 21. The system of claim 20, wherein the at least one processor is configured to execute executable instructions for correcting optical aberrations.

22. 21. The system of claim 20, wherein the at least one processor is configured to execute executable instructions to generate an optical resolution approximately the same as a resolution of the one or more images of the one or more surfaces of the flow cell.

23. The at least one processor is programmed to instruct the system to iteratively perform a sequencing method, the sequencing method comprising: contacting the plurality of primed target nucleic acid sequences bound to the one or more surfaces with a nucleotide conjugate under conditions sufficient to form a binding complex between the primed target nucleic acid sequence of one of the plurality of primed target nucleic acid sequences and a nucleotide moiety of the nucleotide conjugate if the nucleotide moiety is complementary to a nucleotide of the primed target nucleic acid sequence; imaging the one or more surfaces of the flow cell to detect the binding complexes, thereby determining the identity of the nucleotides of the primed target nucleic acid sequence.

24. 24. The system of claim 23, wherein the imaging to detect the binding complex is performed in the absence of incorporation of the nucleotide moiety into the primed nucleic acid sequence.

25. 24. The system of claim 23, wherein the plurality of primed target nucleic acid sequences each comprise a primer sequence comprising a reversible terminator portion sufficient to prevent incorporation of the nucleotide portion into the primed nucleic acid sequence during the imaging.

26. The binding complex comprises: the plurality of target nucleic acid sequences; two or more of the nucleotide moieties attached to the plurality of target nucleic acid sequences; 24. The system of claim 23, optionally comprising two or more DNA polymerase molecules.

27. 24. The system of claim 23, wherein the sequencing method is incorporation sequencing.

28. 24. The system of claim 23, wherein the sequencing-by-incorporation comprises real-time sequencing-by-synthesis (SBS).

29. The optical system of claim 1; the flow cell; a fluidic system configured to deliver one or more reagents to the flow cell.

30. 1. An optical system for sequencing nucleic acids, comprising: an excitation energy source configured to irradiate one or more surfaces of a flow cell, the one or more surfaces being axially displaced from one another along an optical axis of the optical system; and 1.0 mm ２ an objective lens having a field of view (FOV) of more than at least one image sensor configured to acquire one or more images of the one or more surfaces of the flow cell; An optical system, wherein the optical resolution of the optical system is sufficient to resolve two objects immobilized on the one or more surfaces of the flow cell, and the distance between the two objects is at least about 0.5 micrometers (μm) in a plane perpendicular to the optical axis of the optical system.

31. 31. The optical system of claim 30, wherein the distance between the two objects is at least about 400 nm.

32. 31. The optical system of claim 30, wherein the optical system further comprises a numerical aperture (NA) less than 0.

7.

33. 31. The optical system of claim 30, wherein the distance between the two objects is at least about 500 nm.

34. The optical system operates over a 1 mm ２ 31. The optical system of claim 30, configured to acquire the flow cell images in less than 20 minutes per image.

35. 31. The optical system of claim 30, wherein the one or more images of the one or more surfaces of the flow cell are from two or more different color channels.

36. 31. The optical system of claim 30, wherein the one or more images of the one or more surfaces of the flow cell are from four different color channels.

37. 31. The optical system of claim 30, wherein the optical system is configured to complete an imaging cycle in less than six minutes.

38. 31. The optical system of claim 30, further comprising a flow cell, wherein the one or more images of the one or more surfaces of the flow cell include optical signals emanating from samples immobilized on the one or more surfaces of the flow cell.

39. 39. The optical system of claim 38, wherein the sample comprises an in situ sample of cells, tissue, or both.

40. The excitation energy source uniformly illuminates an area and is configured to illuminate less than 1 mm of the one or more surfaces of the flow cell. ２ 31. The optical system of claim 30, configured to produce an illuminated area of ​​more than 100 nm and having an illumination power variation of less than 10% across the illuminated area.

41. 31. The optical system of claim 30, wherein the objective lens comprises an optical aperture stop having an adjustable size configured to change the NA of the optical system.

42. 42. The optical system of claim 41, wherein an optical aperture stop is configured to vary the NA of the optical system in the range of 0.4 to 0.

6.

43. 31. The optical system of claim 30, wherein the one or more images of the one or more surfaces of the flow cell are acquired without moving an optical compensator into the optical path of the optical system.

44. 31. The optical system of claim 30, wherein the flow cell has a top or bottom wall thickness of at least 700 micrometers (μm) along an axis perpendicular to the image plane.

45. 31. The optical system of claim 30, wherein the flow cell has a gap of at least 50 μm in the first fluid channel or the second fluid channel of the flow cell along an axial direction perpendicular to the image plane.

46. 31. The optical system of claim 30, wherein the flow cell has an interposer between a first fluid channel and a second fluid channel of the flow cell that is at least 500 μm along an axial direction perpendicular to the image plane.

47. the optical system of claim 30; a controller comprising at least one processor.

48. 48. The system of claim 47, wherein the at least one processor is configured to process the one or more images of the one or more surfaces of the flow cell to correct for optical aberrations.

49. 48. The system of claim 47, wherein the at least one processor is configured to process the one or more images of the one or more surfaces of the flow cell to produce an optical resolution approximately the same as a resolution of the flow cell image.

50. The at least one processor is programmed to instruct the system to iteratively perform a sequencing method, the sequencing method comprising: contacting the plurality of primed target nucleic acid sequences bound to the one or more surfaces with a nucleotide conjugate to form a binding complex between one primed target nucleic acid sequence of the plurality of primed target nucleic acid sequences and the nucleotide moiety if the nucleotide moiety of the nucleotide conjugate is complementary to a nucleotide of the primed target nucleic acid sequence; imaging the one or more surfaces of the flow cell to detect the binding complexes, thereby determining the identity of the nucleotides of the primed target nucleic acid sequence.

51. 51. The system of claim 50, wherein the imaging to detect the binding complex is performed in the absence of incorporation of the nucleotide moiety into the primed nucleic acid sequence.

52. 51. The system of claim 50, wherein each of the plurality of primed target nucleic acid sequences comprises a primer sequence comprising a reversible terminator portion sufficient to prevent incorporation of the nucleotide portion into the primed nucleic acid sequence during the imaging.

53. The binding complex comprises: the plurality of target nucleic acid sequences; two or more of the nucleotide moieties attached to the plurality of target nucleic acid sequences; 51. The system of claim 50, optionally comprising two or more DNA polymerase molecules.

54. 51. The system of claim 50, wherein the sequencing method is incorporation sequencing.

55. 55. The system of claim 54, wherein the sequencing-by-incorporation comprises real-time sequencing-by-synthesis (SBS).

56. the optical system of claim 30; the flow cell; a fluidic system configured to deliver one or more reagents to the flow cell.

57. 1. An optical system for sequencing nucleic acids, comprising: an excitation energy source configured to irradiate one or more surfaces of a flow cell, the one or more surfaces being axially displaced from one another along an optical axis of the optical system; and 10mm ２ an objective lens having a field of view (FOV) of more than and at least one image sensor configured to acquire one or more images of the one or more surfaces of the flow cell, wherein the excitation energy source illuminates the FOV of the objective lens and has an incident light flux variation of less than 15% across the FOV.

58. 58. The optical system of claim 57, wherein the variation of the incident light flux across the FOV comprises a root mean square of an energy difference.

59. 58. The optical system of claim 57, wherein the variation of the incident light flux across the FOV comprises a ratio of the root mean square of energy difference to average energy level, the ratio being less than 15%.

60. 58. The optical system of claim 57, wherein the optical system further comprises a numerical aperture (NA) less than 0.

7.

61. 58. The optical system of claim 57, wherein the optical resolution of the optical system is sufficient to resolve two objects with a distance between the two objects of at least about 500 nm.

62. The optical system operates over a 1 mm ２ 58. The optical system of claim 57, configured to acquire the flow cell images in less than 20 minutes per image.

63. 58. The optical system of claim 57, wherein the one or more images of the one or more surfaces of the flow cell are from two or more different color channels.

64. 58. The optical system of claim 57, wherein the one or more images of the one or more surfaces of the flow cell are from four different color channels.

65. 58. The optical system of claim 57, wherein the optical system is configured to complete an imaging cycle in less than six minutes.

66. 58. The optical system of claim 57, further comprising a flow cell, wherein the flow cell image comprises an optical signal emanating from a sample immobilized on the one or more surfaces of the flow cell.

67. 67. The optical system of claim 66, wherein the sample comprises an in situ sample of cells, tissue, or both.

68. The excitation energy source is located within 1 mm of the flow cell. ２ 58. The optical system of claim 57, configured to uniformly illuminate an area of ​​more than 10% and to have illumination power vary by less than 10% across the illuminated area.

69. 58. The optical system of claim 57, wherein the objective lens comprises an optical aperture stop having an adjustable size configured to change the NA of the optical system.

70. 70. The optical system of claim 69, wherein an optical aperture stop is configured to vary the NA of the optical system in the range of 0.4 to 0.

6.

71. 58. The optical system of claim 57, wherein the one or more images of the one or more surfaces of the flow cell are acquired without moving an optical compensator into the optical path of the optical system.

72. 58. The optical system of claim 57, wherein the flow cell has a top or bottom wall thickness of at least 700 micrometers (μm) along an axis perpendicular to the image plane.

73. 58. The optical system of claim 57, wherein the flow cell has a gap of at least 50 μm along an axial direction perpendicular to the image plane in the first fluid channel or the second fluid channel of the flow cell.

74. 58. The optical system of claim 57, wherein the flow cell has an interposer between a first fluid channel and a second fluid channel of the flow cell that is at least 500 μm along an axial direction perpendicular to the image plane.

75. 58. The optical system of claim 57; a controller comprising at least one processor.

76. 76. The system of claim 75, wherein the at least one processor is configured to process the one or more images of the one or more surfaces of the flow cell to correct for optical aberrations.

77. 76. The system of claim 75, wherein the at least one processor is configured to process the flow cell image to produce an optical resolution approximately the same as the resolution of the one or more images of the one or more surfaces of the flow cell.

78. The at least one processor is programmed to instruct the system to iteratively perform a sequencing method, the sequencing method comprising: contacting the plurality of primed target nucleic acid sequences bound to the one or more surfaces with a nucleotide conjugate to form a binding complex between a primed target nucleic acid sequence of one of the plurality of primed target nucleic acid sequences and a nucleotide moiety if the nucleotide moiety of the nucleotide conjugate is complementary to a nucleotide of the primed target nucleic acid sequence; imaging the one or more surfaces of the flow cell to detect the binding complexes, thereby determining the identity of the nucleotides of the primed target nucleic acid sequence.

79. 79. The system of Claim 78, wherein said imaging to detect said binding complex is performed in the absence of incorporation of said nucleotide moiety into said primed nucleic acid sequence.

80. 79. The system of Claim 78, wherein each of the plurality of primed target nucleic acid sequences comprises a primer sequence comprising a reversible terminator portion sufficient to prevent incorporation of the nucleotide portion into the primed nucleic acid sequence during the imaging.

81. The binding complex comprises: the plurality of target nucleic acid sequences; two or more of the nucleotide moieties attached to the plurality of target nucleic acid sequences; 79. The system of claim 78, optionally comprising two or more DNA polymerase molecules.

82. 79. The system of claim 78, wherein the sequencing method is incorporation sequencing.

83. 83. The system of claim 82, wherein said sequencing by incorporation comprises real-time sequencing by synthesis (SBS).

84. 58. The optical system of claim 57; the flow cell; a fluidic system configured to deliver one or more reagents to the flow cell.

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