Optical system for nucleic acid sequencing and method thereof

Abstract

A fluorescence imaging system design is described that provides a larger field of view, increased spatial resolution, improved modulation transfer and image quality, higher spatial sampling frequencies, faster image capture-to-image capture transfer upon field of view repositioning, improved imaging system duty cycle, and a more compact system, thereby enabling higher throughput image acquisition and analysis at lower cost in genomics and other imaging applications.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 224,351, filed July 21, 2021, U.S. Provisional Patent Application No. 63 / 334,613, filed April 25, 2022, and U.S. Provisional Patent Application No. 63 / 334,609, filed April 25, 2022, each of 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 real-time, cyclic, or stepwise reaction schemes), dye molecules bound to nucleic acid molecules anchored on a substrate are excited using an excitation light source, causing fluorescence photon signals to be generated at one or more spatially localized locations on the substrate, which are then imaged onto an image sensor by an optical system. An analytical process is then used to analyze the image, locate the labeled molecules (or clusters of clonally amplified molecules) on the substrate, and quantify the fluorescence photon signals with respect to wavelength and spatial coordinates, which can then be correlated with the degree to which a specific chemical reaction, such as a hybridization event or base addition event, occurred at a specific location on the substrate. Imaging-based methods offer massive parallelism and multiplexing capabilities, helping to reduce the cost and availability of such techniques. However, detection errors, for example, resulting from overly dense packing of labeled molecules (or clusters of clonally amplified molecules) within a small area of ​​the substrate surface or due to low contrast-to-noise ratio (CNR) in the image, can lead to errors in attributing fluorescent signals to the correct molecules (or clusters of clonally amplified molecules). Summary of the Invention

[0003] Aspects disclosed herein provide a system comprising: a substrate including a curved surface including at least one binding moiety configured to bind to an analyte; and an optical system including a light source configured to direct light onto the curved surface, the light configured to probe the presence or absence of an analyte bound to the at least one binding moiety. In some embodiments, the analyte comprises a nucleic acid. In some embodiments, the at least one binding moiety comprises at least one nucleic acid configured to bind to the nucleic acid. In some embodiments, the curved surface is a component of a flow cell. In some embodiments, the system further comprises a flow cell including the curved surface. In some embodiments, the curved surface comprises a capillary of the flow cell. In some embodiments, the curved surface comprises glass, a polymer, or a combination thereof. In some embodiments, the light source is configured to probe the curved surface in an epifluorescence configuration. In some embodiments, the light source is configured to probe the curved surface in a transmission configuration. In some embodiments, the light source is a laser, a light emitting diode, a halogen lamp, or an incandescent lamp. In some embodiments, the light source is configured to generate light at a wavelength between about 500 nanometers (nm) and 540 nm, 620 nm and 650 nm, or 460 nm and 500 nm. In some embodiments, the system further comprises a second curved surface. In some embodiments, the system further comprises a focus-shifting assembly configured to move the focal field between the curved surface and the second curved surface. In some embodiments, the focus-shifting assembly comprises at least one movable lens. In some embodiments, the at least one movable lens is disposed within a lens barrel. In some embodiments, the focus-shifting assembly comprises at least one movable prism. In some embodiments, the curved surface and the second curved surface are different portions of a generally cylindrical component of the flow cell. In some embodiments, the second curved surface includes at least one second binding moiety configured to bind to a second analyte. In some embodiments, the optical system is movable relative to the curved surface. In some embodiments, the optical system is rotatable about the curved surface.In some embodiments, the optical system is configured to image a plurality of binding moieties. In some embodiments, the curved surface has a flatness of 25 micrometers (μm). In some embodiments, the curved surface has a flatness greater than the depth of focus of the optical system. In some embodiments, the system further comprises a plurality of sub-optical systems that are not parallel to one another. In some embodiments, each sub-optical system of the plurality of sub-optical systems is disposed perpendicular to a plurality of tangents to the curved surface. In some embodiments, the system further comprises a stage on which the curved surface is disposed. In some embodiments, the stage comprises a tilt stage, a rotation stage, a translation stage, or any combination thereof. In some embodiments, the curved surface comprises a hydrophilic polymer bonded thereto. In some embodiments, at least one binding moiety is bonded to the hydrophilic polymer. In some embodiments, the hydrophilic polymer is polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucan, poly-glycerol ... In some embodiments, the system comprises a coside, streptavidin, or dextran, or any combination thereof. In some embodiments, the system has a numerical aperture of up to about 0.6. In some embodiments, the numerical aperture is up to about 0.25. In some embodiments, the system further comprises an imaging sensor configured to collect light after directing it onto the curved surface. In some embodiments, the system further comprises a heater configured to heat the curved surface. In some embodiments, the heater is an integrated heater. In some embodiments, the heater is an infrared heater.

[0004] Aspects disclosed herein provide a system comprising a flow cell and an optical system comprising: a light source configured to direct a first light toward the flow cell; a filter configured to (i) receive a second light from the flow cell and (ii) transmit a third light, the third light including at least a portion of the second light but not the first light; and a sensor configured to receive the third light from the filter. In some embodiments, the system further comprises a focusing element assembly disposed between the light source and the filter, the focusing element assembly configured to focus the second light from the flow cell and the sensor. In some embodiments, the focusing element assembly comprises a first focusing element and a second focusing element, the first focusing element being disposed between the filter and the second focusing element along an optical path between the light source and the sensor. In some embodiments, the focusing element assembly comprises a wedge block assembly, the first focusing element including a first wedge piece and the second focusing element including a second wedge piece. In some embodiments, the first wedge piece and the second wedge piece are comprised of fused silica. In some embodiments, the first wedge piece and the second wedge piece have a refractive index comprising about 1.5. In some embodiments, the system further comprises a piezo drive coupled to the first wedge piece. In some embodiments, the system further comprises a gap between the first wedge piece and the second wedge piece. In some embodiments, the system further comprises a housing containing the flow cell. In some embodiments, the housing further contains a wedge block and a piezo drive in a wedge block-piezo drive assembly. In some embodiments, the wedge block-piezo drive assembly is disposed between the sensor and the flow cell. In some embodiments, the system further comprises a stage. In some embodiments, the stage is a tilt stage, a rotation stage, a translation stage, or any combination thereof. In some embodiments, the optical system further comprises an autofocus element configured for initial focus. In some embodiments, the system further comprises a lens barrel.In some embodiments, the autofocus element is housed within the lens barrel. In some embodiments, the flow cell includes one or more inner surfaces to which a hydrophilic polymer layer is bonded. In some embodiments, the flow cell further includes a plurality of biopolymers bonded to the hydrophilic polymer layer. In some embodiments, the flow cell includes a first inner surface and a second inner surface, the first inner surface being disposed between the sensor and the second inner surface. In some embodiments, the first inner surface and the second inner surface include biopolymers bonded thereto. In some embodiments, the hydrophilic polymer layer comprises polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, or dextran, or any combination thereof. In some embodiments, the filter comprises a multi-band filter. In some embodiments, the multi-band filter comprises a tri-band stopband filter. In some embodiments, the optical system further comprises imaging optics disposed between the filter and the flow cell. In some embodiments, the imaging optics have a demagnification comprising 1x. In some embodiments, the optical system has a demagnification of 1 millimeter squared (mm). 2) or greater. In some embodiments, the optical system has a numerical aperture (NA) including less than 0.6. In some embodiments, the NA includes about 0.25. In some embodiments, the sensor includes a plurality of imaging sensors configured to capture the FOV. In some embodiments, the light source includes a plurality of light sources including a first light source configured to emit a first light including a first wavelength range, a second light source configured to emit a second light including a second wavelength range, and a third light source configured to emit a third light including a third wavelength range, wherein the first wavelength range, the second wavelength range, and the third wavelength range are different wavelength ranges. In some embodiments, the first fluorophore excited by the first wavelength range of the first light source is different from the second fluorophore excited by the second wavelength range of the second light source. In some embodiments, the first fluorophore excited by the first wavelength range of the first light source is different from the second fluorophore excited by the second wavelength range of the second light source, and the second fluorophore excited by the second wavelength range of the second light source is different from the third fluorophore excited by the third wavelength range of the third light source. In some embodiments, the third fluorophore excited by the third wavelength range of the third light source is different from the first fluorophore excited by the first wavelength range of the first light source. In some embodiments, the first wavelength range of the first light source comprises between about 500 and about 540 nanometers (nm). In some embodiments, the second wavelength range of the second light source comprises between about 620 and about 640 nanometers (nm). In some embodiments, the third wavelength range of the third light source comprises between about 460 and about 500 nanometers (nm). In some embodiments, the flow cell comprises an inner surface comprising a plurality of discrete regions, wherein (i) a first discrete region of the plurality of discrete regions comprises a first set of nucleic acid molecules bound to the inner surface at the first discrete region, and (ii) a second discrete region of the plurality of discrete regions comprises a second set of nucleic acid molecules bound to the inner surface at the second discrete region, wherein the first set of nucleic acid molecules is different from the second set of nucleic acid molecules.In some embodiments, the first set of nucleic acid molecules comprises a first fluorophore bound thereto, and the second set of nucleic acid molecules comprises a second fluorophore bound thereto, wherein the first fluorophore is different from the second fluorophore. In some embodiments, a third discrete region of the plurality of discrete regions comprises a third set of nucleic acid molecules bound to an internal surface at the third discrete region, wherein the third set of nucleic acid molecules is different from the first set of nucleic acid molecules and the second set of nucleic acid molecules. In some embodiments, the third set of nucleic acid molecules comprises a third fluorophore bound thereto, wherein the third fluorophore is different from the first and second fluorophores. In some embodiments, a fourth discrete region of the plurality of discrete regions comprises a fourth set of nucleic acid molecules bound to an internal surface at the fourth discrete region, wherein the fourth set of nucleic acid molecules comprises a first and third fluorophore, wherein the first fluorophore is different from the third fluorophore. In some embodiments, the light source comprises a light-emitting diode (LED) light source. In some embodiments, the optical system further comprises a light delivery component. In some embodiments, the light delivery component comprises a waveguide, a light pipe, an optical fiber, or a combination thereof. In some embodiments, the light source comprises a solid-state light source. In some embodiments, the system further comprises a heater. In some embodiments, the heater is an integrated heater. In some embodiments, the integrated heater is a heater integrated with a transparent heater block. In some embodiments, the heater is an infrared (IR) heater. In some embodiments, the optical system does not include a dichroic. In some embodiments, the optical system does not include a tube lens. In some embodiments, the optical system does not include corrective optics configured to move in and out of the light path between the flow cell and the plurality of imaging sensors. In some embodiments, the optical system does not include a laser. In some embodiments, the optical system does not include any combination of a dichroic, a tube lens, corrective optics configured to move in and out of the light path between the flow cell and the sensors, and a laser. In some embodiments, the flow cell is disposed between the light source and the sensors.

[0005] Aspects disclosed herein provide a system comprising: a light source configured to illuminate a sample; a sensor configured to obtain an image of the illuminated sample; and a focusing element assembly permanently disposed along an optical path between the light source and the sensor, the focusing element assembly comprising a housing, a first focusing element, and a second focusing element, the first focusing element configured to move relative to the second focusing element within the housing without moving the housing relative to the optical path. In some embodiments, the system further comprises a plurality of light sources, each of which emits light of a different wavelength. In some embodiments, the system further comprises a plurality of sensors, each of which is configured to obtain an image of the sample at a different time. In some embodiments, the system further comprises a filter disposed along the optical path between the light source and the sensor, the filter configured to receive light from the sample and transmit another light to the sensor. In some embodiments, the filter comprises a multi-band filter. In some embodiments, the multi-band filter comprises a triple-stopband filter. In some embodiments, the first lens is disposed before the second lens in the optical path. In some embodiments, the first lens is positioned after the second lens in the optical path. In some embodiments, the sample is bound to one or more inner surfaces of the flow cell. In some embodiments, the sample is covalently bound to one or more inner surfaces of the flow cell. In some embodiments, the sample is bound to two or more inner surfaces of the flow cell. In some embodiments, the sample is covalently bound to two or more inner surfaces of the flow cell. In some embodiments, the two or more inner surfaces of the flow cell include a first inner surface and a second inner surface, the first inner surface being disposed along the optical path between the light source and the second inner surface. In some embodiments, one or more inner surfaces include a hydrophilic polymer layer bound thereto. In some embodiments, the one or more inner surfaces include a hydrophilic polymer layer bound thereto. In some embodiments, the sample includes multiple biopolymers bound to the hydrophilic polymer layer.In some embodiments, the hydrophilic polymer layer comprises polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, or dextran, or any combination thereof. In some embodiments, the system has a surface area of ​​1 square millimeter (mm 2 ) or greater. In some embodiments, the system has a numerical aperture (NA) including less than 0.6. In some embodiments, the NA includes about 0.25. In some embodiments, the sensor includes a plurality of imaging sensors configured to capture the FOV. In some embodiments, the focusing element assembly includes a wedge block assembly, wherein the first focusing element includes a first wedge piece and the second focusing element includes a second wedge piece. In some embodiments, the first wedge piece and the second wedge piece are comprised of fused silica. In some embodiments, the first wedge piece and the second wedge piece have a refractive index including about 1.5. In some embodiments, the system further includes a gap between the first focusing element and the second focusing element.

[0006] Aspects disclosed herein provide a method of imaging a sample, the method including: obtaining a system described herein; illuminating a sample bound to one or more inner surfaces of a flow cell with light from a light source; receiving second light from the sample bound to one or more inner surfaces of the flow cell and filtering the second light with a filter by transmitting third light to a sensor; and obtaining an image of the sample with the sensor. In some embodiments, the sample includes biopolymers, wherein a first subset of the biopolymers is bound to a first inner surface of the one or more inner surfaces of the flow cell and a second subset of the biopolymers is bound to a second inner surface of the one or more inner surfaces of the flow cell. In some embodiments, obtaining an image of the sample with the sensor includes imaging the first inner surface and the second inner surface of the flow cell.

[0007] Aspects disclosed herein provide a method of imaging a sample, the method including obtaining a system disclosed herein; illuminating the sample with a light source; focusing light emitted from the sample with a focusing element assembly; and receiving the light from (c) and acquiring an image of the sample with a sensor. In some embodiments, the sample includes biopolymers, and a first subset of the biopolymers are bound to a first inner surface of the flow cell and a second subset of the biopolymers are bound to a second inner surface of the flow cell. In some embodiments, acquiring an image of the sample with the sensor includes imaging the first inner surface and the second inner surface of the flow cell. In some embodiments, the first inner surface and the second inner surface include a hydrophilic polymer layer bound thereto. In some embodiments, acquiring an image of the sample with the sensor includes imaging a 4 mm 2In some embodiments, the method further comprises sequencing the sample. In some embodiments, the sequencing comprises performing sequencing-by-binding or sequencing-by-synthesis. In some embodiments, the sequencing step includes obtaining a detectable nucleotide conjugate comprising (i) a common core, (ii) a plurality of labels, and (iii) a plurality of nucleotides attached to the common core; contacting a plurality of primed nucleic acid sequences with the detectable nucleotide conjugate under conditions that preclude phosphodiester bond formation between one nucleotide of the plurality of nucleotides and a complementary nucleotide of a plurality of primed nucleic acid sequences of a sample, wherein one nucleotide of a first plurality of nucleotides stably binds to a complementary nucleotide in one primed nucleic acid sequence of the plurality of primed nucleic acid sequences; identifying the complementary nucleotide of the primed nucleic acid sequence by detecting a signal from the plurality of labels of the detectable nucleotide conjugate; and identifying another complementary nucleotide in the primed nucleic acid sequence by performing (a)-(c) with a different detectable nucleotide conjugate and detecting a second signal.

[0008] In one aspect, the present disclosure provides a system comprising: a curved substrate including at least one binding moiety configured to bind to an analyte; and an optical system comprising a light source configured to direct light from the light source towards the curved substrate, wherein the light is configured to probe the presence or absence of an analyte bound to the curved substrate.

[0009] In some embodiments, the analyte comprises a nucleic acid. In some embodiments, the at least one binding moiety comprises at least one nucleic acid configured to bind to a nucleic acid. In some embodiments, the curved substrate is a component of a flow cell. In some embodiments, the system further comprises a flow cell comprising the curved substrate. In some embodiments, the curved substrate comprises a capillary of the flow cell. In some embodiments, the curved substrate comprises glass, a polymer, or a combination thereof. In some embodiments, the light source is configured to probe the curved substrate in an epifluorescence configuration. In some embodiments, the light source is configured to probe the curved substrate in a transmission configuration. In some embodiments, the light source is a laser, a light emitting diode, a halogen lamp, or an incandescent lamp. In some embodiments, the light source is configured to generate light at a wavelength between about 500 nanometers (nm) and 540 nm, 620 nm and 650 nm, or 460 nm and 500 nm. In some embodiments, the system further comprises a second curved substrate. In some embodiments, the system further comprises a focus shifting assembly configured to move a focal field between the curved substrate and the second curved substrate. In some embodiments, the focus-shifting assembly includes at least one movable lens. In some embodiments, the at least one movable lens is provided within a lens barrel. In some embodiments, the focus-shifting assembly includes at least one movable prism. In some embodiments, the curved substrate and the second curved substrate are different portions of a generally cylindrical component of the flow cell. In some embodiments, the second curved substrate includes at least one second binding moiety configured to bind to a second analyte. In some embodiments, the optical system is movable relative to the curved substrate. In some embodiments, the optical system is rotatable about the curved substrate. In some embodiments, the optical system is configured to image a plurality of binding moieties. In some embodiments, the curved substrate has a flatness of 25 micrometers (μm). In some embodiments, the curved substrate has a flatness greater than a depth of focus of the optical system.In some embodiments, the system further comprises a plurality of sub-optical systems that are not parallel to one another. In some embodiments, each sub-optical system of the plurality of sub-optical systems is disposed perpendicular to a plurality of tangents of the curved substrate. In some embodiments, the system further comprises a stage on which the curved substrate is disposed. In some embodiments, the stage comprises a tilt stage, a rotation stage, a translation stage, or any combination thereof. In some embodiments, the curved substrate comprises a hydrophilic polymer bonded thereto. In some embodiments, at least one binding moiety is bonded to the hydrophilic polymer. In some embodiments, the hydrophilic polymer is polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glycosyl In some embodiments, the system comprises a fluorophore, streptavidin, or dextran, or any combination thereof. In some embodiments, the system has a numerical aperture of up to about 0.6. In some embodiments, the numerical aperture is up to about 0.25. In some embodiments, the system further comprises an imaging sensor configured to collect light after being directed at the curved substrate. In some embodiments, the system further comprises a heater configured to heat the curved substrate. In some embodiments, the heater is an integrated heater. In some embodiments, the heater is an infrared heater.

[0010] In another aspect, the present disclosure provides a system comprising a curved substrate and an optical system comprising a light source configured to direct light from the light source towards the curved substrate.

[0011] In another aspect, the present disclosure provides a method for manufacturing a substrate and a substrate having an area of ​​at least about 5 square millimeters (mm 2and an optical system configured to image an area of ​​the substrate.

[0012] In some embodiments, the optical system is configured to simultaneously image the regions. In some embodiments, the optical system comprises multiple sub-optical systems. In some embodiments, the multiple sub-optical systems are configured to image the regions of the substrate in parallel. In some embodiments, the optical system comprises a light source configured to provide a light beam and a lens configured to focus the light beam from the light source onto a focal region of the substrate including the regions. In some embodiments, the homogeneity of the light beam on the focal region is at least about 90%. In some embodiments, the regions of the substrate are provided as a hollow cylinder. In some embodiments, the substrate is at least a portion of a capillary flow cell. In some embodiments, the capillary flow cell includes a solid core. In some embodiments, the system further comprises a stage on which the substrate is provided. In some embodiments, the stage includes a tilt stage, a rotation stage, a translation stage, or any combination thereof. In some embodiments, the substrate includes a hydrophilic polymer bonded thereto. In some embodiments, at least one binding moiety is bonded to the hydrophilic polymer. In some embodiments, the hydrophilic polymer comprises polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, or dextran, or any combination thereof. In some embodiments, the system has a numerical aperture of up to about 0.6. In some embodiments, the numerical aperture is up to about 0.25. In some embodiments, the system further comprises an imaging sensor configured to collect the light after directing it onto the substrate. In some embodiments, the system further comprises a heater configured to heat the curved substrate.In some embodiments, the heater is an integrated heater. In some embodiments, the heater is an infrared heater. In some embodiments, the substrate is a curved substrate. In some embodiments, the curved substrate has a flatness of 25 micrometers (μm). In some embodiments, the curved substrate has a flatness greater than the depth of focus of the optical system. In some embodiments, the optical system is configured to image an area of ​​the substrate with a resolution of about 1 μm or less.

[0013] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term in this specification and a term in an incorporated reference, the term in this specification shall control. [Brief explanation of the drawings]

[0014] The novel features of the inventive concept are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood 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.

[0015] [Figure 1A] 1A-1C are schematic diagrams illustrating non-limiting examples of imaging double-sided support structures for presenting sample sites for imaging by the imaging systems disclosed herein. [Figure 1B] 1A-1C are schematic diagrams illustrating non-limiting examples of imaging double-sided support structures for presenting sample sites for imaging by the imaging systems disclosed herein. FIG. 1B illustrates imaging of the front and back exterior surfaces of a substrate. [Figure 2A]2A illustrates a non-limiting example of a multi-channel fluorescence imaging module comprising a dichroic beam splitter for delivering an excitation light beam to a sample, receiving the resulting fluorescence emission, and redirecting it by reflection to four detection channels configured for detection of fluorescence emission at four different corresponding wavelengths or wavelength bands. [Figure 2B] 2A and 2B illustrate a non-limiting example of a multi-channel fluorescence imaging module comprising a dichroic beam splitter for delivering an excitation light beam to a sample, receiving the resulting fluorescence emission, and redirecting it by reflection to four detection channels configured for detection of fluorescence emission at four different corresponding wavelengths or wavelength bands. [Figure 3A] 3A is a plan view illustrating the optical paths within the multi-channel fluorescence imaging module of FIGS. 2A and 2B, comprising a dichroic beam splitter for delivering an excitation light beam to a sample, receiving the resulting fluorescent emission, and redirecting it by reflection to four detection channels configured for detection of fluorescent emission at four different corresponding wavelengths or wavelength bands. [Figure 3B] 3A and 3B are side views illustrating the optical paths within the multi-channel fluorescence imaging module of FIGS. 2A and 2B , each including a dichroic beam splitter for delivering an excitation light beam to a sample, receiving the resulting fluorescence emission, and redirecting it by reflection to four detection channels configured for detection of fluorescence emission at four different corresponding wavelengths or wavelength bands. [Figure 4] 1 is a graph illustrating the relationship between dichroic filter performance and beam incidence angle. [Figure 5] 1 is a graph illustrating the relationship between beam footprint size and beam incidence angle on a dichroic filter. [Figure 6A]6A and 6B are schematic diagrams illustrating exemplary configurations of dichroic filters and detection channels of a multichannel fluorescence imaging module, where the dichroic filter has a reflective surface that is tilted such that the angle between the incident beam (e.g., central angle) and the reflective surface of the dichroic filter is less than 45. FIG. 6A is a schematic diagram of a multichannel fluorescence imaging module including four detection channels. [Figure 6B] 6A and 6B are schematic diagrams illustrating exemplary configurations of dichroic filters and detection channels of a multichannel fluorescence imaging module, where the dichroic filters have reflective surfaces that are tilted such that the angle between the incident beam (e.g., central angle) and the reflective surface of the dichroic filter is less than 45°. Figure 6B is a detailed diagram illustrating the angle of incidence (AOI) of the light beam on the dichroic reflector. [Figure 7] 6A and 6B provide graphs illustrating improved dichroic filter performance corresponding to the imaging module configuration illustrated in FIGS. 6A and 6B. [Figure 8] 6A and 6B provide graphs illustrating improved dichroic filter performance corresponding to the imaging module configuration illustrated in FIGS. 6A and 6B. [Figure 9A] 9A and 9B provide graphs illustrating the reduction in surface deformation resulting from the imaging module configurations of Figures 6A and 6B. Figure 9A shows the effect of fold angle on image degradation induced by adding 1 wave of PV spherical power to the last mirror. [Figure 9B] 9A and 9B provide graphs illustrating the reduction in surface deformation resulting from the imaging module configuration of FIGS. 6A and 6B. FIG. 9B shows the effect of fold angle on image degradation induced by adding 0.1 waves of PV spherical power to the last mirror. [Figure 10A]10A provides graphs illustrating the improvement in excitation filter performance (e.g., a sharper transition between the passband and the surrounding stopband) that results from using s-polarized excitation beam. Figure 10A is the transmission spectrum of an exemplary bandpass dichroic filter at angles of incidence of 40 and 45 degrees, where the incident beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. [Figure 10B] 10A provides graphs illustrating the improvement in excitation filter performance (e.g., a sharper transition between the passband and the surrounding stopband) that results from using s-polarized excitation beam. FIG. 10B is a change in the orientation of the light source relative to the dichroic filter such that the incident beam is s-polarized relative to the plane of the dichroic filter, resulting in a sharper edge between the passband and stopband. [Figure 11A] 11A illustrates the modulation transfer function (MTF) of an exemplary dual-sided imaging system disclosed herein, with a numerical aperture (NA) of 0.3. FIG. 11A is the first surface. [Figure 11B] 11A and 11B illustrate the modulation transfer function (MTF) of an exemplary dual-sided imaging system disclosed herein with a numerical aperture (NA) of 0.3. [Figure 12A] 12A illustrates the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.4. [Figure 12B] 12A and 12B illustrate the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.4. [Figure 13A] 13A illustrates the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.5. [Figure 13B] 13A and 13B illustrate the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.5. [Figure 14A]14A illustrates the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.6. [Figure 14B] 14A and 14B illustrate the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.6. [Figure 15A] 15A illustrates the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.7. [Figure 15B] 15A and 15B illustrate the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.7. [Figure 16A] 16A illustrates the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.8. [Figure 16B] 16A and 16B illustrate the MTF of an exemplary double-sided imaging system disclosed herein with a NA of 0.8. [Figure 17A] 17A provides plots of calculated Strehl ratios for imaging a second flow cell surface through a first flow cell surface. Figure 17A is a plot of the Strehl ratio for imaging a second flow cell surface through a first flow cell surface as a function of intervening fluid layer thickness (fluid channel height) for various objective lenses and / or optical system numerical apertures. [Figure 17B] 17A and 17B provide plots of the calculated Strehl ratio for imaging a second flow cell surface through a first flow cell surface. Figure 17B is a plot of the Strehl ratio as a function of numerical aperture for imaging the first flow cell surface and the second flow cell surface through an intervening layer of water having a thickness of 0.1 mm. [Figure 18] FIG. 1 provides a schematic diagram of the dual wavelength excitation / four channel emission fluorescence imaging system of the present disclosure. [Figure 19]FIG. 1 provides a ray tracing diagram for an objective lens design designed to image the surface opposite a 0.17 mm thick coverslip. [Figure 20] FIG. 20 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface opposite a 0.17 mm thick coverslip. [Figure 21] FIG. 20 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface on the opposite side of a 0.3 mm thick coverslip. [Figure 22] FIG. 20 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface separated from a surface on the other side of a 0.3 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 23] FIG. 20 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface on the opposite side of a 1.0 mm thick coverslip. [Figure 24] FIG. 20 provides a plot of the modulation transfer function for the objective illustrated in FIG. 19 as a function of spatial frequency when used to image a surface separated from a surface on the other side of a 1.0 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 25] FIG. 20 provides a ray tracing diagram for a tube lens design that, when used with the objective illustrated in FIG. 19, improves double-sided imaging with a 1 mm thick coverslip. [Figure 26] FIG. 26 provides a plot of the modulation transfer function for the objective and tube lens combination illustrated in FIG. 25 as a function of spatial frequency when used to image surfaces on opposite sides of a 1.0 mm thick coverslip. [Figure 27]FIG. 26 provides a plot of the modulation transfer function for the objective lens and tube lens combination illustrated in FIG. 25 as a function of spatial frequency when used to image a surface separated from a surface on the other side of a 1.0 mm thick coverslip by a 0.1 mm thick layer of aqueous fluid. [Figure 28] FIG. 1 provides a ray tracing diagram for a tube lens design of the present disclosure (left) that is optimized to provide high-quality double-sided imaging performance. Because the tube lens is no longer corrected to infinity, 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 29] FIG. 1 illustrates a non-limiting example of a single capillary flow cell with two fluidic adapters. [Figure 30] FIG. 1 illustrates a non-limiting example of a flow cell cartridge designed to hold two capillaries, including a chassis, fluidic adapter, and optionally other components. [Figure 31] FIG. 1 illustrates a non-limiting example of a system comprising a single capillary flow cell connected to various fluid flow control components, the single capillary suitable for mounting on a microscope stage or in custom imaging equipment for use in various imaging applications. [Figure 32] FIG. 1 illustrates one non-limiting example of a system comprising a capillary flow cell cartridge with an integrated diaphragm valve to reduce or minimize dead volume and conserve certain critical reagents. [Figure 33] FIG. 1 illustrates a non-limiting example of a system including a capillary flow cell, a microscope setup, and a temperature control mechanism. [Figure 34] FIG. 1 illustrates a non-limiting example of temperature control of a capillary flow cell through the use of a metal plate placed in contact with the flow cell cartridge. [Figure 35]FIG. 1 illustrates one non-limiting approach to temperature control of a capillary flow cell with a non-contact thermal control mechanism. [Figure 36A] Figures 36A and 36B illustrate non-limiting examples of the manufacture of a flow cell device: Figure 36A shows the preparation of a one-piece glass flow cell. [Figure 36B] Figures 36A and 36B illustrate non-limiting examples of fabrication of a flow cell device. Figure 36B shows the preparation of a two-piece glass flow cell. [Figure 36C] Figures 36A and 36B illustrate non-limiting examples of fabrication of a flow cell device. Figure 36C shows the preparation of a three-piece glass flow cell. [Figure 37A] 37A-37C illustrate non-limiting examples of glass flow cell designs. Figure 37A shows a one-piece glass flow cell design. [Figure 37B] 37A and 37B illustrate non-limiting examples of glass flow cell designs. Figure 37B shows a two-piece glass flow cell design. [Figure 37C] 37A-37C illustrate non-limiting examples of glass flow cell designs. Figure 37C shows a three-piece glass flow cell design. [Figure 38] FIG. 1 illustrates visualization of cluster (e.g., polony) amplification in the capillary lumen. [Figure 39] FIG. 1 provides a non-limiting example of a block diagram for a sequencing system disclosed herein. [Figure 40] FIG. 1 provides a non-limiting example of a flow chart for the sequencing methods disclosed herein. [Figure 41] 1A-1C provide non-limiting examples of diagrams for structured lighting systems disclosed herein. [Figure 42] FIG. 1 provides a non-limiting example of a flowchart for acquiring and processing structured illumination images of a flow cell surface as disclosed herein. [Figure 43A]43A provides a non-limiting schematic diagram of a multiplexing readhead as disclosed herein: Figure 43A is a side view of a multiplexing readhead in which individual microfluorophotometers are configured to image a common surface, e.g., the inner surface of a flow cell. [Figure 43B] 43A and 43B provide a non-limiting schematic diagram of a multiplexing readhead as disclosed herein. Figure 43B is a plan view of a multiplexing readhead illustrating the imaging paths acquired by the individual microfluorophotometers of the multiplexing readhead. [Figure 44A] 44A provides a non-limiting schematic diagram of a multiplexed readhead as disclosed herein. Figure 44A is a side view of a multiplexed readhead in which a first subset of a plurality of individual microfluorophotometers (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 microfluorophotometers is configured to image a second surface, e.g., a second inner surface of a flow cell. [Figure 44B] Figure 44B provides a non-limiting schematic diagram of a multiplexing readhead as disclosed herein. Figure 44B is a plan view of the multiplexing readhead of Figure 44A illustrating the imaging paths acquired by the individual microfluorophotometers (4401) of the multiplexing readhead. [Figure 45] 1 illustrates a non-limiting example of an optical imaging system having multiple imaging sensors configured for transmission imaging of a flow cell after 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 tensile force. [Figure 46] FIG. 1 provides a non-limiting schematic diagram of a method of utilizing an optical system for imaging the surface of a flow cell for nucleic acid sequencing, according to some embodiments herein. [Figure 47A] 47A provides a non-limiting cutaway view of an optical system for imaging the surface of a flow cell, according to some embodiments herein. [Figure 47B]47A and 47B provide an optical system according to various embodiments described herein. Figure 47B provides a comparison of the optical system of Figure 47A with an IDEX instrument core. [Figure 48A] FIG. 47C provides a non-limiting example of 424 individual tile flow cells imaged by the IDEX instrument core shown in FIG. 47B. [Figure 48B] 47A-47B provide non-limiting examples of flow cells with fewer than 40 individual tiles that may be imaged by the optical systems described herein (see FIGS. 45, 46, 47A, and 47B). [Figure 49A] 49A and 49B provide non-limiting cutaway views of optical systems configured for dual-sided imaging of a dual-sided flow cell. The optical system shown includes a piezo drive for fast focusing. Figure 49A illustrates an optical system configured to focus on the rear inner surface of the flow cell. [Figure 49B] 49A and 49B provide non-limiting cutaway views of optical systems configured for dual-sided imaging of a dual-sided flow cell. The optical system shown includes a piezo drive for fast focusing. Figure 49B illustrates an optical system configured to focus on the front inner surface of the flow cell. [Figure 50] 1 provides a non-limiting cutaway view of an optical system configured to image a large-area surface, the optical system including multiple optical subsystems, each with an optimized FOV that overlaps with the FOV of each of its adjacent optical subsystems, thereby providing a large-area FOV. [Figure 51A]

[0049] Figures 51A and 51B provide non-limiting cutaway views of focusing lens assemblies. The focusing lens assembly is configured to maintain a fixed position (e.g., optical axis) within an optical path and allow relative movement between at least a first lens and a second lens housed within a lens housing of the focusing lens assembly. Figure 51A shows a focusing lens assembly having a first lens and a second lens. [Figure 51B]

[0013] Figure 51B provides a non-limiting cutaway view of a focusing lens assembly. The focusing lens assembly is configured to maintain a fixed position (e.g., optical axis) within the optical path and allow relative movement between at least a first lens and a second lens housed within a lens housing of the focusing lens assembly. Figure 51B shows the same focusing lens assembly with relative movement of the second lens compared to Figure 51A. [Figure 52] 1 provides a non-limiting cutaway view of an optical system configured to image a curved, large-area surface. The optical system includes multiple optical subsystems, each arranged substantially perpendicular to the surface, and each subsystem's FOV overlapping with the FOV of each adjacent optical subsystem, thereby providing a system for imaging a curved, large-area surface. [Figure 53A]

[0013] Figure 53A provides a non-limiting cutaway view of an optical system configured to image a capillary flow cell. In this example, the optical system configured to image a curved, large-area surface is rotated about and translated along the x-axis to acquire an image of the entire inner surface of the capillary flow cell. Figure 53A illustrates the optical axis of the central optical subsystem aligned with the z-axis. [Figure 53B]

[0062] Figure 53A provides a non-limiting cutaway view of an optical system configured to image a capillary flow cell. In this example, an optical system configured to image a curved, large-area surface is rotated about and translated along the x-axis to acquire an image of the entire inner surface of the capillary flow cell. Figure 53B illustrates the optical axis of the central optical subsystem rotated 90 degrees to align with the y-axis. [Figure 54A] Figure 54A provides a non-limiting cutaway view of an optical system configured to image a capillary flow cell without the need for a stage to rotate the optics about the x-axis. The optical system shown includes a piezo drive for fast focusing. Figure 54A illustrates an optical system configured to focus on the inner surface of the capillary flow cell closest to the light source. [Figure 54B]Figure 54A provides a non-limiting cutaway view of an optical system configured to image a capillary flow cell without the need for a stage to rotate the optics about the x-axis. The optical system shown includes a piezo drive for fast focusing. Figure 54B illustrates an optical system configured to focus on the inner surface of the capillary flow cell away from the light source. [Figure 55] 1 is a bar graph showing the results of capture assays performed by reacting various fluorescently labeled multivalent molecules with the corresponding correct DNA templates. [Figure 56] 1 is a bar graph showing the results of a capture assay in which increasing concentrations of various fluorescently labeled multivalent molecules were reacted with the corresponding correct DNA template. [Figure 57] Figure 1 provides four graphs showing the results of a capture 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 CF680 or CF532 fluorophores. Two different concentrations of multivalent molecules were tested (20 and 80 nM). The graphs show the capture time in seconds (x-axis) and the P90 signal intensity (y-axis). [Figure 58] Figure 1 provides four graphs showing the results of a capture 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 AF647 or CF570 fluorophores. Two different concentrations of multivalent molecules were tested (20 and 80 nM). The graphs show the capture time in seconds (x-axis) and the P90 signal intensity (y-axis). [Figure 59] Figure 1 provides three graphs showing the results of a real-time imaging capture kinetics 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 were tested (15, 7.5, and 2.5 nM). The graphs show capture time in seconds (x-axis) and P90 signal intensity (y-axis). [Figure 60] Figure 14 is a graph showing the results of binding kinetics studies for fluorescently labeled multivalent molecules with nucleotide arms containing linkers 6 or one of 10 to 16. The graph shows multivalent molecule concentration (x-axis, nM) and rate (y-axis). The legend shown in Figure 14 is also applicable to Figure 13. [Figure 61] FIG. 1 is a bar graph showing binding constants (K) determined for fluorescently labeled multivalent molecules with nucleotide arms containing linkers 6 or one of 10-16. [Figure 62] FIG. 1 illustrates an example of a generalized combination system for avidity-based sequencing, according to some embodiments. [Figure 63] FIG. 1 illustrates a computer system programmed or otherwise configured to perform the methods provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0016] There is a need for fluorescence imaging methods and systems that increase optical resolution and improve image quality in genomic applications, resulting in corresponding improvements in the accuracy of genomic testing. Disclosed herein are optical system designs for high-performance fluorescence imaging methods and systems that may provide any one or more of improved optical resolution (including high-performance optical resolution), improved image quality, and higher throughput in fluorescence imaging-based genomic applications. The disclosed optical illumination and imaging system designs may provide any one or more of the following advantages: improved dichroic filter performance, improved uniformity of the dichroic filter frequency response, improved excitation beam filtering, larger field of view, increased 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., of different fields of view), improved imaging system duty cycle, and higher throughput image acquisition and analysis.

[0017] Optical System: In some embodiments herein, an optical system (4500), shown in the non-limiting schematic diagram of FIG. 45, is described, eliminating the need for corrective optics such as dichroics or tube lenses for double-sided imaging of a flow cell. The optical system (4500) disclosed herein can be used as a component of a system 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 imaging sensors (4501)-(4504) configured to image a flow cell (4521). In some embodiments, the imaging sensors (4501)-(4504) may be CCD imaging sensors. In some embodiments, the imaging sensors (4501)-(4504) may be CMOS imaging sensors. In some embodiments, pixel shifters (4505)-(4508) are used to translate the object being imaged relative to the corresponding imaging 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 triple-band fluorescence bandpass filter. In some embodiments, the triple-band fluorescence bandpass filter is referred to as a triple-band notch filter. In some embodiments, the imaging optics (4510)-(4513) are disposed between the imaging sensors (4501)-(4504) and the flow cell (4521). In some embodiments, one imaging optics (4505)-(4508), also referred to as an imaging optics assembly, focuses light emitted from the flow cell (4521) onto one of the imaging 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) dispenses the sample (4515) into the flow cell (4521). In some embodiments, the liquid handling system (4514) dispenses the liquid sample onto 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 interior surface (4528). In some embodiments, the bottom plate comprises a rear interior 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 (4525), (4526), ​​and (4527), respectively, of a different color or spectrum. In some embodiments, the optical system (4500) comprises a heater.

[0018] In some embodiments, a notch filter refers to a bandstop filter. In some embodiments, a notch filter refers to a bandstop filter. In some embodiments, a notch of a filter refers to a bandstop or stopband. In some embodiments, a notch of a filter refers to a bandpass or passband. In some embodiments, a multi-band notch filter refers to a multi-band bandpass filter. In some embodiments, a multi-band notch filter refers to a multi-band bandstop 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 Super, 2mm 2 Super, 4mm 2 Super, 10mm 2 Super, 20mm 2 Super, 36mm 2 Super, 40mm 2 Super, 60mm 2 Super, 80mm 2 Over or 100mm 2The optical system has a field of view (FOV) of greater than 0.6. In some embodiments, the optical system has a numerical aperture (NA) 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, multiple imaging sensors are configured to capture the FOV. In some embodiments, the multiple light sources include 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 biopolymers. In some embodiments, the optical system (4500) does not include a dichroic. 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 analytical applications. Figure 46 provides a schematic diagram of an imaging method (4601) utilizing the optical system (4500) shown in Figure 45 to image a sample (4515) contained within a flow cell (4521), 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 Figure 45. In some embodiments, the sample comprises a biopolymer. In some embodiments, the biopolymer comprises units. In some embodiments, a fluorophore is complementary to a unit of the biopolymer. In some embodiments, a fluorophore is attached to a nucleotide complementary to a unit of the biopolymer. In some embodiments, two or more detectably distinct fluorophores are attached to a nucleotide complementary to a unit of the biopolymer. In some embodiments, the biopolymer is a nucleic acid sequence. In some embodiments, the units are nucleotides complementary to fluorophore-labeled nucleotides. In some embodiments, the multiple light sources emit light that is transmitted through the sample.

[0021] Described herein are various methods for sequencing biopolymers (e.g., nucleic acid molecules). A non-limiting schematic diagram of a sequencing method, instrumentation (4601), and base calling method (4602) is shown in Figure 46. In some embodiments, the method includes illuminating a sample (4515) using an optical system (4500) having 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) to acquire a first image of the sample (4515), the optical system (4500) including a plurality of imaging sensors (4501)-(4504), and further wherein the sample (4515) is illuminated by the plurality of light sources (4522)-(4524). a step of illuminating the sample (4515) using a second light source (4523) of the plurality, the second light source (4523) emitting a second wavelength range (4526) that excites a second fluorophore in the sample (4515) to acquire a second image of the sample (4515); and a step of illuminating the sample (4515) using a third light source (4524) of the plurality, the third light source (4524) emitting a second wavelength range (4526) that excites a third fluorophore in the sample to acquire a second image of the sample (4515). to emit a third wavelength range (4527) to acquire a third image of the sample (4515); combining the first, second, and third images into a composite image; identifying the presence of a first nucleotide by a first signal emitted by a first fluorophore, the first signal being extracted from a first region of interest (ROI) in the composite image; identifying the presence of a second nucleotide by a second signal emitted by a second fluorophore, the second signal being extracted from the second ROI in the composite image; identifying the presence of a third nucleotide by a third signal emitted by a third fluorophore, the third signal being extracted from the third ROI in the composite image; and identifying the presence of a fourth nucleotide by first and third signals emitted by the first and third fluorophores, respectively, the first and third signals being extracted from the fourth ROI in the composite image.In some embodiments, the optical system (4500) further comprises a flow cell (4521), and the flow cell (4521) is disposed in an optical path between the plurality of imaging sensors (4501)-(4504) and the plurality of light sources (4522)-(4524). In some embodiments, the optical system (4500) further comprises at least one pixel shifter (4505)-(4508). In some embodiments, the optical system (4500) further comprises a multi-band bandpass filter (4509) disposed in an optical path between the plurality of imaging sensors (4501)-(4504) and the flow cell (4521). In some embodiments, the method further comprises 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 optical system is 1 mm. 2 Super, 2mm 2 Super, 4mm 2 Super, 10mm 2 Super, 20mm 2 Super, 36mm 2 Super, 40mm 2 Super, 60mm 2 Super, 80mm 2 Over or 100mm 2 In some embodiments, the optical system has a numerical aperture (NA) of less than 0.6. In some embodiments, the NA is 0.25. In some embodiments, the FOV is captured by multiple image sensors (4501)-(4504).

[0022] In some embodiments, the sequencing is avidin-based sequencing. Further discussion of avidin-based sequencing is included 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, 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, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, 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 sulfonylhydrazine, Texas Red ... 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, benzoindolium-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,Examples of suitable dyes include, but are not limited to, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilirin dyes, malachite green, stilbenes, DEG dyes, NR dyes, near-infrared dyes, and other dyes known in the art, such as those described in Haugland, Molecular Probes Handbook (Eugene, Oreg.), 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Edition, Plenum Press, New York (1999); or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or combinations thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms and contain two indolenine, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (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-indol-2-ylidene). or 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(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-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-dimeth- yl Indolenine compounds include, but are not limited to, Cy7 (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, an oxazole derivative rather than an indolenine, and the benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule. In some embodiments, reporter moieties can be FRET-paired so that multiple classifications can 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 biopolymer analysis (e.g., nucleic acid sequencing). In some embodiments, the system (4700) shown in FIGS. 47A-47B is more compact and has higher throughput than existing optical systems. Table 1 and FIGS. 48A-48B provide non-limiting examples comparing sequencing cycle times for a standard flow cell and optical system with the optical systems described herein. Table 1 provides cycle times, run times, and respective calculations for a standard flow cell with 424 individual tiles (e.g., active areas, regions of interest, etc.) shown in FIG. 48A compared to a flow cell with fewer than 40 individual tiles optimized for imaging on the optical systems described herein shown in FIG. 48B. In some embodiments, one image is equivalent to one tile in the area. In some embodiments, when the flow cell (4521), also shown in FIG. 48B, is imaged by the optical system, each tile is subjected to three sequential light pulses from three separate LED light sources, each emitting a different wavelength. In some embodiments, the different wavelengths are matched to the excitation spectra of different fluorophores described herein. In some embodiments, the imaging sensor of the optical system (4500) is synchronized with each excitation pulse to generate an image, imaging the entire area of ​​one tile, and each pixel of the image represents the amount of fluorescence emitted by a fluorophore. In some embodiments, two separate surfaces are imaged in one tile by the optical system (4500). In some embodiments, eight full-images with a total exposure time of 0.3 seconds are acquired by the optical system (4500), (4700), which includes eight imaging modules (e.g., optical subsystems). In Table 1, the row titled "current" and highlighted in blue represents the total time over 322 cycles when imaged with the IDEX optical system shown in Figure 47B, which is 36.17 hours for the standard flow cell shown in Figure 48A.In contrast, the row titled "Sleq" shows total times between 13.63 and 14.28 hours in a Sleq cell (see Figure 48B) when imaged by the optical system (4700) shown in Figures 47A-B. The bottom row of Table 1 shows a total time of 1.11 hours when only 25 cycles were performed. The reduced sequencing time demonstrates the benefit of a larger FOV than allowed by the optical system (4700) described herein.

[0026] [Table 1]

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

[0028] Figure 47A provides a non-limiting cutaway view of an optical system for imaging the surface of a flow cell (4521), according to some embodiments herein. In some embodiments, the optical system comprises 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 imaging 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] Some embodiments herein describe an optical system (4900) shown in the non-limiting schematic diagrams of FIGS. 49A-49B configured for double-sided imaging of a flow cell (4905). 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 imaging sensor (4912) that can be configured to image the flow cell (4905). In some embodiments, the sample flow coincides with the x-axis as shown in FIGS. 49A-49B. In some embodiments, there can be multiple imaging sensors (4912). The imaging sensor (4912) can be a CCD imaging sensor. In some embodiments, the imaging sensor (4912) can be a CMOS imaging 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, the pixel shifter (4911) translates the object being imaged relative to the imaging sensor (4912). In some embodiments, the optical system includes 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 triple-band fluorescence stop-band filter. In some embodiments, a triple-band fluorescence stop-band filter is referred to as a triple-band notch filter. In some embodiments, the system includes imaging optics (4909). In some embodiments, the imaging optics (4909) includes an objective lens.

[0030] In some embodiments, the filter (4910) is disposed between the imaging sensor (4912) and the flow cell (4905). In some embodiments, the imaging optics (4909), also referred to as an imaging optical assembly, focuses light emitted from the flow cell (4909) onto the imaging sensor (4912). In some embodiments, the optical system (49000) 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 a path length of the optical system. In some embodiments, the wedge block (4916) includes a first wedge piece (4907), a second wedge piece (4906), or a combination thereof. In some embodiments, the system comprises a piezo actuator (4908) configured to adjust the optical path length of the optical system by moving the positions of the first wedge piece (4907) and the second wedge piece (4906) relative to each other. In some embodiments, the flow cell (4905) is configured for double-sided imaging (DSI). In some embodiments, the flow cell (4905) includes a front inner surface (4904), a rear inner surface (4905), or a combination thereof. In some embodiments, the front inner surface (4904) and / or the rear inner surface (4903) include the sample site (4902). In some embodiments, the optical system includes an optical axis (4913). In some embodiments, the optical system includes an optimal imaging volume (4915). In certain aspects, the optimal imaging volume (4915) includes a field of view (FOV), an illumination region, an acquisition region, a focal plane, a depth of focus, a region and / or a volume where the sample site (4902) emits brightness at or above an acceptable level, or a combination thereof. Typically in microscopy, the brightness of an object in the center of the FOV may be greatest at the center and decrease toward 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.

[0032] Multivalent molecules The present disclosure provides multivalent molecules comprising a core bound 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 conjugation of the core to the nucleotide arm. In some embodiments, the core can be bound to multiple nucleotide arms. In some embodiments, the core can be bound to about 1 to about 50 nucleotide arms. In some embodiments, the core can be bound to about 2 to about 20 nucleotide arms. In some embodiments, the core can be bound to about 2 to about 4 nucleotide arms. Optionally, the core is linked to about 4 to about 10 nucleotide arms. Optionally, the core is linked to about 10 to about 15 nucleotide arms. Optionally, the core is linked to about 15 to about 20 nucleotide arms. Figures 1, 2, and 3 show the overall structure of the multivalent molecule.

[0033] The present disclosure provides a multivalent molecule comprising a core bound 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 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.

[0034] In some embodiments, the core can include streptavidin- or avidin-type moieties, including streptavidin or avidin proteins, as well as 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 native or recombinant forms, as well as mutant and derivatized molecules. Streptavidin and avidin mutants can include any one or a combination of two or more of amino acid insertions, deletions, substitutions, or truncations. Mutants can also include fusion polypeptides. Many different forms of streptavidin and avidin are commercially available.

[0035] Multivalent molecules can be configured using a streptavidin or avidin core that has a high affinity for the biotin moiety on the biotinylated nucleotide arm, thereby reducing the dissociation of the nucleotide arm from the core. A mixture of multivalent molecules can be prepared, where the mixture contains 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 a high affinity between the core and the nucleotide arm can reduce the undesired dissociation of the nucleotide arm from the core and the exchange of the nucleotide arm between different cores. The exchange of the nucleotide arm during the sequencing reaction can cause inaccurate base calling and reduced sequencing accuracy. In some embodiments, a multivalent molecule with increased stability (e.g., reduced dissociation of biotinylated nucleotide arms) can include dye-labeled streptavidin, where the streptavidin subunit carries a Lys121Arg mutation, which can exhibit reduced dissociation of biotinylated nucleotide arms from the streptavidin core.

[0036] 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 a similar effect. 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 bound to streptavidin at Lys121 can block or inhibit biotin binding to the dye-labeled streptavidin. Multivalent molecules comprising dye-labeled streptavidin possessing a lysine at position 121 can exhibit dissociation of the biotinylated nucleotide arm from the streptavidin core. Multivalent molecules with increased stability can include dye-labeled streptavidin possessing a Lys121Arg mutation, which can exhibit reduced dissociation of the biotinylated nucleotide arm from the streptavidin core.

[0037] In some embodiments, the streptavidin moiety increases the affinity for binding to biotin (e.g., K d About 10 -16 The amino acid substitutions 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 combines increased affinity for binding biotin with improved retention of biotin (increasing the affinity to bind biotin to about 1000 mol / L).

[0038] 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). In some embodiments, avidin can include substitutions of any one of the eight arginine residues (e.g., underlined and bolded in Figure 22 or Figure 23), or any combination thereof. Avidin can include partially deglycosylated forms and non-glycosylated forms. Avidin moieties can include derivatized forms, such as N-acylavidin, e.g., N-acetyl, N-phthalyl, and N-succinyl avidin, as well as commercially available products including EXTRAVIDIN, CAPTAVIDIN (selective nitration of tyrosine residues at the four biotin binding sites to generate avidin that reversibly binds biotin), NEUTRAVIDIN (chemically deglycosylated and containing modified arginine residues), and NEUTRALITE AVIDIN (five of the eight arginine residues are replaced with neutral amino acids, two of the lysine residues are replaced with glutamic acid, and Asp17 is replaced with isoleucine). Amino acids with neutral nonpolar side chains include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and valine. Amino acids with neutral polar side chains include asparagine, cysteine, glutamine, serine, threonine, tryptophan, and tyrosine.

[0039] 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 in the homotetramer can contain at least one lysine residue that can be conjugated to a fluorophore. The labeling reaction can utilize an N-hydroxysuccinimide (NHS) ester-conjugated fluorophore. 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.

[0040] When preparing labeled streptavidin or avidin cores, 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 greater than 6) may exhibit reduced fluorescence due to self-quenching from the conjugated fluorophore. In some embodiments, the predetermined degree of labeling for streptavidin or avidin cores may depend on the dye. Fluorescent dyes include, but are not limited to, CF647, CF680, CF570, and CF532 dyes manufactured by Biotium, AF647, AF680, AF568, and AF532 manufactured by Thermo Fisher Scientific, IFluor 647, IFluor 680, IFlour 568, and IFlour 532 manufactured by AATBio, DY648P1, DY679P1, DY585, and DY530 manufactured by Dyomics, and AFDy 647, IFlour 680LT, AFDye 568, and AFDye 532 manufactured by Fluoroprobes. The predetermined degree of labeling can be about 1-10, about 3-8, about 3.5-7, or about 1.6-4.

[0041] Because red fluorophores are brighter (more intense) than green dyes, color bleeding can occur when imaging both red- and green-labeled multivalent molecules on the same support (e.g., flow cell). To achieve improved signal balance from a mixture of labeled multivalent molecules, the degree of labeling of subpopulations of multivalent molecules can be increased or decreased. For example, the degree of labeling of a subpopulation of multivalent molecules labeled with a red fluorophore can be decreased 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, 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.

[0042] 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 employing a functional assay (e.g., a flow cell capture assay) in which clonal amplified template molecules immobilized on a flow cell are contacted with primers, polymerase, and fluorescently labeled multivalent molecules under conditions suitable for binding to the multiplex polymerase without incorporating nucleotide units into the primers, and the signal intensity can be detected.

[0043] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core bound 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) bound to multiple nucleotide arms or biotinylated nucleotide arms, where all of the bound arms have a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0044] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core bound 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) bound to multiple nucleotide arms or biotinylated nucleotide arms, where at least a first bound arm can have a first nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and a second bound 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.

[0045] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core bound to multiple nucleotide arms. 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) bound to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the bound arms have the same spacer.

[0046] 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 spacers. 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 type of spacer and a second attached arm can have a second type of spacer, and the first spacer unit and the second spacer unit are different. In some embodiments, the first and second types of linkers can be selected from any of the spacers described herein.

[0047] 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 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, in which case all of the attached arms have the same linker. In some embodiments, the linker can be selected from any of the linkers described herein (e.g., Figure 5A (bottom) and Figures 5B-5F).

[0048] 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 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, where 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, and the first and second linker units are different. In some embodiments, the first and second types of linkers can be selected from any of the linkers described herein (e.g., Figure 5A (bottom) and Figures 5B-5F).

[0049] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core bound to multiple e-arms. 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) bound to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the bound 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.

[0050] 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 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, in which case 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.

[0051] In some embodiments, the reactive groups in the linker can be reactive to chemical reagents. For example, the alkyl, alkenyl, alkynyl, and allyl reactive groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The aryl and benzyl reactive groups can be reactive to HPd / C. The amine, amide, keto, isocyanate, phosphate, thio, and disulfide reactive groups can be reactive to phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). The carbonate reactive group can be reactive to potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The reactive groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0052] In some embodiments, the nucleotide arms can have the same type of reactive group in the linker, in which case the reactive group can include an azide, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl group in the linker can be reactive to 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 tri(hydroxyproyl)phosphine (THPP).

[0053] 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 reactive groups in the 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 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, and the first and second reactive groups are different.

[0054] 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.

[0055] In some embodiments, the first and second reactive groups can be reactive to chemical agents. For example, the alkyl, alkenyl, alkynyl, and allyl reactive groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The aryl and benzyl reactive groups can be reactive to HPd / C. The amine, amide, keto, isocyanate, phosphate, thio, and disulfide reactive groups can be reactive to phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). The carbonate reactive group can be reactive to potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The reactive groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0056] In some embodiments, the nucleotide arms can have different types of reactive groups in the linker, where the reactive group can include an azide, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl group in the linker can be reactive to 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 tri(hydroxyproyl)phosphine (THPP).

[0057] The present disclosure provides compositions, systems, methods, and kits comprising multivalent molecules. In some embodiments, the multivalent molecule can comprise a core bound 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) bound to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the bound arms have nucleotide units with the same type of sugar 3'OH group.

[0058] 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, a multivalent molecule can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the attached arms 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 azide, azido, or azidomethyl group.

[0059] In some embodiments, the sugar 3' blocking groups can be reactive to chemical reagents. For example, alkyl, alkenyl, alkynyl, and allyl sugar 3' blocking groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl sugar 3' blocking groups can be reactive to HPd / C. Amine, amide, keto, isocyanate, phosphate, thio, and disulfide sugar 3' blocking groups can be reactive to phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). Carbonate sugar 3' blocking groups can be reactive to potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The sugar 3' blocking groups, urea and silyl, can be made reactive to tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0060] In some embodiments, the sugar 3' blocking group (e.g., azide, azide, and azidomethyl) can be reactive to 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 tri(hydroxyproyl)phosphine (THPP).

[0061] 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.

[0062] 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.

[0063] In some embodiments, the first and second 3'-blocking groups can be reactive to chemical reagents. For example, alkyl, alkenyl, alkynyl, and allyl 3'-blocking groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl 3'-blocking groups can be reactive to HPd / C. Amine, amide, keto, isocyanate, phosphate, thio, and disulfide 3'-blocking groups can be reactive to phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). Carbonate sugar 3'-blocking groups can be reactive to potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The sugar 3' blocking groups, urea and silyl, can be made reactive to tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0064] In some embodiments, the first and second 3' blocking groups (e.g., azide, azide, and azidomethyl) can be reactive to 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 tri(hydroxyproyl)phosphine (THPP).

[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 nucleotide units having a first sugar 3'OH blocking group. In some embodiments, the multiple nucleotide arms can comprise nucleotide units having a second sugar 3'OH blocking group. In some cases, the first and second 3'OH blocking groups can be different. For example, a 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' blocking group, and (c) at least a third arm can comprise a third nucleotide unit having a second blocking group, wherein the first and second 3' blocking groups are different from each other.

[0066] 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.

[0067] In some embodiments, the first and second 3'-blocking groups can be reactive to chemical reagents. For example, alkyl, alkenyl, alkynyl, and allyl 3'-blocking groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl 3'-blocking groups can be reactive to HPd / C. Amine, amide, keto, isocyanate, phosphate, thio, and disulfide 3'-blocking groups can be reactive to phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). Carbonate sugar 3'-blocking groups can be reactive to potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The sugar 3' blocking groups, urea and silyl, can be made reactive to tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0068] In some embodiments, the first and second 3' blocking groups (e.g., azide, azide, and azidomethyl) can be reactive to 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 tri(hydroxyproyl)phosphine (THPP).

[0069] 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.

[0070] A mixture of multivalent molecules having different units in their nucleotide arms, allowing discrimination between different multivalent molecules. In some embodiments, the core of a first multivalent molecule is labeled with a reporter moiety, allowing it to be distinguished 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., 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 represent discriminating units. In some embodiments, the first and second reporter moieties can be spectroscopically distinguishable from each other.

[0071] 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 and the base in the second multivalent molecule are different. In some embodiments, the first and second reporter moieties are spectroscopically distinguishable from one another. 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 a 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 a second multivalent molecule having the second base. The binding event can be a multivalent molecule that binds to the multiple polymerase. The incorporation event can be a nucleotide unit that is incorporated onto the 3' end of an extendible primer in the multiple polymerase, where the nucleotide unit is part of a multivalent molecule.

[0072] Mixtures of multivalent molecules The present disclosure provides separate batches (subpopulations) of labeled multivalent molecules. In some embodiments, the separate batches of labeled multivalent molecules can be prepared using a different reporter moiety for each batch. In some embodiments, the different reporter moieties can correspond to specific bases in the nucleotide arms. A particular batch can be distinguishable from other batches based on the reporter moiety 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 spectroscopically distinguishable multivalent molecules. In some embodiments, at least one batch of multivalent molecules in the mixture can be unlabeled (e.g., dark multivalent molecules).

[0073] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, which may comprise 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. Optionally, the first and second reporter moieties can be different from each other. In some embodiments, the plurality of multivalent molecules can further comprise at least a third subpopulation of multivalent molecules labeled with a third reporter moiety, where the first, second, and third reporter moieties can be different from each other. In some embodiments, the plurality of multivalent molecules can further comprise at least a fourth subpopulation of multivalent molecules labeled with a fourth reporter moiety, where the first, second, third, and fourth reporter moieties can be different from each other. 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, a first subpopulation of multivalent molecules can be labeled with a first fluorophore and a second subpopulation of multivalent molecules can be labeled with a second fluorophore. In some cases, the first fluorophore and the second fluorophore can be different.

[0074] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules, which may comprise a mixture of at least two subpopulations of multivalent molecules labeled with different reporter moieties. In some embodiments, at least a first subpopulation of the 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 the multivalent molecules in the mixture can be unlabeled (e.g., dark multivalent molecules).

[0075] 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 in the mixture can be unlabeled (e.g., dark multivalent molecules). In some embodiments, the first and second reporter moieties can be different from each other.

[0076] 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, where at least the 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, the mixture of multivalent molecules can have at least a second subpopulation, where at least the 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, the mixture of multivalent molecules can have at least a third subpopulation, where at least the third subpopulation of multivalent molecules is labeled with a third reporter moiety. In some embodiments, the mixture of multivalent molecules can have at least a fourth subpopulation, where at least the fourth subpopulation of multivalent molecules can be unlabeled (e.g., dark multivalent molecules). Optionally, the first, second, and third reporter moieties can be different from each other.

[0077] One embodiment includes a mixture of four different types of multivalent molecules, including: (1) a first subpopulation of multivalent molecules, each of which comprises a core labeled with dATP nucleotide units and a first type of fluorophore; (2) a second subpopulation of multivalent molecules, each of which comprises a core labeled with dGTP nucleotide units and a second type of fluorophore; (3) a third subpopulation of multivalent molecules, each of which comprises a core labeled with dCTP nucleotide units and a third type of fluorophore; and (4) a fourth subpopulation of multivalent molecules, each of which comprises a core labeled with dTTP nucleotide units and a fourth type of fluorophore, wherein the first, second, third, and fourth fluorophores can be spectrally distinguishable. In some embodiments, any one of the subpopulations of multivalent molecules can be unlabeled for use as a "dark" multivalent molecule.

[0078] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, where 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.

[0079] 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.

[0080] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising 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 in the plurality. Optionally, 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 have 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 in the plurality. Optionally, 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 and second types of nucleotides are different. In some embodiments, a mixture can include two, three, four, five, or more different types of multivalent molecules with nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, or dUTP, in any combination.

[0081] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, where 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.

[0082] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, where 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.

[0083] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, where 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.

[0084] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising 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, where at least the first multivalent molecule in the plurality 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, where at least the second multivalent molecule in the plurality 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 the first multivalent molecule and at least the second multivalent molecule. Optionally, 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, where 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, and the first and second types of spacers are different.

[0085] 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 include at least a first multivalent molecule, where at least the first multivalent molecule in the plurality comprises a core attached to at least one nucleotide arm having a first type of linker. In some embodiments, the plurality of multivalent molecules can include at least a second multivalent molecule, where at least the second multivalent molecule comprises 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 the first multivalent molecule and at least the second multivalent molecule. Optionally, 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, where the biotinylated arms can have a first type of linker. In some embodiments, the second multivalent molecule can include a core bound to two to five biotinylated nucleotide arms, the biotinylated arms can have a second type of linker, and the first and second types of spacers are different.

[0086] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, wherein each individual 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 on the linker. In some embodiments, each individual multivalent molecule in the plurality can comprise a core attached to two to five nucleotide arms. In some embodiments, each individual multivalent molecule in the plurality 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, or silyl group. In some embodiments, each individual multivalent molecule can comprise a reactive group that can be reactive to a chemical agent. For example, alkyl, alkenyl, alkynyl, and allyl reactive groups are reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl reactive groups can be reactive with HPd / C. Amine, amide, keto, isocyanate, phosphate, thio, and disulfide reactive groups can be reactive with phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). Carbonate reactive groups can be reactive with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). Urea and silyl reactive groups can be reactive with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the reactive group can comprise an azide, azide, or azidomethyl group, hi some embodiments, the azide, azide, or azidomethyl group in the linker can be reactive to 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 tri(hydroxyproyl)phosphine (THPP).

[0087] 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. 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 on the linker. In some embodiments, the plurality of multivalent molecules can have at least a second multivalent molecule (second subpopulation), where at least the second multivalent molecule includes a core attached to at least one nucleotide arm having a second type of reactive group on the linker. Optionally, the first reactive group on the first type of linker in the first subpopulation is different from the second reactive group on 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, where the biotinylated arms can have the first type of reactive group on the linker. 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 reactive group on a linker, where the first reactive group is different from the second reactive group.

[0088] In some embodiments, the first and second reactive groups 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 or second reactive group that can be reactive to a chemical agent. For example, the alkyl, alkenyl, alkynyl, and allyl reactive groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The aryl and benzyl reactive groups can be reactive to HPd / C. The amine, amide, keto, isocyanate, phosphate, thio, and disulfide reactive groups can be reactive to phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). The carbonate reactive group can be reactive to potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The urea and silyl reactive groups can be reactive to tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the first or second reactive group can be selected from the group consisting of azide, azide, or azidomethyl groups, in any combination. In some embodiments, the azide, azide, or azidomethyl reactive groups in the linker can be reactive to 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 tri(hydroxyproyl)phosphine (THPP).

[0089] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e.g., a population) of multivalent molecules, where 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 nucleotide units with the same sugar 3'OH group. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to 2-5 nucleotide arms. In some embodiments, each multivalent molecule in the plurality can comprise a core attached to 2-5 biotinylated nucleotide arms.

[0090] 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 nucleotide units with sugar 3' OH groups substituted with the same 3' blocking 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. 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 multivalent molecule can comprise a 3' blocking group that can be reactive to a chemical agent. For example, 3'-blocking groups such as alkyl, alkenyl, alkynyl, and allyl can be reactive 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 be reactive with HPd / C. 3'-blocking groups such as amine, amide, keto, isocyanate, phosphate, thio, and disulfide can be reactive with phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). 3'-blocking groups such as carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The sugar 3' blocking groups, urea and silyl, can be made reactive to 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, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl 3' blocking group can be reactive to 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 tri(hydroxyproyl)phosphine (THPP).

[0091] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising 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, where at least the first multivalent molecule in the plurality 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 can comprise at least a second multivalent molecule, where at least the second multivalent molecule in the plurality can comprise a core attached to at least one nucleotide arm having a second nucleotide unit with a second type of sugar 3'OH blocking group (chain-terminating moiety). In some embodiments, the plurality can comprise a first multivalent molecule and a second multivalent molecule. In some cases, the first 3' blocking group can be different from the second 3' blocking group. 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 3' blocking group. 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 3' blocking group on a linker, where the first 3' blocking group is different from the second 3' blocking group.

[0092] In some embodiments, the first and second 3' blocking groups 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 or second 3' blocking group that can be reactive to a chemical agent. For example, the alkyl, alkenyl, alkynyl, and allyl 3' blocking groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The aryl and benzyl 3' blocking groups can be reactive to HPd / C. The 3'-blocking groups amine, amide, keto, isocyanate, phosphate, thio, and disulfide can be reactive with phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). The sugar 3'-blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The sugar 3'-blocking groups urea and silyl can be reactive 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 or second 3' blocking group can be selected from the group consisting of azide, azide, or azidomethyl groups, in any combination. In some embodiments, the azide, azide, or azidomethyl 3' blocking group is reactive to 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), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0093] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising 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, where at least the first multivalent molecule in the plurality 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 comprising 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, where 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, where the biotinylated arms can have a first type of 3' blocking group.

[0094] 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 that can be reactive to a chemical agent. For example, the alkyl, alkenyl, alkynyl, and allyl 3' blocking groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The aryl and benzyl 3' blocking groups can be reactive to HPd / C. The 3'-blocking groups amine, amide, keto, isocyanate, phosphate, thio, and disulfide can be reactive with phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). The sugar 3'-blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The sugar 3'-blocking groups urea and silyl can be reactive 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 azide, azide, or azidomethyl groups, in any combination. In some embodiments, the azide, azide, or azidomethyl 3' blocking group can be reactive to 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), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0095] 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. Optionally, the first and second 3' OH blocking groups are different. In some embodiments, a first multivalent molecule can include a core bound to two to five biotinylated nucleotide arms, and 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, and 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, and the biotinylated arms can have a second type of 3' blocking group.

[0096] In some embodiments, the first and second 3' blocking groups 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 or second 3' blocking group that can be reactive to a chemical agent. For example, the alkyl, alkenyl, alkynyl, and allyl 3' blocking groups can be reactive to tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The aryl and benzyl 3' blocking groups can be reactive to HPd / C. The 3'-blocking groups amine, amide, keto, isocyanate, phosphate, thio, and disulfide can be reactive with phosphines or thiol groups, including β-mercaptoethanol or dithiothritol (DTT). The sugar 3'-blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The sugar 3'-blocking groups urea and silyl can be reactive 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, or 3'-O-benzyl groups, in any combination. In some embodiments, the first and second 3' blocking groups can be selected from the group consisting of azide, azide, or azidomethyl groups, in any combination. In some embodiments, the azide, azide, or azidomethyl 3' blocking groups can be reactive to 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), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0097] Wedge Block Assembly Various embodiments of optical systems are described herein. In some embodiments, the optical system is configured for fluorescent readout of a sample. 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) includes 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, 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, the 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 comprised of fused silica. In some embodiments, the first wedge piece (4907) and the second wedge piece (4906) are comprised of fused silica having a refractive index of 1.5. In some embodiments, the first wedge piece (4907) is coupled to a 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 housed within the housing. In some embodiments, the wedge block assembly (4916) and the piezo drive (4908) comprise a wedge block-piezo drive assembly, hi 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).

[0098] 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 upper wedge piece (4907) is aligned with the lower wedge piece (4906), as illustrated in FIG. 49A. In such embodiments, the physical distance of the wedge block assembly (4916) results in a focal plane that is aligned with the rear inner surface. In this case, the sample site (4902) on the rear inner surface is in focus. In some embodiments, the piezo drive (4908) moves the upper wedge piece (4907) to a position relative to the lower wedge piece (4906) such that the physical thickness of the wedge block (4916) in the optical path is greater than the alignment illustrated in FIG. 49A, as illustrated in FIG. 49B. In such embodiments, the focal plane is shifted to be aligned with the front inner surface. In this case, the sample site (4902) on the front inner surface is in focus.

[0099] 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 simultaneously on three different axes, all perpendicular to one another. The stage may be configured to translate simultaneously on three different axes, all perpendicular to one another. The stage may be configured to translate simultaneously on and rotate about three different axes, all perpendicular to one another. As shown in FIG. 50, 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 multiple optical subsystems (5001). The stage may translate a single optical subsystem (4914). As shown in Figures 53A-53B, the stage may rotate the multiple optical subsystems (5001) around the x-axis of the capillary flow cell (5201). As shown in Figures 53A-53B, the stage may translate the multiple optical subsystems (5001) along an x-axis that coincides with the long axis of the capillary flow cell (5201).

[0100] Pixel Shifter Described herein are various embodiments of optical systems including a pixel shifter (4911). In some embodiments, the pixel shifter (4911) enables sub-pixel resolution imaging. In some aspects, the resolution of the optical system can be increased by the use of the pixel shifter (4911) without increasing the actual optical system resolution. In some embodiments, the pixel shifter (4911) effectively increases the resolution of the imaging sensor (4912). In some embodiments, the piezoelectric actuator is configured for pixel shifting in a defined lateral direction that coincides with the image plane (e.g., in the xy 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. Optionally, the tilt stage is configured for pixel shifting in two dimensions. In some embodiments, the optical system with the pixel shifter is configured to image a 3D sample object. Optionally, the optical system with the pixel shifter is configured to image a 2D sample object.

[0101] In some embodiments, the 3D object can include a sample site (4902). In some embodiments, the sample site (4902) is an amplified nucleic acid. In some embodiments, the sample site can include a polony or multiple polonies. In some embodiments, a "polony" can refer to a polymerase colony. In some embodiments, a polony can refer to an isolated clonal amplification of a single nucleic acid. In some embodiments, the 3D object can include a non-biological material. In some embodiments, the 3D object can include an inorganic material. In some embodiments, the 3D object can include a semiconductor. In some embodiments, the polony can be a nucleic acid library molecule that can be clonally amplified (e.g., in solution, on a support, etc.) to generate amplicons. In some embodiments, the amplicons can serve as template molecules for sequencing. The linear library molecules can be circularized to generate circularized library molecules. In some embodiments, the circularized library molecules can be clonally amplified (e.g., in solution, on a support, etc.) to generate concatemers. In some embodiments, the concatemers can serve as nucleic acid template molecules. Optionally, the concatemer can be sequenced. Optionally, the concatemer can be a polony. Optionally, the polony comprises a chain of nucleotides.

[0102] The pixel shifter (4911) may also utilize polarization.

[0103] Autofocus element Described herein are various embodiments of optical systems with autofocus elements.

[0104] 51A-51B provide non-limiting cutaway views of a focusing lens assembly configured to maintain a fixed position (e.g., optical axis) within an optical path and allow relative movement between at least a first lens and a second lens contained within a lens housing of the focusing lens assembly.

[0105] 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 includes a wedge block assembly, a piezo drive, a wedge block-piezo drive assembly, or a combination thereof.

[0106] Multiple Imaging Systems In some embodiments, the optical system shown in FIG. 50 comprises a plurality of optical subsystems (4914). In some embodiments, each optical subsystem (4914) of the plurality (5001) comprises an imaging 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 imaging sensor (4912) is a cell phone camera. In some embodiments, the plurality of optical subsystems (5001) comprises a series of optical subsystems. In some embodiments, a series of optical subsystems can be configured for multiple focal depths, multiple wavelengths, or a combination thereof. In some embodiments, each optical subsystem (4914) of the plurality (5001) is configured for a focal depth, and the focal depths of at least two optical subsystems of the plurality are different. In some embodiments, each optical subsystem of the plurality is configured to detect a wavelength, and the wavelengths detected by at least two optical subsystems of the plurality are different. In some embodiments, the image sensor (4912) of each optical subsystem (4914) of the plurality (5001) includes a series of image sensors (4912). In some embodiments, a high-resolution, low-cost camera is configured for software-compensated imaging. In some embodiments, the optical system includes one optical subsystem (4914), and the optical subsystem (4914) includes one optimal imaging volume, as shown in FIGS. 49A-49B. In FIGS. 49A-49B, the range of the optimal imaging volume (4915) along the x-axis is limited. Certain factors may affect the width of the optimal imaging volume in the x-y plane (e.g., focal plane). The x-y plane, or focal plane, includes a cross-section of the optimal imaging volume and may be referred to as the illumination region, the acquisition region, or a combination thereof. A surface containing a sample site (4902) that extends beyond the optimal FOV may not be optimally illuminated by the light source, may not be optimally captured by the imaging sensor, may not be optimally resolved by the optical system, or a combination thereof.Such non-optimal regions of the surface exhibit non-uniform brightness and non-uniform resolution, as can be observed at the edges and / or corners of the image in Figure 38, where the sample site becomes dimmer and there is less resolution from the center of the image to the edges and / or corners. Figure 50 illustrates 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 an optimal composite imaging volume.

[0107] In some embodiments, the optical system has an optimized FOV of 6 mm x 6 mm. In some embodiments, the optical system has an optimized FOV of about 0.5 mm to about 9 mm. In some embodiments, the optical 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 optical 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 optical 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 optical system has an optimized FOV of at most about 1 mm, about 3 mm, about 6 mm, or about 9 mm.

[0108] In some embodiments, the optical system has an optimized illumination area of ​​6 mm x 6 mm. In some embodiments, the optical system has an optimized illumination area of ​​about 0.5 mm to about 9 mm. In some embodiments, the optical 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 optical 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 optical 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 optical system has an optimized illumination area of ​​at most about 1 mm, about 3 mm, about 6 mm, or about 9 mm.

[0109] In some embodiments, the optical system is configured for rapid imaging of the surface. In some embodiments, the optical system is configured for rapid imaging of the flow cell surface. In some embodiments, the optical system is configured for rapid imaging of the first and second surfaces of the flow cell. In some embodiments, the entire active area (e.g., region of interest, ROI) of surface (4903) or (4904) of the 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, an active area of ​​a surface (e.g., an area of ​​interest) is imaged in about 1 imaging step to about 2 imaging steps, about 1 imaging step to about 3 imaging steps, about 1 imaging step to about 4 imaging steps, about 1 imaging step to about 5 imaging steps, about 1 imaging step to about 6 imaging steps, about 1 imaging step to about 10 imaging steps, about 2 imaging steps to about 3 imaging steps, about 2 imaging steps to about 4 imaging steps, about 2 imaging steps to about 5 imaging steps, about 2 imaging steps to about 6 imaging steps. steps, about 2 imaging steps to about 10 imaging steps, about 3 imaging steps to about 4 imaging steps, about 3 imaging steps to about 5 imaging steps, about 3 imaging steps to about 6 imaging steps, about 3 imaging steps to about 10 imaging steps, about 4 imaging steps to about 5 imaging steps, about 4 imaging steps to about 6 imaging steps, about 4 imaging steps to about 10 imaging steps, about 5 imaging steps to about 6 imaging steps, about 5 imaging steps to about 10 imaging steps, or about 6 imaging steps to about 10 imaging steps. In some embodiments, an active area of ​​a 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., an area of ​​interest) is imaged in a minimum of 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., an area of ​​interest) is imaged in a maximum of 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.

[0110] Optical system method Described herein are various methods for imaging biopolymers, the methods including the steps of obtaining an optical system comprising a plurality of optical subsystems, each optical subsystem comprising a light source configured to separately emit a first wavelength and a second wavelength, the first wavelength being different from the second wavelength, a multi-band filter configured to reject each of the first wavelength and the second wavelength, and an imaging sensor configured to image one or more biopolymers disposed in an optical path between each light source and each imaging sensor; and coupling a first biopolymer of the one or more biopolymers with a first fluorophore of the plurality of fluorophores and a second biopolymer of the one or more biopolymers with a second fluorophore of the plurality of fluorophores. The method includes contacting one or more biopolymers with a plurality of fluorophores under conditions sufficient to image the first biopolymer, the first fluorophore being different from the second fluorophore, imaging the first biopolymer with each imaging sensor, the imaging sensor comprising (i) exciting the first fluorophore by illuminating the first biopolymer with a first wavelength and (ii) acquiring a first image, and imaging a second biopolymer with each imaging sensor, the imaging sensor comprising (i) exciting the second fluorophore by illuminating the second biopolymer with a second wavelength and (ii) acquiring a second image, wherein the one or more biopolymers are disposed on a curved surface and an optical axis of each optical subsystem of the plurality is orthogonal to the curved surface. In some embodiments, the method further includes imaging a third biopolymer of the one or more biopolymers, the imaging sensor comprising (i) illuminating the third biopolymer with a third wavelength to excite a third fluorophore of the plurality of fluorophores and (ii) acquiring a third image. In some embodiments, the method further comprises combining the first image and the second image into a composite image, hi some embodiments, the method further comprises identifying units of the first biopolymer bound by the first fluorophore, comprising analyzing a first region of interest (ROI) of the composite image to detect a first signal emitted by the first fluorophore.In some embodiments, the method further comprises identifying units of a second biopolymer bound by a second fluorophore, comprising analyzing a second ROI of the composite image to detect a second signal emitted by the second fluorophore. In some embodiments, the method further comprises identifying units of a first biopolymer bound by a first fluorophore, comprising analyzing a first ROI of the composite image to detect a first signal emitted by the first fluorophore, and identifying second units of a second biopolymer bound by a second fluorophore, comprising analyzing a second ROI of the composite image to detect a second signal emitted by the first fluorophore. In some embodiments, the method further comprises combining the first, second, and third images into a composite image. In some embodiments, the method further comprises identifying third units of a third biopolymer bound by a third fluorophore, comprising analyzing a third ROI of the composite image to detect a third signal emitted by the third fluorophore. In some embodiments, the method further comprises identifying a first unit of a first biopolymer bound by the first fluorophore, comprising analyzing a first region of interest (ROI) of the composite image to detect a first signal emitted by the first fluorophore; identifying a second unit of a second biopolymer bound by the second fluorophore, comprising analyzing a second ROI of the composite image to detect a second signal emitted by the first fluorophore; identifying a third unit of a third biopolymer bound by a third fluorophore, comprising analyzing a third ROI of the composite image to detect a third signal emitted by the third fluorophore; and identifying a third unit of a third biopolymer bound by the third fluorophore, comprising analyzing the third ROI of the composite image to detect a third signal emitted by the third fluorophore.

[0111] Various methods for using the optical systems described herein for super-resolution imaging are described herein. In some embodiments, the methods include providing a surface further comprising at least one sample site containing clonally amplified sample nucleic acid molecules immobilized to a plurality of bound oligonucleotide molecules, the plurality of immobilized clonally amplified sample nucleic acid molecules being spaced apart by a distance less than λ / (2*NA), where λ is the central wavelength of the excitation energy source and NA is the numerical aperture of the imaging system; simultaneously applying stochastic photoswitching chemistry to the clonally amplified sample nucleic acid molecules to cause the plurality of clonally amplified sample nucleic acid molecules to fluoresce in up to four different color on-off events by stochastic photoswitching; and determining the identity of the nucleotides of the clonally amplified sample nucleic acid molecules by detecting the on-off events in each color channel in real time as the on-off events occur in the plurality of clonally amplified sample nucleic acid molecules. The stochastic photoswitching may include using a dark state of an emitting fluorophore to randomly switch the fluorophore on and off. This allows for the imaging of individual fluorophores, which can then be localized to provide super-resolution images. In some cases, stochastic photoswitching can involve the use of techniques such as stimulated emission depletion (STED) and stochastic optical reconfiguration (STORM).

[0112] In some cases, super-resolution imaging can include imaging at a resolution of up to about 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 nanometers, or lower. In some cases, the resolution of the super-resolution imaging can be controlled by the numerical aperture of the system performing the imaging. In some cases, the resolution of the optical system can be sub-pixel resolution. Sub-pixel resolution can be imaging at a higher resolution than is achievable given the size of the pixels used for imaging (e.g., by computer processing the image, etc.).

[0113] light source In some embodiments, the light source (4901) shown in Figures 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, the light of the first wavelength at a first time, light of the second wavelength at a second time, and light of the third wavelength at a third time are emitted sequentially. In some embodiments, the multiple light sources are configured to deliver sequential colors in a timed pulse sequence. In some embodiments, the multiple optical subsystems (5001) are configured to increase the detection speed. In some embodiments, the solid-state light source is not a laser. In some applications, the light source includes a filter to narrow the spectrum of light emitted by the light source. In some embodiments, the light source is referred to as an excitation source. In some embodiments, the light emitted by the light source is referred to as excitation light.

[0114] Light Delivery Components In some embodiments, the optical system includes 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) 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 by the light delivery component. In some embodiments, the light source delivers light to the flow cell by a light pipe. In some embodiments, the light delivery component is positioned between the light source (4901) and the flow cell (4905). In some embodiments, a second light delivery component is positioned between the flow cell and the image sensor.

[0115] Imaging Channel The optical systems described herein can be configured to image one or more fluorophores. In some embodiments, the optical system is configured to specifically image two, three, or more different fluorophores. In certain aspects, the optical system includes 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, the 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, the imaging channel includes at least one of a light source (4901), a filter (4910), an imaging sensor (4912), or a combination thereof.

[0116] heater Typically, the assay requires heating. 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 a dual-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 heater integrated with a transparent heater block. In some embodiments, the heater is an 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.

[0117] Flow Cell Shape Typically, flow cell geometries are limited by standard microscope systems, which require a flat surface that can reside within the focal depth of the microscope imaging system's FOV. Such limitations restrict flow cell design at its interface, creating gradients in pressure, temperature, viscosity, or a combination thereof. Such gradients may lead to a tendency for bubbles to form, differences in reaction kinetics across cells, or a combination thereof. Furthermore, typical solutions to such problems require flow cell designs that may not be effectively imaged by standard microscope systems. To optimize the imaging performance of non-flat flow cell geometries, infrared (IR) heating, conformal transparent heaters, or a combination thereof may be utilized to reduce gradients in binding, reaction kinetics, or other assay factors. In some embodiments, the surface (5101) may be non-flat and curved, as illustrated in FIG. 52. The surface (5101) may include a concave curve (curving away from the optics) or a convex curve (e.g., curving toward the optics).

[0118] In some embodiments, the flow cell may include a capillary flow cell (5201) as illustrated in Figures 53A-53B and 54A-54B. In Figures 53A-53B, a sample flows through the capillary flow cell (5201), with the flow direction along the x-axis. Figure 53A illustrates a non-limiting example cross-section of the capillary flow cell (5201), with the sample site (4902) located on the inner surface of the capillary flow cell (5201). In some embodiments, a light source (4901) may be directed at the capillary flow cell, where the light is focused to create an optimized imaging volume (4915) containing the sample site (4902) located on the distal side of the inner surface of the capillary flow cell. In some embodiments, multiple optical subsystems (5001), each including a light source (4901), are distributed around the periphery of the capillary flow cell such that their optimal imaging volumes (4915) overlap, thereby enabling optimal imaging of a sample site located over a larger area than that corresponding to a single optimal imaging volume (4915). In some embodiments, the optical subsystems may be rotated around the x-axis of the capillary flow cell (5201), as illustrated in Figures 53A-53B, thereby enabling the overlapping optimized imaging volumes (4915) to be scanned across the entire inner surface of the capillary flow cell (5201). Alternatively, the provided flow cell (5201) may be rotated around the x-axis, and the optical subsystem (4914) may be held in a fixed position during imaging.

[0119] Another method for acquiring images of the sample site (4902) located across the entire inner surface of the capillary flow cell (5201) with uniform image quality is achieved by incorporating a wedge block (4916) into each of the optical subsystems (4914), as illustrated in Figures 53A-53B. In certain embodiments, multiple optical subsystems (4914) are arranged around the x-axis of the capillary flow cell. In such cases, the multiple optical subsystems (4914) can image a portion of the inner surface of the capillary flow cell that is larger than the single optimal imaging volume (4915) of a single optical subsystem (4914) through overlapping optimal imaging volumes (4915) described herein. In certain embodiments, curved surfaces can also be properly imaged by positioning the optical subsystems (4914) so ​​that their corresponding optical axes (4913) are at least approximately perpendicular to the region of the object being imaged, as described herein. In such cases, the multiple optical subsystems (5001) can provide optimized images of the curved, large-area surface. In some embodiments, the wedge block assembly (4916) of each optical subsystem (4914) is adjusted to provide a focus on the half of the interior surface closest to the light source, as illustrated in FIG. 54A. Alternatively, the wedge block assembly (4916) can be adjusted to focus on a sample site (4902) located on the opposite side of the interior surface of the capillary flow cell (5201), as illustrated in FIG. 54B. In such cases, there is no need to rotate the capillary flow cell (5201) or the multiple optical subsystems (5001), because refocusing and acquisition of images using multiple optimal imaging volumes (4914) provides imaging coverage of the entire interior surface of the capillary flow cell. In some embodiments, the capillary flow cell is translated along the x-axis to provide an image along the entire length of the capillary flow cell (5201). In some embodiments, the large surface area can include an area of ​​at least about 5 square millimeters.

[0120] Aberration correction In some embodiments, aberration correction methods may be applied to allow imaging of air bubbles that may appear in the flow cell. In some embodiments, a non-flat flow cell surface allows 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 system may be configured to image a flow cell with circular edges.

[0121] Integrated Field Flattener Typically, the illumination field and / or FOV of a standard fluorescence microscope imaging system is limited to the size of the single lens system and / or single imaging sensor present. Typically, the system's ability to systematically capture brightness across the entire FOV may be referred to as the system's field uniformity. Non-uniformity in brightness and resolution across the FOV is sometimes observed 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 a lens system, usually a single lens system. 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 includes multiple optical subsystems (5001) designed to provide overlapping coverage of the active area of ​​the flow cell surface. If the image of one of the individual optical subsystems (4914) of the plurality (5001) begins to become non-uniform (e.g., increased blurring, loss of intensity at corners and edges), the optimal imaging volume (4915) of the 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.

[0122] In some embodiments, the surface (5101) of the flow cell containing the sample site (4902) is not flat, as shown in Figure 52. In certain aspects, each optical subsystem (4914) of the plurality (5001) is positioned to match the contours of the active area of ​​the flow cell, as shown in Figure 52.

[0123] Optimal System - Super Resolution 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 cases, the super-resolution imaging techniques can include structured illumination.

[0124] In some instances, for example, in double-sided (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or coverslip) thickness greater than 700 μm) and fluidic channels (e.g., fluidic channel height or thickness 50-200 μm), improved imaging performance can be achieved using novel objective lens designs that correct for optical aberrations introduced by imaging surfaces on the opposite side of the thick coverslip and / or fluidic channels from the objective lens.

[0125] In some instances, improved imaging performance in double-sided (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or coverslip) thickness greater than 700 µm) and fluidic channels (e.g., fluidic channel height or thickness 50-200 µm), even when using commercially available, off-the-shelf objective lenses, can be achieved by using novel tube lens designs that, unlike tube lenses in conventional microscopes that simply form an image at an intermediate image plane, correct for optical aberrations induced by the thick flow cell walls and / or intervening fluid layers in combination with the objective lens.

[0126] In some examples, for example, improved imaging performance in multi-channel (e.g., two-color or four-color) imaging applications can be achieved by using multiple tube lenses, one for each imaging channel, where each tube lens design is optimized for the particular wavelength range used for the imaging channel.

[0127] In some examples, for example, improved imaging performance in double-sided (flow cell) imaging applications can be achieved by using an electro-optic phase plate in combination with the objective lens to compensate for optical aberrations induced by the fluid layer separating the upper (proximal) and lower (distal) 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 is moved in and out of the optical path depending on which surface of the flow cell is being imaged.

[0128] Various multichannel fluorescence imaging module designs are disclosed. They may include illumination and imaging optical paths that include a folded optical path (e.g., including one or more beam splitters or beam combiners, such as dichroic beam splitters or combiners) that directs an excitation light beam to an objective lens and directs emission light transmitted through the objective lens to multiple detection channels. Some particularly advantageous features of the fluorescence imaging modules described herein include the specification of dichroic filter angles of incidence that result in a sharper and / or more uniform transition between the passband and stopband wavelength regions of the dichroic filter. Such filters may be enclosed within the folded optics and include dichroic beam splitters or combiners. Further advantageous features of the imaging optical system designs of the present disclosure may include the position and orientation of one or more excitation light sources and one or more detection optical paths relative to the objective lens and the dichroic filters that receive the excitation beam. The excitation beam may also be linearly polarized, with the orientation of the linear polarization being such that s-polarized light is incident on the dichroic reflective surface of the dichroic filter. Such a feature may potentially improve excitation beam filtering and / or reduce wavefront errors introduced into the emission beam due to surface deformations of the dichroic filter. The fluorescence imaging modules described herein may or may not include any of these features and may or may not include any of these advantages.

[0129] 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 can also be useful, for example, for performing sequencing for comparative genomics, tracking gene expression, microRNA sequence analysis, epigenomics, characterization of aptamer and phage display libraries, and other sequencing applications. The devices and systems disclosed herein include various combinations of optical, mechanical, fluidic, thermal, electrical, and computing devices / aspects. Advantages offered by the disclosed flow cell devices, cartridges, and systems include, but are not limited to, (i) reduced complexity and cost in device and system manufacturing, (ii) significantly reduced consumable costs (e.g., compared to currently available nucleic acid sequencing systems), (iii) compatibility with typical flow cell surface functionalization methods, (iv) flexible flow control when combined with microfluidic components such as syringe pumps and diaphragm valves, and (v) flexible system throughput.

[0130] Disclosed herein are capillary flow cell devices and capillary flow cell 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, which may include one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell cartridges (or microfluidic cartridges), a fluid flow controller module, a temperature control module, an imaging module, or any combination thereof.

[0131] Some design features of the disclosed capillary flow cell devices, cartridges, and systems include, but are not limited to: (i) unified flow channel construction; (ii) sealed, reliable, and repeatable switching between reagent streams that can be performed by a simple load / unload mechanism, such that the fluid interface between the system and capillary is reliably sealed, 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 containing multiple flow paths that can be used interchangeably to provide flexible system throughput; and (iv) compatibility with a wide variety of detection methods, such as fluorescence imaging.

[0132] Although the capillary flow cell devices and systems, capillary flow cell cartridges, capillary flow cell-based systems, microfluidic devices and cartridges, and microfluidic chip-based systems of the present disclosure are described primarily in the context of their use for nucleic acid sequencing applications, various aspects of the devices and systems of the present disclosure can be applied not only to nucleic acid sequencing but also to any other type of chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. It should be understood that different aspects of the methods, devices, and systems of the present disclosure can be realized individually, collectively, or in combination with one another. While discussed herein primarily in terms of fluorescence imaging (including, for example, fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, etc.), those skilled in the art will understand that many of the optical design approaches and features of the present disclosure are applicable to other imaging modes, such as bright-field imaging, dark-field imaging, phase-contrast imaging, etc.

[0133] Definitions: Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0134] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Any reference herein to "or" is intended to include "and / or" unless expressly stated otherwise.

[0135] As used herein, a number followed by the term "about" refers to a number that is plus or minus 10% of that number. When used in the context of a range, the term "about" refers to a range of minus 10% of the lowest value and plus 10% of the highest value.

[0136] As used herein, the phrases "imaging module," "imaging unit," "imaging system," "optical imaging module," "optical imaging unit," and "optical imaging system" are used interchangeably and may include components or subsystems of a larger system that may further include, for example, a fluidics module, a temperature control module, a translation stage, robotic fluid dispensing and / or microplate handling, a processor or computer, instrument control software, data analysis and display software, etc.

[0137] As used herein, the term "detection channel" refers to an optical path (and / or optical components therein) within an optical system configured to deliver an optical signal arising from a sample to a detector. In some examples, a detection channel may be configured to monitor a fluorescent signal or other optical signal using a detector such as a photomultiplier tube to perform a spectroscopic measurement. In some examples, a "detection channel" may be an "imaging channel," e.g., an optical path (and / or optical components therein) within an optical system configured to capture and deliver an image to an image sensor.

[0138] As used herein, "detectable label" can refer to any of a variety of detectable labels or tags known to those skilled in the art. Examples include, but are not limited to, chromophores, fluorophores, quantum dots, upconverting phosphors, luminescent or chemiluminescent molecules, radioisotopes, magnetic nanoparticles, mass tags, and the like. In some examples, preferred labels may include fluorophores. Fluorescent moieties that can function as fluorescent labels or fluorophores include fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, 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 sulfonylhydrazine, 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 asLucifer 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, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbenes, DEG dyes, NR dyes, near-infrared dyes, and Molecular Probes Handbook by Haugland, 6th Edition (Eugene, Oreg.), Principles of Fluorescence by Lakowicz. Spectroscopy, 2nd Edition, Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or other fluorescent moieties, or derivatives thereof, or combinations thereof. Cyanine dyes exist in either sulfonated or non-sulfonated forms and may contain two indolenine, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example:Cy3 (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 or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl] Cy5(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-indolin-2-ylidene)penta-1,3-dien-1-yl)-3 ,3-dimethyl-3H-indol-1-ium, or 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 (1-(5-carboxypentyl)-2-[(1 ,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" represents "cyanine" and the first number isThe number of carbon atoms between the two indolenine groups is specified. Cy2, which is an oxazole derivative rather than an indolenine, and the benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0139] As used herein, the term "excitation wavelength" refers to the wavelength of light used to excite a fluorescent indicator (e.g., a fluorophore or dye molecule) and cause it to fluoresce. While the excitation wavelength is typically specified as a single wavelength, e.g., 620 nm, it will be understood by those skilled in the art that this specification refers to a wavelength range or excitation filter bandpass centered around the specified wavelength. For example, in some cases, light of a specific excitation wavelength includes light that is ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or greater, of the specified wavelength. In some instances, the excitation wavelength used may or may not coincide with the absorption peak maximum of the fluorescent indicator.

[0140] As used herein, the term "emission wavelength" refers to the wavelength of light emitted by a fluorescent indicator (e.g., a fluorophore or dye molecule) upon excitation with light of an appropriate wavelength. While the emission wavelength is typically specified as a single wavelength, e.g., 670 nm, it will be understood by those skilled in the art that this specification refers to a wavelength range or emission filter bandpass centered around the specified wavelength. In some cases, light of a specific emission wavelength includes light that is ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or greater, of the specified wavelength. In some cases, the emission wavelength used may or may not coincide with the emission peak maximum of the fluorescent indicator.

[0141] As used herein, fluorescence is "specific" when it arises from a fluorophore that is annealed or otherwise tethered to a surface, such as a fluorescently labeled nucleic acid sequence that has a region of reverse complementarity to and is annealed to a corresponding segment of an oligonucleotide adaptor on the surface. This fluorescence is in contrast to fluorescence arising from a fluorophore that is not tethered to the surface by such an annealing process, or possibly background fluorescence of the surface.

[0142] As used herein, a "nucleic acid" (also referred to as a "nucleic acid molecule," "polynucleotide," "oligonucleotide," ribonucleic acid (RNA), or deoxyribonucleic acid (DNA)) is a linear polymer of two or more nucleotides joined by covalent internucleoside linkages, or variants or functional fragments thereof. In naturally occurring examples of nucleic acids, the internucleoside linkages are typically phosphodiester linkages. However, other examples optionally include other internucleoside linkages, such as phosphorothioate linkages, which may or may not include phosphate groups. Nucleic acids include double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA / RNA hybrids, peptide-nucleic acids (PNAs), hybrids of PNAs with DNA or RNA, and may also include other types of nucleic acid modifications.

[0143] The term "nucleotide" as used herein refers to a molecule comprising an aromatic base, a sugar, and a phosphate. The "nucleotide moiety" referred to herein may be a modified nucleotide or nucleoside, for example, a nucleotide moiety conjugated to a polymer core or linker (e.g., in a nucleotide conjugate, a polymer-nucleotide conjugate, or a particle-nucleotide conjugate). Standard or non-standard nucleotides are consistent with the use of this term. In some instances, the phosphate comprises a monophosphate, a diphosphate, or a triphosphate, or a corresponding phosphate analog. In some embodiments, "nucleotide" refers to a nucleotide, a nucleoside, or an analog thereof. In some cases, the nucleotide is an N- or C-glycoside of a purine or pyrimidine base (e.g., a deoxyribonucleoside containing 2-deoxy-D-ribose, or a ribonucleoside containing D-ribose). Non-limiting examples of other nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and the like.

[0144] The term "non-flat" with respect to a surface as described herein refers to a deviation of the surface from exact flatness in at least one dimension, which may be measured using a flatness gauge or optical methods such as reflectance or interferometry. In some cases, a non-flat surface may include one or more curved portions. In some cases, the curvature of the curved portions may be perceptible to the naked eye. In some cases, a non-flat surface may be a curved surface. For example, curved surfaces described elsewhere herein may be non-flat surfaces. A non-flat substrate may include features that deviate from flatness on a length scale comparable to the surface. For example, a non-flat surface may include one or more features that are at least about 1, 5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60 percent, or more of a dimension (e.g., length, width, thickness, etc.) of the non-flat surface. In some cases, a non-planar surface can include one or more features that are up to about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1 percent, or less of a dimension of the non-planar surface. Example features include, but are not limited to, curves (e.g., a single curve, a wave, etc.), triangular features, square features, other geometric features, etc., or any combination thereof. A non-planar surface can exhibit a variation in surface height (e.g., flatness) of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500 percent, or more, of the length or width of the surface. For example, a semicircular portion of a 5 millimeter wide cylinder may exhibit 100 percent flatness. A non-planar surface may exhibit a variation in surface height (e.g., flatness) of up to about 500, 400, 300, 200, 175, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent, or less, across the length or width of the surface.

[0145] The terms "flat" or "flatness," as used herein in reference to a surface, may refer to average surface flatness or planarity, and may be measured using mechanical gauges or optical methods such as reflectance or interferometry. Optionally, flatness may include an acute angle between tangent directions measured at two different points on the surface, e.g., at least 1 Angstrom, 1 nm, 1 um, 1 mm, 1 cm, or more apart on a non-flat surface, where the acute angle may be greater than 0.1 degrees, greater than 0.5 degrees, greater than 1 degree, greater than 2 degrees, greater than 5 degrees, greater than 10 degrees, 15 degrees, 20 degrees, or more, or within a range defined by any two of the foregoing.

[0146] In some cases, flatness may refer to a property of an object (e.g., a substrate) related to the degree to which the surface height of the object varies across the area of ​​the object. For example, a flat object may exhibit no or substantially no variation in the surface height of the object across the length scale of the object. In another example, a non-flat object may exhibit variation in the surface height of the object relative to the length scale of the object. In some cases, a non-flat surface may have a monotonically varying height (e.g., the height of the object varies in only one direction). For example, a semi-cylindrical object may have a monotonically varying height. In some cases, a non-flat surface may have a non-monotonic varying height. For example, a surface with a sinusoidal height profile may have a non-monotonic varying height.

[0147] The terms "support" or "sample support structure" are used interchangeably herein to include any solid or semi-solid article capable of immobilizing reagents, such as nucleic acids. Nucleic acids can be immobilized on a solid support by any method, including, but not limited to, physical adsorption, ionic or covalent bond formation, or a combination thereof. Solid supports can include polymeric, glass, or metallic materials. Non-limiting examples of solid supports include membranes, flat surfaces, microtiter plates, beads, filters, test strips, slides, coverslips, and test tubes, any solid phase material onto which oligomers can be synthesized, attached, ligated, or otherwise immobilized. Supports can include "resins," "phases," "surfaces," "substrates," "coatings," and / or "supports." Supports can include organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. Supports can also be inorganic, such as glass, silica, controlled-pore-glass (CPG), or reverse-phase silica. The support configuration may be in the form of beads, spheres, particles, granules, gels, or surfaces. The surface may be planar, substantially planar, or non-planar. The support may be porous or non-porous and may have swelling or non-swelling properties. The support may be shaped to include one or more wells, depressions, or other containers, vessels, features, or locations. Multiple supports may be configured in an array with various locations. The support may be addressable (e.g., for robotic delivery of reagents) or addressable by a detection mechanism including scanning with laser illumination and confocal or deflection focusing. Amplification supports (e.g., beads) may be located within or on another support (e.g., within a well of a second support). The support may be a flow cell, such as a nucleic acid sequencing flow cell. In some embodiments, the support may have a surface that is hydrophilic due to the polymeric material of the support.

[0148] 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 localized information about the fluorescent signal emerges at the other end, e.g., at the image sensor. As more information is pumped through this pipeline, some content inevitably gets lost during this transfer process and is never recovered. An example of this is when too many labeled molecules (or clusters of clonally amplified molecules) are present within a small area of ​​the substrate surface that should be clearly resolved in the image. At the image sensor, it becomes difficult to distinguish between photon signals originating from neighboring molecular clusters, increasing the probability of attributing signals to the wrong cluster and resulting in detection errors. In some cases, the clusters are polonies.

[0149] Optical Imaging Module Design: Thus, the goal in designing the optical imaging module is to maximize the flow of information content through this detection pipeline and minimize detection errors. Several key design factors need to be addressed in the design process, including:

[0150] 1) Matching the physical feature density on the substrate surface to be imaged with the overall image quality of the optical imaging system and the pixel sampling frequency of the image sensor used. Mismatching these parameters can result in loss of information or sometimes even the generation of erroneous information; for example, spatial aliasing can occur when the pixel sampling frequency is two times lower than the optical resolution limit.

[0151] 2) Match the size of the imaged area with the overall image quality and focus quality across the field of view of the optical imaging system.

[0152] 3) Match the light collection efficiency, modulation transfer function, and image sensor performance characteristics of the optical system design with the expected fluorescence photon flux in terms of input excitation photon flux, dye efficiency (related to dye extinction coefficient and fluorescence quantum yield), while addressing background signal and system noise characteristics.

[0153] 4) Maximize spectral content separation to reduce crosstalk between fluorescence imaging channels.

[0154] 5) Effective synchronization of the image capture process with repositioning of the specimen or optics between image captures of different fields of view in order to maximize the overall throughput of the image capture process by minimizing downtime of the imaging system (or maximizing the duty cycle).

[0155] This disclosure addresses each of the design elements outlined above and describes a systematic method for creating component-level specifications for imaging systems.

[0156] Improved optical resolution and image quality to improve or maximize information transfer and throughput: One non-limiting design practice can be to start with the optical resolution required to distinguish two adjacent features, specified in terms of the number of line pairs per mm (lp / mm), X, and convert that to a corresponding numerical aperture (NA) requirement. The numerical aperture requirement can then be used to probe the resulting effects on the modulation transfer function and image contrast.

[0157] The standard modulation transfer function (MTF) describes the spatial frequency response for image contrast (modulation) transmitted through an optical system. Image contrast decreases with spatial frequency and increases with increasing NA. This function limits the contrast / modulation that can be achieved for a given NA. Furthermore, because wavefront errors can adversely affect MTF, it is desirable to improve or optimize optical system designs using the true system MTF instead of that predicted by diffraction-limited optics. As used herein, MTF refers to the entire system MTF (including the complete optical path from the coverslip to the image sensor), although it should be noted that design practices may primarily consider the MTF of the objective lens.

[0158] In genomic testing applications, where the target to be imaged is an array of high-density "spots" on a surface (randomly distributed or patterned), one can determine the minimum modulation transfer value required by downstream analysis to resolve two adjacent spots and discriminate between four possible states (e.g., on-off, on-on, off-on, and off-off). For example, assume the spots are small enough to be approximated as point sources. If the detection task is to determine whether two adjacent spots separated by a distance d are on or off (in other words, bright or dark), and the contrast-to-noise ratio (CNR) of the fluorescent signal arising from the spots in the sample plane (or object plane) is C, then sample Assuming that, under ideal conditions, the CNR of the readout signal for two adjacent spots in the image sensor plane is C image is C image =C sampie *Can be closely approximated as MTF(1 / d), where MTF(1 / d) is the MTF value at spatial frequency = (1 / d).

[0159] In a typical design, the value of C can be at least 4 so that a simple threshold method can be used to avoid misclassification of the fluorescent signal. imageAssuming a Gaussian distribution of fluorescent signal intensities around a mean value at , the expected error in accurately classifying a fluorescent signal (e.g., as ON or OFF) is less than 0.035%. The use of unique high CNR sequencing and surface chemistries, such as those described in U.S. Patent Application No. 16 / 363,842, provides sample plane CNRs (C) for clusters of clonally amplified labeled oligonucleotide molecules tethered to a substrate surface when measured against sparse fields (e.g., in low surface density clusters or spots) with MTF values ​​approaching 100%. sampie ) values ​​above 12 (or even much higher) are possible. sample Assuming a sample plane CNR value of greater than 12, a misclassification rate of less than 0.1% (hence, C image >4), in some implementations, a minimum value for M(l / d) can be determined as M(l / d) = 4 / 12 ~ 33%. Thus, a modulation transfer function threshold of at least 33% may be used to preserve the information content of the transfer image.

[0160] Design practice can relate the minimum separation distance d of two features or spots to the optical resolution requirement (specified above in terms of X (lp / mm)) 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, the goal of the design analysis is to increase or maximize the transfer of relevant information, and this design criterion can be relaxed to d = (1 mm) / X / A, where 2 > A > 1. For the same optical resolution of X lp / mm, the value of the minimum resolution spot separation distance d at the sample plane is reduced, allowing for the use of higher feature densities.

[0161] Design practice uses the Nyquist criterion 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 discussed above). When the spatial sampling frequency of a system is close to the Nyquist criterion, aliasing often occurs because the higher frequency information resolved by the optical system cannot be adequately sampled by the image sensor due to the imaging system resolution being greater than S.

[0162] In some of the designs disclosed herein, an oversampling scheme based on the relationship S=B*Y (B is 2 or greater and Y is the true optical system MTF limit) may be used to further improve the information transmission capacity of the imaging system. As noted above, X (lp / mm) corresponds to the actual non-zero (greater than 33%) minimum modulation transfer value, while Y (lp / mm) is the optical resolution limit, so the modulation at Y (lp / mm) is 0. Thus, in the designs disclosed herein, Y (lp / mm) may advantageously be significantly higher than X. For B values ​​greater than or equal to 2, the designs disclosed herein oversample with respect to the sample object frequency X, e.g., S≧B*Y>2*X.

[0163] The above relationship can be used to determine the magnification of the system and can provide an upper limit for the pixel size of the image sensor. The selection of the pixel size of the image sensor is matched to the optical quality of the system, as well as the spatial sampling frequency required to reduce aliasing. The lower limit for the pixel size of the image sensor can be determined based on the photon throughput, as the relative noise contribution increases with smaller pixels.

[0164] However, other design approaches are possible. For example, reducing the NA below 0.6 (e.g., 0.5 or less) can increase the depth of field. Such an increase in depth of field can enable double-sided 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 W or more, can be employed to generate strong signals. 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 ​​less than 20, providing improved signal discrimination for base calling in nucleic acid sequencing applications, etc. 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.

[0165] In various implementations, the optical systems of the present disclosure provide a large field of view (FOV). For example, an FOV of more than 2 or 3 mm can be provided in some optical imaging systems, including, for example, an objective lens and a tube lens. In some cases, the optical imaging systems provide a reduced magnification, for example, a magnification of less than 10x. Such a reduced magnification can facilitate large FOV designs in some implementations. The optical resolution of such systems can remain sufficient despite the reduced magnification because detector arrays with small pixel sizes or pitches can be used. In some implementations, to satisfy the Nyquist theorem, an image sensor including a pixel size that is two times smaller than the optical resolution provided by the optical imaging system (e.g., the objective lens and tube lens) can be used.

[0166] Still other designs are possible. In some optical designs configured to provide double-sided imaging capable of simultaneously imaging two surfaces at different depths, the optical imaging system (e.g., objective lens and / or tube lens) is configured to reduce optical aberrations for imaging the two surfaces (e.g., two planes) at each of these two depths compared 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 each of these two depths through a transparent layer (such as a glass layer (e.g., a coverslip)) on the sample support structure and through a solution (e.g., an aqueous solution) containing the sample or in contact with the sample at at least one of the two surfaces.

[0167] 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 tagged nucleic acid molecules or clusters thereof provided 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 by the sample and deliver an image of the sample (e.g., an image of the substrate surface on which the fluorescently tagged nucleic acid molecules or clusters thereof are provided) to an image sensor or other light detection device. In some examples, the fluorescence imaging system may include two, three, four, or more than four fluorescence excitation light sources and / or an optical path configured to transmit excitation light at two, three, four, or more than four excitation wavelengths (i.e., within two, three, four, or more than four excitation wavelength ranges). In some examples, the fluorescence imaging systems disclosed herein may comprise two, three, four, or more than four fluorescence emission imaging and detection channels configured to collect fluorescence emitted by a sample at two, three, four, or more than four emission wavelengths (i.e., within two, three, four, or more than four emission wavelength ranges) and deliver images of the sample (e.g., images of a substrate surface on which fluorescently tagged nucleic acid molecules or clusters thereof are provided) to two, three, four, or more than four image sensors or other light detection devices.

[0168] Double-Sided Imaging: In some examples, imaging systems disclosed herein, including fluorescent 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 fluorescent imaging systems, can be configured to acquire high-resolution images of two or more sample support structures or substrate surfaces, such as two or more surfaces of a flow cell. 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 such double-sided support structures. Figure 1A shows a double-sided support structure, such as a flow cell, that includes internal channels through which analytes or reagents can flow. The channels can be formed between first and second top and bottom plates, as shown, and / or between first and second top and bottom plates, and / or between front and back layers, such as front and back plates. One or more of the plates may include a glass plate, such as a coverslip. In some implementations, the layers include borosilicate glass, quartz, or plastic. The inner surfaces of these top and bottom layers provide flow channel walls that help confine the flow of analytes or reagents through the flow channels of the flow cell. In some designs, these inner surfaces are planar. Similarly, the top and bottom layers may be planar. In some designs, at least one additional layer (not shown) is provided between the top and bottom layers. This additional layer may have one or more channels cut into it that help define the one or more flow channels and control the flow of analytes or reagents within the flow channels. Further discussion of sample support structures, e.g., flow cells, can be found below.

[0169] 1A schematically illustrates multiple fluorescent sample sites on the first and second upper and lower surfaces and / or the front and rear interior surfaces of a flow cell. In some implementations, sample binding reactions may occur at these sites such that fluorescence is emitted from these sites. (Note that FIG. 1A is a schematic diagram and is not drawn to scale. For example, the size and spacing of the fluorescent sample sites may be smaller than shown.)

[0170] FIG. 1B illustrates another double-sided 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, sample binding reactions may occur at these sites such that fluorescence is emitted from these sites. (Note that FIG. 1B is a schematic representation and is not drawn to scale. For example, the size and spacing of the fluorescent sample sites may be smaller than shown.)

[0171] In some examples, the fluorescence imaging modules and systems described herein can be configured to image fluorescent sample sites on first and second surfaces at different distances from the objective lens. In some designs, only one of the first or second surfaces is focused at a time. Thus, in such designs, one surface is imaged at a first time and the other surface is imaged at a second time. The focus of the fluorescence imaging module can be changed after imaging one surface to image the other surface with equivalent optical resolution, since the images of the two surfaces are not simultaneously focused. 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 two surfaces. The depth of field in such fluorescence imaging configurations may not be large enough to include both the first and second surfaces. In some implementations of the fluorescence imaging modules described herein, both the first and second surfaces can be imaged at once, e.g., simultaneously. For example, the fluorescence imaging module can have a depth of field large enough to include both surfaces. In some examples, such an increase in depth of field may be achieved, for example, by decreasing the numerical aperture of the objective lens (or microscope objective lens), as discussed in more detail below.

[0172] As shown in FIGS. 1A and 1B, the imaging optics (e.g., an objective lens) may be positioned at a suitable distance (e.g., a distance corresponding to the working distance) from the first and second surfaces to form focused images of the first and second surfaces on the image sensor of the detection channel. As shown in the examples of FIGS. 1A and 1B, the first surface may be between the objective lens and the second surface. For example, as illustrated, the objective lens is disposed above both the first and second surfaces, and the first surface is disposed above the second surface. The first and second surfaces may be at different depths, for example. The first and second surfaces may be at different distances from any one or more of the fluorescence imaging module, the illumination and imaging module, the imaging optics, or the objective lens. The first and second surfaces are separated from each other with the first surface spaced apart above the second surface. In the example shown, the first and second surfaces are planar and separated from each other along a direction perpendicular to the first and second planes. Also in the illustrated example, the objective lens has an optical axis, and the first and second surfaces are separated from each other along the direction of the optical axis. Similarly, the separation between the first and second surfaces may correspond to a longitudinal distance, such as along the optical path of the excitation beam and / or along the optical axis passing through the fluorescence imaging module and / or the objective lens. Thus, these two surfaces may be separated from each other by a certain distance in the longitudinal direction (Z), which may be along the direction of the central axis of the excitation beam and / or the optical axis of the objective lens and / or the fluorescence imaging module. This separation may correspond, for example, to a flow path within a flow cell in some implementations.

[0173] In various designs, the objective lens (possibly in combination with another optical component, e.g., a tube lens) has a depth of field and / or depth of focus at least as large as the longitudinal separation (in the Z direction) between the first and second surfaces. Thus, the objective lens, alone or in combination with additional optical components, can simultaneously form focused images of both the first and second surfaces on the image sensors of one or more detection channels, with equivalent optical resolution. In some implementations, the imaging module may or may not need to refocus to capture images of both the first and second surfaces with equivalent optical resolution. In some implementations, the adaptive optics system does not need to be moved in or out of the optical path of the imaging module to form focused images of the first and second surfaces. Similarly, in some implementations, one or more optical elements (e.g., lens elements) in the imaging module (e.g., objective lens and / or tube lens) do not need to be moved longitudinally, e.g., along the first and / or second optical paths (e.g., along the optical axis of the imaging optics), to form a focused image of the first surface, compared to the location of the one or more optical elements when used to form a focused image of the second surface. However, in some implementations, the imaging module includes an autofocus system configured to simultaneously focus on both the first and second surfaces. In various implementations, the sample is focused to sufficiently resolve sample sites that are closely spaced together in the lateral directions (e.g., X and Y directions). Thus, in various implementations, no optical elements are in the optical path between the sample support structure (e.g., between the translation stages supporting the sample support structure) and the image sensor (or photodetector array) in at least one detection channel to form focused images of fluorescent sample sites on the first surface of the sample support structure and the second surface of the sample support structure.Similarly, in various implementations, optical compensation that is not the same as the optical compensation used to form an in-focus image of the fluorescent sample site on the second surface of the sample support structure on the image sensor or photodetector array is not used to form an in-focus image of the fluorescent sample site on the first surface of the sample support structure on the image sensor or photodetector array. Additionally, in certain implementations, optical elements in the optical path between the sample support structure (e.g., between the translation stages supporting the sample support structure) in at least one detection channel and the image sensor are not adjusted differently to form an in-focus image of the fluorescent sample site on the first surface of the sample support structure rather than forming an in-focus image of the fluorescent sample site on the second surface of the sample support structure. Similarly, in some various implementations, optical elements in the optical path between the sample support structure (e.g., between the translation stages supporting the sample support structure) in at least one detection channel and the image sensor are not moved a different amount or in a different direction to form an in-focus image of the fluorescent sample site on the first surface of the sample support structure on the image sensor rather than forming an in-focus image of the fluorescent sample site on the second surface of the sample support structure. Any combination of features is possible. For example, in some implementations, focused images of the upper and lower inner surfaces of the flow cell can be obtained without moving an optical compensator into or out of the optical path between the flow cell and at least one image sensor, and without moving one or more optical elements of the imaging system (e.g., the objective lens and / or the tube lens) along the optical path (e.g., the optical axis) therebetween. For example, focused images of the upper and lower inner surfaces of the flow cell can be obtained without moving one or more optical elements of the tube lens into or out of the optical path or along the optical path (e.g., the optical axis) therebetween.

[0174] Any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at two locations, such as two planes corresponding to two surfaces on a flow cell or other sample support structure on which a 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 relative to other locations or planes, such as the first and second surfaces encompassing the fluorescent sample site on a double-sided flow cell. For example, any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at two depths or planes located at different distances from the objective lens compared to aberrations associated with other depths or planes at other distances from the objective lens. For example, optical aberrations for imaging the first and second surfaces may be less than those at other locations within a range of about 1 to about 10 mm from the objective lens. Additionally, any one or more of the fluorescence imaging module, illumination path, imaging path, objective lens, or tube lens may, in some instances, be configured to compensate for optical aberrations induced by transmission of emission light through one or more portions of a sample support structure, such as a layer including one of the surfaces to which the sample adheres, as well as, optionally, a solution in contact with the sample. This layer (e.g., a coverslip or a wall of a flow cell) may comprise, for example, glass, quartz, plastic, or other transparent material that has a refractive index and introduces optical aberrations.

[0175] Thus, imaging performance can be substantially the same when imaging the first and second surfaces. For example, the optical transfer function (OTF) and / or modulation transfer function (MTF) can be substantially the same when imaging the first and second surfaces. One or both of these transfer functions can 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, the imaging performance criteria can be substantially the same when imaging the upper or lower inner surface of a flow cell without moving an optical compensator into or out of the optical path between the flow cell and the at least one image sensor and without moving one or more optical elements of the imaging system (e.g., the objective lens and / or the tube lens) along the optical path (e.g., the optical axis) therebetween. For example, the imaging performance criteria may be substantially the same for imaging the upper or lower inner surface of a flow cell without moving one or more optical elements of the tube lens into or out of the optical path, or along the optical path therebetween. In some embodiments, the optical path is the optical axis. Further discussion of MTF is contained below and in U.S. Provisional Patent Application No. 62 / 962,723, filed January 17, 2020, which is incorporated herein by reference in its entirety.

[0176] Those skilled in the art will understand that the imaging modules or systems of the present disclosure may, in some instances, be stand-alone 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 functionality and / or image processing functionality. In some instances, they may also include 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, optical fibers, apertures, and image sensors (e.g., complementary metal-oxide semiconductor (CMOS) image sensors and cameras, charge-coupled device (CCD) image sensors and cameras, etc.), as well as mechanical and / or opto-mechanical components such as XY translation stages, XYZ translation stages, piezoelectric focusing mechanisms, electro-optic phase plates, etc. In some instances, they may function as modules, components, subassemblies, or subsystems of larger systems designed, for example, for genomics applications (e.g., genetic testing 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 comprises light-tight and / or other environmentally controlled housings, temperature control modules, flow cells and cartridges, fluidic control modules, fluidic 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 in larger systems, e.g., systems designed for genomic applications, are discussed in more detail below.

[0177] 2A and 2B illustrate a non-limiting example of an illumination and imaging module (100) for multi-channel fluorescence imaging. The illumination and imaging module (100) includes an objective lens (110), an illumination source (115), multiple detection channels (120), and a first dichroic filter 130, which may include a dichroic reflector or beam splitter. Some designs may include an autofocus system, which may include, for example, an autofocus laser (102) that 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).

[0178] 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 discrete wavelength (or line) corresponding to the desired excitation wavelength, or multiple discrete wavelengths (or lines). In some examples, the line may have several very narrow bandwidths. Exemplary light sources that may be suitable for use in the illumination source (115) include, but are not limited to, incandescent filaments, xenon arc lamps, mercury vapor 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, such as the dichroic reflective surfaces of one or more dichroic filters.

[0179] The illumination source (115) may further comprise one or more additional optical components, such as lenses, filters, optical fibers, or any other suitable transmission or reflection optics, to output an excitation light beam with suitable characteristics toward the first dichroic filter (130). Beam-shaping optics, for example, may be included to receive light from, for example, light emitters in the light source, generate the beam, and / or provide desired beam characteristics. Such optics may comprise, for example, a collimating lens configured to reduce divergence of the light, increase collimation, and / or collimate the light.

[0180] In some implementations, multiple light sources are included in the illumination and imaging module (100). In some such implementations, different light sources may generate light with different spectral characteristics, for example, to excite different fluorescent dyes. In some implementations, the light generated by the different light sources may be directed to coincide and form a composite excitation light beam. This composite excitation light beam may be composed of excitation light beams from each of the light sources. The composite excitation light beam has a 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 composite excitation light beam formed from the two individual excitation light beams can 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, which may output excitation light beams that collectively form a composite beam having an optical power that is the sum of the optical powers of the individual beams.

[0181] In some implementations, the light source (115) outputs a large amount of light to generate a sufficiently strong fluorescent emission. The 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 power of the light source and / or the excitation light beam derived therefrom (including the composite excitation light beam) can range from about 0.5 watts (W) to about 5.0 W or more (as discussed in more detail below).

[0182] Referring again to Figures 2A and 2B, a first dichroic filter (130) is positioned relative to the light source to receive light from the light source. The first dichroic filter may 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 within 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 green-red and infrared wavelengths. Other spectral regions or wavelength ranges are also possible.

[0183] 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 containing fluorescently labeled nucleic acid molecules or their complements, relative to the illumination and imaging module (100). Thus, the first optical path extends from the light source to the sample through the first dichroic filter. In various implementations, the sample support structure includes at least one surface on which the sample is provided or to which the sample is attached. In some examples, the sample can be provided in or attached to different regions or sites localized on at least one surface of the sample support structure.

[0184] In some examples, the support structure may include two surfaces on which a sample is provided that are located at different distances from the objective lens (110) (e.g., different positions or depths along the optical axis of the objective lens (110)). As discussed below, for example, a flow cell may include a fluid channel formed at least in part by first and second (e.g., upper and lower) inner surfaces, and the sample may be provided at a localized site on the first inner surface, the second inner surface, or both inner surfaces. The first and second surfaces may be separated by a region corresponding to the fluid channel through which the solution flows, and thus may be at different distances or depths relative to the objective lens (110) of the illumination and imaging module (100).

[0185] 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 may be positioned to focus light from the light source onto the sample, for example, onto the surface of a microscope slide, capillary, flow cell, microfluidic chip, or other substrate or support structure. Similarly, the objective lens (110) may be configured with a suitable focal length, a working distance, and / or may be positioned to collect light reflected, scattered, or emitted from the sample (e.g., fluorescent emission) to form an image of the sample (e.g., a fluorescence image).

[0186] In some implementations, the objective lens (110) may include a microscope objective lens, such as an off-the-shelf objective lens. In some implementations, the objective lens (110) may include a custom objective lens. Examples of custom objective lenses and / or custom objective-tube lens combinations are described below and in U.S. Provisional Patent Application No. 62 / 962,723, filed January 17, 2020, which is incorporated herein by reference in its entirety. The objective lens (110) may be designed to reduce or minimize optical aberrations at two locations, such as two planes corresponding to the two surfaces of a flow cell or other sample support structure. The objective lens (110) may be designed to reduce optical aberrations at selected locations or planes relative to other locations or planes in the optical path, for example, the first and second surfaces of a double-sided flow cell. For example, the objective lens (110) can be designed to reduce optical aberrations at two 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 for imaging the first and second surfaces of the flow cell may be lower than those present elsewhere within a region extending from 1 to 10 mm from the front surface of the objective lens. Additionally, the custom objective lens (110) can be configured to compensate for optical aberrations induced by transmission of fluorescent emission light through one or more portions of the sample support structure, such as, in some instances, a layer comprising one or more of the flow cell surfaces on which the sample is disposed or a layer containing the solution filling the fluidic channels of the flow cell. These layers can include, for example, glass, quartz, plastic, or other transparent materials that have refractive indices and may introduce optical aberrations.

[0187] In some embodiments, the objective lens (110) may have a numerical aperture (NA) of 0.6 or greater (as discussed in more detail below), which may provide reduced depth of focus and / or depth of field, improved background discrimination, and improved imaging resolution.

[0188] In some embodiments, the objective lens (110) may have a numerical aperture (NA) of 0.6 or less (as discussed in more detail below). Such a numerical aperture may provide an increased depth of focus and / or depth of field. Such an increased depth of focus and / or depth of field may improve the ability to image planes separated by a distance, such as the distance separating the first and second surfaces of a double-sided flow cell.

[0189] As discussed above, a flow cell may include first and second layers, each including a first and second inner surface, separated by a fluidic channel through which, for example, an analyte or a reagent can flow. In some implementations, the objective lens (110) and / or the illumination and imaging module (100) may be configured to provide a depth of field and / or depth of focus large enough to image both the first and second inner surfaces of the flow cell, either sequentially by refocusing the imaging module while imaging the first and second 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 the first and second surfaces of the flow cell being imaged, such as the first and second inner surfaces of the flow cell. In some examples, the first and second surfaces, e.g., the first and second inner surfaces of a double-sided flow cell or other sample support structure, may be separated by a distance ranging from, for example, about 10 μm to about 700 μm or more (as discussed in more detail below). Thus, in some examples, the depth of field and / or depth of focus may be in the range from about 10 μm to about 700 μm or more (as discussed in more detail below).

[0190] In some designs, adaptive optics (e.g., "optical compensator" or "compensator") may be moved into or out of an optical path within the imaging module, e.g., the optical path through which light collected by the objective lens (110) is delivered to an image sensor, allowing the imaging module to image the first and second surfaces of a double-sided flow cell. The imaging module may be configured to image the first surface, for example, when the adaptive optics 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. In such designs, the imaging module may be configured to image the second surface when the adaptive optics is removed from or not included in the optical path between the objective lens (110) and an image sensor or photodetector array configured to capture images of the second surface. The need for an optical compensator may be further emphasized when using an objective lens (110) with a high numerical aperture (NA) value, for example, 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 more. In some implementations, the optical compensation system (e.g., an optical compensator or compensator) comprises a refractive optical element such as a lens, a plate of optically transmissive 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 employed to allow the first and second surfaces to be imaged at different times. For example, one or more lenses or optical elements may be configured to be translated in, out of, or along the optical path between the objective lens (110) and the image sensor.

[0191] However, in certain designs, the objective lens (110) is configured to provide a depth of focus and / or depth of field that is large enough to allow the first and second surfaces to be imaged with comparable optical resolution without requiring such adaptive optics to be moved in or out of an optical path within the imaging module, such as an optical path between the objective lens and an image sensor or photodetector array. Similarly, in various designs, the objective lens (110) is configured to provide a depth of focus and / or depth of field that is large enough to allow the first and second surfaces to be imaged with comparable optical resolution without requiring any optical system movement, such as one or more lenses or other optical components being translated along an optical path within the imaging module, such as an optical path between the objective lens and an image sensor or photodetector array. Examples of such objective lenses are described in more detail below.

[0192] 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 can be configured so that the fluorescence imaging module has a magnification of less than 2x to less than 10x (as discussed in more detail below). Such reduced magnification can alter design constraints so that other design parameters can be achieved. For example, the objective lens 110 can also be configured so that the fluorescence imaging module has a large field of view (FOV), for example, in the range of about 1.0 mm to about 5.0 mm (e.g., in diameter, width, length, or longest dimension), as discussed in more detail below.

[0193] In some implementations, the objective lens (110) can be configured to provide the field of view shown above to the fluorescence imaging module such that the FOV has diffraction-limited performance, e.g., an aberration of less than 0.15 waves across at least 60%, 70%, 80%, 90%, or 95% of the field of view, as discussed in more detail below.

[0194] In some implementations, the objective lens (110) can be configured to provide the field of view shown above to the fluorescence imaging module such that the FOV has diffraction-limited performance, e.g., a Strehl ratio of greater than 0.8 over at least 60%, 70%, 80%, 90%, or 95% of the field of view, as discussed in more detail below.

[0195] 2A and 2B, a first dichroic beam splitter or beam combiner is provided 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 provided in one or more second optical paths from the sample to different optical channels used to detect fluorescent emissions. Thus, a first dichroic filter (130) couples the first optical path of the excitation beam emitted by the illumination source (115) and the second optical path of the emission light emitted by the sample specimen into various optical channels through which the light is directed to respective image sensors or photodetector arrays for capturing an image of the sample.

[0196] In various implementations, the first dichroic filter (130), e.g., a first dichroic reflector, beam splitter, or beam combiner, has a passband selected to transmit light from the illumination source (115) only within a specified wavelength band, or possibly within multiple wavelength bands including the desired excitation wavelength. 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, e.g., forming part of the excitation beam. The spectral transmittance response can be configured to not transmit (e.g., instead reflect) light of one or more other wavelengths, 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., 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 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, if possible, reflects light having one or more wavelengths output by the light source not intended to reach the sample. Thus, in some implementations, the dichroic reflector has a spectral transmittance that includes one or more passbands for transmitting light incident on the sample and one or more stopbands that reflect light outside the passbands, such as one or more emission wavelengths and, if possible, one or more wavelengths output by the light source not intended to reach the sample. Similarly, in some embodiments, the dichroic reflector has a spectral reflectance that includes one or more spectral regions configured to reflect one or more emission wavelengths and, if possible, one or more wavelengths output by the light source not intended to reach the sample, and one or more regions that transmit light outside 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 an appropriate spectral transmission and reflection distribution. 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.

[0197] The imaging module (100) shown in Figures 2A and 2B and discussed above is configured such that the excitation beam is transmitted to the objective lens (110) by a first dichroic filter (130); in some designs, the illumination source (115) may be provided 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, optionally, to transmit light having one or more wavelengths output from the light source that are not intended to reach the sample. As discussed below, a design in which fluorescent emission is transmitted instead of reflected can potentially 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 provided in the second optical path to receive fluorescent emission from the sample, at least a portion of which continues to the detection channel (120).

[0198] 3A and 3B illustrate the light paths within the multichannel fluorescence imaging module of FIGS. 2A and 2B. In the example shown in FIGS. 2A and 3A, the detection channel (120) is configured 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 referenced above and described further below, in some designs, the detection channel (120) may be configured 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 comprise optics for receiving at least a portion of the emission light. For example, the detection channel (120) may comprise one or more lenses, such as a tube lens, and 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 comprise, 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. Further discussion of detection channels is included below and in U.S. Provisional Patent Application No. 62 / 962,723, filed January 17, 2020, which is incorporated herein by reference in its entirety. In some examples, improved optical resolution can be achieved using image sensors with relatively high sensitivity, small pixels, and high pixel counts combined with a suitable sampling scheme, which may include oversampling or undersampling.

[0199] 3A and 3B are ray tracing diagrams illustrating the optical paths of the illumination and imaging module (100) of FIGS. 2A and 2B. FIG. 3A corresponds to a plan view of the illumination and imaging module (100). FIG. 3B corresponds to a side view of the illumination and imaging module (100). The illumination and imaging module (100) illustrated in these figures includes four detection channels (120). However, it will be understood that the illumination and imaging modules of the present disclosure may equally 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 as few as one detection channel (120), or 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.

[0200] 3A and 3B includes four detection channels (120), a first dichroic filter (130) that reflects the 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 in the dichroic beam splitters (135) and (140) for splitting the beam (150) among the detection channels are shown oriented at 45 degrees relative to the 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.

[0201] The different detection channels (120) include an imaging device (124), which may include an image sensor or a photodetector array (e.g., a CCD or CMOS detector array). The different detection channels (120) further include an optical system (126), such as a lens (e.g., one or more tube lenses, each including one or more lens elements), configured to focus a portion of the emission light entering the detection channel (120) at a focal plane coincident with the plane of the photodetector array (124). The optical system (126) (e.g., the tube lens) combined with the objective lens (110) is configured to form an image of the sample on the photodetector array (124) to capture an image of the sample, for example, an image of the surface of a flow cell or other sample support structure after the sample has bound to its surface. Thus, such an image of the sample may include multiple fluorescent spots or regions across the spatial extent of the sample support structure from which the sample is fluorescing. The objective lens (110), together with the optical system (126) (e.g., the tube lens), may provide a field of view (FOV) that includes a portion of the sample or the entire sample. Similarly, the photodetector arrays (124) of the different detection channels (120) 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 that image. In some implementations, the photodetector arrays (124) of some or all of the detection channels (120) can detect features in the emission light emitted by the sample 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 an imaging module of the present disclosure (e.g., provided by the combination of the objective lens (110) and the optics (126) and / or (122)), as discussed below, can range, for example, between about 1 mm and 5 mm (e.g., in diameter, width, length, or longest dimension). The FOV can be selected, for example, to provide a balance between the magnification and resolution of the imaging module and / or based on one or more characteristics of the image sensor and / or objective lens. For example, to achieve high throughput, a relatively small FOV can be provided in conjunction with a smaller and faster imaging sensor.

[0202] 3A and 3B , in some implementations, the optics (126) (e.g., tube lenses) in the detection channels 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 different emission wavelengths, the optics (126) (e.g., tube lenses) for different detection channels have different designs that reduce aberrations for the corresponding 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). Similarly, in some implementations, the optical system (126) (e.g., a tube lens) can be configured to reduce aberrations when imaging a first and second surface (e.g., first and second planes, first and second object planes, etc.) on a double-sided sample support structure (e.g., a double-sided flow cell) on which a fluorescent sample site is 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 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 for imaging the first and second surfaces can be less than at other locations within a region of about 1 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 embodiments, be configured to compensate for aberrations induced by transmission of the emission light through one or more portions of the sample support structure, such as a layer comprising one of the surfaces on which the sample is disposed, and possibly a solution adjacent to and in contact with the surface on which the sample is disposed. The layer comprising one of the surfaces on which the sample is disposed can include, for example, glass, quartz, plastic, or other transparent material that has a refractive index and introduces optical aberrations.For example, custom optics (126) (e.g., a tube lens) within the detection channel may, in some implementations, be configured to compensate for optical aberrations induced by the sample support structure, e.g., a coverslip or flow cell wall, or other sample support structure components, as well as, optionally, solutions adjacent to and in contact with the surface on which the sample is placed.

[0203] In some implementations, the optics (126) (e.g., a tube lens) in the detection channel is configured to have a reduced magnification. The optics (126) (e.g., a tube lens) in the detection channel can be configured, for example, so that the fluorescence imaging module has a magnification of less than 10x, as discussed further below. Such reduced magnification can change design constraints so that other design parameters can be achieved. For example, the optics (126) (e.g., a tube lens) can also be configured so that the fluorescence imaging module has a large field of view (FOV), for example, at least 1.0 mm or more (e.g., in diameter, width, length, or longest dimension), as discussed further below.

[0204] In some implementations, the optical system (126) (e.g., a tube lens) can be configured to provide the field of view shown above to the fluorescence imaging module such that the FOV has an aberration wave of less than 0.15 across at least 60%, 70%, 80%, 90%, or 95% of the field of view, as discussed further below.

[0205] Referring again to Figures 3A and 3B, 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 Figures 2A and 2B, a light source, such as a laser source, provides an excitation beam to the sample to stimulate fluorescence. At least a portion of the fluorescent emission is collected by the objective lens (110) as emission light. 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 of which includes 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) on a photodetector array (124).

[0206] As discussed above, in some implementations, the sample support structure includes a flow cell, such as a double-sided flow cell, having two surfaces (e.g., two inner surfaces, a first surface and a second surface, etc.) that contain fluorescent sample sites. These two surfaces can be separated from each other by a certain distance in the longitudinal direction (Z), which is along the direction of the central axis of the excitation beam and / or the optical axis of the objective lens. This separation can correspond, for example, to a flow path within the flow cell. An analyte or reagent may flow through the flow path and contact the first and second inner surfaces of the flow cell, thereby contacting the binding composition such that multiple sites on the first and second inner surfaces emit fluorescence. The imaging optics (e.g., the objective lens (110)) can be positioned at a suitable distance from the sample (e.g., a distance corresponding to the working distance) to form a focused image of the sample on one or more detector arrays (124). As discussed above, in various designs, the objective lens (110) (optionally in combination with the optical system (126)) can have a depth of field and / or depth of focus at least as large as the longitudinal separation between the first and second surfaces. Thus, the objective lens (110) and the optical system (126) (for each detection channel) can simultaneously form images of both the first and second flow cell surfaces on the photodetector array (124), with these first and second surface images both in focus and having equivalent optical resolution (or can be brought into focus with only minor refocusing of the object to obtain images of the first and second surfaces with equivalent 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 and / or second optical paths) to form focused images of the first and second surfaces with equivalent optical resolution. Similarly, in various implementations, one or more optical elements (e.g., lens elements) within the imaging module (e.g., the objective lens (110) or the optical system (126)) do not need to be moved, e.g., longitudinally along the first and / or second optical paths, to form a focused image of the first surface compared to the location of the one or more optical elements when used to form a focused image of the second surface.In some implementations, the imaging module includes an autofocus system configured to rapidly and continuously refocus the imaging module on the first and / or second 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 both the first flow cell surface and the second flow cell surface can be simultaneously focused with equivalent optical resolution without moving an optical compensator into or out of the first and / or second optical paths and without moving one or more lens elements (e.g., the objective lens (110) and / or the optical system (126) (e.g., a tube lens)) longitudinally along the first and / or second optical paths. In some implementations, images of the first and second surfaces, acquired sequentially (e.g., with refocusing of the surfaces) or simultaneously (e.g., without refocusing of the surfaces) using the novel objective and / or tube lens designs disclosed herein, can be further processed using suitable image processing algorithms to increase the effective optical resolution of the images so that they have comparable optical resolution. In various implementations, the sample plane is sufficiently focused to resolve sample sites on the first and / or second flow cell surfaces, and the sample sites are closely spaced laterally (e.g., in the X and Y directions).

[0207] As discussed above, dichroic filters may comprise interference filters that selectively transmit and reflect light of different wavelengths based on the principles of thin-film interference, using layers of optical coatings with different refractive indices and specific thicknesses. Thus, the spectral response (e.g., transmission and / or reflection spectra) of dichroic filters implemented 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 important with respect to dichroic filters in the detection optical path (e.g., dichroic filters (135) and (140) in FIGS. 3A and 3B).

[0208] FIG. 4 is a graph illustrating the relationship between dichroic filter performance and beam angle of incidence (AOI). Specifically, the graph in FIG. 4 illustrates the effect of the angle of incidence on the transition width or spectral span of a dichroic filter, which corresponds to the wavelength range over which the spectral response (e.g., transmission spectrum and / or reflection spectrum) transitions between the passband and stopband regions of the dichroic filter. Thus, a transmission end (or reflection end) with a relatively small spectral span (e.g., a small delta λ value in the graph in FIG. 4) corresponds to a more abrupt transition between the passband and stopband regions or between the transmission and reflection regions (or conversely, between the reflection and transmission regions), while a transmission end (or reflection end) with a relatively large spectral span (e.g., a large delta λ value in the graph in FIG. 4) corresponds to a less abrupt transition between the passband and stopband regions. In various implementations, a more abrupt transition between the passband and stopband regions is generally desirable. Additionally, increased conformance or relatively consistent transition width across all or most of the field of view and / or beam area may also be desirable.

[0209] 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 one example of a dichroic filter, as shown in Figure 4. Because the emission light beam is not collimated and exhibits some divergence, the fluorescence imaging module can have an incidence angle range of approximately 5 degrees between opposing faces of the beam. Thus, as shown in Figure 4, different portions of the emission light beam can be incident on the channel-splitting dichroic filter at various incidence angles between 40 and 50 degrees. This relatively large range of incidence angles corresponds to a transition width range between approximately 40 and 62 nm. This relatively large range of incidence angles thereby results in an increase in the transition width of the dichroic filter within the imaging module. Therefore, the performance of a multichannel fluorescence imaging module can be improved by providing smaller incidence angles throughout the beam, which results in sharper transmission edges and better discrimination between different fluorescence emission bands.

[0210] FIG. 5 is a graph illustrating the relationship between the size of the beam footprint (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 a smaller dichroic filter to be used to split a beam into different wavelength ranges. The use of smaller dichroic filters reduces manufacturing costs and improves the ease of manufacturing a suitably flat dichroic filter. As shown in FIG. 5, an 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 greater than 1.4 times the cross-sectional area of ​​the beam when incident at zero degrees.

[0211] 6A and 6B schematically illustrate non-limiting examples of dichroic filter and detection channel configurations within a multichannel fluorescence imaging module in which the dichroic mirror is disposed at an angle of less than 45 degrees relative to the central beam axis of the emission light or the optical axis of the light path (e.g., of the objective lens and / or tube lens). FIG. 6A depicts an imaging module (500) comprising multiple detection channels (520a), (520b), (520c), and (520d). FIG. 6B is a detailed view of a portion of the imaging module (500) within the circle (5B) shown in FIG. 6A. As discussed in more detail, the configurations illustrated in FIGS. 6A and 6B include numerous 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 with one or a subset of the features described with respect to FIGS. 6A and 6B without departing from the spirit or scope of the present disclosure.

[0212] The imaging module (500) depicted in Figure 6A comprises an objective lens (510) and four detection channels (520a), (520b), (520c), and (520d) configured 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 as well as Figures 3A and 3B, the first dichroic filter (530) (e.g., a dichroic beam splitter or combiner) is configured to reflect light from the light source onto the objective lens (510) and the sample and transmit fluorescent emission from the sample to 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) and (520b) by transmitting a first portion (550a) and reflecting a second portion (550b). Additional dichroic filters (540a) and (540b) are provided to further split the emission light. The dichroic filter (540a) transmits at least a portion of the first portion (550a) of the emission light and reflects a portion (550c) into the third detection channel (520c). The dichroic filter (540b) transmits at least a portion of the second portion (550b) of the emission light and reflects a portion (550d) into the fourth detection channel (520d). Although the imaging module (500) is depicted with four detection channels, in various embodiments, the imaging module (500) may include more or fewer detection channels, with more or fewer dichroic filters being provided accordingly to provide a portion of the emission light to each detection channel. For example, in some embodiments, the features of the imaging module (500) may be implemented with similar advantages to an imaging module that includes only two detection channels (520a), (520b) but omits the additional dichroic filters (540a), (540b). In some implementations, as few as one detection channel may be included. Alternatively, three or more detection channels may be employed.

[0213] The detection channels 520a, 520b, 520c, and 520d illustrated in Figure 6A may include some or all of the same or similar components as the detection channels 120 illustrated in Figures 2A-3B. For example, the different detection channels 520a, 520b, 520c, and 520d may include one or more image sensors or photodetector arrays, and may include transmission and / or reflection optics, such as one or more lenses (e.g., tube lenses), that focus light received by a detection channel onto its corresponding image sensor or photodetector array.

[0214] The objective lens (510) is configured to receive emission light emitted by fluorescence from the specimen. Specifically, the first dichroic filter (530) is configured to receive the emission light collected and transmitted by the objective lens (510). As discussed above and shown in FIG. 6A , in some designs, an illumination source such as a laser source (e.g., the illumination source (115) in FIGS. 2A and 2B ) is configured to provide an excitation beam that is incident on the first dichroic filter (530), such that the first dichroic filter (530) reflects the excitation beam back to the same objective lens (510), which transmits the emission light, for example, in an epifluorescence configuration. In other designs, the illumination 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 a configuration, the first dichroic filter (530) may be omitted.

[0215] 6A, the detection optics (e.g., comprising the detection channels (520a), (520b), (520c), (520d) and any optical components along the optical path between the objective lens (510) and the detection channels (520a), (520b), (520c), (520d), such as the dichroic filters (535), (540a), (540b)) may be located on the transmission path of the first dichroic filter (530) rather than on the reflection path of the first dichroic filter (530). In one exemplary implementation, the objective lens (510) and the detection optics are located such that the objective lens (510) directly transmits the beam of emission light (550) toward the second dichroic filter (535). The wavefront quality of the emission light may be somewhat degraded by the presence of the first dichroic filter (530) along the path of the beam of emission light (550) (e.g., by imparting some wavefront error to the beam (550)). However, the wavefront error introduced by the beam transmitted through the dichroic reflector of the dichroic beamsplitter is generally significantly smaller (e.g., orders of magnitude smaller) than the wavefront error of the beam reflected from the dichroic reflective surface of the dichroic beamsplitter. Therefore, the wavefront quality of the emission light and subsequent imaging quality in a multichannel fluorescence imaging module may be substantially improved by placing detection optics along the transmitted beam path of the first dichroic filter (530) rather than the reflected beam path.

[0216] 6A, within the detection optics of the imaging module 500, dichroic filters 535, 540a, and 540b are provided to split the beam of emission light 550 among the detection channels 520a, 520b, 520c, and 520d. For example, the dichroic filters 535, 540a, and 540b split the beam 550 based on wavelength such that a first wavelength or wavelength band of emission light may be received by the first detection channel 520a, a second wavelength or wavelength band of emission light may be received by the second detection channel 520b, a third wavelength or wavelength band of emission light may be received by the third detection channel 520c, and a fourth wavelength or wavelength band of emission light may be received by the fourth detection channel 520d. In some implementations, multiple separate wavelengths or wavelength bands may be received by a detection channel.

[0217] In contrast to the multichannel fluorescence imaging module designs shown in Figures 2A and 2B as well as Figures 3A and 3B, the imaging module (500) has dichroic filters (535), (540a), and (540b) positioned at angles of incidence less than 45 degrees relative to the central beam axis of the incident beam. As shown in Figure 6B, the different beams (550), (550a), and (550b) have corresponding central beam axes (552), (552a), and (552b). In various implementations, the central beam axes (552), (552a), and (552b) are at the cross-sectional centers 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 in the separate channels, such as the corresponding tube lenses. Additional rays 554, 554a, and 554b of each beam 550, 550a, and 550b are illustrated in Figure 6B to illustrate the diameter of each beam 550, 550a, and 550b. The beam diameter may be defined, for example, as the full-width-at-half-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 as the second moment width, or any other suitable definition of beam diameter.

[0218] 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) on 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 normal to the surface at which the beam is incident, for example, on 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 similarly be described in terms of their central beam axes 552a and 552b, respectively. As noted above, optical axes may alternatively or additionally be used.

[0219] In the exemplary configuration of Figures 6A and 6B, the second dichroic filter (535) is positioned so that the central beam axis (552) of the beam (550) is incident at a 30-degree angle of incidence. Similarly, the additional dichroic filters (540a) and (540b) are positioned so that the central beam axes (552a) and (552b) of the first and second portions (550a) and (550b) of the beam (550) are also incident at a 30-degree angle of incidence. However, in various implementations, these angles of incidence may be other angles less than 45 degrees. In some examples, for example, as discussed further below, the angles of incidence may range between approximately 20 degrees and approximately 45 degrees. Furthermore, the angles of incidence for each of the dichroic filters (535), (540a), and (540b) do not necessarily have to be the same. In some embodiments, some or all of the dichroic filters 535, 540a, and 540b may be arranged such that their incident beams 550, 550a, and 550b have different angles of incidence. As mentioned above, the angles of incidence may be relative to the optical axis of the optics in the imaging module, for example, the objective lens and / or optics (e.g., tube lens) in the detection channel and the dichroic reflective surface in the corresponding dichroic beam splitter. The same ranges and values ​​for the angles of incidence apply when the optical axis is used to identify the AOI.

[0220] The beams of emission light 550, 550a, 550b in a fluorescence imaging module system are typically diverging beams. As noted above, the beams of emission light 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 relative to the angle of incidence of the central beam axis and / or the optical axis of the optical system. In some designs, the objective lens 510 can be configured with an f-number or numerical aperture selected to produce a smaller beam diameter for a given field of view of the microscope, for example. In one example, the f-number or numerical aperture of the objective lens (510) can be selected so that the full diameter of the beams (550), (550a), (550b) is incident on the dichroic filters (535), (540a), (540b) at an angle of incidence that is, for example, within 1 degree, 1.5 degrees, 2 degrees, 2.5 degrees, 3 degrees, 3.5 degrees, 4 degrees, 4.5 degrees, or 5 degrees of the angle of incidence of the central beam axis (552), (552a), (552b).

[0221] In some implementations, the focal length of an objective lens suitable for producing such a narrow beam diameter may be longer than those typically employed in fluorescence microscopes or imaging systems. For example, in some embodiments, the focal length of the objective lens may range between 20 mm and 40 mm, as discussed further below. In one example, an objective lens (510) having a focal length of 36 mm may produce a beam (550) characterized by a divergence so small 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 beam's central axis.

[0222] 7 and 8 are graphs illustrating the performance improvement of dichroic filters according to the embodiments of the imaging module configurations of FIGS. 6A and 6B (or any of the imaging module configurations disclosed herein). The graph in FIG. 7 is similar to the graph in FIG. 4 and shows the effect of incidence angle on the transition width (e.g., the spectral span of the transmission end) of a dichroic filter. FIG. 7 shows an example in which dichroic filters (e.g., dichroic filters 535, 540a, and 540b) and the dichroic reflective surfaces therein are oriented so that the incident beam has a 30-degree incidence angle instead of a 45-degree angle. FIG. 7 illustrates how this reduction in incidence angle significantly improves the sharpness and uniformity of the transition width across the beam diameter. For example, a 45-degree incidence angle at the central beam axis results in a transition width range of between about 40 nm and about 62 nm, while a 30-degree incidence angle at the central beam axis results in a transition width range of between about 16 nm and about 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 the stopband. Furthermore, the variation in transition width across the beam diameter is reduced by nearly 40%, from a 22 nm range to a 14 nm range, indicating a more uniformly sharp transition over the area of ​​the beam.

[0223] FIG. 8 illustrates a further advantage that can be realized by selecting an appropriate f-number or numerical aperture of the objective lens to reduce beam divergence in any of the imaging module configurations disclosed herein. In some implementations, longer focal lengths are used. In the example of FIG. 8, the objective lens (510) has a focal length of 36 mm, which reduces the range of incident angles within the beam (550) from 30°±5° to 30°±2.5° with an appropriate numerical aperture (e.g., less than 5). With this design, the range of transition widths can be reduced to between about 19 nm and about 26 nm. Compared to the improved system of FIG. 7, the average transition width is roughly the same (e.g., a spectral span of about 23 nm), but the variation in transition width across the beam diameter is further reduced to a range of 7 nm, a nearly 70% reduction over the transition width range illustrated in FIG. 4.

[0224] Referring again to FIG. 5, decreasing the angle of incidence from 45 degrees to 30 degrees at the central beam axis is even more advantageous because it reduces the beam spot size on the dichroic filters. As shown in FIG. 5, a 45-degree angle of incidence produces a beam footprint on the dichroic filters with an area greater than 1.4 times the cross-sectional area of ​​the beam. However, a 30-degree angle of incidence produces a beam footprint on the dichroic filters with an area that is only about 1.15 times the cross-sectional area of ​​the beam. Thus, decreasing the angle of incidence on dichroic filters 535, 540a, and 540b from 45 degrees to 30 degrees reduces the beam footprint area on dichroic filters 535, 540a, and 540b by approximately 18%. This reduction in beam footprint area allows for the use of smaller dichroic filters.

[0225] 9A-9B, 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. Overall, larger area optical elements experience increased surface deformations. When a larger area is employed on the dichroic filter, a larger amount of surface deformation is encountered, resulting in more wavefront error being introduced into the beam. FIG. 9A shows the effect of the fold angle on image degradation induced by applying 1 wave of peak-to-valley (PV) spherical power to the last mirror. FIG. 9B shows the effect of the fold angle on image degradation induced by applying 0.1 wave of PV spherical power to the last mirror. As shown in FIGS. 9A and 9B, reducing the angle of incidence to 30 degrees significantly reduces the impact on surface deformations, achieving near-diffraction-limited performance for the detection optics.

[0226] In some implementations of the imaging module of the present disclosure, the polarization state of the excitation beam can be utilized to further improve the performance of the multi-channel fluorescence imaging module disclosed herein. Returning to FIGS. 2A, 2B, and 6A, for example, some implementations of the multi-channel fluorescence imaging module disclosed herein have an epi-fluorescence configuration in which a first dichroic filter (130) or (530) merges the optical paths of the excitation beam and the emission beam so that both excitation and emission light are transmitted through the objective lens (110), (510). As discussed above, the illumination source (115) can include a light source, such as a laser or other light source, that provides light to form the excitation beam. In some designs, the light source includes a linearly polarized light source, and the excitation beam 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. Retarder such as half wave retarders, multiple quarter wave retarders, or retarders with other amounts of retardance may be included in some designs to rotate linear polarization.

[0227] When the linearly polarized excitation beam is incident on any dichroic filter or other planar interface, it may be p-polarized (e.g., have an electric field component parallel to the plane of incidence), s-polarized (e.g., have an electric field component perpendicular to the plane of incidence), or may exhibit 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 that interacts with the excitation beam. 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 an emitter, such as a solid-state laser or laser diode, that can be rotated about its optical axis or central axis of the beam to orient the linearly polarized light output therefrom. Alternatively, or in addition, a retarder may be employed to rotate the linear polarization about the optical axis or central axis of the beam. As discussed above, in some implementations, a polarizer placed in the path of the excitation beam can polarize the excitation beam, for example, if the light source does not output polarized light. In some designs, for example, a linear polarizer is provided in the path of the excitation beam. This polarizer can be rotated to provide the appropriate orientation of the linear polarization to provide s-polarized light.

[0228] In some designs, linearly polarized light is rotated around the optical or central axis of the beam 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 is abrupt, as opposed to when p-polarized light is incident on the dichroic reflector of the dichroic beamsplitter.

[0229] As shown in Figures 10A and 10B, the use of p- or s-polarization states of the excitation beam can significantly affect the narrowband performance of any excitation filter, such as the first dichroic filter (130), (530). Figure 10A shows the transmission spectra between 610 nm and 670 nm of an exemplary bandpass dichroic filter at 40 and 45 degree angles of incidence, where the incident beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. As shown in Figure 10B, changing the orientation of the light source relative to the dichroic filter, such that the incident beam is s-polarized relative to the plane of the dichroic filter, makes the edge between the passband and stopband of the dichroic filter sharper. For this reason, the illumination and imaging modules (100), (500) disclosed herein may advantageously have the illumination source (115) oriented with respect to the first dichroic filter (130), (530) such that the excitation beam is s-polarized with respect to the plane of the first dichroic filter (130), (530). As discussed above, in some implementations, a polarizer, such as a linear polarizer, may be used to polarize the excitation beam. This polarizer may be rotated to provide a linear polarization orientation corresponding to s-polarization. Also, as discussed above, in some implementations, other techniques for rotating 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 configurations are possible.

[0230] As discussed elsewhere herein, decreasing the numerical aperture (NA) of the fluorescence imaging module and / or objective lens can increase the depth of field and allow equivalent imaging of the two surfaces. Figures 11A-16B show how the MTF is similar at a first and second surface separated by 1 mm of glass for a small NA rather than a large NA.

[0231] 11A and 11B show the MTF at the first surface (FIG. 11A) and the second surface (FIG. 11B) when the NA is 0.3.

[0232] 12A and 12B show the MTF at the first surface (FIG. 12A) and the second surface (FIG. 12B) when the NA is 0.4.

[0233] 13A and 13B show the MTF at the first surface (FIG. 13A) and the second surface (FIG. 13B) when the NA is 0.5.

[0234] 14A and 14B show the MTF at the first surface (FIG. 14A) and the second surface (FIG. 14B) when the NA is 0.6.

[0235] 15A and 15B show the MTF at the first surface (FIG. 15A) and the second surface (FIG. 15B) when the NA is 0.7.

[0236] Figures 16A and 16B show the MTF at the first surface (Figure 16A) and the second surface (Figure 16B) 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.

[0237] Figures 17A-17B provide plots of the calculated Strehl ratio (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. Figure 17A shows 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 (fluid channel height) for various objective and / or optical system numerical apertures. As shown, the Strehl ratio decreases as the separation between the first and second surfaces increases. Therefore, one surface may have degraded image quality as the separation between the two surfaces increases. The degradation in second surface imaging performance with increasing separation distance between the two surfaces is less for imaging systems with smaller numerical apertures compared to imaging systems with larger numerical apertures. Figure 17B 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 layer of water with a thickness of 0.1 mm. The loss in imaging performance at higher numerical apertures can be attributed to the increase in optical aberrations induced by the fluid in second-surface imaging. As the numerical aperture increases, the increase in optical aberrations introduced by the fluid in second-surface imaging significantly degrades image quality. However, as the numerical aperture of the optical system as a whole decreases, the achievable resolution decreases. 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 fluorescent emission of labeled nucleic acid clusters and / or reduce background fluorescent emission. In some examples, a sample support structure including, for example, a hydrophilic substrate material and / or a hydrophilic coating may be employed. In some examples, such hydrophilic substrates and / or hydrophilic coatings can reduce background noise.Further discussion of sample support structures, hydrophilic surfaces and coatings, and methods for improving contrast-to-noise ratios in, for example, nucleic acid sequencing applications can be found below.

[0238] In some implementations, 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 are configured to have a reduced magnification, such as a magnification of less than 10x, as discussed further below. Such reduced magnification may adjust design constraints so that other design parameters can be achieved. For example, any one or more of the fluorescence microscope, illumination and imaging module (100), imaging optics (e.g., optical system (126)), objective lens, or tube lens may also be configured, for example, such that the fluorescence imaging module has a large field of view (FOV), such as a field of view (e.g., in diameter, width, height, or longest dimension) of at least 3.0 mm or more, as discussed further below. 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 to provide the fluorescence microscope with a field of view such that the FOV has an aberration of less than 0.1 waves 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 an FOV and is diffraction-limited or diffraction-limited across such FOV.

[0239] As discussed above, in various implementations, the optical system of the present disclosure provides a large field of view (FOV). In some implementations, obtaining an increased FOV is facilitated in part by the use of a larger image sensor or photodetector array. As discussed further below, the photodetector array may have an active area with a diagonal of, for example, at least 15 mm or more. As discussed above, in some implementations, the optical imaging system of the present disclosure provides a reduced magnification of less than 10x, which may facilitate, for example, large 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. As discussed...

Claims

1. A method for imaging a sample, comprising: (a) obtaining an optical system, wherein the optical system comprises: (1) a light source that illuminates the sample with a first light; (2) a sensor that obtains an image of the sample while the sample is illuminated by the first light from the light source; and (3) a focusing element assembly provided along the optical path from the light source to the sensor, the focusing element assembly comprising (i) a first focusing element and (ii) a second focusing element, wherein the first focusing element is movable relative to the second focusing element without removing the first focusing element from the optical path; and (b) illuminating the sample with the first light from the light source; (c) focusing the second light emitted from the sample by the focusing element assembly; (d) receiving the second light from (c) and obtaining one or more images of the sample by the sensor.

2. The method according to claim 1, wherein the optical system further comprises a plurality of the light sources, and two or more of the plurality of light sources emit the first light of different wavelengths.

3. The method according to claim 1, wherein the optical system further comprises a plurality of sensors including the sensor, and two or more of the plurality of sensors obtain the one or more images of the sample at different times.

4. The method according to claim 1, wherein the optical system further comprises a filter provided along the optical path from the light source to the sensor, the filter being configured to receive the second light from the sample and transmit another light to the sensor.

5. The method according to claim 4, wherein the filter includes a multi-band filter.

6. The method according to claim 5, wherein the multi-band filter includes a three-band stop-band filter.

7. The method according to claim 1, wherein the optical system further comprises a piezo drive unit coupled to the first focusing element, the piezo drive unit controlling the movement of the first focusing element relative to the second focusing element.

8. The method according to claim 1, wherein the optical system further comprises a housing, the housing including the focusing element assembly.

9. The method according to claim 1, wherein the first focusing element and the second focusing element each have a refractive index of approximately 1.

5. **Claim 10**: The method according to claim 1, wherein the optical system further comprises a gap between the first focusing element and the second focusing element, and the gap has a width that remains constant when the first focusing element moves relative to the second focusing element. **Claim 11**: The method according to claim 1, wherein the focusing element assembly comprises a wedge block assembly, the first focusing element includes a first wedge piece of the wedge block assembly, and the second focusing element includes a second wedge piece of the wedge block assembly. **Claim 12**: The method according to claim 1, wherein the sample includes a plurality of biological polymers, a first subset of the plurality of biological polymers is bound to a first inner surface of the flow cell, a second subset of the plurality of biological polymers is bound to a second inner surface of the flow cell, and the step of obtaining the one or more images of the sample includes obtaining one or more images of the first inner surface and the second inner surface of the flow cell. **Claim 13**: Obtaining the one or more images of the first inner surface comprises (a) obtaining a first image of the first inner surface; (b) moving the first focusing element relative to the second focusing element without removing the first focusing element from the optical path, thereby adjusting the depth of focus of the optical system to focus on the second inner surface; and (c) obtaining a second image of the second inner surface. The method according to claim 12. **Claim 14**: The method according to claim 12, wherein the first inner surface and the second inner surface of the flow cell include a hydrophilic polymer layer bound thereto. **Claim 15**: The method according to claim 14, wherein the plurality of biological polymers are bound to the hydrophilic polymer layer. **Claim 16**: The method according to claim 1, wherein the optical system has a field of view (FOV) greater than 1 square millimeter (mm2). **Claim 17**: The method according to claim 16, wherein the optical system has a field of view (FOV) greater than 2.5 mm2. **Claim 18**: The method according to claim 1, wherein the optical system includes a numerical aperture (NA) less than 0.

6. **Claim 19**: The method according to claim 1, wherein the first focusing element and the second focusing element include fused silica. **Claim 20**: The method according to claim 1, wherein the optical system is a fluorescence imaging system.

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