Single color fluorescent optical systems

Abstract

Fluorescence imaging systems designs and methods of are described herein that enable imaging of sequencing samples with a light source of a single color. The optical systems herein provides higher signal efficiency, simpler optical filter design, and lower costs for DNA sequencing analysis and other imaging applications.

Description

SINGLE COLOR FLUORESCENT OPTICAL SYSTEMSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 645,443, filed May 10, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled 52933-762_601.XML, created on May 8, 2025, which is 2,810 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0003] In fluorescence-based genomic testing assays, e.g. , genotyping or nucleic acid sequencing (using either real time, cyclic, or stepwise reaction schemes), dye molecules that are attached to nucleic acid molecules tethered on a substrate are excited using an excitation light source, a fluorescence photon signal can be generated in one or more spatially-localized positions on the substrate, and the fluorescence can be subsequently imaged through an optical system onto an image sensor. An analysis process can then used to analyze the images, find the positions of labeled molecules (or clonally amplified clusters of molecules) on the substrate, and quantify the fluorescence photon signal in terms of wavelength and spatial coordinates, which may then be correlated with the degree to which a specific chemical reaction, e.g. , a hybridization event or base addition event, occurred in the specified locations on the substrate. Imagingbased methods can provide large scale parallelism and multiplexing capabilities, which can help to drive down the cost and accessibility of such technologies. However, detection errors that arise from, for example, overly dense packing of labeled molecules (or clonally-amplified clusters of molecules) within a small region of the substrate surface, or due to low contrast-to-noise ratio (CNR) in the image, may lead to errors in attributing the fluorescence signal to the correct molecules (or clonally amplified clusters of molecules).SUMMARY

[0004] Described herein are sequencing systems with imaging modules and methods of using thereof for analyzing biological analytes such as DNA. In some embodiments, the biological analytes are nucleic acids. In some embodiments, the methods for use of the devices and systems comprise sequencing nucleic acids. The optical systems and methods described herein can advantageously utilize a single laser or a light source with a single wavelength to excite the biological analytes and detect emission light of differait wavelengths in response to the excitation. Comparing with existing optical system that can only function with multiple lasers or otherwise light source of multiple wavelengths, the optical systems herein are more compact in size, lower in cost, simpler in filter design, and more efficient in collecting emission signals. The optical systems herein also advantageously allow better separation of emission light according to theirwavelength, thus reducing color cross-talk and improve photon collection efficiency which in turn can result in generating more accurate sequencing analysis results. Further, the optical systems and methods described herein advantageously allow homogenous illumination over an wide field of view (FOV) of at least 10 mm2with less than 10% energy variance across the FOV. The systems and methods disclosed herein can enable simultaneous sequencing of morphological, RNA, and / or protein targets within traditional 2D samples or in situ samples. The systems and methods disclosed herein can enable imaging and sequencing of such targets simultaneously within a same sequencing run or even within the same sequencing cycle(s) with significantly reduced hands-on time of a user. Furthermore, the optical systems and methods described herein is advantageously capable of imaging two, three, four, or even more surfaces of the flow cell devices that are axially-displaced from each other, therefore advantageously achieving improved sequencing throughput within a set system run time. As such, the optical systems and methods herein can increase effectiveness and efficiency of sequencing analysis. The devices, systems, and methods herein can enable imaging of three or more axially-displaced surfaces of the flow cell device without moving any optical compensator into, out of, or along the optical path, therefore providing simpler and more conveniently optical systems that are also less prone to errors due to vibrations. Further, the devices, systems, and methods herein can advantageously allow conveniently switching between imaging traditional flow cells, e.g. , with one or dual surfaces, and the multiple surface flow cells herein with three or more axially-displaced surfaces. Such switching does not require adding or removing any optical elements in the system in and out of the optical path, e.g., an optical compensator. The devices, systems, and methods herein also enable imaging with a numerical aperture (NA) of less than 0.6 with sufficient image qualities for accurate sequencing analysis, allow imaging of the multiple axially-displaced surfaces independently, and allow adjustment of the objective lens to alter the NA to a desired value less than 0.6. The devices, systems, and methods herein can reduce reagent consumption needed for sequencing analysis of a same amount of samples than existing flow cell devices and optical systems.

[0005] In an aspect, the present disclosure provides a system, comprising: a flow cell configured to contain a sample; a light source configured to illuminate the sample with an illumination light, wherein illuminating the sample generates an emission light comprising an emission bandwidth; an imaging system comprising a detector configured to receive the emission light, wherein the imaging system has a field of view of at least 1 square millimeters (mm2); and an emission filter disposed in an optical path between the flow cell and the detector, wherein the emission filter does not comprise a filter region for the illumination light within the emission bandwidth of the emission light.

[0006] In some embodiments, the filter region for the illumination light is configured to reject the illumination light. In some embodiments, the system further comprises an excitation filter disposed in an optical path between the illumination source and the flow cell. In some embodiments, the excitation filter is configuredto filter a portion of the illumination light, thereby generating a filtered illumination light. In some embodiments, the illumination light is in acontinuous wavelength range. In some embodiments, the illumination light has a bandwidth of at least about 5 nanometers (nm). In some embodiments, the illumination light has a bandwidth of at least about 50 nm. In some embodiments, the illumination light has a bandwidth of at least about 100 nm. In some embodiments, the illumination light has a wavelength range from about 400 nm to about 530 nm. In some embodiments, the illumination light has a wavelength range from about 530 nm to about 750 nm. In some embodiments, the light source comprises a single excitation laser and does not comprise a second excitation laser. In some embodiments, the light source consists of a single excitation laser. In some embodiments, the single excitation laser is configured to generate the emission light in a single color. In some embodiments, the light source comprises a plurality of excitation lasers. In some embodiments, a first peak of the emission light is separated from a second peak of the emission light by at least about 40 nm. In some embodiments, the first peak and the second peak are generated by a first and second label, respectively. In some embodiments, the emission filter comprises one or more of a dichroic filter, a notch filter, a single bandpass filter, and a single step filter. In some embodiments, the emission filter comprises a single step dichroic filter and one or more single bandpass filters. In some embodiments, the emission filter comprises a single step dichroic filter and a single step notch filter or a single pass notch filter. In some embodiments, the emission filter comprises one or more pass-bands. In some embodiments, the one or more pass-bands comprise at least about three passbands. In some embodiments, the one or more pass-bands comprise at least two pass-bands that do not overlap with one another. In some embodiments, the one or more pass -bands have a continuous range of about 500 nm to about 1,200 nm. In some embodiments, the one or more pass-bands haveabandwidth of atleast about 10 nm. In some embodiments, the one or more passbands have a bandwidth of at least about 100 nm. In some embodiments, the system does not comprise one or more second pass-bands outside of a wavelength range of the illumination light. In some embodiments, the illumination light and the emission light do not overlap in a wavelength range. In some embodiments, a photon collection efficiency of the emission filter is at least about 80%. In some embodiments, the system does not comprise one or more second pass -bands. In some embodiments, the field of view is atleast about 10 mm2. In some embodiments, the detector has a size of at least about 10 mm2. In some embodiments, the sample is immobilized on a surface of the flow cell. In some embodiments, the flow cell comprises a plurality of surfaces. In some embodiments, the plurality of surfaces are displaced from one another along an optical axis of the system. In some embodiments, the sample comprises in situ cells. In some embodiments, the sample comprises a tissue sample. In some embodiments, the emission light has a bandwidth of at least about 100 nm. In some embodiments, the emission light has a bandwidth of at least about 250 nm. In some embodiments, the imaging system has a numerical aperture of atleast about 0.25.In some embodiments, the imaging system has a numerical aperture of at most about 0.5. In some embodiments, the imaging system has an ability to resolve features that are at least about 400 nm in size. In some embodiments, the system is configured to complete imaging for a sequencing cycle within an imaging cycle time of at most about 120 seconds per imaging cycle. In some embodiments, the system is configured to complete a sequencing cycle within a cycle time of at most about 120 seconds per sequencing cycle. In some embodiments, the light source is configured to illuminate an area of the flow cell of at least about 1 mm2with an energy variation of less than about 10% across the area of the flow cell. In some embodiments, the sample comprises one or more nucleic acid molecules. In some embodiments, the detector comprises a pixel having a pixel size such that a spatial sampling frequency of the imaging system is at least about twice an optical resolution of the system. In some embodiments, the system further comprises using the emission light to generate an image of the sample. In some embodiments, the sample is disposed in a surface of the flow cell at a density of at least about 10,000 nucleic acid polonies / mm2. In some embodiments, the system further comprises a hydrophobic coating layer on a surface of the flow cell. In some embodiments, the system is configured to image two or more fields of view ata same focus setting on at least one surface of the flow cell. In some embodiments, the imaging system has a diffraction limited resolution over the field of view. In some embodiments, the detector has an active area with a diagonal length of at least about 15 mm. In some embodiments, the system is configured to sequence the sample and identify a number of morphological features, a number of ribonucleic acid features, a number of protein features, or a combination thereof. In some embodiments, the sequencing and the identifying the number of morphological features, the number of ribonucleic acid features, the number of protein features, or the combination thereof occurs with no more than two sequencing cycles.

[0007] In an aspect, the present disclosure provides a system, comprising: a flow cell configured to contain a biological sample; a single light source configured to illuminate the biological sample, wherein illuminating the sample with the single light source generates emission light within at least three emission bands; and an imaging system comprising a detector configuredto receive the emission light, wherein the imaging system has a field of view of at least about 1 square millimeter.

[0008] In some embodiments, the emission light within the at least three emission bands is from at least three types of fluorophores. In some embodiments, the system further comprises an excitation filter disposed in an optical path between the illumination source and the flow cell. In some embodiments, the excitation filter is configured to filter a portion of the illumination light, thereby generating a filtered illumination light. In some embodiments, the illuminating is using a continuous wavelength range. In some embodiments, the single light source has a bandwidth ofat least about 5 nanometers (nm). In some embodiments, the single light source has a bandwidth of at least about 50 nm. In some embodiments, the single light source has a bandwidth of at least about 100 nm. In some embodiments, the single light source has a wavelength range from about 400 nm to about 530 nm. In some embodiments, the single light source has a wavelength range from about 530 nm to about 750 nm. In some embodiments, the single light source comprises a single excitation laser and does not comprise a second excitation laser. In some embodiments, the single light source consists of a single excitation laser. In some embodiments, a first peak of the emission light is separated from a second peak of the emission light by at least about 40 nm. In some embodiments, the first peak and the second peak are generated by a first and second label, respectively. In some embodiments, the emission filter comprises one or more of a dichroic filter, a notch filter, a single bandpass filter, and a single step filter. In some embodiments, the emission filter comprises a single step dichroic filter and one or more single bandpass filters. In some embodiments, the emission filter comprises a single step dichroic filter and a single step notch filter or a single pass notch filter. In some embodiments, light generated by the single light source and the emission light do not overlap in a wavelength range. In some embodiments, the field of view is at least about 10 mm2. In some embodiments, the detector has a size of at least about 10 mm2. In some embodiments, the sample is immobilized on a surface of the flow cell. In some embodiments, the flow cell comprises a plurality of surfaces. In some embodiments, the plurality of surfaces are displaced from one another along an optical axis of the system. In some embodiments, the sample comprises in situ cells. In some embodiments, the sample comprises a tissue sample. In some embodiments, the emission light has a bandwidth of at least about 100 nm. In some embodiments, the emission light has a bandwidth of at least about 250 nm. In some embodiments, the imaging system has a numerical aperture of at least about 0.25. In some embodiments, the imaging system has a numerical aperture of at most about 0.5. In some embodiments, the imaging system has an ability to resolve features that are at least about 400 nm in size. In some embodiments, the system is configured to complete imaging for a sequencing cycle within an imaging cycle time of at most about 120 seconds per imaging cycle. In some embodiments, the system is configured to complete a sequencing cycle within a cycle time of at most about 120 seconds per sequencing cycle. In some embodiments, the single light source is configured to illuminate an area of the flow cell of at least ab out 1 mm2with an energy variation of less than about 10% across the area of the flow cell. In some embodiments, the sample comprises one or more nucleic acid molecules. In some embodiments, the detector comprises a pixel having a pixel size such that a spatial sampling frequency of the imaging system is at least about twice an optical resolution of the system. In some embodiments, the system further comprises using the emission light to generate an image of the sample. In some embodiments, thesample is disposed in a surface of the flow cell at a density of at least about 10,000 nucleic acid polonies / mm2. In some embodiments, the system further comprises a hydrophobic coating layer on a surface of the flow cell. In some embodiments, the system is configured to image two or more fields of view ata same focus setting on at least one surface of the flow cell. In some embodiments, the imaging system has a diffraction limited resolution over the field of view. In some embodiments, the detector has an active area with a diagonal length of at least about 15 mm. In some embodiments, the system is configured to sequence the sample and identify a number of morphological features, a number of ribonucleic acid features, a number of protein features, or a combination thereof. In some embodiments, the sequencing and the identifying the number of morphological features, the number of ribonucleic acid features, the number of protein features, or the combination thereof occurs with no more than two sequencing cycles.

[0009] In an aspect, the present disclosure provides a system, comprising: a flow cell configured to contain a biological sample; a single light source configured to illuminate the biological sample with an illumination light to generate an emission light; an imaging system comprising a detector configured to receive the emission light, wherein the imaging system has a field of view of at least about 1 square millimeter; and an emission filter disposed in an optical path between the flow cell and the detector, wherein the emission filter has a transmissivity of at least 70%, 80%, or 90% over a range of wavelengths of at least 350 nanometers.

[0010] In some embodiments, the system further comprises an excitation filter disposed in an optical path between the illumination source and the flow cell. In some embodiments, the excitation filter is configured to filter a portion of the illumination light, thereby generating a filtered illumination light. In some embodiments, the illumination light is in a continuous wavelength range. In some embodiments, the illumination light has a bandwidth of at least about 5 nanometers (nm). In some embodiments, the illumination light has a bandwidth of at least about 50 nm. In some embodiments, the illumination light has a bandwidth of at least about lOO nm. In some embodiments, the illumination light has a wavelength range from about 400 nm to about 530 nm. In some embodiments, the illumination light has a wavelength range from about 530 nm to about 750 nm. In some embodiments, the light source comprises a single excitation laser and does not comprise a second excitation laser. In some embodiments, the light source consi sts of a single excitation laser. In some embodiments, the single excitation laser is configured to generate the emission light in a single color. In some embodiments, a first peak of the emission light is separated from a second peak of the emission light by at least about40 nm. In some embodiments, the first peak and the second peak are generated by a first and second label, respectively. In some embodiments, the emission filter comprises one or more of a dichroic filter, a notch filter, a single bandpass filter, and a single step filter. In some embodiments, the emission filter comprises asingle step dichroic filter and one or more single bandpass filters. In some embodiments, the emission filter comprises a single step dichroic filter and a single step notch filter or a single pass notch filter. In some embodiments, the emission filter comprises one or more pass-bands. In some embodiments, the one or more pass-bands comprise at least about three pass-bands. In some embodiments, the one or more pass-bands comprise at least two pass-bands that do not overlap with one another. In some embodiments, the one or more pass-bands have a continuous range of about 500 nm to about 1,200 nm. In some embodiments, the one or more pass-bands have a bandwidth of at least about 10 nm. In some embodiments, the one or more pass -bands have a bandwidth of at least about 100 nm. In some embodiments, the system does not comprise one or more second pass-bands outside of a wavelength range of the illumination light. In some embodiments, the illumination light and the emission light do not overlap in a wavelength range. In some embodiments, a photon collection efficiency of the emission filter is at least about 80%. In some embodiments, the system does not comprise one or more second pass-bands. In some embodiments, the field of view is at least about 10 mm2. In some embodiments, the detector has a size of at least about 10 mm2. In some embodiments, the sample is immobilized on a surface of the flow cell. In some embodiments, the flow cell comprises a plurality of surfaces. In some embodiments, the plurality of surfaces are displaced from one another along an optical axis of the system. In some embodiments, the sample comprises in situ cells. In some embodiments, the sample comprises a tissue sample. In some embodiments, the emission light has a bandwidth of at least about 100 nm. In some embodiments, the emission light has a bandwidth of at least about 250 nm. In some embodiments, the imaging system has a numerical aperture of atleast about 0.25. In some embodiments, the imaging system has a numerical aperture of at most about 0.5. In some embodiments, the imaging system has an ability to resolve features that are at least about 400 nm in size. In some embodiments, the system is configured to complete imaging for a sequencing cycle within an imaging cycle time of at most about 120 seconds per imaging cycle. In some embodiments, the system is configured to complete a sequencing cycle within a cycle time of at most about 120 seconds per sequencing cycle. In some embodiments, the single light source is configured to illuminate an area of the flow cell of at least about 1 mm2with an energy variation of less than about 10% across the area of the flow cell. In some embodiments, the sample comprises one or more nucleic acid molecules. In some embodiments, the detector comprises a pixel having a pixel size such that a spatial sampling frequency of the imaging system is at least about twice an optical resolution of the system. In some embodiments, the system further comprises using the emission light to generate an image of the sample. In some embodiments, the sample is disposed in a surface of the flow cell at a density of at least about 10,000 nucleic acid polonies / mm2. In some embodiments, the system further comprises a hydrophobic coating layeron a surface of the flow cell. In some embodiments, the system is configured to image two or more fields of view ata same focus setting on at least one surface of the flow cell. In some embodiments, the imaging system has a diffraction limited resolution over the field of view. In some embodiments, the detector has an active area with a diagonal length of at least about 15 mm. In some embodiments, the system is configured to sequence the sample and identify a number of morphological features, a number of ribonucleic acid features, a number of protein features, or a combination thereof. In some embodiments, the sequencing and the identifying the number of morphological features, the number of ribonucleic acid features, the number of protein features, or the combination thereof occurs with no more than two sequencing cycles.

[0011] In an aspect, the present disclosure provides a sequencing system, comprising: the system of the present disclosure; and a processor, reconfigurable logic device, or both configured to process images comprising the emission light.

[0012] In some embodiments, the reconfigurable logic device comprises one or more field programmable gate array units, artificial intelligence chips, or neural processing units. In some embodiments, the reconfigurable logic device comprises one or more field programmable gate array units. In some embodiments, the processor comprises one or more of a central processing unit, graphics processing unit, tensor processing unit, neural processing unit, field programmable gate array, or artificial intelligence chip. In some embodiments, the processor or the reconfigurable logic device is configured to detect a fluorescent labeled composition comprising one or more nucleic acids disposed on one or more surfaces of the flow cell to determine an identity of a nucleotide of the one or more nucleic acids.

[0013] In an aspect, the present disclosure provides a sequencing system, comprising: the system of the present disclosure; and one or more reagent dispensing systems configured to dispense one or more reagents to the flow cell.

[0014] In an aspect, the present disclosure provides a method, comprising: (a) providing the system of the present disclosure, wherein the sample comprises an optical label and the sample is immobilized to the a flow cell of the the system; (b) exciting, using the illumination light source, the sample, thereby generating the emission light; (c) directing the emission light through the emission filter, thereby generating filtered emission light; and (d) detecting, using the imaging system, the filtered emission light, thereby generating a detected light signal.

[0015] In some embodiments, the method further comprises processing the detected light signal to generate an image of the flow cell. In some embodiments, the method further comprises processingthe image or the detected light signal to sequence a biomolecule of the sample. In some embodiments, the sequencing comprises one or more of sequencing-by-avidity, sequencing-by- nucleotide base-pairing, sequencing-by-nucleotide binding, or sequencing-by-nucleotideincorporation reaction. In some embodiments, the biomolecule is a nucleic acid. In some embodiments, the method further comprises a plurality of optical labels comprising the optical label. In some embodiments, the plurality of optical labels generate a plurality of wavelengths of light. In some embodiments, the plurality of optical labels are a plurality of fluorophores.INCORPORATION BY REFERENCE

[0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application fde contains at least one drawing executed in color. Copies of this patait or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0019] FIGS. 1A-1B schematically illustrate non-limiting examples of imaging multiple surface support structures for presenting sample sites for imaging by the imaging systems disclosed herein. FIG. 1A: illustration of imaging front and rear interior surfaces of a flow cell. FIG. IB: illustration of imaging front and rear exterior surfaces of a substrate.

[0020] FIGS. 2A-2D illustrate a non-limiting example of a multi-channel fluorescence imaging module comprising a dichroic beam splitter for transmitting an excitation light beam to a sample, and for receiving and redirecting by reflection the resultant fluorescence emission to four detection channels configured for detection of fluorescence emission at four different respective wavelengths or wavelength bands. FIG 2A top isometric view. FIG. 2B: bottom isometric view. FIGS. 2C-2D illustrate the optical paths within the multi-channel fluorescence imaging module of FIGS. 2A and 2B comprising a dichroic beam splitter for transmitting an excitation light beam to a sample, and for receiving and redirecting by reflection a resultant fluorescence emission to four detection channels for detection of fluorescence emission at four differait respective wavelengths or wavelength bands. FIG. 2C: top view. FIG. 2D: side view.

[0021] FIG. 3 is a graph illustrating a relationship between dichroic filter performance and beam angle of incidence.

[0022] FIG. 4 is a graph illustrating a relationship between beam footprint size and beam angle of incidence on a dichroic filter.

[0023] FIGS. 5A-5B schematically illustrate an example configuration of dichroic filters and detection channels of a multi-channel fluorescence imagi”'1"U^rejndjchroic filters have reflectivesurface tilted such that the angle between the incident beam (e.g. , the central angle) and the reflective surface of the dichroic filter is less than 45. FIG. 5 A: schematic illustration of a multichannel fluorescence imaging module comprising four detection channels. FIG. 5B: detail view illustrating the angle of incidence (AOI) of a light beam on a dichroic reflector.

[0024] FIG. 6 provides a graph illustrating improved dichroic filter performance corresponding to the imaging module configuration illustrated in FIGS. 5 A and 5B.

[0025] FIG. 7 provides a graph illustrating improved dichroic filter performance corresponding to the imaging module configuration illustrated in FIGS. 5 A and 5B.

[0026] FIGS. 8A-8B provide graphs illustrating reduced surface deformation resulting from the imaging module configuration of FIGS 5 A and 5B. FIG. 8 A illustrates the effect of folding angle on image quality degradation induced by the addition of 1 wave of PV spherical power to the last mirror. FIG. 8B illustrates the effect of folding angle on image quality degradation induced by the addition of 0. 1 wave of PV spherical power to the last mirror.

[0027] FIGS. 9A-9B provide graphs illustrating improved excitation filter performance (e.g, sharper transitions between pass-bands and surrounding stop bands) resulting from use of s-polarization of the excitation beam. FIG. 9A: transmission spectra for an example bandpass dichroic filter at angles of incidence of 40 degrees and 45 degrees, where the incident beam is linearly polarized and is p-polarized with respect to the plane of the dichroic filter. FIG. 9B: changing the orientation of the light source with respect to the dichroic filter, such that the incident beam is s-polarized with respect to the plane of the dichroic filter, results in a substantially sharper edge between the pass-band and the stopband.

[0028] FIGS . 10A- 10B illustrate the modulation transfer function (MTF) of an example multiple surface imaging system disclosed herein having a numerical aperture (NA) of 0.3. FIG. 10A: first surface. FIG. 10B: second surface.

[0029] FIGS . 11 A- 11 B illustrate the MTF of an example multiple surface imaging system disclosed herein having an NA of 0.4. FIG. 11 A: first surface. FIG. 11B: second surface.

[0030] FIGS. 12A-12B illustrate the MTF of an example multi pie surf ace imaging system disclosed herein having an NA of 0.5. FIG. 12A: first surface. FIG. 12B: second surface.

[0031] FIGS. 13A-13B illustrate the MTF of an example multi pie surf ace imaging system disclosed herein having an NA of 0.6. FIG. 13 A: first surface. FIG. 13B: second surface.

[0032] FIGS. 14A-14B illustrate the MTF of an example multi pie surf ace imaging system disclosed herein having an NA of 0.7. FIG. 14A: first surface. FIG. 14B: second surface.

[0033] FIGS . 15 A- 15B illustrate the MTF of an example multiple surface imaging system disclosed herein having an NA of 0.8. FIG. 15 A: first surface. FIG. 15B: second surface.

[0034] FIGS. 16A-16B provide plots of the calculated Strehl ratio for imaging a second flow cell surface through a first flow cell surface. FIG. 16A: plot of the Strehl ratios for imaging a second flow cell surface through a first flow cell surface as a function of the thickness of the intervening fluid layer (fluid channel height) for different objective lens and / or optical system numerical apertures. FIG. 16B: plot of the Strehlratio as a function of numerical aperture for imaging a second flow cell surface through a first flow cell surface and an intervening layer of water having a thickness of 0. 1 mm.

[0035] FIG. 17 provides a schematic illustration of a dual -wavelength excitation / four channel emission fluorescence imaging system of the present disclosure.

[0036] FIG. 18 provides an optical ray tracing diagram for an obj ective lens design that has been designed for imaging a surface on the opposite side of a 0. 17 mm thick coverslip.

[0037] FIG. 19 provides a plot of the modulation transfer function for the obj ective lens illustrated in FIG. 18 as a function of spatial frequency when used to image a surface on the opposite side of a 0. 17 mm thick coverslip.

[0038] FIG. 20 provides a plot of the modulation transfer function for the obj ective lens illustrated in FIG. 18 as a function of spatial frequency when used to image a surface on the opposite side of a 0.3 mm thick coverslip.

[0039] FIG. 21 provides a plot of the modulation transfer function for the obj ective lens illustrated in FIG. 18 as a function of spatial frequency when used to image a surface that is separated from that on the opposite side of a 0.3 mm thick coverslip by a 0. 1 mm thick layer of aqueous fluid.

[0040] FIG. 22 provides a plot of the modulation transfer function for the obj ective lens illustrated in FIG. 18 as a function of spatial frequency when used to image a surface on the opposite side of a 1.0 mm thick coverslip.

[0041] FIG. 23 provides a plot of the modulation transfer function for the obj ective lens illustrated in FIG. 18 as a function of spatial frequency when used to image a surface that is separated from that on the opposite side of a 1.0 mm thick coverslip by a 0. 1 mm thick layer of aqueous fluid.

[0042] FIG. 24 provides a ray tracing diagram for a tube lens design which, if used in conjunction with the obj ective lens illustrated in FIG. 18, provides for improved multiple-side imaging through a 1 mm thick coverslip.

[0043] FIG. 25 provides a plot of the modulation transfer function for the combination of objective lens and tube lens illustrated in FIG. 24 as a function of spatial frequency when used to image a surface on the opposite side of a 1.0 mm thick coverslip.

[0044] FIG. 26 provides a plot of the modulation transfer function for the combination of objective lens and tube lens illustrated in FIG. 24 as a function of spatial frequency when used to image a surface that is separated from that on the opposite side of a 1.0 mm thick coverslip by a 0. 1 mm thick layer of aqueous fluid.

[0045] FIG. 27 provides ray tracing diagrams for tube lens design (left) of the present disclosure that has been optimized to provide high-quality, multiple-side imaging performance. Because the tube lens is no longer infinity -corrected, an appropriately 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.

[0046] FIG. 28 illustrates one non-limiting example of a single capillary flow cell having 2 fluidic adaptors.

[0047] FIG. 29 illustrates one non-limiting example of a flow cell cartridge comprising a chassis, fluidic adapters, and optionally other components, which is designed to hold two capillaries.

[0048] FIG. 30 illustrates one non-limiting example of a system comprising a single capillary flow cell connected to various fluid flow control components, where the single capillary is compatible with mounting on a microscope stage or in a custom imaging instrument for use in various imaging applications.

[0049] FIG. 31 illustrates one non-limiting example of a system that comprises a flow cell having integrated diaphragm valves to reduce or minimize dead volume and conserve certain key reagents.

[0050] FIG. 32 illustrates one non-limiting example of a system that comprises a flow cell, a microscope setup, and a temperature control mechanism.

[0051] FIG. 33 illustrates one non-limiting example for temperature control of the flow cells through the use of a metal plate that is placed in contact with the flow cell cartridge.

[0052] FIG. 34 illustrates one non-limiting approach for temperature control of the flow cells that comprises a non-contact thermal control mechanism.

[0053] FIGS. 35A-35C illustrate non-limiting examples of flow cell device fabrication. FIG. 35A shows the preparation of a one-piece glass flow cell. FIG. 35B shows the preparation of a two-piece glass flow cell. FIG. 35C shows the preparation of a three-piece glass flow cell.

[0054] FIGS. 36A-36C illustrate non-limiting examples of glass flow cell designs. FIG. 36Ashows aone- piece glass flow cell design. FIG. 36B shows a two-piece glass flow cell design. FIG. 36C shows a three- piece glass flow cell design.

[0055] FIG. 37 illustrates visualization of cluster (e.g., polony) amplification in a capillary lumen.

[0056] FIG. 38 provides a non-limiting example of a block diagram for a sequencing system as disclosed herein.

[0057] FIG. 39 provides a non-limiting example of a flow chart for a sequencing method as disclosed herein.

[0058] FIGS. 40A-40B provide non-limiting examples of a kit as disclosed herein. FIG. 40A shows a kit comprising a flow cell and a plurality of numbered reagent wells, according to some embodiments. FIG. 40B shows the ornamental appearance of a kit, according to some embodiments.

[0059] FIG. 41 provides a non-limiting example of a schematic for a structured illumination system as disclosed herein.

[0060] FIG. 42 provides a non-limiting example of a flow chart for acquiring and processing structured illumination images of a flow cell as disclosed herein.

[0061] FIGS. 43A-43B provide non-limiting schematic illustrations of a multiplexed read-head as disclosed herein. FIG. 43 A: side view of a multiplexed read-head in which individual microfluorometers are configured to image a common surface, e.g. , the interior surface of a flow cell. FIG. 43B: top view of a multiplexed read-head illustrating the imaging paths acquired by individual microfluorometers of the multiplexed read-head.

[0062] FIGS. 44A-44B provide non-limiting schematic illustrations of a multiplexed read -head as disclosed herein. FIG. 44A: side view of a multiplexed read-head in which a first subset of a plurality ofindi victual microfluorometers 4401 is configured to image a first surface, e.g. , a first interior surface of a flow cell, and a second subset of the plurality of individual microfluorometers is configured to image a second surface, e.g. , a second interior surface of a flow cell. FIG. 44B: top view of the multiplexed readhead of FIG. 44A illustrating the imaging paths acquired by individual microfluorometers 4401 of the multiplexed read-head.

[0063] FIG. 45 illustrates a non-limiting example of an optical imaging system having multiple imaging sensors configured for transmission imaging a flow cell upon sequential illumination by multiple light sources, each light source emitting a different color, according to some embodiments herein. Liquid samples are introduced to the flow cell on a hydrophobic pad and flow through the flow cell by a pulling force.

[0064] FIG. 46 provides a non-limiting schematic illustration of a method utilizing an optical system for imaging the surface of a flow cell for nucleic acid sequencing, according to some embodiments herein.

[0065] FIGS . 47A-47B provides optical systems according to various embodiments described herein. FIG. 47A provides anon-limiting cut-away illustration of an optical system for imaging the surfaces of a flow cell, according to some embodiments herein. FIG. 47B provides a comparison of the optical system of FIG. 47A with IDEX instrument core.

[0066] FIGS. 48A-48B provide non-limiting comparisons of flow cells and optical system imaging areas. FIG. 48A provides a non-limiting example of a flow cell with 424 individual tiles, imaged by the IDEX instrument core shown in FIG. 47 B. FIG. 48B provides a non-limiting example of a flow cell with < 40 individual tiles, imaged by the optical system described herein (see FIGS. 45, 46, 47A-47B).

[0067] FIGS. 49A-49B provide a non-limiting cut-away illustration of an optical system configured for multiple side imaging of a multiple sided flow cell. The optical system as shown comprises a piezo driven wedge block for rapid focusing. FIG. 49A illustrates the optical system configured to focus on the back- interior surface of the flow cell. FIG. 49B illustrates the optical system configured to focus on the frontinterior surface of the flow cell.

[0068] FIG. 50 provides anon-limiting cut-away illustration of an optical system configured for imaging a large area surface. The optical system comprises multiple optical subsystems, wherein the optimized FOV of each subsystem overlaps the FOV of each neighboring optical subsystem, thereby providing a large area FOV.

[0069] FIGS. 51A-51B provides a non-limiting cut-away illustration of a focusing lens assembly. The focusing lens assembly is configured to maintain a fixed position within the optical path (e.g., optical axis) and to allow for relative motion between at least a first lens and second lens contained within a lens housing of the focusing lens assembly. FIG. 51A shows a focusing lens assembly with a first lens and a second lens. FIG. 5 IB shows the same focus lens assembly with the relative movement of the second lens as compared with FIG. 51 A.

[0070] FIG. 52 provides anon-limiting cut-away illustration of an optical system configured for imaging a curved, large area surface. The optical system comprises multiple optical subsystems wherein each system is placed approximately orthogonal to the surface and wherein the FOV of each subsystem overlapsthe FOV of each neighboring optical subsystem, thereby providing a system for imaging curved, large area surfaces.

[0071] FIGS. 53A-53B provides a non-limiting cut away illustration of an optical system configured to image a capillary flow cell. In this example, the optical system configured to image curved, large area surfaces are rotated about the x-axis and translated along the x-axis to obtain images of the entire interior surface of the capillary flow cell. FIG. 53A illustrates the optical axis of the center optical subsystem aligned with the z-axis. FIG. 53B illustrates the optical axis of the center optical subsystem rotated 90 degrees to align with the y-axis.

[0072] FIGS. 54A-54B provides a non-limiting cut away illustration of an optical system configured to image a capillary flow cell without the need for a stage to rotate the optical system about the x-axis. The optical system as shown comprises a piezo driven wedge block for rapid focusing. FIG. 54A illustrates the optical system configured to focus on the interior surface of the capillary flow cell closest to the light sources. FIG. 54B illustrates the optical system configured to focus on the interior surface of the capillary flow cell further from the light sources.

[0073] FIG. 55 is a bar graph showing the results of a trapping assay conducted by reacting various fluorescently-labeled multivalent molecules with a corresponding correct DNA template.

[0074] FIG. 56 is a bar graph showing the results of a trapping assay in which increasing concentrations of various fluorescently-labeled multivalent molecules were reacted with corresponding correct DNA templates.

[0075] FIG. 57 presents four graphs showing the results of a trapping assay comparing the signal intensity of fluorescently-labeled multivalent molecules carrying nucleotide arms comprising either an 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 trap time in seconds (x-axis) and P90 signal intensity (y-axis).

[0076] FIG. 58 presents four graphs showing the results of atrapping assay comparing the signal intensity of fluorescently-labeled multivalent molecules carrying nucleotide arms comprising either an 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 trap time in seconds (x-axis) and P90 signal intensity (y-axis).

[0077] FIG. 59 presents three graphs showing the results of real-time imaging trapping kinetics assays comparing signal intensity of fluorescently-labeled multivalent molecules carrying nucleotide arms comprising one of Linkers 6 or 10-16. Three different concentrations of the multivalent molecules were tested (15, 7.5 and 2.5 nM). The graphs show trap time in second (x-axis) and signal intensity (y-axis).

[0078] FIG. 60 is a graph showing the results of a binding kinetic study of fluorescently -labeled multivalent molecules carrying nucleotide arms comprising one of Linkers 6 or 10-16. The graph shows multivalent molecule concentration (x-axis, nM) and rate (y-axis). The legend shown in FIG. 60 is also applicable to FIG. 59.

[0079] FIG. 61 is a bar graph showing the binding constant (K) determined for fluorescently -labeled multivalent molecules carrying nucleotide arms comprising one of Linkers 6 or 10-16.

[0080] FIG. 62 shows an example of a sequencing system, according to some embodiments.

[0081] FIG. 63 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0082] FIG. 64A shows an exemplary spectrum of three different excitation wavelength ranges that can be used to excite the sample(s) using the optical systems disclosed herein or other existing optical systems. The emission light from the sample in response to the excitation can include four different peak emission wavelengths that are different from each other.

[0083] FIG. 64B shows an exemplary emission wavelength range that an emission filter or emission filter set disclosed herein allows emission light to pass through (top) when excited with a first excitation wavelength range; an example of three pass-bands, each pass-band at least partly overlaps with another pass-band (middle); and an example of three pass-bands that do not overlap partly or completely with each other (bottom). One or more pass-bands may overlap with second and third excitation wavelength range(s) used by existing optical systems (mid and bottom) but not the first excitation wavelength range.

[0084] FIGS. 65A-65B shows exemplary diagrams of the filter set in the optical systems herein that are used to filter the excitation light and / or the emission light. The structural element of the filter set can be place along the excitation path and / or the emission path.

[0085] FIG. 66 shows a non-limiting example of the illumination system of the optical assembly herein, which includes an illumination subsystem and a light beam delivery subsystem.

[0086] FIG. 67 shows a non-limiting example of the illumination subsystem herein.

[0087] FIGS. 68A-68C show uniformity in the illumination power density by the illumination system of FIG. 66 herein. FIG. 68A shows an example image of an illumination field and the associated illumination intensity. FIG. 68B shows a line trace of the illumination intensity along the long axis of FIG. 68A. FIG. 68C shows a line trace of the illumination intensity along the short axis of FIG. 68A.

[0088] FIG. 69 illustrates a non-limiting example of the illumination subsystem and the light beam delivery system of the optical assembly.

[0089] FIG. 70 illustrates anon-limiting example of the illumination subsystem of the optical assembly.

[0090] FIG. 71 shows a despeckler and its relative position to a collimator of the light beam delivery subsystem.

[0091] FIG. 72 shows a non-limiting example of the optical fiber and the light beam delivery subsystem

[0092] FIG. 73 shows a non-limiting example of the liquid light guide and the light beam delivery subsystem.

[0093] FIGS. 74A-74D show non-limiting examples of the despeckler, in this case, a mechanical vibration source that is loosely or fixedly attached to at least a portion of the optical fiber(s). FIG. 74A shows a wound portion of an optical fiber, according to some embodiments. FIG. 74B shows a portion of an optical fiber round around a vibration source, according to some embodiments. FIG. 74C shows a portion of an optical fiber round around a fan vibration source, according to some embodiments. FIG.74D shows a portion of an optical fiber round around a fan vibration source, according to some embodiments.

[0094] FIG. 75 shows a table of different despeckler configurations in relation to the optical fiber and their corresponding speckle noise levels.

[0095] FIG. 76 illustrates a block diagram of a sequencing system for imaging DNA sample(s) during DNA sequencing reactions, according to some embodiments.

[0096] FIG. 77 is a schematic of various examples of configurations of multivalent molecules. Left (Class I): schematics of multivalent molecules having a “starbursf ’ or “helter-skelter” configuration. Center (Class II): a schematic of a multivalent molecule having a dendrimer configuration. Right (Class III): a schematic of multiple multivalent molecules formed by reacting streptavidin with 4 -arm or 8-arm PEG-NHS with biotin and dNTPs. Nucleotide units are designated ‘N’, biotin is designated ‘B’, and streptavidin is designated ‘SA’.

[0097] FIG. 78 is a schematic of an example of a multivalent molecule comprising a generic core attached to a plurality of nucleotide-arms.

[0098] FIG. 79 is a schematic of an example of a multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide-arms.

[0099] FIG. 80 shows a schematic of an example of a multivalent molecule comprising a core attached to a plurality of nucleotide-arms, where the nucleotide arms comprise biotin, a spacer, a linker and a nucleotide unit.

[0100] FIG. 81 is a schematic of an example of a nucleotide-arm comprising a core attachment moiety, a spacer, a linker and a nucleotide unit.

[0101] FIG. 82 shows the chemical structure of an example of a spacer (top), and the chemical structures of various examples of linkers, including an 11 -atom linker, 16-atom linker, 23-atom linker and an N3 linker (bottom).

[0102] FIG. 83 shows the chemical structures of various examples of linkers, including linkers 1 -9.

[0103] FIG. 84 shows the chemical structures of various examples of linkers joined / attached to nucleotide units.

[0104] FIG. 85 shows the chemical structures of various examples of linkers joined / attached to nucleotide units.

[0105] FIG. 86 shows the chemical structures of various examples of linkers joined / attached to nucleotide units.

[0106] FIG. 87 shows the chemical structures of various examples of linkers joined / attached to nucleotide units.

[0107] FIG. 88 shows the chemical structure of an example of a biotinylated nucleotide-arm. In this example, the nucleotide unit is connected to the linker via a propargyl amine attachment at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0108] FIG. 89 shows a flow chart of a method of analyzing a biological molecule, according to some embodiments.

[0109] FIG. 90 shows a flow chart of a method for analyzing a biological sample, according to some embodiments.

[0110] FIG. 91 illustrates a perspective view of a non-limiting example of an imaging module or optical assembly.

[0111] FIG. 92 illustrates a cross-sectional view of the non-limiting example of an imaging module or optical assembly.

[0112] FIG. 93 shows a cross-sectional view of the non-limiting example of the single channel time- sequential color imaging module in FIGS. 91-92.

[0113] FIGS. 94A - 94B show examples of an external actuator coupling, according to some embodiments. FIG. 94A shows a detail view of an external actuator and optical assembly, according to some embodiments. FIG. 94B shows a far view of an external actuator and the optical assembly, according to some embodiments.

[0114] FIG. 95 shows an example of the optical elements of an optical assembly and the associated focus paths, according to some embodiments.

[0115] FIG. 96 shows an example of the optical elements of an optical assembly and the associated focus paths, according to some embodiments.

[0116] FIGS. 97A - 97B provide diffraction modulation transfer functions (MTFs) for optical systems, according to some embodiments. FIG. 97A shows an example MTF for an objective based optical system. FIG. 97B shows an example MTF for an optical system not comprising an objective.

[0117] FIGS. 98A - 98B show wavefront analysis calculations for an optical system of the present disclosure, according to some embodiments. FIG. 98A shows an example wavefront analysis calculation at position 1. FIG. 98B shows an example wavefront analysis calculation at position 2.

[0118] FIG. 99 shows a top surface optical performance curve, according to some embodiments.

[0119] FIG. 100 shows a bottom surface optical performance curve, according to some embodiments.

[0120] FIG. 101 shows a plot of an MTF of an optical system, according to some embodiments.

[0121] FIG. 102 shows a plot of a cumulative probability of achieving a given wavefront error, according to some embodiments.

[0122] FIG. 103 is a schematical illustration of arotatory stage for moving the sample(s) relative to the objective lens of the optical system for imaging sequencing reactions.DETAILED DESCRIPTION

[0123] There is a need for increased throughput and flexibility in next generation sequencing (NGS) analysis systems. Disclosed herein are systems, kits, compositions, and methods that may provide any one or more of the following advantages: higher system throughput for fluorescence imaging-based genomics applications, compatibility with traditional flow cell devices and / or optical systems, flexibility in analysis or comparison of samples (e.g., larger sample volume and / or increased sample variety), improved opticalresolution (including high performance optical resolution), and improved image quality. The disclosed optical illumination and imaging system designs may provide any one or more of the following advantages: improved dichroic filter performance, increased uniformity of dichroic filter frequency response, improved excitation beam filtering, larger fields-of-view, increased spatial resolution, improved modulation transfer, contrast-to-noise ratio, and image quality, higher spatial sampling frequency, faster transitions between image capture 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. The disclosed flow cells can provide enhanced throughput and sample processing speed while maintaining low background noise.SYSTEMS

[0124] Provided herein, in some embodiments, are systems useful for nucleic acid analysis. In some embodiments, the system comprises an imaging system. In some embodiments, the system comprises a fluidic control system. In some embodiments, the system comprises a multivalent molecule, or mixture of multivalent molecules. In some embodiments, the system comprises a solid phase for performing a nucleic acid analysis. In some embodiments, the solid phase is a flow cell disclosed herein. In some embodiments, the systems disclosed herein comprises one or more kits disclosed herein. In some aspects, the kits can be kits as described in International Patent Application No. PCT / US2021 / 057441, which is incorporated by reference in its entirety.Imaging Systems

[0125] Disclosed herein, in some embodiments, are imaging systems useful for imaging one or more surfaces to perform nucleic acid analysis. In some embodiments, the imaging system comprises an adjustable numerical aperture. In some cases, the imaging system does not comprise a compensatory. In some cases, the imaging system comprises a compensator. Imaging systems with a wide field of view can provide for enhanced throughput and decreased sample imaging times.

[0126] In some embodiments, the imaging systems disclosed herein are optical systems. In some embodiments, the optical system of the present disclosure is provided in FIG. 45. In some embodiments, the optical system 4500 eliminates a need for dichroic, or corrective optics, such as a tube lens for multipleside imaging of a flow cell. The multiple-side can be dual-side, three-side, quad-side, or even more sides. The optical system 4500 disclosed herein may be used as components of systems designed for a variety of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. As shown in FIG. 45, the optical system comprises multiple imaging sensors 4501-4504 that are configured for imaging a flow cell 4521 , in some embodiments. In some embodiments, an imaging sensor 4501 -4504 may be a CCD imaging sensor. In some embodiments, the imaging sensor 4501 -4504 may be a CMOS imaging sensor. In some embodiments, pixel shifters 4505-4508 are used to translate the object being imaged relative to a corresponding imaging sensor 4505-4508. In some embodiments, the optical system comprises 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 tri -band fluorescence bandpass filter. In some embodiments, the tri -band fluorescence bandpass filter is referred toas a tri-band notch filter. In some embodiments, imaging optics 4510-4513 are positioned between the imaging sensors 4501-4504 and the flow cell 4521. In some embodiments, one imaging optic 4505-4508, also referred to as an imaging optic assembly, focuses light emitted from the flow cell 4521 to one of the imaging sensors, for e.g., 4501, 4502, 4503, or 4504. In some embodiments, the optical system comprises an integrated field flattening assembly. In some embodiments, the optical system comprises aberration correction. In some embodiments, the optical system lacks bandpass filters. In some embodiments, the optical system lacks cutoff filters. In some embodiments, the optical system lacks dichroic mirrors. In some embodiments, a liquid handling system 4514 dispenses a sample 4515 to the flow cell 4521. In some embodiments, the liquid handling system 4514 dispenses a liquid sample to a hydrophobic pad 4516 attached to the flow cell 4521. In some embodiments, the liquid handling system 4514 is a drop dispensing system. In some embodiments, the drop dispensing system 4514 delivers the sample 4515 as a droplet 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 enclosed by a bottom plate 4519 and a top plate 4520. In some embodiments, the top plate 4520 and 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 back interior surface 4529. In some embodiments, the sample, present in the interior channel 4517 of the flow cell 4521 is illuminated by a plurality of light sources 4522, 4523 or 4524. In some embodiments, each of the individual light sources 4522, 4523 and 4524 emit a different color or spectrum of light, 4525, 4526 and 4527 respectively. In some embodiments, the optical system 4500 comprises a heater.

[0127] Although the flow cell 4521 is shown with two interior surfaces in FIG. 45, in other embodiments, the flow cell 4521 can include two or more axially displaced channels and three or more axially displaced interior surfaces. As used herein, “axially displaced” can be used to refer to the relative positioning of surfaces. In some cases, two surfaces may be axially displaced, when a normal vector extending from one surface intersects with the other surface. In some cases, two surfaces may be axially displaced, when a normal vector extending from one surface is colinear with a normal vector of the other surface. In some cases, two surfaces may be axially displaced, when the two surfaces are displaced along an axis of an optical path of an optical system comprising the two surfaces.

[0128] In some embodiments, a notch filter refers to a band stop filter. In some embodiments, a notch filter refers to aband stop filter. In some embodiments, the notch of a filter refers to a band stop or stopband. In some embodiments, the notch of a filter refers to a band pass or pass-band. In some embodiments, a multiband notch filter refers to a multiband bandpass filter. In some embodiments, a multiband notch filter refers to a multiband band stop filter.

[0129] In some embodiments, an imaging optic 4510 of the optical system 4500 comprises a reduction of lx. In some embodiments, the optical system has a field-of-view(FOV) of greater than 1 mm2, greater than 2 mm2, greater than 4 mm2, greater than 10 mm2, greater than 20 mm2, greater than 36 mm2, greater than 40 mm2, greater than 60 mm2, greater than 80 mm2, or greater than 100 mm2. In some embodiments, theoptical system has a field-of-view (FOV) ofless than 2 mm2, less than 4 mm2, less than 10 mm2, less than 20 mm2, less than 36 mm2, less than 40 mm2, less than 60 mm2, less than 80 mm2, or less than 100 mm2. In some embodiments, the optical system has a numerical aperture (NA) of less than 0.6. In some embodiments, the NA is between about 0.1 to about 0.50, about 0.20 to about 0.40, or about 0.30. In some embodiments, the NA is 0.25. In some embodiments, the NA is about 0.1, 0. 15, 0.20, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or 0.60. In some embodiments, the NA is greater than 0.1. In some embodiments, the plurality of imaging sensors is configured to capture the FOV. In some embodiments, a plurality of light sources comprises: 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, a first fluorophore is excited by the first excitation wavelength range 4525 of the first light source 4522. In some embodiments, a second fluorophore is excited by the second excitation wavelength range 4526 of the second light source 4523. In some embodiments, a third fluorophore is excited by the third excitation wavelength range 4527 of the third light source 4524. In some embodiments, a sample comprises a plurality of biological polymers. In some embodiments, the optical system 4500 does not comprise a dichroic filter. In some embodiments, the optical system 4500 does not comprise a tube lens.

[0130] Described herein are various methods for a variety of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis application. FIG. 46 provides a schematic illustration of an imaging method 4601 utilizing the optical system 4500 shown in FIG. 45 for imaging a sample 4515 contained within the 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, the interior channel 4517 of a flow cell 4521, as shown in FIG. 45. In some embodiments, the sample comprises a biological polymer. In some embodiments, the biological polymer comprises units. In some embodiments, a fluorophore is complementary to a unit of the biological polymer. In some embodiments, the fluorophore is attached to a nucleotide that is complementary to a unit of the biological polymer. In some embodiments, two or more detectably distinct fluorophores are attached to a nucleotide that is complementary to a unit of the biological polymer. In some embodiments, the biological polymer is a nucleic acid sequence. In some embodiments, the unit is a nucleotide complementary to the fluorophore labeled nucleotide. In some embodiments, the multiple light sources emit light, transmitting through the sample.

[0131] Described herein are various methods for sequencing a biological polymer (e.g., nucleic acid molecule). A non-limiting schematic illustration of the sequencing method and instrumentation 4601 and the base calling method 4602 is shown in FIG. 46. In some embodiments, the method comprises: illuminating a sample 4515 using an optical system 4500 comprising a first light source 4522 of a plurality of light sources, wherein the first light source 4522 emits a first wavelength range 4525 exciting a first fluorophore of the sample 4515 and acquiring a first image of the sample 4515, wherein the optical system 4500 comprises a plurality of imaging sensors 4501-4504, further wherein the sample 4515 is disposed in an optical path between the plurality of light sources 4522-4524 and the plurality of imaging sensors 4501-4504; illuminating the sample 4515 using a second light source 4523 of the plurality, wherein the second light source 4523 emits a second wavelength range 4526 exciting a second fluorophore of the sample 4515 and acquiring a second image the sample 4515; illuminating the sample 4515 using a third light source 4524 of the plurality, wherein the third light source 4524 emits a third wavelength range 4527 exciting a third fluorophore of the sample and acquiring a third image of the sample 4515; combining the first image, the second image, and the third image into a composite image; identifying the presence of a first nucleotide via a first signal emitted by the first fluorophore, wherein the first signal is extracted from a first region of interest (ROI) of the composite image; identifying the presence of a second nucleotide via a second signal emitted by the second fluorophore, wherein the second signal is extracted from a second ROI of the composite image; identifying the presence of a third nucleotide via a third signal emitted by the third fluorophore, wherein the third signal is extracted from a third ROI of the composite image; and identifying the presence of a fourth nucleotide via the first and third signals emitted by the first and third fluorophores, respectively, wherein the first and third signals are extracted from a fourth ROI of the composite image. In some embodiments, the optical system 4500 further comprises a flow cell 4521, wherein the flow cell 4521 is disposed in the 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 the 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 the optical path between the multi-band bandpass filter 4509 and the flow cell 4521. In some embodiments, the optical system 4500 has a reduction of lx. In some embodiments, the optical system 4500 has a magnification of lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 8x, or lOx. In some embodiments, the optical system has a field-of-view (FOV) of greater than or equal to about 1 mm2, greater than or equal to about 2 mm2, greater than or equal to about 4 mm2, greater than or equal to about 10 mm2, greater than or equal to about 20 mm2, greater than or equal to about 36 mm2, greater than or equal to about 40 mm2, greater than or equal to about 60 mm2, greater than or equal to about 80 mm2, or greater than or equal to about 100 mm2. In some embodiments, the optical system has a numerical aperture (NA) of less than 0.6. In some embodiments, the optical system has a numerical aperture (NA) of about 0.6. In some embodiments, the optical system has a numerical aperture (NA) that is greater than 0.6. In some embodiments, the NA is 0.25. In some embodiments, the NA is about 0.25. In some embodiments, the NA is greater than or equal to about 0.25. In some embodiments, the NA is less than or equal to about 0.25. In some embodiments, the NA is 0.4 to 0.5. In some embodiments, the NA is about 0.4 to 0.5. In some embodiments, the NA is greater than or equal to about 0.4. In some embodiments, theNA is less than or equal to about 0.5. In some embodiments, the FOV is captured by the plurality of image sensors 4501 -4504.

[0132] In some embodiments, the sequencing is described in U.S. Patent No. 10,768, 173 filed on September 23, 2019, which is hereby incorporated by reference in its entirety. In some embodiments, the first fluorophore is associated with a first nucleotide conjugate. In some embodiments, the nucleotide conjugate is a polymer-nucleotide conjugate. In some embodiments, the multivalent molecule disclosedelsewhere herein is or comprises a nucleotide conjugate or polymer nucleotide conjugate disclosed herein. In some embodiments, the second fluorophore is associated with a second nucleotide conjugate. In some embodiments, the 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.

[0133] In some embodiments, fluorophores which may serve as a first fluorophore, a second fluorophore, and / or a third fluorophore include, but are not limited to fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS -fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescdn, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS -rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DO A 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, cyanine 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, Cy 3, 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 derivatives thereof, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes and others known in the art such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms, and comprise two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5- dioxopyrrolidin- 1 -yloxy)-6-oxohexyl] -3,3-dimethyl- 1 ,3-dihy dr o-2H-indol -2-ylidene} prop-1 -en- 1-yl)- 3,3-dimethyl-3H-indolium or l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5- dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene}prop-l-en- l-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may comprise l-(6-((2,5-dioxopyrrolidin-l- yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta- 1.3-dien- l - l)-3.3-dimethyl-3H-indol- l -ium or l-(6-((2,5-dioxopyrrolidin-l- yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl- 5-sulfoindolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and Cy7 (which may comprise l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-l,3-dihydro-2H-indol-2-ylidene)hepta- l,3,5-trien-l-yl]-3H-indolium or l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-5-sulfo-l,3-dihydro- 2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium-5-sulfonate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo -derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule. In some embodiments, the reporter moiety can be aFRET pair, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[0134] Described herein are optical systems 4700 for imaging samples in a flow cell where no focusing step is included.

[0135] Described herein are optical systems 4700 for imaging samples in a flow cell for analysis of biological polymers (e.g., nucleic acid sequencing). In some embodiments, such systems 4700 as shown in FIGS. 47A-47B are more compact and have higher throughput than previous optical systems. Table 1 and FIGS . 48A-48B provide anon-limiting example comparing sequencing cycle times for a standard flow cell and optical system versus the optical system as described herein. Table 1 provides cycle and run times and the respective calculations for a standard flow cell with 424 individual tiles (e.g., active area (e.g., the area undergoing active illumination), region of interest (e.g., the region undergoing active detection), etc.) as shown in FIG. 48A, compared with a flow cell with < 40 individual tiles optimized for imaging on the optical system described herein is shown in FIG. 48B. In some embodiments, one image is equivalent to one tile in area. In some embodiments, when the flow cell 4521, also shown in FIG. 48B, is imaged by the optical system, each tile is exposed to three sequential pulses of light from three separate LED light sources, where each LED light source emits a different wavelength. In some embodiments, each different wavelength is matched to the excitation spectra of a different fluorophore as described herein. In some embodiments, the imaging sensors of the optical system 4500 are synchronized with each excitation pulse to generate an image, wherein one image the entire area of one tile, and further wherein the pixels of the image each represent the amount of fluorescence emitted by the fluorophore. In some embodiments, 2 separate surfaces are imaged in one tile by the optical system 4500. In some embodiments, 8 total images having a total exposure time of 0.3 seconds are acquired by the optical system 4500, 4700 comprising 8 imaging modules (e.g., optical subsystems). In Table 1 the row titled “current” and highlighted in blue represent the total time, over 322 cycles, to be 36. 17 hours for the standard flow cell shown in FIG. 48A when imaged with the IDEX optical system shown in FIG. 47B. In comparison, the rows titled “Sleq” show total times between 13.63 and 14.28 hours for the Sleq Cell (see FIG. 48B) when imaged with the optical system 4700 as shown in FIGS. 47A-47B. The bottom row of Table 1 displays a total time of 1. 11 hours when only 25 cycles are performed. The decreased sequencing times demonstrate the advantage of a larger FOV allowed by the optical system 4700 as described herein.

[0136] TABLE 1. Cycle and run times for the previous system versus the system according to some embodiments.

[0137] FIG. 48A provides an illustration of an imaging area of a flow cell described herein with 424 individual tiles. FIG. 48B provides an illustration of an imaging area of a flow cell described herein with less than 40 tiles.

[0138] FIG. 47A provides a non-limiting cut-away illustration of an optical system for imaging the surfaces of a flow cell 4521. In some embodiments, the optical system comprises an LED bank heat sink 4701, light pipe illuminators 4702, a flow cell 4521, sections of imaging optics 4703, one or more pixel shifters 4704, and a plurality of imaging sensors 4705. As shown in FIG. 47B, the optical system 4700 is smaller than a comparable instrument, such as the IDEX instrument core. Advantages of a smaller optical instrument include, but are not limited to reduced cabling requirements, reduction in the number of available failure modes, reduced heat exchange requirements, as well as a reduced benchtop footprint.

[0139] Described herein, in some embodiments, is an optical system 4900, as shown in the non-limiting schematic of FIGS. 49A-49B, configured for multiple side imaging of allow cell 4905. The multiple-side can be dual-side, three-side, quad-side, or even more sides. The optical system 4900 disclosed herein marbe used in systems designed for a variety of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. As shown in FIGS. 49A-49B, the optical system comprises an imaging sensor 4912 that may be configured for imaging a flow cell 4905. In some embodiments, the sample flow is coincident with the x-axis as shown in FIGS. 49A-49B. In some embodiments, there may be a plurality of imaging sensors 4912. The imaging sensor 4912 may be a CCD imaging sensor. In some embodiments, the imaging sensor 4912 may be aCMOS imaging sensor. In some embodiments, the optical system 4900 comprises a pixel shifter 4911. The pixel shifter 4911 may 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 comprises a filter 4910. In some embodiments, the filter 4910 is a multi-band filter. In some embodiments, the filter 4509 is a multi-band stopband filter. In some embodiments, the filter 4910 is a tri -band fluorescence stopband filter. In someembodiments, the tri-band fluorescence stopband filter is referred to as a tri -band notch filter. In some embodiments, the system comprises imaging optics 4909. In some embodiments, the imaging optics 4909 comprise an objective lens.

[0140] In some embodiments, the filter 4910 is positioned between the imaging sensor 4912 and the flow cell 4905. In some embodiments, the imaging optics 4909, also referred to as an imaging optic assembly, focuses light emitted from the flow cell 4909 to the imaging sensor 4912. In some embodiments, theoptical system 4900 comprises an integrated field flattening assembly. In some embodiments, the optical system comprises an aberration correction module. In some embodiments, the optical system comprises a wedge block 4916, configured for adj usting the pathlength of the optical system. In some embodiments, the wedge block 4916 comprises a first wedge piece 4907, a second wedge piece 4906, or a combination thereof. In some embodiments, the system comprises a piezo drive 4908 configured to move the position of the first wedge piece 4907 and the second wedge piece 4906 relative to each other, therefore adjusting the optical path length of the optical system. In some embodiments, the first wedge piece 4907 may be actuated to move in a range from 0. 1 um to 1000 um relative to the second wedge piece 4906. In some embodiments, the first wedge piece 4907 may be actuated to move in a range from 100 um to 800 um relative to the second wedge piece 4906. In some embodiments, the first wedge piece 4907 may be actuated to move in a range from 300 um to 700 um relative to the second wedge piece 4906. The relative movement of the wedge pieces 4907, 4906 may be achieved by moving the first wedge piece 4907 and / or the second wedge piece 4906 relative to the sample or sample stage that stays still during imaging of one or more images. In some embodiments, the movement of the wedge pieces 4907, 4906 may be at least in the X-Y plane. In some embodiments, the movement of the wedge pieces 4907, 4906 may be only in the X-Y plane. In some embodiments the flow cell 4905 is configured for multiple sided imaging (DSI) . In some embodiments, the flow cell 4905 comprises a front interior surface 4904, a back-interior surface 4905, or a combination thereof. In some embodiments the front interior surface 4904 and / or the back-interior surface 4903 comprise sample sites 4902. In some embodiments, the optical system comprises an optical axis 4913. In some embodiments, the optical system comprises an optimal imaging volume 4915. In certain aspects, the optimal imaging volume 4915 comprises the field-of-view (FOV), the area of illumination, the area of acquisition, the focal plane, the focal depth, the region and / or volume where sample sites 4902 emit a brightness at or above an acceptable level, or a combination thereof. Typically, in the art of microscopy, the brightness of objects in the center of the FOV may be maximum at the center and decrease toward the comers and / or edges.

[0141] In some embodiments, the optical system lacks bandpass filters. In some embodiments, the optical system lacks cutoff filters. In some embodiments, the optical system lacks dichroic mirrors or dichroic filters.Wedge-block A ssembly

[0142] Described herein are various embodiments of an optical system comprising a focusing element assembly.

[0143] In some embodiments, the focusing element assembly is an autofocus element. As an example, FIGS. 51A-51B provides a non-limiting cut-away illustration of a focusing lens assembly. The focusing lens assembly is configured to maintain a fixed position within the optical pathway (e.g., optical axis) and to allow for relative motion between at least a first lens and second lens contained within a lens housing of the focusing lens assembly.

[0144] In some embodiments, the autofocus element is configured for initial focus. In some embodiments, the autofocus element is contained within a 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 contained within the lens barrel of the lens assembly. In some embodiments, the autofocus element is configured to improve reliability, reduces mechanical footprint of the optical system. In some embodiments the autofocus element comprises the wedge block assembly, the piezo drive, the wedge block-piezo drive assembly, or a combination thereof.

[0145] In some embodiments, the focusing element assembly comprises a first focusing element, and a second focusing element. In some embodiments, the first and second focusing elements are housed in a housing comprising the first and second focusing assembly. In some embodiments, the first focusing element is configured to move relative to the second focusing element to focus between a first surface of a flow cell and second surface of the flow cell, respectively. In some embodiments, the first and / or second focusing elements is a lens. In some embodiments, the first and / or second focusing elements is a prism In some embodiments, the focusing element assembly is a wedge block assembly, wherein the first and second focusing elements are wedge blocks that are configured to move relative to the other. In some embodiments, the optical system comprises a wedge block assembly 4916 as shown in FIGS. 49A-49B. In certain aspects, the wedge block assembly 4916 comprises a first wedge piece 4907 and a second wedge piece 4906. In some embodiments, the wedge block assembly 4916 comprises 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, the relative movement of the first wedge piece 4907 to the second wedge piece 4906 causes the optical path length of the wedge block assembly 4916 to change due to the change in the physical thickness wedge block assembly 4916 as shown in FIGS. 49A to 49B. In some embodiments, the wedge block assembly 4916 comprises 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 position 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 piezo drive 4908 are contained within the housing. In some embodiments, the wedge block assembly 4916 and piezo drive 4908 comprise a wedge block-piezo drive assembly . In some embodiments, the second wedge piece 4906 of the wedge block assembly 4916 contactsthe housing. In some embodiments, the second wedge piece 4906 of the wedge block assembly 4916 contacts the flow cell 4905.

[0146] 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., z axis). In some embodiments, the top wedge piece 4907 is aligned with the bottom wedge piece 4906, as illustrated in FIG. 49A. In such an embodiment, the physical distance of the wedge block assembly 4916 results in the focal plane aligning with the back-interior surface. In this case, the sample sites 4902 of the back-interior surface are in focus. In some embodiments, the piezo drive 4908 moves the top wedge piece 4907 to a position relative to the bottom wedge piece 4906, as illustrated in FIG. 49B, such that the physical thickness of the wedge block 4916 within the optical path is greater than in the aligned state illustrated in FIG. 49A. In such an embodiment, the focal plane is shifted to align with the front-interior surface. In this case, the sample sites 4902 of the front-interior surface are in focus.Stage

[0147] Described here are various embodiments of an optical system comprising a stage. The stage may be a tilt stage. The stage may be a tip-tilt stage. The stage may allow for rotation. The stage may be configured to translate in three different axes, simultaneously, wherein all axes are perpendicular to each other. The stage may be configured to translate in three different axes, simultaneously, wherein all axes are perpendicular to each other. The stage may be configured to translate in, and rotate about, three different axes, simultaneously, wherein all axes are perpendicular to each other. The stage may translate the plurality of optical subsystems 5001 relative to the flow cell 4905 as shown in FIG. 50. The stage may translate the flow cell 4905 relative to the plurality of optical subsystems 5001 as shown in FIG. 50. The stage may translate a single optical subsystem 4914. The stage may rotate the plurality of optical subsystems 5001 about the x-axis of the capillary flow cell 5201 as shown in FIG. 53A-53B. The stage may translate the plurality of optical subsystems 5001 along the x-axis, coincident to the long axis of the capillary flow cell 5201 as shown in FIG. 52A-53B.Pixel Shifter

[0148] Described herein are various embodiments of an optical system comprising a pixel shifter 4911. In some embodiments, the pixel shifter 4911 enables sub-pixel resolution imaging. In certain aspects, the resolution of the optical system may be increased by use of the pixel shifter 4911 without increasing the actual optical system resolution. In some embodiments, the pixel shifter 4911, effectively multiplies the resolution of the imaging sensor 4912. In some embodiments, a piezoelectric actuator is configured for defined lateral pixel shifts coincident to the image plane (e.g., in the x-y plane). In some embodiments, a piezoelectric actuator is configured for pixel shifting in the optical axis 4913 (e.g., z-axis or z-axis containing planes). In some embodiments, tilt stage is configured for pixel shifts in X-Z or Y-Z or X-Y-Z. In some cases, the tilt stage is configured for pixel shifts in two dimensions. In some embodiments, the optical system comprising the pixel shifter is configured for imaging the 3D sample objects. In some cases, the optical system comprising the pixel shifter is configured for imaging a 2D sample object.

[0149] In some embodiments, the 3D objects may comprise sample sites 4902. In some embodiments, sample sites 4902 are amplified nucleic acids. In some embodiments, a sample site may comprise a polony or multiple polonies.

[0150] The pixel-shifter 4911 may utilize polarization.Multiple imaging systems

[0151] In some embodiments, the optical system as seen 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 driven - wedge block assembly, a light source 4901, or a combination thereof. In some embodiments, the imaging sensor 4912 is a cellphone-style camera. In some embodiments, the plurality of optical subsystems 5001 comprises an array of optical subsystems. In some embodiments, the array of optical subsystems may be configured for multiple focal depths, multiple wavelengths, or a combination thereof. In some embodiments, each optical subsystem 4914 of the plurality 5001 is configured for a focal depth, wherein 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, wherein the wavelengths detected by at least two optical subsystems of the plurality are different. In some embodiments, an image sensor 4912 of each optical subsystem 4914 of the plurality 5001 comprise an array of image sensors 4912. In some embodiments, high resolution low-cost cameras are configured to provide imaging, with aberrations compensated by software. In some embodiments, the optical system comprises one optical subsystem 4914, wherein the optical subsystem 4914 comprises one optimal imaging volume as shown in FIGS. 49A-49B. In FIGS. 49A-49B the extent 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 xy plane (e.g. , the focal plane). The xy plane, or focal plane, comprises a cross section of the optimal imaging volume and may comprise referred to as the area of illumination, area of acquisition, or a combination thereof. Surfaces comprising sample sites 4902 that extend beyond the optimal FOV are not optimally illuminated by the light source, not optimally captured by the imaging sensor, not optimally resolved by the optics, or a combination thereof. Such non- optimal regions of the surface exhibit non-uniform brightness and non-uniform resolution as may be observed in the edges and / or corners of the image in FIG. 37. In FIG. 37 the sample sites become dimmer and less resolved from the center to the edges and / or corners of the image. FIG. 50 illustrates an embodiment where the sample site 4902 covered surface extends beyond the optimal imaging volume 4915 of one optical subsystem 4916 and where overlapping optimal imaging volumes 4915 overlap to provide a composite optimal imaging volume.

[0152] In some embodiments, the optical system has an optimized FOV of 6 mm x 6 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm to about 9 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm to about 1 mm, about 0.5 mm to about 3 mm, about 0.5 mm to about 6 mm, about 0.5 mm to about 9 mm, about 1 mm to about 3 mm, about 1 mm to about 6 mm, about 1 mm to about 9 mm, about 3 mm to about 6 mm, about 3 mm to about 9 mm, or about 6 mm to about 9 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm, about 1 mm, about3 mm, about 6 mm, or about 9 mm. In some embodiments, the system has an optimized FOV of at least about 0.5 mm, about 1 mm, about 3 mm, or about 6 mm. In some embodiments, the system has an optimized FOV of at most about 1 mm, about 3 mm, about 6 mm, or about 9 mm.

[0153] In some embodiments, the optical system has an optimized area of illumination, of 6 mm x 6 mm In some embodiments, the system has an optimized area of illumination, of about 0.5 mm to about 9 mm In some embodiments, the system has an optimized area of illumination, of about 0.5 mm to about 1 mm, about 0.5 mm to about 3 mm, about 0.5 mm to about 6 mm, about 0.5 mm to about 9 mm, about 1 mm to about 3 mm, about 1 mm to about 6 mm, about 1 mm to about 9 mm, about 3 mm to about 6 mm, about 3 mm to about 9 mm, or about 6 mm to about 9 mm. In some embodiments, the system has an optimized area of illumination, of about 0.5 mm, about 1 mm, about 3 mm, about 6 mm, or about 9 mm. In some embodiments, the system has an optimized area of illumination, of at least about 0.5 mm, about 1 mm, about 3 mm, or about 6 mm. In some embodiments, the system has an optimized area of illumination, of at most about 1 mm, about 3 mm, about 6 mm, or about 9 mm.

[0154] 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 surface of the flow cell. In some embodiments, the optical system is configured for rapid imaging of a first surface and a second surface of the flow cell. In some embodiments, the entire active area (e.g., region of interest, ROI) of the surface 4903 or 4904 of the flow cell 4905 is imaged in 5 imaging steps. In some embodiments, the active area (e.g, region of interest) of the surface 4903 or 4904 is imaged in about 1 imaging step to about 10 imaging steps. In some embodiments, the active area (e.g., region of interest) of the surface 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 about4 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, 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, the active area (e.g., region of interest) of the surface 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, the active area (e.g. , region of interest) of the surface is imaged in at least about 1 imaging step, about 2 imaging steps, about 3 imaging steps, about 4 imaging steps, about 5 imaging steps, or about 6 imaging steps. In some embodiments, the active area (e. g. , region of interest) of the surface is imaged in at most about 2 imaging steps, about 3 imaging steps, about 4 imaging steps, about 5 imaging steps, about 6 imaging steps, or about 10 imaging steps.

[0155] In some embodiments, each imaging step includes imaging at least a portion of the entire active area or ROI. In some embodiments, each imaging step includes imaging at least an overlapping portion of the ROI, and the overlapping portion in the ROI can also be imaged in a different imaging step. In some embodiments, the complete ROI can be imaged, a portion at a time in an imaging step (with or without some overlapping area), in multiple imaging steps.

[0156] In some embodiments, the image acquired using the optical systems herein is a flow cell image. The flow cell image can include a FOV that covers at least a portion of the entire active area or ROI on a surface of a flow cell or otherwise a different sample support structure. The flow cell images can be aligned with each other to cover the entire ROI of a surface of the flow cell.Light delivery component

[0157] In some embodiments, the optical system comprises a light delivery component. In some embodiments, the light delivery component is a waveguide. In some embodiments, the light delivery component is alight pipe 4702 as shown in FIG. 47. In some embodiments, the light delivery component is a fiber optic. 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.Imaging channels

[0158] The optical system as described herein may be configured for imaging one or more fluorophores. In some embodiments, the optical system is configured to distinctly image two, three, or more different fluorophores. In certain aspects, the optical system comprises one or more imaging channels. In some embodiments, a first imaging channel of the one or more imaging channels is configured to image a first fluorophore of the one or more fluorophores. In some embodiments, a second imaging channel of the one or more imaging channels is configured to image a second fluorophore of the one or more fluorophores. In some embodiments, a third imaging channel of the one or more imaging channels is configured to image a third fluorophore of the one or more fluorophores. In some embodiments, an imaging channel comprises at least one of a light source 4901, a filter 4910, an imaging sensor 4912, or a combination thereof.Heater

[0159] Described herein, in some embodiments are systems and devices comprising a heater. In some instances, the flow cell 4905 further comprises a heater. In some embodiments, the heater is integrated with the flow cell. In some embodiments, the heater is integrated with a multiple surface imaging flow cell 4905. In some embodiments, the heater is integrated with the capillary flow cell 5201. In some embodiments, the integrated heater is a transparent heater block integrated heater. In some embodiments, the heater is an infrared (IR) heater. In some embodiments, the transparent heater conforms to the surface of the flow cell. In some embodiments, a transparent heater conforms to and fully surrounds a flow cell with a non- rectangular cross section. In some embodiments, a transparent heater conforms to and fully surrounds a flow cell with a round cross section. In some embodiments, a transparent heater conforms to and fullysurrounds a capillary flow cell 5201. In some embodiments, the transparent heater is transparent in all image channels of the one or more image channels of the optical system.Aberration correction

[0160] In some embodiments, aberration correction methods may be applied to allow for imaging through air bubbles that may appear within the flow cell. In some embodiments, non-flat flow cell surfaces enable right angle or off-axis illumination. In some embodiments, the optical system described herein may comprise magnetic positioning of various elements. In some embodiments, the optical system may be configured to image flow cells with round edges. Information regarding imaging rounded edges or curved surfaces may be found in International Patent Application No. PCT / US2022 / 037831, which is hereby incorporated by reference in its entirety.

[0161] In some embodiments, one or more flow cell images acquired using the optical system herein, eg, the imaging module with a single light source (for excitation of the sample(s)), may have aberration within a same flow cell image. In some embodiments, flow cell images of the one or more samples acquired using the optical system with a single light source may include at least one image with signals of two or more different wavelengths or different wavelength ranges. Such two different wavelengths or wavelength ranges may cause different signal spots, e.g., polonies or clusters, to shift spatially by a different amount relative from the positions if no aberration occurred. For example, in sequencing cycle 15 of a sequencing run of in situ cells, polony 1 within an in situ cell is labeled with a first dye that emits green color when excited by a single color excitation light, e.g., blue light, and polony 2 is labeled with a second dye that emits red color when excited by the same single color blue light. Both green and red light signals are detected using the same imaging module disclosed herein to generate a flow cell image. The polonies are bright spots in the flow cell images in greyscale. The polonies are adjacent to each other on the flow cell device, and the distance between the two centers of the polonies is 900 urns. Due to aberration, differait spatial shifting of the green and red color in the same flow cell image may occur, polony 1 and polony 2 are 600 urns away from each other in the flow cell image, and the shifting causes polony 2 to overlap with a polony 3, which is not overlapping on the flow cell device with polony 2. With chromatic aberration correction, the relative distance between polonies can be restored to be consistent to what the distance is on the flow cell device. Without correction, aberration in such flow cell images may cause errors in subsequent sequencing analysis, e.g., base calling in polony 2 and polony 3.

[0162] In some embodiments, the aberration includes spherical aberration. In some embodiments, the aberration includes chromatic aberration. In some embodiments, the one or more flow cell images acquired using the optical system, e.g., the imaging module with a single light source, may include signals at two different wavelengths or wavelength ranges. In some embodiments, polonies or signal spots of differait wavelengths or wavelength ranges may experience different spatial offsets causing chromatic aberration In some embodiments, chromatic aberration within an image may be unique to the imaging module with single light source. In some embodiments, such chromatic aberration within an image may not be present in different optical systems, e. g., traditional optical systems in NSG sequencers. In some embodiments, the aberration in the flow cell images may be improved by physically adjusting one or more optical elementsof the imaging module. In some embodiments, the aberration may be corrected using software or algorithms after flow cell images are generated. The chromatic aberration may be corrected using various algorithms, e.g., using machine learning or artificial intelligence models. In some embodiments, the chromatic aberration may be corrected using fiducial markers on the flow cell device or a calibration device spatially separated from the flow cell device. Embodiments of the fiducial markers or the calibration device is disclosed in Patent Application No. PCT / IB2024 / 059112, and is incorporated herein by reference in its entirety.Integrated Field Flattener

[0163] The area of illumination and / or FOV of an existing fluorescence microscope imaging system can be limited to the size of the single lens system and / or single imaging sensor present. The ability of a system to systematically capture brightness across the FOV may be referred to as field uniformity of the system Non-uniformity of brightness and resolution across the FOV is, in some cases, observed from the center to the edge of the FOV. In some instances, illumination non-uniformity is caused by non-uniform field curvature effects of a lens the system, usually these are single lens systems. Systems, devices and methods designed to improve field uniformity are sometimes referred to as field flatteners or field flattening, respectively. The optical system described herein can comprise a field flattener. In some instances, the field flattener comprises a plurality of optical subsystems 5001 designed to provide overlapping coverage of the ‘active area of the flow cell surface. Where one image of an individual optical subsystem system 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 a second optical subsystem 4914 may overlap. In some instances, the optimal imaging volume 4915 of a first optical subsystem overlaps with a second optical subsystem and a third optical subsystem.

[0164] In some embodiments, the surface 5101 of the flow cell comprising sample sites 4902 is not flat as shown in FIG. 52. In certain aspects, each optical subsystem 4914 of the plurality 5001 is positioned to match the contour of the active area of the flow cell as shown in FIG. 52.Multivalent Molecules

[0165] The present disclosure provides a multivalent molecule that serves as a reagent for labeled a nucleotide in a nucleic acid template for a nucleic acid sequencing reaction. In some embodiments, the multivalent molecule comprises a core attached to at least one nucleotide-arm. In some embodiments, the at least one nucleotide-arm can comprise a core attachment moiety. In some embodiments, the at least one nucleotide-arm can comprise a spacer. In some embodiments, the at least one nucleotide-arm can comprise a linker. In some embodiments, the at least one nucleotide-arm can comprise a nucleotide unit. In some embodiments, the at least one nucleotide-arm can comprise a core attachment 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 attached to a plurality of nucleotide-arms. In some cases, the core can be attached to between about 1 to about 50 nucleotide arms.In some cases, the core is attached to between about 2 to about 20 nucleotide-arms. In some cases, the core is attached to between about 2 to about 4 nucleotide-arms. In some cases, the core is attached to between about 4 to about 10 nucleotide-arms. In some cases, the core is attached to between about 10 to about 15 nucleotide-arms. In some cases, the core is attached to between about 15 to about 20 nucleotide-arms.

[0166] The present disclosure provides a multivalent molecule comprising a core attached to at least one biotinylated nucleotide-arm. In some embodiments, the at least one biotinylated nucleotide-arm can comprise a core attachment moiety. In some embodiments, the at least one biotinylated nucleotide-arm can comprise a spacer. In some embodiments, the at least one biotinylated nucleotide-arm can comprise a linker. In some embodiments, the at least one biotinylated nucleotide-arm can comprise a nucleotide unit. In some embodiments, the at least one biotinylated nucleotide-arm can comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit. In some embodiments the core can comprise a streptavidin -type or avidin-type moiety, and the biotin unit of the biotinylated nucleotide-arm can mediate conjugation of the core to the biotinylated nucleotide-arm. A streptavidin-type or avidin-type core can be a tetrameric biotin-binding protein that can bind one, two, three or up to four biotinylated nucleotide-arms.

[0167] In some embodiments, the core can comprise a streptavidin-type or avidin-type moiety, including streptavidin or avidin protein, as well as any derivatives, analogs and other non -native forms of streptavidin or avidin that can bind to at least one biotin moiety. The streptavidin or avidin moiety can comprise native or recombinant forms, as well as mutant versions and derivatized molecules. Mutant versions of streptavidin and avidin can comprise any one or any combination of two or more of amino acid insertions, deletions, substitutions, or truncations. Mutant versions can also include fusion polypeptides. Many different forms of streptavidin and avidin are commercially available.

[0168] The multivalent molecules can be configured using a streptavidin or avidin core having a high affinity for the biotin moiety on a biotinylated nucleotide-arm to reduce dissociation of the nucleotide-arms from the core. A mixture of multivalent molecules can be prepared, where the mixture contains two or more sub-populations of multivalent molecules and each sub-population contains multivalent molecules having one type of nucleotide units (e.g., dATP, dGTP, dCTP, d iTP or dUTP). Multivalent molecules that are configured to have high affinity between the core and nucleotide-arms can reduce undesirable dissociation of nucleotide-arms from the core, and exchange of nucleotide arms between different cores. Exchange of nucleotide arms during a sequencing reaction can lead to incorrect based calling and reduced sequencing accuracy. In some embodiments, multivalent molecules having increased stability (e.g, reduced dissociation of biotinylated nucleotide-arms) can comprise a dye labeled streptavidin, where the streptavidin subunits carry aLys!21Arg mutation which can exhibit reduced dissociation of a biotinylated nucleotide-arm from the streptavidin core.

[0169] The streptavidin moiety can comprise full-length or truncated forms having a high affinity for binding biotin. For example, the streptavidin moiety can exhibit a dissociation constant (Kd) of about 1044mol / L, or about I 0-15mol / L. In some embodiments, the streptavidin moiety can comprise any amino acid substitution mutation at a site that can be labeled with a dye. For example, the dye-labeling site can comprise lysine at position 121 which may overlap with a biotin binding site. In some embodiments, a dyeattached to streptavidin at Lysl21 may block or inhibit biotin binding to the dye-labeled streptavidin A multivalent molecule comprising a dye labeled streptavidin carrying lysine at position 121 may exhibit dissociation of a biotinylated nucleotide- arm from the streptavidin core (e.g., SEQ ID 1). A multivalent molecule having increased stability can comprise a dye labeled streptavidin carrying aLysl21 Arg mutation which can exhibit reduced dissociation of a biotinylated nucleotide-arm from the streptavidin core.

[0170] In some embodiments, the streptavidin moiety can comprise any amino acid substitution that increases the affinity for binding biotin (e.g, increases the Ka to about 10'16mol / L), improves retention of biotin at temperatures up to about 60 °C, or about 65 °C, or about 70 °C or about 80 °C, or a combination of increases the affinity for binding biotin and improves retention of biotin.

[0171] The avidin moiety can comprise full-length or truncated forms having ahigh affinity for binding biotin. For example, the avidin moiety can exhibit a dissociation constant (Ka) of about 10'14mol / L, or about 10-15mol / L. In some embodiments, the avidin can comprise substitutions of any one or any combination of the eight arginine residues. The avidin can comprise partially de-glycosylated forms and non-glycosylated forms. The avidin moiety can include derivatized forms, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin, and the commercially-available products including EXTRA VIDIN, CAPTAVIDIN (selective nitration of tyrosine residues at the four biotin -binding sites to generate avidin that reversibly binds biotin), NEUTRAVIDIN (having chemically de-glycosylated and include 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 Asp 17 is replaced with isoleucine, e.g., SEQ ID 2). Amino acids having neutral non-polar side chains include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline and valine. Amino acids having neutral polar side chains include asparagine, cysteine, glutamine, serine, threonine, tryptophan and tyrosine.

[0172] In some embodiments, the core can be labeled with a detectable reporter moiety. The core can be streptavidin or avidin which are homo-tetramers. Each subunit in the homo-tetramer can include at least one lysine residue which can be conjugated to a fluorophore. A labeling reaction can employ N- hydroxysuccinimide (NHS) ester-conj ugated fluorophores. The maximum number of fluorophores that can be attached to a streptavidin or avidin subunit can be dictated by the number of lysine residues in the subunit.

[0173] 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 a lower degree of labeling will exhibit weaker fluorescent intensities. Dye-core conjugates with very high degree of labeling (e.g., DoL > 6) may exhibit reduced fluorescence due to self-quenching from the conjugated fluorophore. In some embodiments, the predetermined degree of labeling for streptavidin or avidin cores may depend upon the dye. Fluorescent dyes include but are not limited to: CF647, CF680, CF570 and CF532 dyes from Biotium; AF647, AF680, AF568 and AF532 from Thermo Fisher Scientific; IFluor 647, IFluor 680, IFlour 568 and IFlour 532 from AATBio; DY648P1, DY679P1, DY585 and DY530 from Dyomics; and AFDy647, IRFlour 680LT, AFDye 568 and AFDye 532 from Fluoroprobes. The predetermined degree of labeling can be about 1 - 10, or about 3 - 8, or about 3.5 - 7, or about 1.6 - 4.

[0174] Red fluorophores can be brighter (higher intensity) than green dyes, which can cause color bleeding when imaging both red-labeled and green-labeled multivalent molecules on the same support (e.g., flow cell). The degree of labeling of a sub-population of multivalent molecules can be increased or decreased to achieve improved signal balance from a mixture of labeled multivalent molecules. For example, the degree of labeling of a sub-population of multivalent molecules labeled with ared fluorophore can be decreased compared to the degree of labeling of a sub-population of multivalent molecules labeled with a green fluorophore. In some embodiments, the degree of labeling of a sub -population of multivalent molecules labeled with a red fluorophore can be about 1 -3, or about 2 - 3, or about 3 - 6. In some embodiments, the degree of labeling of a sub-population of multivalent molecules labeled with a green fluorophore can be about 4 - 7.

[0175] Solution fluorescence measurements can be used to determine the relative brightness of the labeled streptavidin or avidin cores. Alternatively, the degree of labeling can be determined by employing a functional assay (e.g. , a flow cell trap assay) in which clonally-amplified template molecules immobilized on a flow cell are contacted with primers, polymerases and fluorescently -labeled multivalent molecules, under a condition suitable for binding the multivalent molecules to complexed polymerases without incorporating the nucleotide units into the primer, and signal intensity can be detected.

[0176] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms. In some embodiments, the plurality of nucleotide-arms can comprise the same type of nucleotide units. For example, a multivalent molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have a nucleotide unit selected from a group consisting of dATP, dGTP, dCTP, dl IP and dUTP.

[0177] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms. In some embodiments, the plurality of nucleotide-arms can comprise different types of nucleotide units. For example, a multivalent molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first nucleotide unit selected from a group consisting of dATP, dGTP, dCTP, dl IP and dUTP, and a second attached arm can have a second nucleotide unit selected from a group consisting of dATP, dGTP, dCTP, dl IP and dUTP, where the first and second nucleotide units are different.

[0178] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms, wherein a nucleotide-arm of the plurality comprises a spacer. In some embodiments, the plurality of nucleotide-arms can comprise the same type of spacer. For example, a multivalent molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have the same spacer.

[0179] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms, wherein a nucleotide-arm of the plurality comprises a spacer. In some embodiments, the plurality of nucleotide-arms can comprise different types of spacers. For example, a multivalent molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of 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, where the first and second spacer units are different.

[0180] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms, where a nucleotide-arm of the plurality comprises a linker. In some embodiments, the plurality of nucleotide-arms can comprise the same type of linker. For example, a multivalent molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have the same linker. In some embodiments, the first and second type of linker can be selected from any of the spacers described herein.

[0181] The present disclosure provides compositions, systems, methods, and kits comprising a multivalent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms, where a nucleotide-arm of the plurality comprises a linker. In some embodiments, the plurality of nucleotide-arms can comprise different types of linkers. For example, a multivalent molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of 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, where the first and second linker units are different. In some embodiments, the first and second type of linker can be selected from any of the linkers described herein In some embodiments, the first and second type of linker can be selected from any of the spacers described herein.

[0182] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms, where a nucleotide-arm of the plurality comprises a linker and a spacer. In some embodiments, the plurality of nucleotide-arms can comprise the same type of spacer and linker. For example, a multivalent molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have the same spacer and linker. In some embodiments, the spacer and linker can be selected from any of the spacers and linkers described herein.

[0183] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms, wherein a nucleotide-arm of the plurality comprises a reactive group. In some embodiments, the plurality of nucleotide-arms can comprise the same type of reactive group. For example^ a multivalent molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have the same reactivegroup. In some embodiments, the reactive group can comprise an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group.

[0184] In some embodiments, the reactive group is in the linker. In some embodiments, the reactive group in the linker can be reactive with a chemical reagent. For example, the reactive groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-l,4-benzo-quinone (DDQ). The reactive groups aryl and benzyl can be reactive with H2 Pd / C. The reactive groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). The reactive group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The reactive groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with tri ethylamine trihydrofluoride.

[0185] In some embodiments, the nucleotide- arms can have the same type of reactive group in the linker where the reactive group can comprise an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl group in the linker can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2- carboxyethyl)phosphine (TCEP),bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0186] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms, wherein a nucleotide arm of the plurality comprises a linker that comprises a reactive group. In some embodiments, the plurality of nucleotide-arms can comprise different types of reactive groups in the linkers. For example, a multivalent molecule can comprise a core (e.g. , streptavidin or avidin core) attached to a plurality of 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, where the first and second reactive groups are different.

[0187] 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 in any combination from a group consisting of an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, and silyl group.

[0188] In some embodiments, the first and second reactive groups can be reactive with a chemical agent. For example, the reactive groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-1.4-benzo-quinone(DDQ). The reactive groups aryl and benzyl can be reactivewith H2 Pd / C. The reactive groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). The reactive group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with tri ethylamine in pyridine, or with Zn in acetic acid (AcOH). The reactive groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0189] In some embodiments, the nucleotide-arms can have the different types of reactive groups in the linkers where the reactive group can comprise an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl group in the linker can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2- carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0190] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms. In some embodiments, the plurality of nucleotide-arms can comprise a nucleotide unit with the same type of sugar 3’OH group. For example, amultival ent molecule can comprise a core (e.g, streptavidin or avidin core) attached to aplurality of nucleotide arms or biotinylated nucleotide arms, where all of the attached arms have a nucleotide unit having the same type of sugar 3’OH group.

[0191] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms. In some embodiments, the plurality of nucleotide-arms can comprise a nucleotide unit with the same type of sugar 3 ’ blocking group (e. g. , chain terminating moiety F or example, a multivalait molecule can comprise a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, where all of the attached arms can have a nucleotide unit having the same type of sugar 3’ blocking group. In some embodiments, the sugar 3’ blocking group can comprise an allyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. In some embodiments, the sugar 3’ blocking group can comprise a 3'-O-alkyl hydroxylamino 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.

[0192] In some embodiments, the sugar 3’ blocking group can be reactive with a chemical reagent. For example, the sugar 3’ blocking groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-1.4-benzo-quinone (DDQ). The sugar 3’ blocking groups aryl and benzyl can be reactive with H2 Pd / C. The sugar 3’ blocking groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). The sugar 3’blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with tri ethylamine in pyridine, or with Zn in acetic acid (AcOH). The sugar 3’ blocking groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0193] In some embodiments, the sugar 3’ blocking group (e.g., azide, azido and azido methyl) can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri-alkyl phosphine moiety or a derivatized tri -aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0194] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms. In some embodiments, the plurality of nucleotide-arms can comprise a nucleotide unit with different sugar 3’ blocking groups. For example, a multivalent molecule can comprise a core (e.g, streptavidin or avidin core) attached to aplurality of nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first nucleotide unit having a first 3’ blocking group, and a second attached arm can have a second nucleotide unit having a second 3’ blocking group, where the first and second 3’ blocking groups are different.

[0195] 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 in any combination from a group consisting of an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. 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 in any combination from a group consisting of an 3’-O-alkyl hydroxylamino group, a 3’ -phosphor othioate group, a 3’-O- malonyl group, or a 3’-O-benzyl group. 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 in any combination from a group consisting of an azide, azido or azidomethyl group.

[0196] In some embodiments, the first and second 3’ blocking groups can be reactive with a chemical reagent. For example, the 3’ blocking groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano- 1,4-benzo-quinone (DDQ). The 3’ blocking groups aryl and benzyl can be reactive with H2 Pd / C. The 3’ blocking groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta- mercaptoethanol or dithiothritol (DTT). The 3’ blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The 3’ blocking groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0197] In some embodiments, the first and second 3’ blocking groups (e. g. , azide, azido and azido methyl) can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri -alkyl phosphine moiety or a derivatized tri -aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0198] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. In some embodiments, the multivalent molecule can comprise a core attached to a plurality of nucleotide-arms. In some embodiments, the plurality of nucleotide-arms can comprise a nucleotide unit with a first sugar 3’ OH blocking groups. In some embodiments, the plurality of nucleotide-arms can comprise a nucleotide unit with a second 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, streptavidin or avidin core) attached to aplurality of nucleotide arms or biotinylated nucleotide arms, where (a) at least a first arm can comprise a first nucleotide unit having a sugar moiety which includes a 3 ’OH group, (b) at least second arm can comprise a second nucleotide unit having a first 3’ blocking group, and (c) at least 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.

[0199] 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 in any combination from a group consisting of an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group. 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 in any combination from a group consisting of an 3’-O-alkyl hydroxylamino group, a 3’ -phosphor othioate group, a 3’-O- malonyl group, or a 3’-O-benzyl group. 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 in any combination from a group consisting of an azide, azido or azidomethyl group.

[0200] In some embodiments, the first and second 3’ blocking groups can be reactive with a chemical reagent. For example, the 3’ blocking groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano- 1,4-benzo-quinone (DDQ). The 3’ blocking groups aryl and benzyl can be reactive with H2 Pd / C. The 3’ blocking groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). The 3’ blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The 3’ blocking groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride.

[0201] In some embodiments, the first and second 3’ blocking groups (e. g. , azide, azido and azido methyl) can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri -alkyl phosphine moiety or a derivatized tri -aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0202] The present disclosure provides compositions, systems, methods, and kits comprising amultival ent molecule. 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 1 -6 or more reporter moi eties. In some embodiments, the reporter moiety can comprise a fluorophore.

[0203] A mixture of multivalent molecules having different units in their nucleotide-arms, where distinction between the different multivalent molecules can be achieved. In some embodiments, the core of a first multivalent molecule can be labeled with a reporter moiety to distinguish it from a second labeled (or non-labeled) multivalent molecule. For example, a unit in a nucleotide-arm of the labeled first multivalent molecule can differ from a unit in a nucleotide-arm 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 differ from a corresponding unit in the second multivalent molecule, where the first and second reporter moieties correspond to the differentiating unit. In some embodiments, the first and second reporter moieties can be spectrally distinguishable from each other.

[0204] In some embodiments, the core of a first multivalent molecule can be labeled with a first reporter moiety that corresponds to the base (e.g., dATP, dGTP, dCTP, diTP or dUTP) in the attached nucleotide- arms, and the core of a second multivalent molecule can be labeled with a second reporter moiety that corresponds to the base (e.g., dATP, dGTP, dCTP, dl lP or dUTP) in the attached nucleotide-arms, 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 spectrally distinguishable from each other. In some embodiment, detection of the first reporter moiety indicates a binding event, an incorporation event, or a combination of binding and incorporation events of the first multivalent molecule having the first base, and detection of the second reporter moiety indicates a binding event, an incorporation event, or a combination of binding and incorporation events of the second multivalent molecule having the second base. The binding event can be a multivalent molecule binding to a complexed polymerase. The incorporation event can be a nucleotide unit incorporating into the terminal 3’ end of an extendible primer in a complexed polymerase, where the nucleotide unit is part of a multivalent molecule.Mixture of multivalent molecules

[0205] The present disclosure provides separate batches (sub-populations) 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 moietyreporter moiety can correspond to a particular base 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 (sub-populations) can be mixed together to form a plurality of labeled multivalent molecules comprising two or more sub-populations of spectrally distinguishable multivalent molecules. In some embodiments, at least one batch of multivalent molecules in the mixture can be non-labeled (e.g, dark multivalent molecules).

[0206] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules which can comprise a mixture of at least two sub-populations of multivalent molecules labeled with different reporter moieties. In some embodiments, at least a first sub-population of multivalent molecules can be labeled with a first reporter moiety that corresponds to a first nucleotide unit on the nucleotide-arms. In some embodiments, at least a second sub-population of multivalent molecules can be labeled with a second reporter moiety that corresponds to a second nucleotide unit on the nucleotide- arms. In some cases, the first and second reporter moieties can differ from each other. In some embodiments, the plurality of multivalent molecules can further comprise at least a third sub-population of multivalent molecules which is labeled with a third reporter moiety, wherein the first, second and third reporter moieties can differ from each other. In some embodiments, the plurality of multivalent molecules can further comprise at least a fourth sub-population of multivalent molecules which is labeled with a fourth reporter moiety, wherein the first, second, third and fourth reporter moieties can differ from each other. In some embodiments, additional sub-populations (e.g., fifth, sixth, seventh, eighth, nineth, tenth or more) of labeled multivalent molecules can be added into the mixture. In some embodiments, the reporter moiety can be a fluorophore. In some embodiments, a first sub-population of multivalent molecules can be labeled with a first fluorophore and a second fluorophore of multivalent molecules can be labeled with a second fluorophore. In some cases, the first fluorophore and the second fluorophore can be different.

[0207] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules which can comprise a mixture of at least two sub-populations of multivalent molecules labeled with different reporter moieties. In some embodiments, at least a first sub-population of multivalent molecules can be labeled with a first reporter moiety that corresponds to a first nucleotide unit on the nucleotide-arms. In some embodiments, at least a second sub-population of multivalent molecules can be non-labeled (e.g., a dark multivalent molecule).

[0208] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules which comprises a mixture of at least three sub-populations of multivalent molecules labeled with different reporter moieties. In some embodiments, at least a first sub-population of multivalent molecules can be labeled with a first reporter moiety that corresponds to a first nucleotide unit on the nucleotide-arms. In some embodiments, at least a second sub-population of multivalent molecules can be labeled with a second reporter moiety that corresponds to a second nucleotide unit on the nucleotide- arms. In some embodiments, at least a third sub-population of multivalent molecules can be non-labeled (e. g. , a dark multivalent molecule). In some embodiments, the first and second reporter moieties can differ from each other.

[0209] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules which comprises a mixture of at least four sub-populations of multivalent molecules labeled with different reporter moieties. In some embodiments, the mixture of multivalent molecules can have at least a first sub-population of multivalent molecules can be labeled with a first reporter moiety that corresponds to a first nucleotide unit on the nucleotide-arms. In some embodiments, the mixture of multivalent molecules can have at least a second sub-population of multivalent molecules can be labeled with a second reporter moiety that corresponds to a second nucleotide unit on the nucleotide- arms. In some embodiments, the mixture of multivalent molecules can have at least a third sub-population of multivalent molecules is labeled with a third reporter moiety. In some embodiments, the mixture of multivalent molecules can have at least a fourth sub-population of multivalent molecules can be nonlabeled (e.g., a dark multivalent molecule). In some cases, the first, second and third reporter moieties can differ from each other.

[0210] An embodiment comprises: a mixture of four different types of multivalent molecules comprising (1) a first sub-population of multivalent molecules each comprising a dATP nucleotide unit and a core labeled with a first type of fluorophore, (2) a second sub-population of multivalent molecules each comprising a dGTP nucleotide unit and a core labeled with a second type of fluorophore, (3) a third subpopulation of multivalent molecules each comprising a dCTP nucleotide unit and a core labeled with a third type of fluorophore, and (4) afourth sub-population of multival entmolecules each comprising ad I IP nucleotide unit and a core labeled with a fourth type of fluorophore, where the first, second, third and fourth fluorophores can be spectrally distinguishable. In some embodiments, any one of the sub-populations of multivalent molecules can be non-labeled for use as “dark” multivalent molecules.

[0211] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e. g. , a population) of multivalent molecules, wherein individual multivalent molecules in the plurality can comprise a core bound to at least one nucleotide-arm. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 nucleotide-arms. In some embodiments, individual multivalent molecules in the plurality can comprise a streptavidin or avidin core bound to 2-5 biotinylated nucleotide-arms.

[0212] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e. g. , a population) of multivalent molecules, wherein individual multivalent molecules in the plurality can comprise a core bound to at least one nucleotide-arm having one type of nucleotide unit comprising dATP, dGTP, dCTP, dl lP or dUTP. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 nucleotide-arms, where the nucleotide-arms have one type of nucleotide unit comprising dATP, dGTP, dCTP, dl lP or dUTP. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 biotinylated nucleotide-arms, where the biotinylated nucleotide-arms have one type of nucleotide unit comprising dATP, dGTP, dCTP, d lTP or dUTP.

[0213] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising a mixture (sub-populations) of two or more different types of multivalent molecules. In some embodiments, the plurality of multivalent molecules can have at least afirst multivalent molecule in the plurality. In some cases, the at least the first multivalent molecule can comprise a core bound to at least one nucleotide-arm having afirsttype of nucleotide selected from a 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 a first multivalent molecule in the plurality and at least a second multivalent molecule. In some cases, the at least second multivalent molecule can comprise a core bound to at least one nucleotide-arm having a second type of nucleotide that differs from the first nucleotide in the first multivalent molecule. In some embodiments, the first multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a first type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dl lP or dUTP. In some embodiments, the second multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a second type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dl lP or dUTP, where the first and second type of nucleotides are different. In some embodimaits, the mixture can comprise two, three, four, five, or more different types of multivalent molecules having nucleotides selected in any combination from a group consisting of dATP, dGTP, dCTP, d lTP or dUTP.

[0214] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e. g. , a population) of multivalent molecules, wherein individual multivalent molecules in the plurality can comprise a core bound to at least one nucleotide-arm. In some embodiments, the at least one nucleotide arm that are bound to a core can have the same spacer. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 nucleotide-arms. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 biotinylated nucleotide- arms.

[0215] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e. g. , a population) of multivalent molecules, wherein individual multivalent molecules in the plurality can comprise a core bound to at least one nucleotide-arm. In some embodiments, the at least one nucleotide- arm that are bound to a core can have the same linker. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 nucleotide-arms. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 biotinylated nucleotide- arms.

[0216] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e. g. , a population) of multivalent molecules, wherein individual multivalent molecules in the plurality can comprise a core bound to at least one nucleotide-arm In some embodiments, all of the nucleotide arms that are bound to a core can have the same spacer and linker. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 nucleotide-arms. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 biotinylated nucleotide- arms.

[0217] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising a mixture (sub-populations) of two or more different types ofmultivalent molecules. In some embodiments, the plurality of multivalent molecules can comprise at least a first multivalent molecule in the plurality can comprise a core bound to at least one nucleotide-arm having a first type of spacer. In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule can comprise a core bound to at least one nucleotide-arm having a second type of spacer. In some embodiments, the plurality of multivalent molecules can comprise a mixture of the at least the first multivalent molecule and the at least the second multivalent molecule. In some cases, the second type of spacer in the second multivalent molecule can differ from the first spacer in the first multivalent molecule. In some embodiments, the first multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a first type of spacer. In some embodiments, the second multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a second type of spacer, where the first and second type of spacers are different.

[0218] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising a mixture (sub-populations) 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 in the plurality comprises a core bound to at least one nucleotide-arm having a first type of linker. In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule comprises a core bound 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 at least the first multivalent molecule and the at least the second multivalent molecule. In some cases, the second type of linker in the second multivalent molecule can differ from the first linker in the first multivalent molecule. In some embodiments, the first multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a first type of linker. In some embodiments, the second multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a second type of linker, where the first and second type of spacers are different.

[0219] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e. g. , a population) of multivalent molecules, wherein individual multivalent molecules in the plurality can comprise a core bound to at least one nucleotide-arm In some embodiments, all of the nucleotide arms that are bound to a core can have the same reactive group in the linker. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 nucleotide-arms. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 biotinylated nucleotide-arms. In some embodiments, the reactive group can comprise alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl group. In some embodiments, the individual multivalent molecules can comprise a reactive group that can be reactive with a chemical agent. For example, the reactive groups alkyl, alkenyl, alkynyl and allyl are reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano-l,4-benzo-quinone (DDQ). The reactive groups aryl and benzyl can be reactivewith H2 Pd / C. The reactive groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta- mercaptoethanol or dithiothritol (DTT). The reactive group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The reactive groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride. In some embodiments, the reactive group can comprise an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl group in the linker can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri -alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0220] The present disclosure provides compositions, systems and kits comprising a plurality of multivalent molecules comprising a mixture (sub-populations) of two or more different types of multi valent molecules. In some embodiments, the plurality of multivalent molecules can have at least a first multivalait molecule (a first subpopulation) in the plurality. In some embodiments, the at least the first subpopulation can comprise a core bound to at least one nucleotide-arm having a first type of reactive group in the linker. In some embodiments, the plurality of multivalent molecules can have at least a second multivalent molecule (a second subpopulation) comprises a core bound to at least one nucleotide-arm having a second type of reactive group in the linker. In some cases, the first reactive group in the first type of linker in the first sub-population differ from the second reactive group in the second type of linker in the second subpopulation. In some embodiments, the first multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a first type of reactive group in the linker. In some embodiments, the second multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a second type of reactive group in the linker, where the first reactive group differs from the second reactive group.

[0221] In some embodiments, the first and second reactive can be selected, in any combination, from a group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, and silyl group. In some embodiments, the individual multivalent molecules can comprise a first or second reactive group that can be reactive with a chemical agent. For example, the reactive groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano- l,4-benzo-quinone(DDQ). The reactive groups aryl and benzyl can be reactivewith H2 Pd / C. The reactive groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta-mer captoethanol or dithiothritol (DTT). The reactive group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with tri ethylamine in pyridine, or with Zn in acetic acid (AcOH). The reactive groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride. In some embodiments, thefirst or second reactive can be selected, in any combination, from a group consisting of an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl reactive group in the linker can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri-alkyl phosphine moiety or a derivatized tri -aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0222] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e. g. , a population) of multivalent molecules, wherein individual multivalent molecules in the plurality can comprise a core bound to at least one nucleotide-arm, wherein all of the nucleotide arms that are bound to a core can have a nucleotide unit having the same sugar 3 ’OH group. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 nucleotide-arms. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 biotinylated nucleotide-arms.

[0223] The present disclosure provides compositions, systems, methods, and kits comprising a plurality (e. g. , a population) of multivalent molecules, wherein individual multivalent molecules in the plurality can comprise a core bound to at least one nucleotide-arm, wherein all of the nucleotide arms that are bound to a core can have a nucleotide unit having the sugar 3 ’ OH group substituted with the same 3 ’ blocking group. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 nucleotide-arms. In some embodiments, individual multivalent molecules in the plurality can comprise a core bound to 2-5 biotinylated nucleotide-arms. In some embodiments, the sugar 3 ’blocking group can comprise alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl group. In some embodiments, the individual multivalent molecules can comprise a 3’ blocking group that can be reactive with a chemical agent. For example, the 3’ blocking groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd PPhsty) with piperidine, or with 2,3-Dichloro-5,6-dicyano-l,4-benzo-quinone (DDQ). The 3’ blocking groups aryl and benzyl can be reactive with H2 Pd / C. The 3’ blocking groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including betamercaptoethanol or dithiothritol (DTT). The 3’ blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The 3’ blocking groups urea and silyl can be reactive with tetrabutyl ammonium fluoride, pyridine-HF, with ammonium fluoride, or with tri ethylamine trihydrofluoride. In some embodiments, the 3’ blocking group can comprise a 3’-O-alkyl hydroxylamino group, a 3’ -phosphor othioate group, a 3’-O-malonyl group, or a 3’-O-benzyl group. In some embodiments, the 3’ blocking group can comprise an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl 3’ blocking group can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri-alkyl phosphine moiety or a derivatized tri -aryl phosphine moiety. In some embodiments, the phosphine compound cancomprise Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0224] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising a mixture (sub-populations) 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 in the plurality can comprise a core bound to at least one nucleotide-arm having a first nucleotide unit with a first type of sugar 3’OH blocking group (chain terminating moiety). In some embodiments, the plurality of multivalent molecules can comprise at least a second multivalent molecule comprises a core bound to at least one nucleotide-arm having a second nucleotide unit having a second type of sugar 3’ blocking group (chain terminating moiety). In some embodiments, the plurality can comprise the first multivalent molecule and the second multivalent molecule. In some cases, the first 3’ blocking group can differ from the second 3’ blocking group. In some embodiments, the first multi v al ait molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have afirsttypeof 3’ blocking group. In some embodiments, the second multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a second type of 3’ blocking group, where the first 3’ blocking group differs from the second 3’ blocking group.

[0225] In some embodiments, the first and second 3’ blocking group can be selected, in any combination, from a group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate^ phosphate, thio, disulfide, carbonate, urea, and silyl group. In some embodiments, the individual multivalent molecules can comprise a first or second 3 ’ blocking group that can be reactive with a chemical agent. For example, the 3’ blocking groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano- 1,4-benzo-quinone (DDQ). The 3’ blocking groups aryl and benzyl can be reactive with H2 Pd / C. The 3’ blocking groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta- mercaptoethanol or dithiothritol (DTT). The 3’ blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The 3’ blocking groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride. In some embodiments, the first and second 3’ blocking group can be selected, in any combination, from a group consisting of a 3’ -O-alkyl hydroxylamino group, a 3’ -phosphorothioate group, a 3’-O-malonyl group, and a 3’-O-benzyl group. In some embodiments, the first or second 3’ blocking group can be selected, in any combination, from agroup consisting of an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl 3’ blocking group is reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri -alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2- carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0226] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising a mixture (sub-populations) 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 in the plurality can comprise a core bound 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 bound to at least one nucleotide-arm having a second nucleotide unit having a first type of sugar 3’ blocking group. In some embodiments, the plurality of multivalent molecules can comprise the first multivalent molecule and the second multivalent molecule. In some embodiments, the first multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a sugar 3’ OH group. In some embodiments, the second multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a first type of 3’ blocking group.

[0227] In some embodiments, the first 3’ blocking group can be selected, in any combination, from a group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, and silyl group. In some embodiments, the individual multivalent molecules can comprise a first 3’ blocking group that can be reactive with a chemical agent. For example, the 3’ blocking groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano- 1,4-benzo-quinone (DDQ). The 3’ blocking groups aryl and benzyl can be reactive with H2 Pd / C. The 3’ blocking groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta- mercaptoethanol or dithiothritol (DTT). The 3’ blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The 3’ blocking groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride. In some embodiments, the first 3’ blocking group can be selected, in any combination, from a group consisting of a 3’-O-alkyl hydroxylamino group, a 3’ -phosphor othioate group, a 3’-O- malonyl group, and a 3 ’ -O-benzyl group. In some embodiments, the first 3 ’ blocking group can be selected, in any combination, from a group consisting of an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl 3’ blocking group can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2- carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).

[0228] The present disclosure provides compositions, systems, methods, and kits comprising a plurality of multivalent molecules comprising a mixture (sub-populations) 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 comprisea core bound 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 bound to at least one nucleotide-arm having a second nucleotide unit having 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 third multivalent molecule can comprise a core bound to at least one nucleotide-arm having a third nucleotide unit having a second type of sugar 3’ blocking group. In some cases, the first and second 3’ blocking groups are different. In some embodiments, the first multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a sugar 3’ OH group. In some embodiments, the second multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a first type of 3’ blocking group. In some embodiments, the third multivalent molecule can comprise a core bound to 2-5 biotinylated nucleotide arms, where the biotinylated-arms can have a second type of 3’ blocking group.

[0229] In some embodiments, the first and second 3 ’ blocking groups can be selected, in any combination, from a group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate^ phosphate, thio, disulfide, carbonate, urea, and silyl group. In some embodiments, the individual multivalent molecules can comprise a first or second 3 ’ blocking group that can be reactive with a chemical agent. For example, the 3’ blocking groups alkyl, alkenyl, alkynyl and allyl can be reactive with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-Dichloro-5,6-dicyano- 1,4-benzo-quinone (DDQ). The 3’ blocking groups aryl and benzyl can be reactive with H2 Pd / C. The 3’ blocking groups amine, amide, keto, isocyanate, phosphate, thio, disulfide can be reactive with phosphine or with a thiol group including beta- mercaptoethanol or dithiothritol (DTT). The 3’ blocking group carbonate can be reactive with potassium carbonate (K2CO3) in MeOH, with triethylamine in pyridine, or with Zn in acetic acid (AcOH). The 3’ blocking groups urea and silyl can be reactive with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride, or with triethylamine trihydrofluoride. In some embodiments, the first and second 3’ blocking groups can be selected, in any combination, from a group consisting of a 3’ -O-alkyl hydroxylamino group, a 3’ -phosphorothioate group, a 3’-O-malonyl group, and a 3’-O-benzyl group. In some embodiments, the first and second 3’ blocking groups can be selected, in any combination, from a group consisting of an azide, azido or azidomethyl group. In some embodiments, the azide, azido or azidomethyl 3’ blocking group can be reactive with a chemical agent. In some embodiments, the chemical agent can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri -alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound can comprise Tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or Tri(hydroxyproyl)phosphine (THPP).Light sources

[0230] In some embodiments, the light source, e.g. , the single light source for exciting the sample(s) to be sequenced, comprises a solid-state laser, a dye laser, a diode laser, an arc lamps, or a tungsten-halogai lamp. Any of a variety of light sources may be used to provide the imaging or excitation light, including but not limited to, tungsten lamps, tungsten-halogen lamps, arc lamps, lasers, light emitting diodes (LEDs), or laser diodes. In some instances, a combination of one or more light sources, and additional optical components, e.g., lenses, filters, apertures, diaphragms, mirrors, and the like, may be configured as an illumination system (or sub-system). In some embodiments, the light source 4901 as shown in FIGS. 49A- 53B is a solid-state light source. In some embodiments, the solid-state light source is a light emitting diode (LED). In some embodiments, the light source 4901 is configured to emit a plurality of wavelengths. In some embodiments, the light source comprises a plurality of light sources. In some embodiments, each light source of the plurality is configured to emit a different wavelength of light. In some embodiments, the light source 4901 is configured to emit: a first wavelength of light at a first time; a second wavelength of light at a second time and a third wavelength of light at a third time. In some embodiments, the first wavelength of light at a first time, the second wavelength of light at the second time and the third wavelength of light at the third time are emitted in a sequence. In some embodiments, the plurality of light sources is configured to be delivered for timed pulse sequences in sequential colors. In some embodiments, the plurality of optical subsystems 5001 are configured to increase speed of detection. In some embodiments, the solid-state light source is not a laser. For some applications, the light source comprises a filter to narrow the spectrum of the light emitted by the light source. In some embodiments, the light source is referred to as the excitation source. In some embodiments, the light emitted by the light source is referred to as excitation light. In some cases, a light source that generates light in the wavelength ranges of the present disclosure may be used.Fluorescence imaging viewed as an information pipeline

[0231] A useful abstraction of the role that fluorescence imaging systems plays in typical genomic assay techniques (including nucleic acid sequencing applications) is as an information pipeline, where the photon signal enters at one end of the pipeline, e.g. , the objective lens used for imaging, and location specific information regarding the fluorescence signal emerges at the other end of the pipeline, e.g. , at the position of the image sensor. When more information is pumped through this pipeline, some content, inevitably, will be lost during this transfer process and never recovered. An example of this case is when too many labeled molecules (or clonally-amplified clusters of molecules) are present within a small region of a substrate surface to be clearly resolved in the image; at the position of the image sensor, it becomes difficult to differentiate photon signals arising from adjacent clusters of molecules, thus increasing the probability of attributing the signal to the wrong cluster and leading to detection errors. In some cases, the clusters are polonies.Design of imaging modules

[0232] The goal of designing an optical imaging module is thus to maximize the flow of information content through this detection pipeline and to minimize detection errors. The inventors of the present disclosure considered several design elements,

[0233] 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 A mismatch of these parameters may result in loss of information or sometimes even the generation of false information, e.g. , spatial aliasing may arise when pixel sampling frequency is lower than twice the optical resolution limit.

[0234] 2) Matching the size of the area to be imaged with the overall image quality of the optical imaging system and focus quality across the entire field-of-view.

[0235] 3) Matching the optical collection efficiency, modulation transfer function, and image sensor performance characteristics of the optical system design with the fluorescence photon flux expected for the input excitation photon flux, dye efficiency (related to dye extinction coefficient and fluorescence quantum yield), while accounting for background signal and system noise characteristics.

[0236] 4) Maximizing the separation of spectral content to reduce cross talk between fluorescence imaging channels.

[0237] 5) Effective synchronization of image acquisition steps with repositioning of the sample or optics between image capture of different fields-of-view to minimize the down time (or maximize the duty cycle) of the imaging system and thus maximize the overall throughput of the image capture process.

[0238] This disclosure describes a systematic way to address each of the design elements outlined above and to create component level specifications for the imaging system.Improved optical resolution and image quality to improve or maximize information transfer and throughput

[0239] One non-limiting design practice may be to start with the optical resolution required to distinguish two adjacent features as specified in terms of a number, X, of line pairs per mm (Ip / mm) and translate it to a corresponding numerical aperture (NA) requirement. The numerical aperture requirement can then be used to assess the resulting impact on modulation transfer function and image contrast.

[0240] The standard modulation transfer function (MTF) describes the spatial frequency response for image contrast (modulation) transferred through an optical system; image contrast decreases as a function of spatial frequency and increases with increasing NA. This function limits the contrast / modulation that can be achieved for a given NA. Furthermore, wave front error can negatively impact the MTF, thus making it desirable to improve or optimize the optical system design using the true system MTF instead of that predicted by diffraction-limited optics. Note that, as used herein, MTF will refer to the total system MTF (including the complete optical path from coverslip to image sensor) although design practice may primarily consider the MTF of the objective lens.

[0241] In genomic testing applications, where the target to be imaged is an array of high density “spots” on a surface (either 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 that the spots are small enough to be approximated as point sources of light. Assuming that the detection task is to determine if the two adjacent spots separated by a distance, d, are ON or OFF (in other words, bright ordark), and that the contrast -to-noise ratio (CNR) for the fluorescence signals arising from the spots at the sample plane (or object plane) is Csampie, then under ideal conditions the CNR of the readout signal for the two adjacent spots at the image sensor plane, Cimage, can be closely approximated as Cimage = Csampie * MTF(l / d), where MTF(l / d) is the MTF value at spatial frequency = (1 / d).

[0242] In a typical design, the value of Cimage may be at least 4 so that a simple threshold method can be used to avoid misclassification of fluorescence signals. Assuming a Gaussian distribution of fluorescence signal intensities around a mean value, at Cimage > 4, the expected error in correctly classifying fluorescence signals (e.g. , as being ON or OFF) is <0.035%. The use of high CNR sequencing and surface chemistry is described in U.S. Patent Nos. 10,876,148, 10,704,094, each of which is incorporated by reference in its entirety. In some embodiments, the high CNR sequencing and surface chemistry allows one to achieve sample plane CNR (Csampie) values for clusters of clonally-amplified, labeled oligonucleotide molecules tethered to a substrate surface of greater than 12 (or even much higher) when measured for a sparse field (e. g. , at a low surface density of clusters or spots) where the MTF has a value of close to 100%. Assuming a sample plane CNR value of Csampie > 12 and targeting a classification error rate of <0. 1% (thus, Cimage > 4), in some implementations the minimum value for M(l / d) can be determined as M(l / d) = 4 / 12 ~ 33%. Thus, amodulation transfer function threshold of at least 33% may be used to retain the information contort of the transferred image.

[0243] Design practice can relate the minimum separation distance of two features or spots, , to the optical resolution requirement (specified as noted above in terms of A (Ip / mm)) as <7 = (lmm) / A, e.g, is the minimum separation distance between two features or spots which can be fully resolved by the optical system. In some designs disclosed herein, where the objective of the design analysis is to increase or maximize relevant information transfer, this design criterion can be relaxed to d = (lmm) / A / A, where 2 > A > 1. For the same optical resolution of A Ip / mm. the value of , the minimum resolvable spot separation distance at the sample plane, is reduced, thereby enabling the use of higher feature densities.

[0244] Design practice determines the minimum spatial sampling frequency at the sample plane using the Nyquist criteria, where spatial sampling frequency 5 > 2*A (and where X is the optical resolution of the imaging system specified in terms of X Ip / mm as noted above). When the system spatial sampling frequency is close to the Nyquist criteria, as is often the case, imaging system resolution of greater than 5 results in aliasing as the higher frequency information resolved by the optical system cannot be sufficiaitly sampled by the image sensor.

[0245] In the some of the designs disclosed herein, an oversampling scheme based on the relationship 5 = B*Y (where B > 2 and Y is the true optical system MTF limit) may be used to further improve the information transfer capacity of the imaging system. As indicated above, X (Ip / mm) corresponds to a practical, non-zero (>33%) minimum modulation transfer value, whereas Y (Ip / mm) is the limit of optical resolution so modulation at Y(lp / mm) is 0. Thus, in the disclosed designs, Y (Ip / mm) may advantageously be significantly greater than A. For values of B > 2, the disclosed designs are oversampling for the sample object frequency A, e.g. , S > B*Y > 2*A

[0246] The above relationship can be used to determine the system magnification and may provide an upper bound for image sensor pixel size. The choice of image sensor pixel size is matched to the system optical quality as well to the spatial sampling frequency required to reduce aliasing. The lower bound of image sensor pixel size can be determined based on photon throughput, as relative noise contributions increase with smaller pixels.

[0247] Other design approaches are, however, also possible. For example, reducing the NA to less than 0.6 (e.g. , 0.5 or less,) may provide increased depth of field. Such increased depth of field may enable multiple surface imagining wherein two surfaces at different depths can be imaged at the same time with or without refocusing. As discussed above, reducing NA may reduce optical resolution. In some implementations, use of higher excitation beam power, e.g, 1 Watt or higher, may be employed to produce strong signal. An inherently high contrast sample (e.g. , comprising a sample surface that exhibits strong foreground signal and dramatically reduced background signal, may also be used to facilitate acquisition of high contrast-to-noise ratio (CNR) images, e.g. , having CNR values of > 20, that provide for improved signal discrimination for base-calling in nucleic acid sequencing applications, etc. In some optical system designs disclosed herein, sample support structures such as flow cells having hydrophilic surfaces are used to reduce background noise.

[0248] In various implementations, a large field-of-view (FOV) is provided by the disclosed optical systems. For example, a FOV of greater than 2 or 3 mm may be provided with some optical imaging systems comprising, e.g. , an objective lens and a tube lens. In some cases, the optical imaging system provides a reduced magnification, for example, a magnification of less than lOx. Such reduced magnification may in some implementations facilitate large F OV designs. Despite a reduced magnification, the optical resolution of such systems can still be sufficient as detector arrays having small pixel size or pitch may be used. In some implementations, image sensors comprising a pixel size that is smaller than twice the optical resolution provided by the optical imaging system (e.g. , objective and tube lens) may be used to satisfy the Nyquist theorem.

[0249] Still other designs are also possible. In some optical designs configured to provide for multiple surface imaging where two surfaces at different depths can be imaged at the same time, the optical imaging system (e.g. , the objective lens and / or tube lens) is configured to reduce optical aberration for imaging said two surfaces (e.g , two planes) at those two respective depths more than at other locations (e.g. , other planes) at other depths. Additionally, the optical imaging system may be configured to reduce aberration for imaging said two surfaces (e.g. , two planes) at those two respective depths through a transmissive layer on said sample support structure (such as a layer of glass (e.g. , a cover slip) and through a solution (e.g, an aqueous solution) comprising the sample or in contact with a sample on at least one of said two surfaces.

[0250] Multichannel fluorescence imaging modules and systems:

[0251] In some instances, the imaging modules or optical systems disclosed herein may comprise fluorescence imaging modules or systems. In some instances, the fluorescence imaging systems disclosed herein may comprise a single fluorescence excitation light source (for providing excitation light at a single wavelength or within a single excitation wavelen"11' an optical path configured to deliver theexcitation light to a sample (e.g. , fluorescently -tagged nucleic acid molecules or clusters thereof disposed on a substrate surface). In some instances, the fluorescence imaging systems disclosed herein may comprise 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 a substrate surface on which fluorescently -tagged nucleic acid molecules or clusters thereof are disposed) to an image sensor or other photodetection device. In some instances, the fluorescence imaging systems may comprise two, three, four, or more than four fluorescence excitation light sources and / or optical paths configured to deliver excitation light at two, three, four, or more than four excitation wavelengths (or within two, three, four, or more than four excitation wavelength ranges). In some instances, 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 the sample at two, three, four, or more than four emission wavelengths (or within two, three, four, or more than four emission wavelength ranges and deliver an image of the sample (e.g. , an image of a substrate surface on which fluorescently -tagged nucleic acid molecules or clusters thereof are disposed) to two, three, four, or more than four image sensors or other photodetection devices.Multiple surface imaging

[0252] In some instances, the imaging systems disclosed herein, including fluorescence imaging systems, may be configured to acquire high-resolution images of a single sample support structure or substrate surface. In some instances, the imaging systems disclosed herein, including fluorescence imaging systems, may be configured to acquire high-resolution images of two or more sample support structures or substrate surfaces, e.g. , two, three, four, or more surfaces of a flow cell. The multiple surfaces of a sample support structure or a flow cell device can be axially displaced from each other, along the axial or z direction. The multiple surfaces of a sample support structure or a flow cell device can be interior surfaces facing fluidic channel(s) disclosed herein. The fluidic channels or capillaries of the sample support structure or flow cell can be axially displaced from each other, along the axial or z direction. In an example, the two or more surfaces can be a top and bottom of a first channel and a top and bottom of a second channel axially displaced from the first channel. In this example, there may be an interposer between the first and second channels. The multiple surfaces of a sample support structure or a flow cell can be surfaces of a curved surface (e.g., as described elsewhere herein). For example, the curved surface can be a round surface. A portion of allow cell (e.g., atop surface, an interposer layer, a bottom surface, etc.) may have a thickness of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or more millimeters. A portion of a flow cell (e.g., a top surface, an interposer layer, a bottom surface, etc.) may have a thickness of at most about 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, or fewer millimeters.

[0253] In some instances, the high-resolution images provided by the disclosed imaging systems may be used to monitor reactions occurring on the two or more surfaces of the flow cell (e.g , nucleic acid hybridization, amplification, and / or sequencing reactions) as various reagents flow through the flow cellor around a flow cell substrate. FIG. 1 A and FIG. IB provide schematic illustrations of a multiple surface support structures.

[0254] FIGS. 1A-1B shows the multiple surface support structure relative to the objective lens 102 of the optical systems disclosed herein. The optical system may have an optical axis 101 orthogonal to the sample plane, i. e. , XY plane, in this embodiment. The optical axis is along the z axis. The multiple surface support structure, e.g., flow cell device, may have layers 103, 104 defining a channel 107 in between. The layers may have interior surfaces 103 104, and samples may be immobilized on the surface(s) at sample sites 105, 108.

[0255] FIG. 1 A shows a multiple surface support structure such as a flow cell that includes an internal flow channel through which an analyte or reagent can be flowed. The flow channel may be formed between first and second, top and bottom, and / or front and back layers such as first and second, top and bottom, and / or front and back plates as shown. One or more of the plates may include a glass plate, such as a coverslip, or the like. In some implementations, the layer comprises borosilicate glass, quartz, or plastic. Interior surfaces of these top and bottom layers provide walls of the flow channel that assist in confining the flow of analyte or reagent through the flow channel of the flow cell. In some designs, these interior surfaces are planar. Similarly, the top and bottom layers may be planar. In some designs, at least one additional layer (not shown) is disposed between the top and bottom layers. This additional layer may have one or more pathways cut therein that assist in defining one or more flow channels and controlling the flow of the analyte or reagent within the flow channel. Additional discussion of sample support structures, e.g, flow cells, can be found below.

[0256] FIG. 1 A schematically illustrates a plurality of fluorescing sample sites on the first and second, top and bottom, and / or front and back interior surfaces of the flow cell. In some implementations, reactions may occur at these sites to bind sample such that fluorescence is emitted from these sites (note that FIG. 1 A is schematic and not drawn to scale; for example, the size and spacing of the fluorescing sample sites may be smaller than shown).

[0257] FIG. IB shows another multiple surface support structure having two surfaces containing fluorescing sample sites to be imaged. The sample support structure comprises 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, the analyte or reagent is flowed across these first and second exterior surfaces. FIG. IB schematically illustrates a plurality of fluorescing sample sites on the first and second, top and bottom, and / or front and back exterior surfaces of the sample support structure. In some implementations, reactions may occur at these sites to bind sample such that fluorescence is emitted from these sites (note that FIG. IB is schematic and not drawn to scale; for example, the size and spacing of the fluorescing sample sites may be smaller than shown). Support structures with one or more surfaces can have similar sample site distributions as shown in FIGS. 1A or IB on each of the surfaces.

[0258] In some instances, the fluorescence imaging modules and systems described herein may be configured to image such fluorescing sample sites on each of the multiple surfaces at different distances from the objective lens. In some designs, onl -rmultiple surfaces is in focus at a time.Accordingly, in such designs, one of the surfaces is imaged at a first time, and the other surface is imaged at a second time. The focus of the fluorescence imaging module may be changed after imaging one of the surfaces in order to image a next surface with comparable optical resolution, as the images of the multiple surfaces are not simultaneously in focus. In some designs, an optical compensation element may be introduced into the optical path between the sample support structure and the image sensor in order to image one of the surfaces. The depth of field in such fluorescence imaging configurations may not be sufficiently large to include two or more surfaces of the multiple surfaces. In some implementations of the fluorescence imaging modules described herein, two or more surfaces may be imaged at the same time. e.g. , simultaneously. For example, the fluorescence imaging module may have a depth of field that is sufficiently large to include two or more surfaces. In some instances, this increased depth of field may be provided by, for example, reducing the numerical aperture of the objective lens (or microscope objective) as will be discussed in more detail below.

[0259] As shown in FIGS. 1A and IB, 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 surfaces to form in-focus images of the surfaces on an image sensor of a detection channel. The first surface may be between the objective lens and the second surface. For example, as illustrated in FIGS. 1A and IB, the objective lens is disposed above the multiple surfaces, the first surface is disposed above the second surface. The multiple surfaces can be at different depths. The surfaces are at different distances from any one or more of the fluorescence imaging module, the illumination and imaging module, imaging optics, or the objective lais. The multiple surfaces are separated from each other along the z direction. The surfaces can be planar surfaces and are separated from each other along a direction normal to the planar surfaces. In some embodiments, the objective lens has an optical axis and the surfaces are separated from each other along the direction of the optical axis. Similarly, the separation between the surfaces may correspond to the axial distance such as along the optical path of the excitation beam and / or along an optical axis through the fluorescence imaging module and / or the objective lens. Accordingly, these surfaces may be separated by a distance from each other in the axial (Z) direction, 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 aflow channel within a flow cell in some implementations between the first and second surfaces or between the third and fourth surfaces. This separation may correspond, for example, to an interposer substrate, within a flow cell in some implementations between the second and third surface.

[0260] In various designs, the objective lens (possibly in combination with another optical component, e.g. , a tube lens) have a depth of field and / or depth of focus that is at least as large as the axial separation (in the Z direction) between two adjacent surfaces of the multiple surfaces. In some embodiments, the depth of filed and / or depth of focus that is at least as large as the axial separation (in the z direction) between the first and the last (e. g. , the fourth) surfaces along the optical path from the obj ective lens. The obj ective lens, alone or in combination with the additional optical component, may thus simultaneously form infocus images of at least two adjacent surfaces on an image sensor of one or more detection channels wherethese images have comparable optical resolution. In some implementations, the imaging module may or may not need to be re-focused to capture images of at least two adjacent surfaces with comparable optical resolution. In some implementations, compensation optics need not be moved into or out of an optical path of the imaging module to form in-focus images of the surfaces. Similarly, in some implementations, one or more optical elements (e.g. , lens elements) in the imaging module (e.g, the objective lens and / or a tube lens) neednot be moved, for example, in the axial direction along the first and / or second optical paths (e.g, along the optical axis of the imaging optics) to form in-focus images of one of the surfaces, e.g., the first surface, in comparison to the location of said one or more optical element when used to form in-focus images of another one of the surfaces, e.g., the second, third, or fourth surface. In some implementations, however, the imaging module includes an autofocus system configured to provide at least two adjacent surfaces in focus at the same time. In various implementations, the sample is in focus to sufficiently resolve the sample sites, which are closely spaced together in lateral directions (e.g , the X and Y directions). Accordingly, in various implementations, no optical element enters an optical path between the sample support structure (e. g. , between a translation stage that supports the sample support structure) and an image sensor (or photodetector array) in the at least one detection channel in order to form in-focus images of fluorescing sample sites on one surface of the sample support structure and on two other surfaces of the sample support structure. In various implementations, no optical compensation is used to form an in-focus image of fluorescing sample sites on one surface, e.g. , the first surface, of the sample support structure on the image sensor or photodetector array that is not identical to optical compensation used to form an infocus image of fluorescing sample sites on another surface, e.g., the second, third, or fourth surface, of the sample support structure on the image sensor or photodetector array. Additionally, in certain implementations, no optical element in an optical path between the sample support structure (e.g. , betweal a translation stage that supports the sample support structure) and an image sensor in the at least one detection channel is adjusted differently to form an in-focus image of fluorescing sample sites on one surface, e.g., the first surface of the sample support structure than to form an in-focus image of fluorescing sample sites on another surface, e.g., the second, third, or fourth surface of the sample support structure. Similarly, in some various implementations, no optical element in an optical path between the sample support structure (e. g. , between a translation stage that supports the sample support structure) and an image sensor in the at least one detection channel is moved a different amount or a different direction to form an in-focus image of fluorescing sample sites on one surface, e.g., the first surface, of the sample support structure on the image sensor than to form an in-focus image of fluorescing sample sites on another surface, e. g. , the second, third, fourth surface of the sample support structure on the image sensor. Any combination of the features herein can be possible. For example, in some implementations, in-focus images of the first interior surface and the second interior surface of the flow cell can be obtained without moving an optical compensator into or out of an 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 and / or tube lens) along the optical path (e.g. , optical axis) therebetween. For example, in-focus images of the first interior surface and the second, third interior surfaces of the flow cell can be obtained without moving one or moreoptical elements of the tube lens into or out of the optical path, or without moving one or more optical elements of the tube lens along the optical path (e.g. , optical axis) therebetween.

[0261] Any one or more of the fluorescence imaging module, the illumination optical path, the imaging optical path, the objective lens, or the tube lens may be designed to reduce or minimize optical aberration at multiple locations such as the planes corresponding to the multiple surfaces on a flow cell or other sample support structure, for example, where fluorescing sample sites are located. Any one or more of the fluorescence imaging module, the illumination optical path, the imaging optical path, the objective lens, or the tube lens may be designed to reduce or minimize optical aberration at the selected locations or planes relative to other locations or planes, such as the surfaces containing fluorescing sample sites on a flow cell. For example, any one or more of the fluorescence imaging module, the illumination optical path, the imaging optical path, the objective lens, or the tube lens may be designed to reduce or minimize optical aberration at two depths or planes located at different distances from the obj ective lens as compared to the aberrations associated with other depths or planes at other distances from the objective lens. For example^ optical aberration may be less for imaging the surfaces than elsewhere in a region ranging from about 1 to about 10 mm from the objective lens. Additionally, any one or more of the fluorescence imaging module, the illumination optical path, the imaging optical path, the objective lens, or the tube lens may, in some instances, be configured to compensate for optical aberration induced by transmission of emission light through one or more portions of the sample support structure such as a layer that includes one of the surfaces on which sample adheres as well as possibly a solution that is in contact with the sample. This layer (e.g , a coverslip or the wall of a flow cell) may comprise, e.g. , glass, quartz, plastic, or other transparent material having a refractive index and that introduces optical aberration.

[0262] Accordingly, the imaging performance may be substantially the same when imaging the multiple surfaces, e.g., three or four surfaces. For example, the optical transfer functions (OTF) and / or modulation transfer functions (MTF) may be substantially the same for imaging of the multiple surfaces. Either or both of these transfer functions may, for example, be within 20%, within 15%, within 10%, within 5%, within 2.5%, or within 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 arange of spatial frequencies. Accordingly, an imaging performance metric may be substantially the same for imaging each surface of the multiple surfaces of the flow cell without moving an optical compensator into or out of an optical path between the flow cell and the at least one image sensor, and without moving one or more optical elements of the imaging system (e.g. , the objective and / or tube lens) along the optical path (e.g. , optical axis) therebetween. For example, an imaging performance metric may be substantially the same for imaging the first, second, third, and fourth surfaces of the flow cell without moving one or more optical elements of the tube lens into or out of the optical path or without moving one or more optical elements of the tube lens along the optical path therebetween. In some embodiments, the optical path is an optical axis. Additional discussion of MTF is included below and in U.S. Provisional Application No. 62 / 962,723 filed January 17, 2020, which is incorporated herein by reference in its entirety.

[0263] It will be understood by those of skill in the art that the disclosed imaging modules or systems may, in some instances, be stand-alone optical systems designed for imaging a sample or substrate surface. In some instances, they may comprise one or more processors or computers. In some instances, they may comprise one or more software packages that provide instrument control functionality and / or image processing functionality. In some instances, in addition to optical components such as light sources (e.g, solid-state lasers, dye lasers, diode lasers, arc lamps, tungsten-halogen lamps, etc.), lenses, prisms, mirrors, dichroic reflectors, beam splitters, optical filters, optical bandpass filters, light guides, optical fibers, apertures, and image sensors (e.g. , complementary metal oxide semiconductor (CMOS) image sensors and cameras, charge-coupled device (CCD) image sensors and cameras, etc. ), they may also include mechanical and / or optomechanical components, such as X-Y translation stages, X-Y-Z translation stages, piezoelectic focusing mechanisms, electro-optical phase plates, and the like. In some instances, they may function as modules, components, sub-assemblies, or sub-systems of larger systems designed for, e.g, genomics applications (e.g. , genetic testing and / or nucleic acid sequencing applications). For example, in some instances, they may function as modules, components, sub-assemblies, or sub-systems of larger systems that further comprise light-tight and / or other environmental control housings, temperature control modules, flow cells and cartridges, fluidics control modules, fluid dispensing robotics, cartridge- and / or microplate-handling (pick-and-place) robotics, one or more processors or computers, one or more local and / or cloud-based software packages (e.g, instrument / system control software packages, image processing software packages, data analysis software packages), data storage modules, data communication modules (e.g. , Bluetooth, WiFi, intranet, or internet communication hardware and associated software), display modules, etc. , or any combination thereof. These additional components of larger systems, e.g. , systems designed for genomics applications, will be discussed in more detail below.

[0264] FIGS. 2A and 2B illustrate a non-limiting example of an illumination and imaging module 200 for multi-channel fluorescence imaging. The illumination and imaging module 200 includes an objective lais 210, an illumination source 215, aplurality of detection channels 220, and a first dichroic filter 230, which may comprise a dichroic reflector or beam splitter. An autofocus system, which may include an autofocus laser 202, for example, which projects a spot the size of which is monitored to determine when the imaging system is in-focus may be included in some designs. Some or all components of the illumination and imaging module 200 may be coupled to a baseplate 205.

[0265] The illumination or light source 215 may include any suitable light source configured to produce light of at least a desired excitation wavelength (discussed in more detail below). The light source may be a broadband source that emits light within one or more excitation wavelength ranges (or bands). The light source may be a narrowband source that emits light within one or more narrower wavelength ranges. In some instances, the light source may produce a single isolated wavelength (or line) corresponding to the desired excitation wavelength, or multiple isolated wavelengths (or lines). In some instances, the lines may have some very narrow bandwidth. Example light sources that may be suitable for use in the illumination source 115 include, but are not limited to, an incandescent filament, xenon arc lamp, mercury -vapor lamp, a light-emitting diode, a laser source such as a laser diode or a solid-state laser, or other types of lightsources. As discussed below, in some designs, the light source may comprise a polarized light source such as a linearly polarized light source. In some implementations, the orientation of the light source is such that s-polarized light is incident on one or more surfaces of one or more optical components such as the dichroic reflective surface of one or more dichroic filters.

[0266] The illumination or light source 215 may further include one or more additional optical components such as lenses, filters, optical fibers, or any other suitable transmissive or reflective optics as appropriate to output an excitation light beam having suitable characteristics toward a first dichroic filter 230. For example, beam shaping optics may be included, for example, to receive light from a light emitter in the light source and produce a beam and / or provide a desired beam characteristic. Such optics may, for example, comprise a collimating lens configured to reduce the divergence of light and / or increase collimation and / or to collimate the light.

[0267] In some implementations, multiple light sources are included in the illumination and imaging module 200. In some such implementations, different light sources may produce light having differait spectral characteristics, for example, to excite different fluorescence dyes. In some implementations, light produced by the different light sources may be directed to coincide and form an aggregate 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 will have more optical power than the individual beams that overlap to form the composite beam. For example, in some implementations that include two light sources that produce two excitation light beams, the composite excitation light beam formed from the two individual excitation light beams may have optical power that is the sum of the optical power of the individual beams. Similarly, in some implementations, three, four, five or more light sources may be included, and these light sources may each output excitation light beams that together form a composite beam that has an optical power that is the sum of the optical power of the individual beams.

[0268] In some implementations, the light source 215 outputs a sufficiently large amount of light to produce sufficiently strong fluorescence emission. Stronger fluorescence emission can increase the signal- to-noise ratio (SNR) and the contrast-to-noise ratio (CNR) of images acquired by the fluorescence imaging module. In some implementations, the output of the light source and / or an excitation light beam derived therefrom (including a composite excitation light beam) may range in power from about 0.5 watts (W) to about 5.0 W, or more (as will be discussed in more detail below).

[0269] Referring again to FIGS. 2A and 2B, the first dichroic filter 230 is disposed with respect to the light source to receive light therefrom. The first dichroic filter may comprise a dichroic mirror, dichroic reflector, dichroic beam splitter, or 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, e.g , one or more spectral bands in the ultraviolet and blue wavelength ranges. Similarly, a second spectral region may include one or more spectral bands, e.g. , one or more spectral bands extending from the green to red and infrared wavelengths. Other spectral regions or wavelength ranges are also possible.

[0270] In some implementations, the first dichroic filter may be configured to transmit light from the light source to a sample support structure such as to a microscope slide, a capillary, a flow cell, a microfluidic chip, or other substrate or support structure. The sample support structure supports and positions the sample, e.g. , a composition comprising a fluorescently-labeled nucleic acid molecule or complement thereof, with respect to the illumination and imaging module 200. Accordingly, a first optical path extends from the light source to the sample via the first dichroic filter. In various implementations, the sample support structure includes at least one surface on which the sample is disposed or to which the sample binds. In some instances, the sample may be disposed within or bound to different localized regions or sites on the at least one surface of the sample support structure.

[0271] In some instances, the support structure may include two, three, four, or even more surfaces located at different distances from objective lens 210 (e.g, at different positions or depths along the optical axis of objective lens 210 or an axial direction) on which the sample is disposed. As discussed below, for example, the flow cell may comprise a fluid channel formed at least in part by first and second e.g. , upper and lower) interior surfaces, and the sample may be disposed at localized sites on the first interior surface, the second interior surface, or both interior surfaces. The first and second surface may be separated by the region corresponding to the fluid channel through which a solution flows, and thus be at different distances or depth with respect to objective lens 210 of the illumination and imaging module 200. The flow cell may comprise a second fluid channel, axially displaced from a first channel, that is formed at least in part by the third and fourth (e.g. , upper and lower) interior surfaces, and the sample may be disposed at localized sites on the third interior surface, the fourth interior surface, or both. The third and fourth surface may be separated by the region corresponding to the second fluid channel through which a solution flows, and thus be at different distances or depth with respect to objective lens 210 of the illumination and imaging module 200. The first and the second fluidic channel may be separated axially by an interposer substrate disposed in between.

[0272] The obj ective lens 210 may be included in the first optical path between the first dichroic filter and the sample. This objective lens may be configured, for example, to have a focal length, working distance, and / or be positioned to focus light from the light source(s) onto the sample, e.g , onto a surface of the microscope slide, capillary, flow cell, microfluidic chip, or other substrate or support structure. Similarly, the objective lens 210 may be configured to have suitable focal length, working distance, and / or be positioned to collect light reflected, scattered, or emitted from the sample (e.g. , fluorescence emission) and to form an image of the sample (e.g. , a fluorescence image).

[0273] In some implementations, objective lens 210 may comprise a microscope objective such as an off- the-shelf objective. In some implementations, objective lens 210 may comprise a custom objective. An example of a custom objective lens and / or custom objective - tube lens combination is described below and in U.S. Patent Application No. 17 / 999,023, which is incorporated herein by reference in its entirety. The objective lens 210 may be designed to reduce or minimize optical aberration at two, three, four, or more locations. For example, such locations can include planes corresponding to the multiple surfaces of a flow cell. The obj ective lens 210 may be designed to reduce the optical aberration at the selected locationsor planes, e.g. , the first and second surfaces of a multiple surface flow cell, or the first, second, third, and fourth surfaces of a quad surface flow cell, relative to other locations or planes in the optical path. For example, the objective lens 210 may be designed to reduce the optical aberration at two, three, or four depths or planes located at different distances from the objective lens as compared to the optical aberrations associated with other depths or planes at other distances from the obj ective. For example, in some instances, optical aberration may be less for imaging the surfaces of a flow cell than that exhibited elsewhere in a region spanning from 1 to 10 mm from the front surface of the objective lens. Additionally, a custom objective lens 210 may in some instances be configured to compensate for optical aberration induced by transmission of fluorescence emission light through one or more portions of the sample support structure, such as a layer that includes one or more of the flow cell surfaces on which a sample is disposed, or a layer comprising a solution filling the fluid channel of a flow cell. These layers may comprise, e. g. , glass, quartz, plastic, or other transparent material having a refractive index, and which may introduce optical aberration

[0274] In some implementations, objective lens 210 may have a numerical aperture (NA) of 0.6 or more (as discussed in more detail below). Such a numerical aperture may provide for reduced depth of focus and / or depth of field, improved background discrimination, and increased imaging resolution.

[0275] In some implementations, objective lens 210 may have a numerical aperture (NA) of 0.6 or less (as discussed in more detail below). Such a numerical aperture may provide for increased depth of focus and / or depth of field. Such increased depth of focus and / or depth of field may increase the ability to image planes separated by a distance such as the first and second surfaces, the second and third surface, or the third and fourth surfaces. In some implementations, obj ective lens 210 may have a numerical aperture (NA) of 0.5 or less, e. g. , 0.4. Such a numerical aperture may provide for lower optical aberration that needs to be compensated by the optical system than higher NA values. Such a numerical aperture may provide for the capability of focusing and imaging additional image planes, e.g., the planes of the third or fourth surfaces of the flow cell, with minimal changes to the optical system design (e.g., with no requirement for adding or removing a compensator from the optical path from the objective lens to the flow cell being imaged) that is configured to image one or multiple surfaces of the flow cell.

[0276] As discussed above, a flow cell may comprise, for example, first and second layers comprising first and second interior surfaces respectively that are separated by a fluid channel through which an analyte or reagent can flow. The flow cell may also include third and fourth layers comprising third and fourth interior surfaces respectively that are separated by a second fluid channel through which an analyte or reagent can flow. In some implementations, the obj ective lens 210 and / or illumination and imaging module 200 may be configured to provide a depth of field and / or depth of focus sufficiently large to image at least two adjacent surfaces of the flow cell, either sequentially by re-focusing the imaging module between imaging the at least two surfaces, or simultaneously by ensuring a sufficiently large depth of field and / or depth of focus, with comparable optical resolution. In some instances, the depth of field and / or depth of focus may be at least as large or larger than the distance separating the two adj acent surfaces of the flow cell to be imaged. In some instances, the two adjacent surfaces, e.g. , the first and second interior surfaces of a dual surface flow cell or the third and fourth surfaces of a quad surface flow cell, may be separated,for example, by a distance ranging from about 10 pm to about 700 pm, or more (as will be discussed in more detail below). In some instances, the depth of field and / or depth of focus may thus range from about 10 pm to about 700 pm, or more (as will be discussed in more detail below).

[0277] In some designs, compensation optics (e.g. , an “optical compensator” or “compensator”) may be moved into or out of an optical path in the imaging module, for example, an optical path by which light collected by the objective lens 210 is delivered to an image sensor, to enable the imaging module to image the surfaces of the flow cell. The imaging module may be configured, for example, to image one surface, e. g. , the first surface, when a first compensation optics is included in the optical path between the obj ective lens and an image sensor or photodetector array configured to capture an image of the first surface. The imaging module may be configured, for example, to image another surface, e.g. , the second surface whai a second compensation optics is included in the optical path between the obj ective lens and an image sensor or photodetector array configured to capture an image of the second surface. In such a design, the imaging module may be configured to image yet another surface, e.g., the third surface, when the first and the second compensation optics is removed from or not included in the optical path between the objective lais 210 and the image sensor or photodetector array configured to capture an image of the third surface. The need for an optical compensator may be more pronounced when using an objective lens 210 with a high numerical aperture (NA) value, e.g, for numerical aperture values of at least 0.6, least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, or higher. In some implementations, the optical compensation optics (e.g., an optical compensator or compensator) comprises a refractive optical element such as a lens, a plate of optically -transparent material such as glass, a plate of optically -transparent material such as glass, or in the case of polarized light beams, a quarter-wave plate or half-wave plate, etc. Other configurations may be employed to enable the surfaces to be imaged at different times. For example, one or more lenses or optical elements may be configured to be translated in and out of, or along, an optical path between the objective lens 210 and the image sensor.

[0278] In certain designs, the objective lens 210 is configured to be adjusted to change the NA of the optical system. In some embodiments, the objective lens2 can be adjusted so that the NA of the optical system can be adjusted within the range of 0.25 to 0.6. In some embodiments, the objective lens 210 can be adjusted so that the NA of the optical system can be adjusted within the range of 0.35 to 0.55. In some embodiments, the objective lens 210 can be adjusted so that the NA of the optical system can be adjusted within the range of 0.4 to 0.5. In some embodiments, the NA of the optical system can be adjusted by changing the objective lens 210. In some embodiments, the NA of the optical system can be adjusted by only changing the objective lens 210 without moving any optical compensator in, out of, or along the optical path from the objective lens to the sample. In some embodiments, the NA of the optical system can be adjusted by changing an optical element within the objective lens 210, without removing an existing objective lens and adding a new objective lens.

[0279] In some embodiments, the objective lens 210 can include an aperture stop, and adjustment of the aperture stop’s size can result in change of the NA of the optical system. In some embodiments, changing the aperture stop’s size does not involve translating the objective lens or any other optical elements in, outof, or along an optical path between the objective lens and the image sensor. As a non -limiting example^ changing the aperture stop’s size comprises rotating an optical element or part of the objective lens about an axial axis or a longitudinal axis of the objective lens. As another non-limiting example, changing the aperture stop’s size comprises moving an optical element or part of the objective lens orthogonal to an axial axis or a longitudinal axis of the objective lens.

[0280] In some embodiments, the NA of the optical system can be adjusted without changing the aperture’s stop’s size. Instead, the objective lens and / or the tube lens can be redesigned from an optical system with a first NA, e.g., NA of 0.5, to make the NA of the optical system to a predetermined different value, e.g., 0.4. The redesign can involve changing one or more characteristics of the objective lens and / or the tube lens including but is not limited to: a diameter, a size, a magnification, a length, a cover thickness, a working distance, and a lens design that functions to correct aberration.

[0281] In some embodiments, the NA of the optical system can be adjusted, from the NA used in imaging traditional one or dual surface flow cells (e.g. , NA of 0.5 to NA of 0.4), to image multiple surface flow cell with three or more axially-displaced surfaces with predetermined image quality. In some embodiments, the NA of the optical system can remain unaltered from the NA used in imaging traditional one or dual surface flow cells (e. g. , NA of 0.4), to image multiple surface flow cell with three or more axially-displaced surface with predetermined image quality.

[0282] The optical systems herein can allow the adjustment of the objective lens to change the NA (e.g, between 0.5 and 0.4 or in the range from 0.4 to 0.5). The optical system also can allow usage of the same NA, e.g., NA= 0.4 to image flow cell devices with a total thickness that is within a matching range of the NA, e.g., a range from about 220 um to about 360 um. As such, the optical systems herein can provide flexibility and compatibility to image both (1) traditional flow cells with one or dual surfaces and (2) the multiple surface flow cells herein (e.g., with three, four, or even more axially displaced surfaces) with sufficient image quality, e.g., a CNR of at least 5, 10, 15, or 20.

[0283] In certain designs, however, the obj ective lens 210 is configured to provide sufficiently large depth of focus and / or depth of field to enable the surfaces to be imaged with comparable optical resolution without such compensation optics moving into and / or out of an optical path in the imaging module, such as an optical path between the objective lens and the image sensor or photodetector array. In various designs, the objective lens 210 is configured to provide sufficiently large depth of focus and / or depth of field to enable the surfaces to be imaged with comparable optical resolution without optics being moved, such as one or more lenses or other optical components being translated along an optical path in the imaging module, such as an optical path between the objective lens and the image sensor or photodetector array. Examples of such objective lenses will be described in more detail below.

[0284] In some implementations, the objective lens (or microscope objective) 210 may be configured to have reduced magnification. The objective lens 210 may be configured, for example, such that the fluorescence imaging module has a magnification of from less than 2x to less than 1 Ox (as will be discussed in more detail below). Such reduced magnification may alter design constraints such that other design parameters can be achieved. For example, the objective lens 2110 may also be configured such that thefluorescence imaging module has a large field-of-view (FOV) ranging, for example, from about 1.0 mm to about 5.0 mm (e.g. , in diameter, width, length, or longest dimension) as will be discussed in more detail below.

[0285] In some implementations, the objective lens 210 may be configured to provide the fluorescence imaging module with a field-of-view as indicated above such that the FOV has diffraction-limited performance, e.g. , less than 0. 15 waves of aberration over at least 60%, 70%, 80%, 90%, or 95% of the field, as will be discussed in more detail below.

[0286] In some implementations, the objective lens 210 may be configured to provide the fluorescence imaging module with a field-of-view as indicated above 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% ofthe field, as will be discussed in more detail below.

[0287] Referring again to FIGS. 2A and 2B, the first dichroic beam splitter or beam combiner is disposed in the first optical path between the light source and the sample so as to illuminate the sample with one or more excitation beams. This first dichroic beam splitter or combiner is also in one or more second optical path(s) from the sample to the different optical channels used to detect the fluorescence emission Accordingly, the first dichroic filter 230 couples the first optical path of the excitation beam emitted by the illumination source 115 and second optical path of the emission light emitted by a sample specimen to the various optical channels where the light is directed to respective image sensors or photodetector arrays for capturing images of the sample.

[0288] In various implementations, the first dichroic filter 230, e.g. , first dichroic reflector or beam splitter or beam combiner, has a pass-band selected to transmit light from the illumination source 115 only within a specified wavelength band or possibly a plurality of wavelength bands that include the desired excitation wavelength or wavelengths. For example, the first dichroic beam splitter 130 includes a reflective surface comprising a dichroic reflector that has spectral transmissivity response that is, e.g., configured to transmit light having at least some of the wavelengths output by the light source that form part of the excitation beam. The spectral transmissivity response may be configured not to transmit (e.g, instead to reflect) light of one or more other wavelengths, for example, of one or more other fluorescence emission wavelengths. In some implementations, the spectral transmissivity response may also be configured notto transmit (eg, instead to reflect) light of one or more other wavelengths output by the light source. Accordingly, the first dichroic filter 230 may be utilized to select which wavelength or wavelengths of light output by the light source reach the sample. Conversely, the dichroic reflector in the first dichroic beam splitter 130 has a spectral reflectivity response that reflects light having one or more wavelengths corresponding to the desired fluorescence emission from the sample and possible reflects light having one or more wavelengths output from the light source that is not intended to reach the sample. Accordingly, in some implementations, the dichroic reflector has a spectral transmissivity that includes one or more pass-bands to transmit the light to be incident on the sample and one or more stop bands that reflects light outside the pass-bands, for example, light at one or more emission wavelengths and possibly one or more wavelengths output by the light source that are not intended to reach the sample. Likewise, in some implementationsthe dichroic reflector has a spectral reflectivity that includes one or more spectral regions configured to reflect one or more emission wavelengths and possible one or more wavelengths output by the light source that are not intended to reach the sample and includes one or more regions that transmit light outside these reflection regions. The dichroic reflector included in the first dichroic filter 230 may comprise a reflective filter such as an interference filter (e.g. , a quarter- wave stack) configured to provide the appropriate spectral transmission and reflection distributions. FIGS. 2A and 2B also show a dichroic filter 230, which may comprise for example a dichroic beam splitter or beam combiner, which may be used to direct the autofocus laser 102 though the objective and to the sample support structure.

[0289] In some embodiments, the dichroic filter e.g., 230, 530, can include one or more spectral pass- band(s) that increase intensity thus SNR of the light signal(s) being passed by the filter. As such, the dichroic filter(s) herein can provide increased uniformity of frequency response than traditional dichroic filter(s) with narrower spectral pass-bands.

[0290] Although the imaging module 200 shown in FIGS. 2A and 2B and discussed above is configured such that theexcitation beamis transmitted by the first dichroic filter 230tothe objective lens 210, in some designs the illumination source 215 may be disposed with respect to the first dichroic filter 230 and / or the first dichroic filter is configured (e.g. , oriented) such that the excitation beam is reflected by the first dichroic filter 230 to the objective lens 210. Similarly, in some such designs, the first dichroic filter 230 is configured to transmit fluorescence emission from the sample and possibly transmit light having one or more wavelengths output from the light source that is not intended to reach the sample. As will be discussed below, a design where the fluorescence emission is transmitted instead of reflected may potentially reduce wavefront error in the detected emission and / or possibly have other advantages. In either case, in various implementations the first dichroic reflector 130 is disposed in the second optical path so as to receive fluorescence emission from the sample, at least some of which continues on to the detection channels 220.

[0291] FIGS. 2C and2D illustrate the optical paths within the multi-channel fluorescence imaging module ofFIGS. 2Aand2B. In the example shown in FIG. 2AandFIG. 2C, the detection channels 220 aredisposed to receive fluorescence emission from a sample specimen that is transmitted by the objective lens 210 and reflected by the first dichroic filter 230. As referred to above and described more below, in some designs the detection channels 220 may be disposed to receive the portion of the emission light that is transmitted, rather than reflected, by the first dichroic filter. In either case, the detection channels 220 may include optics for receiving at least a portion of the emission light. For example, the detection channels 220 may include one or more lenses, such as tube lenses, and may include one or more image sensors or detectors such as photodetector arrays (e.g. , CCD or CMOS sensor arrays) for imaging or otherwise producing a signal based on the received light. The tube lenses may, for example, comprise one or more lens elements configured to form an image of the sample onto the sensor or photodetector array to capture an image thereof. Additional discussion of detection channels is included below and in U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020, which is incorporated herein by reference in its entirety. In some instances, improved optical resolution may be achieved using an image sensor having relatively highsensitivity , small pixels, and high pixel count, in conjunction with a suitable sampling scheme, which main elude oversampling or undersampling.

[0292] FIGS . 2C and 2D are ray tracing diagrams illustrating optical paths of the illumination and imaging module 200 of FIGS. 2A and 2B. FIG. 2C corresponds to a top view of the illumination and imaging module 200. FIG. 2D corresponds to a side view of the illumination and imaging module 200. The illumination and imaging module 200 illustrated in these figures includes four detection channels 220. However, it will be understood that the disclosed illumination and imaging modules may equally be implemented in systems including more or fewer than four detection channels 220. For example, the multichannel systems disclosed herein may be implemented with as few as one detection channel 220, or as many as two detection channels 220, three detection channels 220, four detection channels 220, five detection channels 220, six detection channels 220, seven detection channels 220, eight detection channels 220, or more than eight detection channels 220, without departing from the spirit or scope of the present disclosure.

[0293] The non-limiting example of imaging module 200 illustrated in FIGS. 2C and 2D includes four detection channels 220, a first dichroic filter 230 that reflects a beam 250 of emission light, a second dichroic filter (e.g. , a dichroic beam splitter) 235 that splits the beam 250 into a transmitted portion and a reflected portion, and two channel-specific dichroic filters (e.g. , dichroic beam splitters) 240 that further split the transmitted and reflected portions of the beam 250 among individual detection channels 220. The dichroic reflecting surface in the dichroic beam splitters 235 and 240 for splitting the beam 250 among detection channels are shown disposed at 45 degrees relative to a central beam axis of the beam 250 or an optical axis of the imaging module. However, as discussed below, an angle smaller than 45 degrees may be employed and may offer advantages such as sharper transitions from pass-band to stop band.

[0294] The different detection channels 220 includes imaging sensors / detectors 224, which may include an image sensor or photodetector array (e.g , a CCD or CMOS detector array). The different detection channels 220 further include optics 226 such as lenses (e.g. , one or more tube lenses each comprising one or more lens elements) disposed to focus the portion of the emission light entering the detection channel 220 at a focal plane coincident with a plane of the photodetector array 224. The optics 226 (e.g , a tube lens) combined with the objective lens 210 are configured to form an image of the sample onto the photodetector array 224 to capture an image of the sample, for example, an image of a surface on the flow cell or other sample support structure after the sample has bound to that surface. Accordingly, such an image of the sample may comprise a plurality of fluorescent emitting spots or regions across a spatial extent of the sample support structure where the sample is emitting fluorescence light. The objective lens 210 together with the optics 226 (e.g. , tube lens) may provide a field-of-view (FOV) that includes a portion of the sample or the entire sample. Similarly, the photodetector array 224 of the different detection channels 220 may be configured to capture images of a full field-of-view (FOV) provided by the objective lens and the tube lens, or a portion thereof. In some implementations, the photodetector array 224 of some or all detection channels 220 can detect the emission light emitted by a sample disposed on the sample support structure, e.g. , a surface of the flow cell, or a portion thereof and record electronic data representing animage thereof. In some implementations, the photodetector array 224 of some or all detection channels 220 can detect features in the emission light emitted by a specimen without capturing and / or storing an image of the sample disposed on the flow cell surface and / or of the full field-of-view (FOV) provided by the objective lens and optics 226 and / or 222 (e.g. , elements of atube lens). In some implementations, the FOV of the disclosed imaging modules (e.g. , that provided by the combination of objective lens 210 and optics 226 and / or 222) may range, for example, between about 1 mm and 5 mm (e.g. , in diameter, width, length, or longest dimension) as will be discussed below. The FOV may be selected, for example, to provide a balance between magnification and resolution of the imaging module and / or based on one or more characteristics of the image sensors and / or objective lenses. For example, arelatively smaller FOV mac be provided in conjunction with a smaller and faster imaging sensor to achieve high throughput.

[0295] Referring again to FIGS. 2C and 2D, in some implementations, the optics 226 in the detection channel (e.g. , the tube lens) may be configured to reduce optical aberration in images acquired using optics 226 in combination with objective lens 210. In some implementations comprising multiple detection channels for imaging at different emission wavelengths, the optics 226 (e.g. , the tube lens) for differait detection channels have different designs to reduce aberration for the respective emission wavelengths at which that particular channel is configured to image. In some implementations, the optics 226 (e.g. , the tube lens) may be configured to reduce aberrations when imaging a specific surface (e.g. , a plane, object plane, etc.) on the sample support structure comprising fluorescing sample sites disposed thereon as compared to other locations (e.g. , other planes in object space). In some implementations, the optics 226 (e.g. , the tube lens) may be configured to reduce aberrations when imaging the multiple surfaces (e.g, first and second planes, first and second object planes, etc. ) on a sample support structure (e.g. , a dual or quad surface flow cell) having fluorescing sample sites disposed thereon as compared to other locations (e.g, other planes in object space). For example, the optics 226 in the detection channel (e.g. , tube lens) may be designed to reduce the aberration at two, three, or more depths or planes located at different distances from the objective lens as compared to the aberrations associated with other depths or planes at other distances from the objective. For example, optical aberration may be less for imaging the multiple surfaces than elsewhere in a region from about 1 to about 10 mm from the objective lens. Additionally, custom optic 126 in the detection channel (e.g , a tube lens) may in some embodiments be configured to compensate for aberration induced by transmission of emission light through one or more portions of the sample support structure such as a layer that includes one of the surfaces on which the sample is disposed as well as 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 may comprise, e.g. , glass, quartz, plastic, or other transparent material having a refractive index, and which introduces optical aberration. Custom optic 126 in the detection channel (e.g , the tube lens), for example, may in some implementations be configured to compensate for optical aberration induced by a sample support structure, e.g, a coverslip or flow cell wall, or other sample support structure components, as well as possibly a solution adjacent to and in contact with the surface on which the sample is disposed.

[0296] In some implementations, the optics 226 in the detection channel (e. g. , a tube lens) are configured to have reduced magnification. The optics 226 in the detection channel (e.g. , a tube lens) may be configured, for example, such that the fluorescence imaging module has a magnification of less than, for example, lOx, as will be discussed further below. Such reduced magnification may alter design constraints such that other design parameters can be achieved. For example, the optics 226 (e.g., a tube lens) may also be configured such that the fluorescence imaging module has a large field-of-view (FOV), for example^ of at least 1.0 mm or larger (e. g. , in diameter, width, length, or longest dimension), as will be discussed further below.

[0297] In some implementations, the optics 226 (e.g , a tube lens) may be configured to provide the fluorescence imaging module with a field-of-view as indicated above such that the FOV has less than 0.15 waves of aberration over at least 60%, 70%, 80%, 90%, or 95% of the field, as will be discussed further below.

[0298] Referring again to FIGS. 2C and 2D, in various implementations, a sample is located at or near a focal position 212 of the objective lens 210. As described above with reference to FIGS. 2Aand 2B, alight source such as a laser source provides an excitation beam to the sample to induce fluorescence. At least a portion of fluorescence emission is collected by the objective lens 210 as emission light. The objective lais 210 transmits the emission light toward the first dichroic filter 230, which reflects some or all of the emission light as the beam 250 incident upon the second dichroic filter 235 and to the different detection channels, each comprising optics 226 that form an image of the sample (e.g , a plurality of fluorescing sample sites on a surface of a sample support structure) onto a photodetector array 224.

[0299] As discussed above, in some implementations, the sample support structure comprises a flow cell such as a flow cell having multiple surfaces (e.g. , two or more interior surfaces) containing sample sites that emit fluorescent emission. These surfaces may be separated by a distance from each other in the longitudinal (Z) direction along the direction of the central axis of the excitation beam and / or the optical axis of the objective lens. This separation may correspond, for example, to one or more flow channels within the flow cell. Analytes or reagents may be flowed through the flow channel(s) and contact the surfaces of the flow cell, which may thereby be contacted with a binding composition such that fluorescence emission is radiated from a plurality of sites on the surfaces. The imaging optics (e.g, objective lens 210) may be positioned at a suitable distance (e.g. , a distance corresponding to the working distance) from the sample to form in-focus images of the sample on one or more detector arrays 124. As discussed above, in various designs, the objective lens 210 (possibly in combination with the optics 226) may have a depth of field and / or depth of focus that is at least as large as the longitudinal separation between two adjacent surfaces or any two surfaces of the multiple surfaces. The objective lens 210 and the optics 226 (of each detection channel) can thus simultaneously form images of multiple surfaces on the photodetector array 224, and these images of the surfaces are in focus and have comparable optical resolution (or may be brought into focus with only minor refocusing of the objects to acquire images that have comparable optical resolution). In various implementations, compensation optics need not be moved into or out of an optical path of the imaging module (e.g. , into or out of the first and / or second opticalpaths) to form in-focus images of the surfaces that are of comparable optical resolution. Similarly, in various implementations, one or more optical elements (e.g. , lens elements) in the imaging module (e.g, the objective lens 210 or optics 226) need not be moved, for example, in the longitudinal direction along the first and / or second optical paths to form in-focus images of the first surface in comparison to the location of said one or more optical elements when used to form in-focus images of the second surface. In some implementations, the imaging module includes an autofocus system configured to quickly and sequentially refocus the imaging module on one or more of the multiple surfaces such that the images have comparable optical resolution. In some implementations, objective lens 210 and / or optics 226 are configured such that at least two of the multiple surfaces, e.g., two adjacent surfaces, are in focus simultaneously with comparable optical resolution without moving an optical compensator into or out of the first and / or second optical path, and without moving one or more lens elements (e.g. , objective lens 210 and / or optics 226 (such as atube lens) longitudinally along the first and / or second optics path. In some implementations, images of the surfaces, acquired either sequentially (e.g , with refocusing between surfaces) or simultaneously (e.g , without refocusing between surfaces) using the novel objective lens and / or tube lens designs disclosed herein, may be further processed using a suitable image processing algorithm to enhance the effective optical resolution of the images such that the images have comparable optical resolution. In various implementations, the sample plane is sufficiently in focus to resolve sample sites on the flow cell surfaces, the sample sites being closely spaced in lateral directions (e.g. , in the X and Y directions).

[0300] As discussed above, the dichroic filters may comprise interference filters that selectively transmit and reflect light of different wavelengths based on the principle of thin-film interference, using layers of optical coatings having different refractive indices and particular thickness. Accordingly, the spectral response (e.g , transmission and / or reflection spectra) of the dichroic filters implemented within multichannel fluorescence imaging modules may be at least partially dependent upon the angle of incidence, or range of angles of incidence, at which the light of the excitation and / or emission beams are incident upon the dichroic filters. Such effects may be especially significant with respect to the dichroic filters of the detection optical path (e.g , the dichroic filters 235 and 240 of FIGS. 2C and 2D).

[0301] FIG. 3 is a graph illustrating a relationship between dichroic filter performance and beam angle of incidence (AOI). Specifically, the graph of FIG. 3 illustrates the effect of angle of incidence on the transition width or spectral span of a dichroic filter, which corresponds to the range of wavelengths where the spectral response (e. g. , transmission spectrum and / or reflection spectrum) transitions between the passband and stopband regions of a dichroic filter. Thus, a transmission edge (or reflection edge) having a relatively small spectral span (e.g. , a small delta X value in the graph of FIG. 3) corresponds to a sharper transition between pass-band and stopband regions or the transmission and reflection regions (or conversely between reflection and transmission regions), while a transmission edge (or reflection edge) having a relatively large spectral span (e. g. , a large delta_ X value in the graph of FIG. 3) corresponds to a less sharp transition between pass-band and stopband regions. In various implementations, sharper transitions between pass-band and stopband regions are generally desirable. Moreover, it may also bedesirable to have increased consistency or a relatively consistent transition width across all or most of the field-of-view and / or beam area.

[0302] Fluorescence imaging modules, in which the dichroic mirrors are disposed at 45 degrees relative to a central beam axis of the emission light or the optical axis of the optical paths (e.g. , of the objective lens and / or tube lens), accordingly can have a transition width of roughly 50 nm for an example dichroic filter, as shown in FIG. 3. Because the emission light beam is not collimated and has some degree of divergence, fluorescence imaging modules may have a range of angles of incidence of approximately 5 degrees between opposing sides of the beam. Thus, as shown in FIG. 3, different portions of the beam of emission light may be incident upon a channel splitting dichroic filter at various angles of incidence between 40 degrees and 50 degrees. This range of relatively large angles of incidence corresponds to a range of transition widths between about 40 nm and about 62 nm. This range of relatively large angles of incidence thereby leads to an increase in transition width of the dichroic filter in the imaging module. Performance of multi-channel fluorescence imaging modules may thus be improved by providing smaller angles of incidence across the full beam, thereby making the transmission edge sharper and allowing better discrimination between different fluorescence emission bands.

[0303] FIG. 4 is a graph illustrating a relationship between beam footprint size (DBS) and beam angle of incidence (DBS angle) on a dichroic filter. In some instances, a smaller beam footprint may be desirable. For example, a small beam footprint allows smaller dichroic filters to be used to split a beam into different wavelength ranges. The use of smaller dichroic filters in turn reduces manufacturing costs and improves the ease of manufacturing suitably flat dichroic filters. As shown in FIG. 4, any angle of incidence greater than 0 degrees (e. g. , perpendicular to the surface of the dichroic filter) results in an elliptical beam footprint having an area larger than the cross-sectional area of the beam. An angle of incidence of 45 degrees 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.

[0304] FIGS. 5A and 5B schematically illustrate a non-limiting example configuration of dichroic filters and detection channels in a multi-channel fluorescence imaging module where the dichroic mirrors are disposed at an angle of less than 45 degrees relative to a central beam axis of the emission light or the optical axis of the optical paths (e.g. , of the objective lens and / or tube lens). FIG. 5A depicts an imaging module 500 including a plurality of detection channels 520a, 520b, 520c, 520d. FIG. 5B is a detailed view of the portion of the imaging module 500 within the circle 5B as shown in FIG. 5A. As will be described in greater detail, the configuration illustrated in FIGS. 5 A and 5B includes a number of aspects that may result in significant improvements over conventional multi-channel fluorescence imaging module designs. In some instances, fluorescence imaging modules and systems of the present disclosure may, however, may be implemented with one or a subset of the features described with respect to FIGS . 5 A and 5B without departing from the spirit or scope of the present disclosure.

[0305] The imaging module 500 depicted in FIG. 5A includes an objective lens 510 and four detection channels 520a, 520b, 520c, and 520d disposed to receive and / or image emission light transmitted by the objective lens 510. A first dichroic filter 530 is provided to couple the excitation and detection opticalpaths. In contrast to the design shown in FIGS. 2A and 2B, as well as in FIGS. 2C and 2D, the first dichroic filter 530 (e.g. , a dichroic beam splitter or combiner) is configured to reflect light from the light source to the objective lens 510 and sample, and transmit fluorescence emission from the sample to the detection channels 520a, 520b, 520c, and 520d. A second dichroic filter 535 splits abeam of emission light among at least two detection channels 520a, 520b by transmitting a first portion 550a and reflecting a second portion 550b. Additional dichroic filters 540a, 540b are provided to further split the emission light Dichroic filter 540a transmits at least a portion of the first portion 550a of the emission light and reflects a portion 550cto a third detection channel 520c. Dichroic filter 540b transmits at least a portion of the second portion 550b of the emission light and reflects a portion 550d to a fourth detection channel 520d. Although the imaging module 500 is depicted with four detection channels, in various embodiments the imaging module 500 may include more or fewer detection channels, with a correspondingly larger or smaller number of dichroic filters as appropriate 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 advantageous effects in an imaging module including only two detection channels 520a, 520b, and omitting additional dichroic filters 540a, 540b. In some implementations, only one detection channel mac be included. Alternatively, three or more detection channels may be employed.

[0306] The detection channels 520a, 520b, 520c, 520d illustrated in FIG. 5A may include some or all of the same or similar components to those of the detection channels 220 illustrated in FIGS. 2A - 3B. For example, different detection channel 520a, 520b, 520c, 520d may include one or more image sensors or photodetectors arrays, and may include transmissive and / or reflective optics such as one or more lenses (e.g. , tube lenses) that focus the light received by the detection channel onto its respective image sensor or photodetector array.

[0307] The objective lens 510 is disposed to receive emission light emitted by fluorescence from a specimen. In particular, the first dichroic filter 530 is disposed to receive the emission light collected and transmitted by the objective lens 510. As discussed above and shown in FIG. 5 A, in some designs, an illumination source (e.g. , the illumination source 115 of FIGS. 2A and 2B) such as a laser source or the like is disposed to provide an excitation beam which is incident on the first dichroic filter 530 such that the first dichroic filter 530 reflects the excitation beam into the same objective lens 510 that transmits the emission light, for example, in an epifluorescence configuration. In some other designs, the illumination source may be directed to the specimen by other optical components along a different optical path that does not include the same obj ective lens 510. In such configurations, the first dichroic filter 530 may be omitted

[0308] Similarly, as discussed above and shown in FIG. 5A, the detection optics (e.g , including the detection channels 520a, 520b, 520c, 520d and any optical components such as dichroic filters 535, 540a, 540b along the optical path between the objective lens 510 and the detection channels 520a, 520b, 520c. 520d) may be disposed on the transmission path of the first dichroic filter 530, rather than on the reflected path of the first dichroic filter 530. In one example implementations, the objective lens 510 and detection optics aredisposed such that the objectivelens 510 transmits the beam 550 ofemission light directly toward the second dichroic filter 535. The wavefront quality of the emission light may be degraded somewhat bythe presence of the first dichroic filter 530 along the path of the beam 550 of emission light (e.g. , by imparting some wavefront error to the beam 550). However, the wavefront error introduced by a beam transmitted through a dichroic reflector of a dichroic beam splitter is generally significantly smaller than the wavefront error of abeam reflected from the dichroic reflecting surface of a dichroic beam splitter (e. g. , an order of magnitude smaller). Thus, the wavefront quality and subsequent imaging quality of the emission light in a multi-channel fluorescence imaging module may be substantially improved by placing the detection optics along the transmitted beam path of the first dichroic filter 530 rather than along the reflected beam path.

[0309] Still referring to FIG. 5A, within the detection optics of the imaging module 500, dichroic filters 535, 540a, and 540b are provided to split the beam 550 of emission light among the detection channels 520a, 520b, 520c, 520d. For example, the dichroic filters 535, 540a, and 540b split the beam 550 on the basis of wavelength, such that a first wavelength or wavelength band of the emission light can be received by the first detection channel 520a, a second wavelength or wavelength band of the emission light can be received by the second detection channel 520b, a third wavelength or wavelength band of the emission light can be received by the third detection channel 520c, and a fourth wavelength or wavelength band of the emission light can be received by the fourth detection channel 520d. In some implementations, multiple separated wavelengths or wavelength bands can be received by the detection channel.

[0310] In contrast to the multi-channel fluorescence imaging module design shown in FIGS. 2A and 2B, as well as FIGS. 2C and 2D, the imaging module 500 has dichroic filters 535, 540a, and 540b disposed at angles of incidence of less than 45 degrees with respect to the central beam axis of the incident beams. As shown in FIG. 5B, the different beams 550, 550a, 550b have respective central beam axes 552, 552a, 552b. In various implementations, the central beam axes 552, 552a, 552b is at the center of a cross-section of the beam orthogonal to the propagation direction of the beam. These central beam axes 552, 552a, 552b mac correspond to the optical axis of the objective lens and / or the optics within the separate channels, for example, the optical axes of the respective tube lenses. Additional rays 554, 554a, 554b of each beam 550, 550a, 550b are illustrated in FIG. 5B to indicate the diameter ofeach beam 550, 550a, 550b. Beam diameter may be defined, for example, as a full width at half maximum diameter, a D4o (e.g., 4 times o, where o is the standard deviation of the horizontal or vertical marginal distribution of the beam respectively) or second-moment width, or any other suitable definition of beam diameter.

[0311] The central beam axis 552 of the beam 550 of emission light may serve as a reference point for defining the angle of incidence of the beam 550 on the second dichroic filter 535. Accordingly, the “angle of incidence” (AOI) of a beam 550 may be the angle between the central beam axis 552 of the incident beam 550 and a lineN normal to the surface the beam is incident on, for example, the dichroic reflective surface. When the beam 550 of emission light is incident upon the dichroic reflective surface of the second dichroic filter 535 at an angle of incidence AOI, the second dichroic filter 535 transmits a first portion 550a of the emission light (e.g , the portion having wavelengths within the pass-band region of the second dichroic filter 535) and reflects a second portion 550b of the emission light (e.g , the portion having wavelengths within the stopband region of the second dichroic filter 535). The first portion 550a and thesecond portion 550b may each be similarly described in terms of a central beam axis 552a, 552b. As referred to above, the optical axis may alternatively or additionally be used.

[0312] In the example configuration of FIGS. 5A and 5B, the second dichroic filter 535 is disposed such that the central beam axis 552 of the beam 550 is incident at an angle of incidence of 30 degrees. Similarly, the additional dichroic filters 540a, 540b are disposed such that the central beam axes 552a, 552b of the first and second portions 550a, 550b of the beam 550 are also incident at angles of incidence of 30 degrees. However, in various implementations these angles of incidence may be other angles smaller than 45 degrees. In some instances, for example, the angles of incidence may range between about 20 degrees and about 45 degrees, as will be discussed further below. Moreover, the angles of incidence on each of the dichroic filters 535, 540a, 540b need not necessarily be the same. In some embodiments, some or all of the dichroic filters 535, 540a, 540b may be disposed such that their incident beams 550, 550a, 550b have different angles of incidence. As described above, the angle of incidence may be with respect to the optical axis of the optics within the imaging module, for example, the objective lens and / or the optics in the detection channels (e.g. , the tube lenses) and the dichroic reflective surface in the respective dichroic beam splitter. The same ranges and values for the angle of incidence apply to the case when the optical axis is used to specify the AOI.

[0313] The beams 550, 550a, 550b of emission light in a fluorescence imaging module system are typically diverging beams. As noted above, the beams of emission light can have a beam divergence large enough that regions of the beam within the beam diameter are incident upon the dichroic filters at angles of incidence that differ by up to 5 degrees or more relative to the angle of incidence of the central beam axis and / or optical axis of the optics. In some designs, the objective lens 510 may be configured, for example, to have an f-number or numerical aperture selected to produce a smaller beam diameter for a given field-of-view of the microscope. In one example, the f-number or numerical aperture of the obj ective lens 510 may be selected such that the full diameter of the beams 550, 550a, 550b are incident upon dichroic filters 535, 540a, 540b at angles of incidence within, for example, 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 axes 552, 552a, 552b.

[0314] In some implementations, the focal length of the obj ective lens that is 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 implementations, the focal length of the objective lens may range between 20 mm and 40 mm, as will be 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 small enough that light across the full diameter of the beam 550 is incident upon the second dichroic filter 535 at angles within 2.5 degrees of the angle of incidence of the central beam axis.

[0315] FIG. 6 and FIG. 7 provide graphs illustrating improved dichroic filter performance due to aspects of the imaging module configuration of FIGS. 5 A and 5B (or any of the imaging module configurations disclosed herein). The graph in FIG. 6 is similar to that of FIG. 3 and illustrates the effect of angle of incidence on the transition width (e.g. , the spectral span of the transmission edge) of a dichroic filter. FIG.6 shows an example where the orientation of a dichroic filter (e.g. , dichroic filters 535, 540a, and 540b) and the dichroic reflective surface therein is such that its incident beam has an angle of incidence of 30 degrees, rather than 45 degrees. FIG. 6 shows how this reduced angle of incidence significantly improves the sharpness and the uniformity of the transition width across the full beam diameter. For example, while an angle of incidence of 45 degrees at the central beam axis results in a range of transition widths between about 40 nm and about 62 nm, an angle of incidence of 30 degrees at the central beam axis results in a range of transition widths between about 16 nm and about 30 nm. In this example, the average transition width is reduced from about 51 nm to about 23 nm, indicating a sharper transition between pass-band and stopband. Moreover, the variation in transition widths across the beam diameter is reduced by nearly 40% from a 22 nm range to a 14 nm range, indicating a more uniform sharpness of the transition over the area of the beam.

[0316] FIG. 7 illustrates additional advantages that may be realized by selecting the appropriate f -number or numerical aperture for the objective lens to reduce beam divergence in any of the imaging module configurations disclosed herein. In some implementations, a longer focal length is used. In the example of FIG. 7, the objective lens 510 has a focal length of 36 mm, which with the appropriate numerical aperture (e.g. , less than 5), reduces the range of angles of incidence within the beam 550 from 30 degrees± 5 degrees to 30 degrees ± 2.5 degrees. With this design, the range of transition widths may be reduced to between about 19 nm and about 26 nm. When compared to the improved system of FIG. 6, although the average transition width is substantially the same (e.g. , a spectral span of roughly 23 nm), the variation in transition widths across the beam diameter is further reduced to a 7 nm range, representing a reduction of nearly 70% relative to the range of transition widths illustrated in FIG. 3.

[0317] Referring again to FIG. 4, the reduction in angle of incidence from 45 degrees to 30 degrees at the central beam axis is further advantageous because it reduces the beam spot size on the dichroic filter. As shown in FIG. 4, an angle of incidence of 45 degrees results in a beam footprint on the dichroic filter having an area greater than 1.4 times the cross-sectional area of the beam. However, an angle of incidence of 30 degrees results in a beam footprint on the dichroic filter having an area only about 1.15 times the cross- sectional area of the beam. Thus, reducing the angle of incidence at the dichroic filters 535, 540a, 540b from 45 degrees to 30 degrees results in a reduction of about 18% in the area of the beam footprint on the dichroic filters 535, 540a, 540b. This reduction in beam footprint area allows smaller dichroic filters to be used.

[0318] Referring now jointly to FIGS. 8A-B, the reduction in angle of incidence from 45 degrees to 30 degrees may also provide improved performance with regard to surface deformation caused by the dichroic filters in any of the imaging module configurations disclosed herein, as indicated by improvements in the modulation transfer function. In general, the amount of surface deformation increases with larger area optical elements. If a larger area on the dichroic filter is employed, a larger amount of surface deformation is encountered, thereby introducing more wavefront error into the beam. FIG. 8A illustrates the effect of folding angle on image quality degradation induced by the addition of 1 wave of peak-to-valley (PV) spherical power to the last mirror. FIG. 8B illustrates the effect of folding angle on image qualitydegradation induced by the addition of 0. 1 wave of PV spherical power to the last mirror. As shown in FIGS. 8A and 8B, the reduction in angle of incidence to 30 degrees significantly reduces the effect of surface deformation to achieve close to diffraction-limited performance of the detection optics.

[0319] In some implementations of the disclosed imaging modules, the polarization state of the excitation beam may be utilized to further improve the performance of the multi-channel fluorescence imaging modules disclosed herein. Referring back to FIGS. 2A, 2B, and 5 A, for example, some implementations of the multi-channel fluorescence imaging modules disclosed herein have an epifluorescence configuration in which a first dichroic filter 230 or 530 merges the optical paths of the excitation beam and the beam of emission light such that both the excitation and emission light are transmitted through the objective lens 210, 510. As discussed above, the illumination source 115 may include a light source such as a laser or other source which provides the light that forms the excitation beam. In some designs, the light source comprises a linearly polarized light source and the excitation beam may be linearly polarized. In some designs, polarization optics are included to polarize the light and / or rotate the polarization of the light. For example, a polarizer such as a linear polarizer may be included in an optical path of the excitation beam to polarize the excitation beam. Retarders such as half wave retarders or a plurality of quarter wave retarders or retarders having other amounts of retardance may be included to rotate the linear polarization in some designs.

[0320] The linearly polarized excitation beam, when it is incident upon any dichroic filter or other planar interface, may be p-polarized (e.g. , having an electric field component parallel to the plane of incidence), s-polarized (e g , having an electric field component normal to the plane of incidence), or may have a combination of p-polarization and s-polarization states within the beam. The p- or s-polarization state of the excitation beam may be selected and / or changed by selecting the orientation of the illumination source 115 and / or one or more components thereof with respect to the first dichroic filter 230, 530 and / or with respect to any other surfaces with which the excitation beam will interact. In some implementations where the light source outputs linearly polarized light, the light source can be configured to provide s-polarized light. For example, the light source may comprise an emitter such as a solid-state laser or a laser diode that may be rotated about its optical axis or the central axis of the beam to orient the linearly polarized light output therefrom. Alternatively, or in addition, retarders may be employed to rotate the linear polarization about the optical axis or the central axis of the beam. As discussed above, in some implementations, for example when the light source does not output polarized light, a polarizer disposed in the optical path of the excitation beam can polarize the excitation beam. In some designs, for example, a linear polarizer is disposed in the optical path of the excitation beam. This polarizer may be rotated to provide the proper orientation of the linear polarization to provide s-polarized light.

[0321] In some designs, the linear polarization is rotated about the optical axis or the central axis of the beam such that s-polarization is incident on the dichroic reflector of the dichroic beam splitter. When s- polarized light is incident on the dichroic reflector of the dichroic beam splitter the transition between the pass-band and the stop band is sharper as opposed to when p-polarized light is incident on the dichroic reflector of the dichroic beam splitter.

[0322] As shown in FIGS. 9A and 9B, use of the p- or s-polarization state of the excitation beam may significantly affect the narrowband performance of any excitation filters such as the first dichroic filter 230, 530. FIG. 9Aillustratesatransmissionspectrumbetween610nm and670nmfor an example bandpass dichroic filter at angles of incidence of 40 degrees and 45 degrees, where the incident beam is linearly polarized and is p-polarized with respect to the plane of the dichroic filter. As shown in FIG. 9B, changing the orientation of the light source with respect to the dichroic filter, such that the incident beam is s- polarized with respect to the plane of the dichroic filter, results in a substantially sharper edge between the pass-band and the stopband of the dichroic filter. Thus, the illumination and imaging modules 200, 500 disclosed herein may advantageously have an illumination light source 215 oriented relative to the first dichroic filter 230, 530 such that the excitation beam is s-polarized with respect to the plane of the first dichroic filter 230, 530. As discussed above, in some implementations, a polarizer such as a linear polarizer may be used to polarize the excitation beam. This polarizer may be rotated to provide an orientation of the linearly polarized light corresponding to s-polarized light. Also as discussed above, in some implementations, other approaches to rotating the linearly polarized light may be used. For example, optical retarders such as half wave retarders or multiple quarter wave retarders may be used to rotate the polarization direction. Other arrangements are also possible.

[0323] As discussed elsewhere herein, reducing the numerical aperture (NA) of the fluorescence imaging module and / or of the objective lens may increase the depth of field to enable the comparable imaging of multiple surfaces, e.g., 3, 4, or more surfaces. FIGS. 10A-15B, showhowthe MTF is more similar at first and second surfaces separated by 1 mm of glass for lower numerical apertures than for larger numerical apertures.

[0324] FIGS. 10A and 10B show the MTF at first (FIG. 10A) and second (FIG. 10B) surfaces for an NA of 0.3.

[0325] FIGS. 11 A and 1 IB show the MTF at first (FIG. 11 A) and second (FIG. 1 IB) surfaces for an NA of 0.4.

[0326] FIGS. 12A and 12B show the MTF at first (FIG. 12A) and second (FIG. 12B) surfaces for an NA of 0.5.

[0327] FIGS. 13 A and 13B show the MTF at first (FIG. 13 A) and second (FIG. 13B) surfaces for an NA of 0.6.

[0328] FIGS. 14A and 14B show the MTF at first (FIG. 14A) and second (FIG. 14B) surfaces for an NA of 0.7.

[0329] FIGS. 15 A and 15B show the MTF at first (FIG. 15 A) and second (FIG. 15B) surfaces for an NA of 0. 8. The first and second surfaces in each of these figures correspond to, e. g. , the top and bottom surfaces of a flow cell.

[0330] FIGS. 16A-B provide plots of the calculated Strehl ratio (e.g , the ratio of peak light intensity focused or collected by the optical system versus that focused or collected by an ideal optical system and point light source) for imaging a second flow cell surface through a first flow cell surface. FIG. 16A shows a plot of the Strehl ratios for imaging a second flow cell surface through a first flow cell surface as afunction of the thickness of the intervening fluid layer (fluid channel height) for different objective lens and / or optical system numerical apertures. In some embodiments, the Strehl ratio decreases with increasing separation between two adjacent surfaces, e.g., the first and second surfaces. One of the surfaces can thus have deteriorated image quality with increasing separation between the two surfaces. The decrease in surface imaging performance with increased separation distance between the two adjacent surfaces is reduced for imaging systems having smaller numerical apertures, e.g., NA of 0.5 or 0.4 as compared to those having larger numerical apertures. FIG. 16B shows aplot of the Strehl ratio as a function of numerical aperture for imaging a second flow cell surface through a first flow cell surface and an intervening layer of water having a thickness of 0. 1 mm. The loss of imaging performance at higher numerical apertures may be attributed to the increased optical aberration induced by the fluid for the second surface imaging. With increasing NA, the increased optical aberration introduced by the fluid for the second, third, or fourth surface imaging can degrade the image quality significantly. In general, however, reducing the numerical aperture of the optical system reduces the achievable resolution. This loss of image quality can be at least partially offset by providing an increased sample plane (or object plane) contrast-to-noise ratio, for example, by using chemistries for nucleic acid sequencing applications that enhance the fluorescence emission for labeled nucleic acid clusters and / or that reduce background fluorescence emission. In some instances, for example, sample support structures comprising hydrophilic substrate materials and / or hydrophilic coatings may be employed. In some cases, such hydrophilic substrates and / or hydrophilic coatings may reduce background noise. Additional discussion of sample support structures, hydrophilic surfaces and coatings, and methods for enhancing contrast-to-noise ratios, e.g., for nucleic acid sequencing applications, can be found below.

[0331] In some implementations, any one or more of the fluorescence imaging system, the illumination and imaging module 200, the imaging optics (e.g. , optics 226), the objective lens, and / or the tube lens is configured to have reduced magnification, such as a magnification of less than lOx, as will be discussed further below. Such reduced magnification may adjust design constraints such that other design parameters can be achieved. For example, any one or more of the fluorescence microscope, illumination and imaging module 200, the imaging optics (e. g. , optics 226), the obj ective lens or the tube lens may also be configured such that the fluorescence imaging module has a large field-of-view (FOV), for example, a field-of-view of at least 3.0 mm or larger (e.g. , in diameter, width, height, or longest dimension), as will be discussed further below. Any one or more of the fluorescence imaging system, the illumination and imaging module 200, the imaging optics (e.g , optics 226), the objective lens and / or the tube lens may be configured to provide the fluorescence microscope with such a field-of-view such that the FOV has less than, e.g. , 0.1 waves of aberration over at least 80% of field. Similarly, any one or more of the fluorescence imaging system, illumination and imaging module 200, the imaging optics (e.g , optics 226), the objective lens and / or the tube lens may be configured such that the fluorescence imaging module has such a FOV and is diffraction limited or is diffraction limited over such an FOV.

[0332] As discussed above, in various implementations, a large field-of-view (FOV) is provided by the disclosed optical systems. In some implementations, obtaining an increased FOV is facilitated in part bythe use of larger image sensors or photodetector arrays. The photodetector array, for example, may have an active area with a diagonal of at least 15 mm or larger, as will be discussed further below. As discussed above, in some implementations the disclosed optical imaging systems provide a reduced magnification, for example, of less than lOx which may facilitate large FOV designs. Despite the reduced magnification, the optical resolution of the imaging module may still be sufficient as detector arrays having small pixel size or pitch may be used. The pixel size and / or pitch may, for example, be about 5 pm or less, as will be discussed in more detail below. In some implementations, the pixel size is smaller than twice the optical resolution provided by the optical imaging system (e.g. , objective and tube lens) to satisfy the Nyquist theorem. Accordingly, the pixel dimension and / or pitch for the image sensor(s) may be such that a spatial sampling frequency for the imaging module is at least twice an optical resolution of the imaging module. For example, the spatial sampling frequency for the photodetector array may be is at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, or at least 5 times the optical resolution of the fluorescence imaging module (e.g. , the illumination and imaging module, the objective and tube lens, the object lens and optics 226 in the detection channel, the imaging optics between the sample support structure or stage configured to support the sample support stage and the photodetector array) or any spatial sampling frequency in a range between any of these values.

[0333] Although a wide range of features are discussed herein with respect to fluorescence imaging modules, any of the features and designs described herein may be applied to other types of optical imaging systems including without limitation bright-field and dark-field imaging, and may apply to luminescence or phosphorescence imaging.Dual wavelength excitation / four channel imaging system

[0334] FIG. 17 illustrates a dual excitation wavelength / four channel imaging system for dual -side or quad-side imaging applications that includes an objective and tube lens combination that is scanned in a direction perpendicular to the optical axis to provide for large area imaging, e.g. , by tiling several images to create a composite image having a total field-of-view (FOV) that is much larger than that for each individual image. The system comprises two excitation light sources, e.g. , lasers or laser diodes, operating at different wavelengths and an autofocus laser. The two excitation light beams and autofocus laser beam are combined using a series of mirrors and / or dichroic reflectors and delivered to the surfaces of the flow cell through the objective. Fluorescence that is emitted by labeled oligonucleotides (or other biomolecules) tethered to one of the flow cell surfaces is collected by the objective, transmitted through the tube lens, and directed to one of four imaging sensors according to the wavelength of the emitted light by a series of intermediate dichroic reflectors. Autofocus laser light that has been reflected from the flow cell surface is collected by the objective, transmitted through the tube lens, and directed to an autofocus sensor by a series of intermediate dichroic reflectors. The system allows accurate focus to be maintained (e.g. , by adjusting the relative distance between the flow cell surface and the objective using a precision linear actuator, translation stage, or microscope turret-mounted focus adjustment mechanism, to reduce or minimize the reflected light spot size on the autofocus image sensor) while the objective / tube lens combination is scanned in a direction perpendicular to the optical axis of the objective. Dual wavelength excitation usedin combination with four channel (e.g, four wavelength) imaging capability provides for high-throughput imaging of the multiple surfaces of the flow cell.Optical systems with super -resolution

[0335] For imaging very small sample site features present in high surface densities, such as nucleic acid polonies (e.g., spots comprising amplified target nucleic acids) super resolution imaging techniques as described herein may be used. In some embodiments, stochastic photo-switching techniques as described herein may be used to improve image resolution. Alternatively, structured illumination techniques as described herein may be used to improve image resolution in the optical system. In some cases, the super resolution imaging technique can comprise structured illumination.

[0336] In some instances, improvements in imaging performance, e.g , for multiple-side (flow cell) imaging applications comprising the use of thick flow cell walls (e.g. , wall (or coverslip) thickness > 700 pm) and fluid channels (e.g. , fluid channel height or thickness of 50 - 200 pm) may be achieved using novel objective lens designs that correct for optical aberration introduced by imaging surfaces on the opposite side of thick coverslips and / or fluid channels from the objective.

[0337] In some instances, improvements in imaging performance, e.g , for multiple-side (flow cell) imaging applications comprising the use of thick flow cell walls (e.g. , wall (or coverslip) thickness > 700 pm) and fluid channels (e. g. , fluid channel height or thickness of 50 - 200 pm) may be achieved even when using commercially-available, off-the-shelf objectives by using a novel tube lens design that, unlike the tube lens in a conventional microscope that simply forms an image at the intermediate image plane, corrects for the optical aberrations induced by the thick flow cell walls and / or intervening fluid layer in combination with the objective.

[0338] In some instances, improvements in imaging performance, e.g, for multichannel (e.g. , two-color or four-color) imaging applications, may be achieved by using multiple tube lenses, one for each imaging channel, where each tube lens design has been optimized for the specific wavelength range used in that imaging channel.

[0339] In some instances, improvements in imaging performance, e.g , for multiple-side (flow cell) imaging applications, may be achieved by using an electro -optical phase plate in combination with an objective lens to compensate for the optical aberrations induced by the layer of fluid separating the upper (near) and lower (far) interior surfaces of a flow cell. In some instances, this design approach may also compensate for vibrations introduced by, e.g. , a motion-actuated compensator that is moved in or out of the optical path depending on which surface of the flow cell is being imaged.

[0340] Various multichannel fluorescence imaging module designs are disclosed that may include illumination and imaging optical paths comprising folded optical paths (e.g , comprising one or more beam splitters or beam combiners, such as dichroic beam splitters or combiners) that direct an excitation light beam to an obj ective lens, and direct emission light transmitted through the obj ective lens to a plurality of detection channels. Some particularly advantageous features of the fluorescence imaging modules described herein include specification of dichroic filter incidence angles that result in sharper and / or more uniform transitions between pass-band and stopband wavelength regionsof the dichroic filters. Such filtersmay be included within the folded optics and may comprise dichroic beam splitters or combiners. Further advantageous features of the disclosed imaging optics designs may include the position and orientation of one or more excitation light sources and one or more detection optical paths with respect to the objective lens and to a dichroic filter that receives the excitation beam. The excitation beam may also be linearly- polarized and the orientation of the linear polarization may be such that s-polarized light is incident on the dichroic reflective surface of the dichroic filter. Such features may potentially improve excitation beam filtering and / or reduce wavefront error introduced into the emission light beam due to surface deformation of dichroic filters. 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.

[0341] Also described herein are devices and systems configured to analyze large numbers of differait nucleic acid sequences by imaging, e.g. , arrays of immobilized nucleic acid molecules or amplified nucleic acid clusters formed on flow cell surfaces. The devices and systems described herein can also be useful in, e.g. , performing sequencing for comparative genomics, tracking gene expression, performing micro RNA sequence analysis, epigenomics, aptamer and phage display library characterization, and for performing other sequencing applications. The devices and systems disclosed herein comprise various combinations of optical, mechanical, fluidic, thermal, electrical, and computing devices / aspects. The advantages conferred by the disclosed flow cell devices, cartridges, and systems include, but are not limited to: (i) reduced device and system manufacturing complexity and cost, (ii) significantly lower consumable costs (e. g. , as compared to those for currently available nucleic acid sequencing systems), (iii) compatibility with typical flow cell surface functionalization methods, (iv) flexible flow control when combined with microfluidic components, e.g , syringe pumps and diaphragm valves, etc. , and (v) flexible system throughput.

[0342] Disclosed herein are capillary flow-cell devices and capillary flow cell cartridges that are constructed from off-the-shelf, disposable, single lumen (e.g , single fluid flow channel) or multi-lumai capillaries that may also comprise fluidic adaptors, cartridge chassis, one or more integrated fluid flow control components, or any combination thereof. Also disclosed herein are capillary flow cell-based systems that may comprise one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell cartridges (or microfluidic cartridges), fluid flow controller modules, temperature control modules, imaging modules, or any combination thereof.

[0343] The design features of some disclosed capillary flow cell devices, cartridges, and systems include^ but are not limited to, (i) unitary flow channel construction, (ii) sealed, reliable, and repetitive switching between reagent flows that can be implemented with a simple load / unload mechanism such that fluidic interfaces between the system and capillaries are reliably sealed, thereby facilitating capillary replacement and system reuse, and enabling precise control of reaction conditions such as reagent concentration, pH, and temperature, (iii) replaceable single fluid flow channel devices or capillary flow cell cartridges comprising multiple flow channels that can be used interchangeably to provide flexible system throughput, and (iv) compatibility with a wide variety of detection methods such as fluorescence imaging.

[0344] Although the disclosed capillary flow cell devices and systems, capillary flow cell cartridges, capillary flow cell-based systems, microfluidic devices and cartridges, and microfluidic chip-based systems, are described primarily in the context of their use for nucleic acid sequencing applications, various aspects of the disclosed devices and systems may be applied not only to nucleic acid sequencing but also to any other type of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis application. It shall be understood that different aspects of the disclosed methods, devices, and systems can be appreciated individually, collectively, or in combination with each other. Although discussed herein primarily in the context of fluorescence imaging (including, e. g. , fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, and the like), it will be understood by those of skill in the art that many of the disclosed optical design approaches and features are applicable to other imaging modes, e.g. , bright-held imaging, dark-field imaging, phase contrast imaging, and the like. Multiplexed optical read-heads

[0345] In some instances, miniaturized versions of any of the imaging modules described herein may be assembled to create a multiplexed read-head that may be translated in one or more directions horizontally relative to a sample surface, e.g. , an interior surface of a flow cell, to image several sections of the surface simultaneously. A non-limiting example of a multiplexed read-head has recently been described in U.S. Patent Application No. 16 / 735,001, which is incorporated by reference in its entirety.

[0346] In some instances, for example, a miniaturized imaging module may comprise a “microfluorometer” comprising an illumination or excitation light source such as an LED or laser diode (or the tip of an optical fiber connected to an external light source), one or more lenses for collimating or focusing the illumination or excitation light, one or more dichroic rehectors, one or more optical filters, one or more mirrors, beam-splitters, prisms, apertures, etc. , one or more objectives, one or more custom tube lenses for enabling multiple surface imaging with minimal focus adjustment as described elsewhere herein, one or more image sensors, or any combination thereof, as described elsewhere herein. In some instances, a miniaturized imaging module (e.g., a “microfluorometer”) may further comprise an autofocus mechanism, a microprocessor, power and data transfer connectors, a light-tight housing, etc. The resulting miniaturized imaging module may thus comprise an integrated imaging package or unit having a small form factor. In some instances, the shortest dimension (e. g. , width or diameter) of the miniaturized imaging module may be less than 5 cm, less than 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, less than 1.2 cm, less than 1 cm, less than 0.8 cm, or less than 0.6 cm. In some instances, the longest dimension (e.g. , height or length) of the miniaturized imaging module may be less than 16 cm, less than 14 cm, less than 12 cm, less than 10 cm, less than 9 cm, less than 8 cm, less than 7 cm, less than 5 cm, less than 5 cm, less than 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, less than 1.2 cm, or less than 1 cm. In some instances, one or more individual miniaturized imaging modules within the multiplexed read -head may comprise an autofocus mechanism.

[0347] In some instances, multiplexed read-heads as described herein may comprise an assembly of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 miniaturized imaging modules or microfluorometers held infixed position relative to each other. In some instances, the optical design specifications and performance properties of the individual miniaturized imaging modules or microfluorometers, e.g., for numerical aperture, field-of-view, depth-of-field, image resolution, etc. , may be the same as described elsewhere herein for other ...

Claims

CLAIMSWhat is claimed is:

1. A system, comprising: a flow cell configured to contain a sample; a light source configured to illuminate said sample with an illumination light, wherein illuminating said sample generates an emission light comprising an emission bandwidth; an imaging system comprising a detector configured to receive said emission light, wherein said imaging system has a field of view of at least 1 square millimeters (mm2); and an emission filter disposed in an optical path between said flow cell and said detector, wherein said emission filter does not comprise a filter region for said illumination light within the emission bandwidth of said emission light.

2. The system of claim 1 , wherein said filter region for said illumination light is configured to reject said illumination light.

3. The system of claim 1, further comprising an excitation filter disposed in an optical path between said illumination source and said flow cell.

4. The system of claim 2, wherein said excitation filter is configured to filter a portion of said illumination light, thereby generating a filtered illumination light.

5. The system of claim 1, wherein said illumination light is in a continuous wavelength range.

6. The system of claim 1, wherein said illumination light has a bandwidth of at least about 5 nanometers (nm).

7. The system of claim 6, wherein said illumination light has a bandwidth of at least about 50 nm.

8. The system of claim 7, wherein said illumination light has a bandwidth of at least about 100 nm.

9. The system of claim 1, wherein said illumination light has a wavelength range from about 400 nm to about 530 nm.

10. The system of claim 1, wherein said illumination light has a wavelength range from about 530 nm to about 750 nm.11 . The system of claim 1, wherein said light source comprises a single excitation laser and does not comprise a second excitation laser.

12. The system of claim 11, wherein said light source consists of a single excitation laser.

13. The system of claim 11, wherein said single excitation laser is configured to generate said emission light in a single color.

14. The system of claim 1, wherein said light source comprises a plurality of excitation lasers.

15. The system of claim 1 , wherein a first peak of said emission light is separated from a second peak of said emission light by at least about 40 nm.

16. The system of claim 13, wherein said first peak and said second peak are generated by a first and second label, respectively.

17. The system of claim 1, wherein said emission filter comprises one or more of a dichroic filter, a notch filter, a single bandpass filter, and a single step filter.

18. The system of claim 17, wherein said emission filter comprises a single step dichroic filter and one or more single bandpass filters.

19. The system of claim 17, wherein said emission filter comprises a single step dichroic filter and a single step notch filter or a single pass notch filter.

20. The system of claim 1, wherein said emission filter comprises one or more pass-bands.21 . The system of claim 20, wherein said one or more pass-bands comprise at least about three pass-bands.

22. The system of claim 20, wherein said one or more pass-bands comprise at least two passbands that do not overlap with one another.

23. The system of claim 20, wherein said one or more pass-bands have a continuous range of about 500 nm to about 1,200 nm.

24. The system of claim 20, wherein said one or more pass-bands have a bandwidth of at least about 10 nm.

25. The system of claim 24, wherein said one or more pass-bands have a bandwidth of at least about 100 nm.

26. The system of claim 20, wherein said system does not comprise one or more second passbands outside of a wavelength range of said illumination light.

27. The system of claim 1, wherein said illumination light and said emission light do not overlap in a wavelength range.

28. The system of claim 1, wherein a photon collection efficiency of said emission filter is at least about 80%.

29. The system of claim 1, wherein said system does not comprise one or more second passbands.

30. The system of claim 1, wherein said field of view is at least about 10 mm2.31 . The system of claim 1, wherein said detector has a size of at least about 10 mm2.

32. The system of claim 1, wherein said sample is immobilized on a surface of said flow cell.

33. The system of claim 1, wherein said flow cell comprises a plurality of surfaces.

34. The system of claim 33, wherein said plurality of surfaces are displaced from one another along an optical axis of said system.

35. The system of claim 1, wherein said sample comprises in situ cells.

36. The system of claim 1, wherein said sample comprises a tissue sample.

37. The system of claim 1, wherein said emission light has a bandwidth of atleast about 100 nm.

38. The system of claim 37, wherein said emission light has a bandwidth of at least about 250 nm.

39. The system of claim 1, wherein said imaging system has a numerical aperture of at least about 0.25.

40. The system of claim 39, wherein said imaging system has a numerical aperture of at most about 0.5.

41. The system of claim 1, wherein said imaging system has an ability to resolve features that are at least about 400 nm in size.

42. The system of claim 1, wherein said system is configured to complete imaging for a sequencing cycle within an imaging cycle time of at most about 120 seconds per imaging cycle.

43. The system of claim 1, wherein said system is configured to complete a sequencing cycle within a cycle time of at most about 120 seconds per sequencing cycle.

44. The system of claim 1, wherein said light source is configured to illuminate an area of said flow cell of at least about 1 mm2with an energy variation of less than about 10% across said area of said flow cell.

45. The system of claim 1, wherein said sample comprises one or more nucleic acid molecules.

46. The system of claim 1, wherein said detector comprises a pixel having a pixel size such that a spatial sampling frequency of said imaging system is at least about twice an optical resolution of said system.

47. The system of claim 1, further comprising using said emission light to generate an image of said sample.

48. The system of claim 1, wherein said sample is disposed in a surface of said flow cell at a density of at least about 10,000 nucleic acid polonies / mm2.

49. The system of claim 1, further comprising a hydrophobic coating layer on a surface of said flow cell.

50. The system of claim 1, wherein said system is configured to image two or more fields of view at a same focus setting on at least one surface of said flow cell.51 . The system of claim 1, wherein said imaging system has a diffraction limited resolution over said field of view.

52. The system of claim 1, wherein said detector has an active area with a diagonal length of at least about 15 mm.

53. The system of claim 1, wherein said system is configured to sequence said sample and identify a number of morphological features, a number of ribonucleic acid features, a number of protein features, or a combination thereof.

54. The system of claim 53, wherein said sequencing and said identifying said number of morphological features, said number of ribonucleic acid features, said number of protein features, or said combination thereof occurs with no more than two sequencing cycles.

55. A system, comprising: a flow cell configured to contain a biological sample; a single light source configured to illuminate said biological sample, wherein illuminating said sample with said single light source generates emission light within at least three emission bands; and an imaging system comprising a detector configured to receive said emission light, wherein said imaging system has a field of view of at least about 1 square millimeter.

56. The system of claim 55, wherein said emission lightwithin said at least three emission bands is from at least three types of fluorophores .

57. The system of claim 55, further comprising an excitation filter disposed in an optical path between said illumination source and said flow cell.

58. The system of claim 57, wherein said excitation filter is configured to filter a portion of said illumination light, thereby generating a filtered illumination light.

59. The system of claim 55, wherein said illuminating is using a continuous wavelength range.

60. The system of claim 55, wherein said single light source has a bandwidth of at least about 5 nanometers (nm).61 . The system of claim 60, wherein said single light source has a bandwidth of at least about 50 nm.

62. The system of claim 61, wherein said single light source has a bandwidth of at least about 100 nm.

63. The system of claim 55, wherein said single light source has a wavelength range from about400 nm to about 530 nm.

64. The system of claim 55, wherein said single light source has a wavelength range from about 530 nm to about 750 nm.

65. The system of claim 55, wherein said single light source comprises a single excitation laser and does not comprise a second excitation laser.

66. The system of claim 65, wherein said single light source consists of a single excitation laser.

67. The system of claim 55, wherein a first peak of said emission light is separated from a second peak of said emission light by at least about 40 nm.

68. The system of claim 67, wherein said first peak and said second peak are generated by a first and second label, respectively.

69. The system of claim 55, wherein said emission filter comprises one or more of a dichroic filter, a notch filter, a single bandpass filter, and a single step filter.

70. The system of claim 69, wherein said emission filter comprises a single step dichroic filter and one or more single bandpass filters.

71. The system of claim 69, wherein said emission filter comprises a single step dichroic filter and a single step notch filter or a single pass notch filter.

72. The system of claim 55, wherein light generated by said single light source and said emission light do not overlap in a wavelength range.

73. The system of claim 55, wherein said field of view is at least about 10 mm2.

74. The system of claim 55, wherein said detector has a size of at least about 10 mm2.

75. The system of claim 55, wherein said sample is immobilized on a surface of said flow cell.

76. The system of claim 55, wherein said flow cell comprises a plurality of surfaces.

77. The system of claim 76, wherein said plurality of surfaces are displaced from one another along an optical axis of said system.

78. The system of claim 55, wherein said sample comprises in situ cells.

79. The system of claim 55, wherein said sample comprises a tissue sample.

80. The system of claim 55, wherein said emission light has a bandwidth of at least about 100 nm.81 . The system of claim 80, wherein said emission light has a bandwidth of at least about 250 nm.

82. The system of claim 55, wherein said imaging system has a numerical aperture of at least about 0.25.

83. The system of claim 82, wherein said imaging system has a numerical aperture of at most about 0.5.

84. The system of claim 55, wherein said imaging system has an ability to resolve features that are at least about 400 nm in size.

85. The system of claim 55, wherein said system is configured to complete imaging for a sequencing cycle within an imaging cycle time of at most about 120 seconds per imaging cycle.

86. The system of claim 55, wherein said system is configured to complete a sequencing cycle within a cycle time of at most about 120 seconds per sequencing cycle.

87. The system of claim 55, wherein said single light source is configured to illuminate an area of said flow cell of at least about 1 mm2with an energy variation of less than about 10% across said area of said flow cell.

88. The system of claim 55, wherein said sample comprises one or more nucleic acid molecules.

89. The system of claim 55, wherein said detector comprises a pixel having a pixel size such that a spatial sampling frequency of said imaging system is at least about twice an optical resolution of said system.

90. The system of claim 55, further comprising using said emission lightto generate an image of said sample.91 . The system of claim 55, wherein said sample is disposed in a surface of said flow cell at a density of at least about 10,000 nucleic acid polonies / mm2.

92. The system of claim 55, further comprising a hydrophobic coating layer on a surface of said flow cell.

93. The system of claim 55, wherein said system is configured to image two or more fields of view at a same focus setting on at least one surface of said flow cell.

94. The system of claim 55, wherein said imaging system has a diffraction limited resolution over said field of view.

95. The system of claim 55, wherein said detector has an active area with a diagonal length of at least about 15 mm.

96. The system of claim 55, wherein said system is configured to sequence said sample and identify a number of morphological features, a number of ribonucleic acid features, a number of protein features, or a combination thereof.

97. The system of claim 96, wherein said sequencing and said identifying said number of morphological features, said number of ribonucleic acid features, said number of protein features, or said combination thereof occurs with no more than two sequencing cycles.

98. A system, comprising: a flow cell configured to contain a biological sample; a single light source configured to illuminate said biological sample with an illumination light to generate an emission light;an imaging system comprising a detector configured to receive said emission light, wherein said imaging system has a field of view of at least about 1 square millimeter, and an emission filter disposed in an optical path between said flow cell and said detector, wherein said emission filter has a transmissivity of at least 70%, 80%, or 90% over a range of wavelengths of at least 350 nanometers.

99. The system of claim 98, further comprising an excitation filter disposed in an optical path between said illumination source and said flow cell.

100. The system of claim 99, wherein said excitation filter is configured to filter a portion of said illumination light, thereby generating a filtered illumination light.

101. The system of claim 98, wherein said illumination light is in a continuous wavelength range.

102. The system of claim 98, wherein said illumination light has a bandwidth of at least about 5 nanometers (nm).

103. The system of claim 102, wherein said illumination light has a bandwidth of at least about 50 nm.

104. The system of claim 103, wherein said illumination light has a bandwidth of at least about 100 nm.

105. The system of claim 98, wherein said illumination light has a wavelength range from about 400 nm to about 530 nm.

106. The system of claim 98, wherein said illumination light has a wavelength range from about 530 nm to about 750 nm.

107. The system of claim 98, wherein said light source comprises a single excitation laser and does not comprise a second excitation laser.

108. The system of claim 107, wherein said light source consists of a single excitation laser.

109. The system of claim 107, wherein said single excitation laser is configured to generate said emission light in a single color.

110. The system of claim 98, wherein a first peak of said emission light is separated from a second peak of said emission light by at least about 40 nm.

111. The system of claim 110, wherein said first peak and said second peak are generated by a first and second label, respectively.

112. The system of claim 98, wherein said emission filter comprises one or more of a dichroic filter, a notch filter, a single bandpass filter, and a single step filter.

113. The system of claim 112, wherein said emission filter comprises a single step dichroic filter and one or more single bandpass filters.

114. The system of claim 112, wherein said emission filter comprises a single step dichroic filter and a single step notch filter or a single pass notch filter.

115. The system of claim 98, wherein said emission filter comprises one or more pass-bands.

116. The system of claim 115, wherein said one or more pass-bands comprise at least about three pass-bands.

117. The system of claim 115, wherein said one or more pass-bands comprise at least two pass-bands that do not overlap with one another.

118. The system of claim 115, wherein said one or more pass-bands have a continuous range of about 500 nm to about 1,200 nm.

119. The system of claim 115, wherein said one or more pass-bands have a bandwidth of at least about 10 nm.

120. The system of claim 119, wherein said one or more pass-bands have a bandwidth of at least about 100 nm.

121. The system of claim 115, wherein said system does not comprise one or more second pass-bands outside of a wavelength range of said illumination light.

122. The system of claim 98, wherein said illumination light and said emission light do not overlap in a wavelength range.

123. The system of claim 98, wherein a photon collection efficiency of said emission filter is at least about 80%.

124. The system of claim 98, wherein said system does not comprise one or more second pass-bands.

125. The system of claim 98, wherein said field of view is at least about 10 mm2.

126. The system of claim 98, wherein said detector has a size of at least about 10 mm2.

127. The system of claim 98, wherein said sample is immobilized on a surface of said flow cell.

128. The system of claim 98, wherein said flow cell comprises a plurality of surfaces.

129. The system of claim 128, wherein said plurality of surfaces are displaced from one another along an optical axis of said system.

130. The system of claim 98, wherein said sample comprises in situ cells.

131. The system of claim 98, wherein said sample comprises a tissue sample.

132. The system of claim 98, wherein said emission light has a bandwidth of at least about100 nm.

133. The system of claim 132, wherein said emission light has a bandwidth of at least about 250 nm.

134. The system of claim 98, wherein said imaging system has a numerical aperture of at least about 0.25.

135. The system of claim 134, wherein said imaging system has a numerical aperture of at most about 0.5.

136. The system of claim 98, wherein said imaging system has an ability to resolve features that are at least about 400 nm in size.

137. The system of claim 98, wherein said system is configured to complete imaging for a sequencing cycle within an imaging cycle time of at most about 120 seconds per imaging cycle.

138. The system of claim 98, wherein said system is configured to complete a sequencing cycle within a cycle time of at most about 120 seconds per sequencing cycle.

139. The system of claim 98, wherein said single light source is configured to illuminate an area of said flow cell of at least about 1 mm2with an energy variation of less than about 10% across said area of said flow cell.

140. The system of claim 98, wherein said sample comprises one or more nucleic acid molecules.

141. The system of claim 98, wherein said detector comprises a pixel having a pixel size such that a spatial sampling frequency of said imaging system is at least about twice an optical resolution of said system.

142. The system of claim 98, further comprising using said emission light to generate an image of said sample.

143. The system of claim 98, wherein said sample is disposed in a surface of said flow cell at a density of at least about 10,000 nucleic acid polonies / mm2.

144. The system of claim 98, further comprising a hydrophobic coating layer on a surface of said flow cell.

145. The system of claim 98, wherein said system is configured to image two or more fields of view at a same focus setting on at least one surface of said flow cell.

146. The system of claim 98, wherein said imaging system has a diffraction limited resolution over said field of view.

147. The system of claim 98, wherein said detector has an active area with a diagonal length of at least about 15 mm.

148. The system of claim 98, wherein said system is configured to sequence said sample and identify a number of morphological features, a number of ribonucleic acid features, a number of protein features, or a combination thereof.

149. The system of claim 148, wherein said sequencing and said identifying said number of morphological features, said number of ribonucleic acid features, said number of protein features, or said combination thereof occurs with no more than two sequencing cycles.

150. A sequencing system, comprising: the system of any one of the preceding claims; and a processor, reconfigurable logic device, or both configured to process images comprising said emission light.

151. The system of claim 150, wherein said reconfigurable logic device comprises one or more field programmable gate array units, artificial intelligence chips, or neural processing units.

152. The system of claim 151, wherein said reconfigurable logic device comprises one or more field programmable gate array units.

153. The system of claim 150, wherein said processor comprises one or more of a central processing unit, graphics processing unit, tensor processing unit, neural processing unit, field programmable gate array, or artificial intelligence chip.

154. The system of claim 150, wherein said processor or said reconfigurable logic device is configured to detect a fluorescent labeled composition comprising one or more nucleic acids disposed on one or more surfaces of said flow cell to determine an identity of a nucleotide of said one or more nucleic acids.

155. A sequencing system, comprising: the system of any one of the preceding claims; and one or more reagent dispensing systems configured to dispense one or more reagents to said flow cell.

156. A method, comprising:(a) providing said system of any one of the preceding claims, wherein said sample comprises an optical label and said sample is immobilized to the a flow cell of the said system;(b) exciting, using said illumination light source, said sample, thereby generating said emission light;(c) directing said emission light through said emission filter, thereby generating filtered emission light; and(d) detecting, using said imaging system, said filtered emission light, thereby generating a detected light signal.

157. The method of claim 156, further comprising processing said detected light signal to generate an image of said flow cell.

158. The method of claim 157, further comprising processing said image or said detected light signal to sequence a biomolecule of said sample.

159. The method of claim 158, wherein said sequencing comprises one or more of sequencing-by-avidity, sequencing-by-nucleotide base-pairing, sequencing-by-nucleotide binding, or sequencing-by-nucleotide incorporation reaction.

160. The method of claim 158, wherein said biomolecule is a nucleic acid.

161. The method of claim 156, further comprising a plurality of optical labels comprising said optical label.

162. The method of claim 161, wherein said plurality of optical labels generate a plurality of wavelengths of light.

163. The method of claim 161, wherein said plurality of optical labels are a plurality of fluorophores.

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