Flow cell devices and optical systems for in situ nucleic acid sequencing.

Abstract

This specification describes a fluorescence imaging system design, flow cell device, and method that enables imaging of surfaces displaced in three or more axial directions without the use of any optical compensator. The optical systems and flow cell devices described herein provide lower cost and higher throughput analysis for genomics and other imaging applications.

Description

[Background technology]

[0001] In a typical fluorescence-based genome test assay, e.g., genotyping or nucleic acid sequencing (using real-time, cyclic, or stepwise reaction schemes), dye molecules attached to tethered nucleic acid molecules on a substrate are excited using an excitation light source, generating fluorescence photon signals at one or more spatially localized locations on the substrate, which are then imaged onto an image sensor via an optical system. An analytical process is then used to analyze the image, detect the location of the labeled molecules (or clone-amplified clusters of molecules) on the substrate, and quantify the fluorescence photon signal in terms of wavelength and spatial coordinates. This can then correlate to the extent to which a particular chemical reaction (e.g., hybridization or base addition event) occurred at a specified location on the substrate. Image-based methods offer large-scale parallel processing and multiplexing capabilities, which helps reduce the cost and accessibility of such techniques. However, detection errors resulting from, for example, excessively high-density packing of labeled molecules (or clone-amplified clusters of molecules) within small regions of the substrate surface, or detection errors due to a low contrast-to-noise ratio (CNR) in the image, can lead to errors in attributing the fluorescence signal to the correct molecule (or clone-amplified cluster of molecules).

[0002] Built-in by reference This application claims the benefits of U.S. Provisional Application No. 63 / 469,027, filed on 25 May 2023, which is incorporated herein by reference in its entirety. [Overview of the Initiative]

[0003] Described herein are sequencing systems, including flow cell devices and optical systems, and methods for sequencing nucleic acids. The flow cell devices described herein may include multiple axially displaced fluid channels and three, four, or more axially displaced surfaces facing the channels, so that the surfaces advantageously allow for the placement of more samples (e.g., increased sample volume and / or sample varieties) on a single flow cell than in conventional flow cells. The optical systems and methods described herein can image two, three, four, or more surfaces of a flow cell device axially displaced from each other, thus advantageously achieving improved sequencing throughput within a set system run time. The optical systems and methods described herein advantageously have an energy variation of less than 10% across the field of view (FOV) and at least 10 mm 2It enables uniform illumination across a wide FOV. The systems and methods disclosed herein can enable simultaneous sequencing of morphological, RNA, and / or protein targets in conventional 2D samples or in situ samples. The systems and methods disclosed herein can enable simultaneous imaging and sequencing of such targets within the same sequencing run or further within the same sequencing cycle, significantly reducing the user's actual working time. Thus, the systems and methods disclosed herein can increase the effectiveness and efficiency of sequencing analysis. The devices, systems, and methods disclosed herein can enable imaging of axially displaced surfaces of a flow cell device without moving any optical compensator in, out of, or along the optical path, thus providing a simpler, more convenient optical system that is less susceptible to vibration-induced errors. Furthermore, the devices, systems, and methods disclosed herein can advantageously enable convenient switching between, for example, imaging of a conventional flow cell having one or two surfaces and imaging of the multi-surface flow cell disclosed herein having three or more axially displaced surfaces. Such switching does not require the addition or removal of any optical elements in the system, such as an optical compensator. The devices, systems, and methods herein also enable imaging at numerical apertures (NA) less than 0.6 with sufficient image quality for accurate sequencing analysis, enable independent imaging of multiple axially displaced surfaces, and allow adjustment of the objective lens to change the NA to a desired value less than 0.6. The devices, systems, and methods herein can reduce the amount of reagents required for sequencing analysis of the same amount of sample compared to existing flow cell devices and optical systems.

[0004] In one embodiment, the Disclosure provides a system for in situ biomolecular analysis, comprising an imaging system, the imaging system comprising: a flow cell configured to immobilize cells or tissues, wherein the cells or tissues comprise a plurality of analytes of different kinds; a light source configured to illuminate the cells or tissues and thereby generate a plurality of signals corresponding to the plurality of analytes; a detector configured to image the plurality of signals; and one or more processors communicatively coupled to the imaging system, wherein one or more processors are programmed to (a) use the light source to illuminate the cells or tissues and thereby generate a plurality of signals corresponding to the plurality of analytes; (b) use the detector to detect the plurality of signals; and (c) use one or more computer processors to determine the identity or arrangement of the plurality of analytes using the plurality of signals.

[0005] In some embodiments, the cells or tissue are in situ cells or tissue samples. In some embodiments, the light source is a flow cell and the cells or tissues, approximately 20 square millimeters (mm) 2 The light source is configured to illuminate the flow cell and cells or tissue with a maximum peak-to-valley variation of approximately 5%. In some embodiments, the light source illuminates the flow cell and cells or tissue with a maximum RMS wavefront error of approximately 0.09λ, approximately mm 2It is configured to illuminate the super. In some embodiments, the imaging system has a composite root mean square error of less than about 0.05. In some embodiments, the cells or tissues are whole cells or whole tissues. In some embodiments, the imaging system does not include an objective lens positioned in the optical path of the light source or detector. In some embodiments, the imaging system does not include an objective lens. In some embodiments, the imaging system does not include a tube lens. In some embodiments, the illumination has an illuminance of at least about 40 milliwatts / square meter. In some embodiments, the cells or tissues are transmission-treated. In some embodiments, the multiple signals are multiple fluorescence signals. In some embodiments, the multiple signals are detected with a Q score of at least about 30. In some embodiments, the cells or tissues are illuminated in a plane perpendicular to the optical axis of the imaging system with at most about 10 illumination fields. In some embodiments, the field of view of the detector is at least about 10 mm 2 In some embodiments, cells or tissues are imaged with a resolution of at least about 1 micrometer. In some embodiments, the flow cell is configured to allow one or more reagent flows to come into contact with the cells or tissues. In some embodiments, the cells or tissues are cultured cells or cultured tissues. In some embodiments, the cells or tissues are isolated cells or isolated tissues. In some embodiments, the image fidelity of the cells or tissues is at least about 0.1 micrometers. In some embodiments, the multiple analytes include nucleic acid molecules. In some embodiments, the nucleic acid molecules are deoxyribonucleic acid molecules. In some embodiments, the nucleic acid molecules are ribonucleic acid molecules. In some embodiments, the multiple analytes include proteins. In some embodiments, the multiple analytes include carbohydrates.

[0006] In another aspect, the present disclosure provides a method for imaging an in situ sample, comprising: (a) providing an in situ sample comprising a plurality of different types of analytes; (b) illuminating the plurality of different types of analytes to generate a plurality of signals associated with the plurality of analytes; and (c) imaging the plurality of signals.

[0007] In some embodiments, illuminating multiple different types of analytes is sequential illumination of multiple different types of analytes. In some embodiments, illuminating multiple different types of analytes is simultaneous illumination of multiple different types of analytes. In some embodiments, the multiple different types of analytes are selected from the group consisting of deoxyribonucleic acid molecules, ribonucleic acid molecules, proteins, and phosphorylated proteins. In some embodiments, the method further comprises applying multiple sequencing reagents to an in-situ sample, each configured to sequence a different analyte from the multiple different types of analytes. In some embodiments, illumination is performed on an area of ​​approximately 20 square millimeters (mm²). 2 Over the flow cell region exceeding ), there is a peak-to-valley variation of at most about 5%. In some embodiments, illumination is performed with an RMS wavefront error of at most about 0.09λ, and the flow cell has a peak-to-valley variation of at least about 1 mm. 2 It extends to about 20 square millimeters (mm). In some embodiments, the illumination covers an area of ​​about 20 square millimeters (mm). 2 Over the flow cell region exceeding ), there is a peak-to-valley variation of at most about 5%. In some embodiments, illumination is performed with an RMS wavefront error of at most about 0.09λ, and the flow cell has a peak-to-valley variation of at least about 1 mm. 2extends over. In some embodiments, the method further includes (d) using a computer processor operably coupled to the detector to analyze a plurality of signals. In some embodiments, analyzing the plurality of signals includes determining the sequence of nucleic acid molecules within an in-situ sample. In some embodiments, the sequence of the nucleic acid molecules is determined with at least about 95% accuracy, sensitivity, or specificity. In some embodiments, the sequence of the nucleic acid molecules is determined without destroying the in-situ sample. In some embodiments, the in-situ sample has a length, width, or height of at least about 10 micrometers. In some embodiments, the in-situ sample includes tissue. In some embodiments, the in-situ sample includes a plurality of cultured cells. In some embodiments, the in-situ sample includes a plurality of isolated cells. In some embodiments, the in-situ sample is imaged with a maximum of about 10 images in a plane perpendicular to the optical axis of the optical assembly. In some embodiments, the plurality of signals are a plurality of fluorescent signals. In some embodiments, the plurality of signals are detected with at least about 30 Q-scores. In some embodiments, the in-situ sample includes nucleic acid molecules. In some embodiments, the nucleic acid molecules are deoxyribonucleic acid molecules. In some embodiments, the nucleic acid molecules are ribonucleic acid molecules. In some embodiments, the field of view of the optical assembly is at least about 10 mm 2 is. In some embodiments, the in-situ sample is imaged with a resolution of at least about 1 micrometer. In some embodiments, the in-situ sample is imaged within a maximum of about 24 hours. In some embodiments, the fidelity of imaging the plurality of images of the in-situ sample is at least about 0.1 micrometer.

[0008] In another aspect, the present disclosure provides an optical assembly for in-situ imaging, comprising a flow cell configured to contain an in-situ sample, a light source configured to illuminate the in-situ sample within the flow cell, thereby generating a signal related to the characteristics of the in-situ sample, and a detector configured to image the signal.

[0009] In some embodiments, approximately 20 square millimeters (mm) 2 Illumination over the flow cell region exceeding ) has a peak-to-valley variation of at most about 5%. In some embodiments, illumination is at least about 1 square millimeter (mm 2 The optical assembly has a maximum root-mean-square (RMS) wavefront error of about 0.09λ over the region. In some embodiments, the optical assembly further includes a processor configured to analyze the signal to characterize the in-situ sample. In some embodiments, the in-situ sample has a length, width, or height of at least about 10 micrometers. In some embodiments, the optical assembly does not include an objective lens. In some embodiments, the system does not include an objective lens. In some embodiments, the optical assembly does not include a tube lens. In some embodiments, the system does not include an objective lens. In some embodiments, the in-situ sample includes tissue. In some embodiments, the in-situ sample includes a plurality of cultured cells. In some embodiments, the in-situ sample includes a plurality of isolated cells. In some embodiments, the in-situ sample is imaged in at most about 10 images in a plane perpendicular to the optical axis of the optical assembly. In some embodiments, the signal is a fluorescence signal. In some embodiments, the signal is detected with a Q score of at least about 30. In some embodiments, the in-situ sample includes a nucleic acid molecule. In some embodiments, the nucleic acid molecule is a deoxyribonucleic acid molecule. In some embodiments, the nucleic acid molecule is a ribonucleic acid molecule. In some embodiments, the field of view of the optical assembly is at least about 10 mm. 2 In some embodiments, the in-situ sample is imaged with a resolution of at least about 1 micrometer. In some embodiments, the in-situ sample is imaged within a maximum of about 24 hours. In some embodiments, the fidelity for capturing multiple images of the in-situ sample is at least about 0.1 micrometer.

[0010] In another aspect, the present disclosure provides a method for imaging an in-situ sample, comprising: (a) providing the in-situ sample in a flow cell contained within a system comprising an optical assembly comprising a light source and a detector; (b) illuminating the in-situ sample to generate a signal related to an analyte of the in-situ sample; and (c) imaging the signal using a detector.

[0011] In some embodiments, the area to be illuminated is approximately 20 square millimeters (mm). 2 Over the flow cell region exceeding ), there is a peak-to-valley variation of at most about 5%. In some embodiments, illumination is performed with an RMS wavefront error of at most about 0.09λ, and the flow cell has a peak-to-valley variation of at least about 1 mm. 2The method extends to (d) a computer processor operably coupled to the detector to analyze the signal. In some embodiments, analyzing the signal includes determining the sequence of nucleic acid molecules in an in-situ sample. In some embodiments, the sequence of nucleic acid molecules is determined with at least about 95% accuracy, sensitivity, or specificity. In some embodiments, the sequence of nucleic acid molecules is determined without destroying the in-situ sample. In some embodiments, the in-situ sample has a length, width, or height of at least about 10 micrometers. In some embodiments, the optical assembly does not include an objective lens. In some embodiments, the system does not include an objective lens. In some embodiments, the optical assembly does not include a tube lens. In some embodiments, the system does not include an objective lens. In some embodiments, the in-situ sample includes tissue. In some embodiments, the in-situ sample includes a plurality of cultured cells. In some embodiments, the in-situ sample includes a plurality of isolated cells. In some embodiments, the in-situ sample is imaged in at most about 10 images in a plane perpendicular to the optical axis of the optical assembly. In some embodiments, the signal is a fluorescence signal. In some embodiments, the signal is detected with a Q score of at least about 30. In some embodiments, the in-situ sample contains a nucleic acid molecule. In some embodiments, the nucleic acid molecule is a deoxyribonucleic acid molecule. In some embodiments, the nucleic acid molecule is a ribonucleic acid molecule. In some embodiments, the field of view of the optical assembly is at least about 10 mm. 2 In some embodiments, the in-situ sample is imaged with a resolution of at least about 1 micrometer. In some embodiments, the in-situ sample is imaged within a maximum of about 24 hours. In some embodiments, the fidelity for capturing multiple images of the in-situ sample is at least about 0.1 micrometer.

[0012] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by whole to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by whole. In the event of any conflict between terms used herein and terms used in incorporated references, the terms defined herein shall prevail.

[0013] Novel features of the concept of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are used, and to the appended drawings. [Brief explanation of the drawing]

[0014] [Figure 1A] A non-limiting example of imaging a multi-surface support structure is schematically shown to present a sample area for imaging with the imaging system disclosed herein. The figure shows imaging of the front and rear inner surfaces of a flow cell. [Figure 1B] A non-limiting example of imaging a multi-surface support structure is schematically shown to present a sample area for imaging with the imaging system disclosed herein. The figure shows imaging of the front and rear outer surfaces of the substrate. [Figure 2A] A non-limiting example of a multichannel fluorescence imaging module is shown, which includes a dichroic beam splitter for transmitting an excitation light beam through a sample, receiving the resulting fluorescence emission, and redirecting it via reflection to four detection channels configured to detect fluorescence emission at four different wavelengths or wavelength bands. Top isometric view. [Figure 2B] A non-limiting example of a multichannel fluorescence imaging module is shown, which includes a dichroic beam splitter for transmitting an excitation light beam through a sample, receiving the resulting fluorescence emission, and redirecting it via reflection to four detection channels configured to detect fluorescence emission at four different wavelengths or wavelength bands. Bottom isometric view. [Figure 3A]Figures 2A and 2B show the optical path within the multichannel fluorescence imaging module, which includes a dichroic beam splitter for transmitting an excitation light beam through the sample, receiving the resulting fluorescence emission, and redirecting it via reflection to four detection channels for detecting fluorescence emission at four different wavelengths or wavelength bands. (Top view.) [Figure 3B] Figures 2A and 2B show the optical path within the multichannel fluorescence imaging module, which includes a dichroic beam splitter for transmitting an excitation light beam through the sample, receiving the resulting fluorescence emission, and redirecting it via reflection to four detection channels for detecting fluorescence emission at four different wavelengths or wavelength bands. (Side view.) [Figure 4] This graph shows the relationship between dichroic filter performance and beam incidence angle. [Figure 5] This graph shows the relationship between the beam footprint size on a dichroic filter and the beam incidence angle. [Figure 6A] A schematic diagram of an exemplary configuration of the dichroic filter and detection channels in a multi-channel fluorescence imaging module is shown. The dichroic filter has a reflective surface tilted such that the angle between the incident beam (e.g., central angle) and the reflective surface of the dichroic filter is less than 45 degrees. A schematic diagram of a multi-channel fluorescence imaging module including four detection channels is shown. [Figure 6B] A schematic diagram illustrates an exemplary configuration of the dichroic filter and detection channel of a multi-channel fluorescence imaging module. The dichroic filter has a reflective surface inclined such that the angle between the incident beam (e.g., central angle) and the reflective surface of the dichroic filter is less than 45 degrees. A detailed diagram showing the angle of incidence (AOI) of the light beam on the dichroic reflector is also provided. [Figure 7] Figures 6A and 6B provide graphs showing the improved dichroic filter performance corresponding to the imaging module configuration shown. [Figure 8] Figures 6A and 6B provide graphs showing the improved dichroic filter performance corresponding to the imaging module configuration shown. [Figure 9A] Figures 6A and 6B provide graphs illustrating the reduction of surface deformation due to the imaging module configuration. They also show the effect of the bending angle on image quality degradation induced by applying a single wavelength of PV spherical power to the final mirror. [Figure 9B] Figures 6A and 6B provide graphs illustrating the reduction in surface deformation caused by the imaging module configuration. They also show the effect of the bending angle on image quality degradation induced by applying a 0.1 wavelength PV spherical power to the final mirror. [Figure 10A] This provides graphs demonstrating improved excitation filter performance (e.g., sharper transitions between the passband and the surrounding stopband) resulting from the use of s-polarization of the excitation beam. Exemplary bandpass dichroic filter transmission spectra at incidence angles of 40 and 45 degrees are also shown. The incident beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. [Figure 10B] This graph demonstrates improved excitation filter performance (e.g., a sharper transition between the passband and the surrounding stopband) resulting from the use of s-polarization of the excitation beam. Changing the orientation of the light source relative to the dichroic filter so that the incident beam is s-polarized relative to the plane of the dichroic filter significantly sharpens the edge between the passband and the stopband. [Figure 11A] The modulation transfer function (MTF) of an exemplary multi-surface imaging system disclosed herein, having a numerical aperture (NA) of 0.3, is shown. The first surface. [Figure 11B] The modulation transfer function (MTF) of an exemplary multi-surface imaging system disclosed herein having a numerical aperture (NA) of 0.3 is shown. The second surface. [Figure 12A] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.4 is shown. The first surface. [Figure 12B] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.4 is shown. Second surface. [Figure 13A]The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.5 is shown. The first surface. [Figure 13B] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.5 is shown. Second surface. [Figure 14A] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.6 is shown. The first surface. [Figure 14B] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.6 is shown. Second surface. [Figure 15A] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.7 is shown. The first surface. [Figure 15B] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.7 is shown for the second surface. [Figure 16A] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.8 is shown. The first surface. [Figure 16B] The MTF of an exemplary multi-surface imaging system disclosed herein having an NA of 0.8 is shown for the second surface. [Figure 17A] This provides plots of calculated Strehl ratios when imaging a second flow cell surface through a first flow cell surface. It also provides plots of the Strehl ratio as a function of the thickness of the intervening fluid layer (fluid channel height) when imaging a second flow cell surface through a first flow cell surface for different numerical apertures of objective lenses and / or optical systems. [Figure 17B] Provides a plot of the calculated Strehl ratio when imaging the second flow cell surface through the first flow cell surface. Plot of the Strehl ratio as a function of numerical aperture when imaging the second flow cell surface through the first flow cell surface and an intervening aqueous layer having a thickness of 0.1 mm. [Figure 18] This disclosure provides a schematic diagram of the dual-wavelength excitation / 4-channel emission fluorescence imaging system. [Figure 19]This provides a ray tracing diagram for an objective lens design intended to image the opposite surface of a 0.17 mm thick coverslip. [Figure 20] Figure 19 provides a plot of the modulation transfer function as a function of spatial frequency when imaging the opposite surface of a 0.17 mm thick coverslip using the objective lens shown. [Figure 21] Figure 19 provides a plot of the modulation transfer function as a function of spatial frequency when imaging the opposite surface of a 0.3 mm thick coverslip using the objective lens shown. [Figure 22] Figure 19 provides a plot of the modulation transfer function as a function of spatial frequency when imaging a surface separated by a 0.1 mm thick aqueous fluid layer from the opposite surface of a 0.3 mm thick coverslip using the objective lens shown. [Figure 23] Figure 19 provides a plot of the modulation transfer function as a function of spatial frequency when the objective lens shown is used to image the opposite surface of a 1.0 mm thick coverslip. [Figure 24] Figure 19 provides a plot of the modulation transfer function as a function of spatial frequency when imaging a surface separated from the opposite surface of a 1.0 mm thick coverslip by a 0.1 mm thick aqueous fluid layer using the objective lens shown. [Figure 25] This provides a ray tracing diagram for a tube lens design that, when used in conjunction with the objective lens shown in Figure 19, improves multi-plane imaging through a 1 mm thick coverslip. [Figure 26] Figure 25 provides a plot of the modulation transfer function as a function of spatial frequency when imaging the opposite surface of a 1.0 mm thick coverslip using the objective lens and tube lens combination shown. [Figure 27] Figure 25 provides a plot of the modulation transfer function as a function of spatial frequency when imaging a surface separated from the opposite surface of a 1.0 mm thick coverslip by a 0.1 mm thick aqueous fluid layer using the objective lens and tube lens combination shown. [Figure 28] A ray tracing diagram of the tube lens design of this disclosure (left), optimized to provide high-quality multi-plane imaging performance, is provided. Since the tube lens is not infinity corrected, a well-designed null lens (right) may be used in combination with the tube lens for manufacturing and testing purposes to compensate for the non-infinity corrected tube lens. [Figure 29] This shows a non-limiting example of a single capillary reflow cell with two fluid adapters. [Figure 30] A non-limiting example of a flow cell cartridge is shown, including a chassis, fluid adapter, and optionally other components, designed to hold two capillaries. [Figure 31] This is a non-limiting example of a system containing a single capillary flow cell connected to various fluid flow control components. The single capillary is compatible with mounting on a microscope stage or on custom imaging equipment for use in various imaging applications. [Figure 32] A non-limiting example of a system including a capillary flow cell cartridge with an integrated diaphragm valve is shown to reduce or minimize dead volume and conserve certain key reagents. [Figure 33] A non-limiting example of a system including a capillary flow cell, microscope, and temperature control mechanism is shown. [Figure 34] This presents a non-limiting example of temperature control for a capillary flow cell using a metal plate positioned in contact with the flow cell cartridge. [Figure 35] This paper presents one non-restrictive approach to temperature control of a capillary flow cell, including a non-contact thermal control mechanism. [Figure 36A] This shows a non-limiting example of the manufacturing of a flow cell device. It demonstrates the preparation of a one-piece glass flow cell. [Figure 36B] This shows a non-limiting example of the fabrication of a flow cell device. It demonstrates the preparation of a two-piece glass flow cell. [Figure 36C]This shows a non-limiting example of the manufacturing of a flow cell device. It demonstrates the preparation of a three-piece glass flow cell. [Figure 37A] This shows a non-restrictive example of glass flow cell design. It demonstrates the design of a one-piece glass flow cell. [Figure 37B] This shows a non-restrictive example of glass flow cell design. It demonstrates the design of a two-piece glass flow cell. [Figure 37C] This shows a non-limiting example of glass flow cell design. It demonstrates the design of a three-piece glass flow cell. [Figure 38] This shows a visualization of cluster (or Polonie) amplification in a capillary lumen. [Figure 39] This specification provides non-limiting examples of block diagrams of sequencing systems disclosed herein. [Figure 40] This specification provides non-limiting examples of flowcharts for the sequencing methods disclosed herein. [Figure 41] This specification provides non-limiting examples of schematic diagrams of lighting systems disclosed herein. [Figure 42] This specification provides non-limiting examples of flowcharts for acquiring and processing structured illumination images of flow cell surfaces. [Figure 43A] This specification provides a non-limiting schematic diagram of a multiplexed readout head disclosed herein. A side view of a multiplexed readout head in which individual micro-fluorometers are configured to image a common surface (e.g., the inner surface of a flow cell). [Figure 43B] This specification provides a non-limiting schematic diagram of a multiplexing read head disclosed herein. A top view of the multiplexing read head showing the imaging path acquired by the individual micro-fluorometers of the multiplexing read head. [Figure 44A]This specification provides a non-limiting schematic diagram of a multiplexed read head disclosed herein. A side view of a multiplexed read head in which a first subset of a plurality of individual micro-fluorometers 4401 is configured to image a first surface (e.g., a first inner surface of a flow cell), and a second subset of a plurality of individual micro-fluorometers is configured to image a second surface (e.g., a second inner surface of a flow cell). [Figure 44B] This specification provides a non-limiting schematic diagram of the multiplexing read head disclosed herein. Figure 44A is a top view of the multiplexing read head showing the imaging path acquired by the individual micro-fluorometers 4401 of the multiplexing read head. [Figure 45] Some embodiments of this specification illustrate non-limiting examples of optical imaging systems having multiple imaging sensors configured to transmit images through a flow cell during sequential illumination by multiple light sources (each emitting a different color). A liquid sample is introduced into a flow cell on a hydrophobic pad and flows through the flow cell by tensile force. [Figure 46] This specification provides non-limiting schematic diagrams of methods utilizing optical systems for imaging the surface of a flow cell for nucleic acid sequencing, according to several embodiments thereof. [Figure 47A] This specification provides optical systems according to various embodiments described herein. It also provides non-limiting cross-sectional views of optical systems for imaging the surface of a flow cell according to some embodiments of this specification. [Figure 47B] This specification provides optical systems according to various embodiments described herein. A comparison of the optical system in Figure 47A with the IDEX instrument core is provided. [Figure 48A] Figure 47B provides a non-limiting example of a flow cell having 424 individual tiles, imaged by the IDEX instrument core shown. [Figure 48B] This specification provides non-limiting examples of flow cells having fewer than 40 individual tiles, imaged by the optical systems described herein (see Figures 45, 46, 47A–47B). [Figure 49A]A non-limiting cross-sectional view of an optical system configured to image a multi-faceted flow cell is provided. The shown optical system includes a piezo-driven wedge block for high-speed focusing. The optical system is shown configured to focus on the inner surface of the back of the flow cell. [Figure 49B] A non-limiting cross-sectional view of an optical system configured to image a multi-faceted flow cell is provided. The shown optical system includes a piezo-driven wedge block for high-speed focusing. The optical system is shown configured to focus on the inner surface of the front of the flow cell. [Figure 50] This provides an unrestricted cross-sectional view of an optical system configured to image a wide area of ​​a surface. The optical system includes multiple optical subsystems. The optimized field of view (FOV) of each subsystem overlaps with the FOV of each adjacent optical subsystem, thereby providing a wide area of ​​FOV. [Figure 51] Figures A and B provide non-limiting cross-sectional views of a focusing lens assembly. The focusing lens assembly is configured to maintain a fixed position within the optical path (e.g., the optical axis) and to allow relative movement between at least a first lens and a second lens contained within the lens housing of the focusing lens assembly. Figure A shows a focusing lens assembly with a first lens and a second lens. Figure B shows the same focusing lens assembly with the second lens moved relative to A. [Figure 52] This provides a non-limiting cross-sectional view of an optical system configured to image a curved, wide-area surface. The optical system includes multiple optical subsystems. Each system is positioned substantially orthogonal to the surface, and the field of view (FOV) of each subsystem overlaps with the FOV of each adjacent optical subsystem, thereby providing a system for imaging a curved, wide-area surface. [Figure 53A]This provides a non-limiting cross-sectional view of an optical system configured to image a capillary reflow cell. In this example, the optical system is configured to acquire an image of the entire inner surface of the capillary reflow cell by rotating a curved, wide area surface around the x-axis and moving along the x-axis. The optical axis of the central optical subsystem is shown aligned with the z-axis. [Figure 53B] This provides a non-limiting cross-sectional view of an optical system configured to image a capillary reflow cell. In this example, the optical system is configured to acquire an image of the entire inner surface of the capillary reflow cell by rotating a curved, wide area of ​​surface around the x-axis and moving along the x-axis. The optical axis of the central optical subsystem is shown rotated 90 degrees to align with the y-axis. [Figure 54A] This provides a non-limiting cross-sectional view of an optical system configured to image a capillary reflow cell without requiring a stage to rotate the optical system around the x-axis. The shown optical system includes a piezo-driven wedge block for high-speed focusing. The optical system is shown configured to focus on the inner surface of the capillary reflow cell closest to the light source. [Figure 54B] This provides a non-limiting cross-sectional view of an optical system configured to image a capillary reflow cell without requiring a stage to rotate the optical system around the x-axis. The shown optical system includes a piezo-driven wedge block for high-speed focusing. It shows an optical system configured to focus on the inner surface of a capillary reflow cell at a greater distance from the light source. [Figure 55] This bar graph shows the results of a trapping assay performed by reacting various fluorescently labeled polyvalent molecules with the corresponding correct DNA template. [Figure 56] This bar graph shows the results of a trapping assay in which various fluorescently labeled polyvalent molecules were reacted with the corresponding correct DNA template by increasing their concentrations. [Figure 57]Four graphs are presented showing the results of a trapping assay comparing the signal intensity of fluorescently labeled polyvalent molecules carrying nucleotide arms containing either an N3 linker, linker 6, linker 8, or propargyl linker. The polyvalent molecules were labeled with CF680 or CF532 fluorophores. Two different concentrations (20 nM and 80 nM) of polyvalent molecules were tested. The graphs show trap time in seconds (x-axis) and P90 signal intensity (y-axis). [Figure 58] Four graphs are presented showing the results of a trapping assay comparing the signal intensity of fluorescently labeled polyvalent molecules carrying nucleotide arms containing either an N3 linker, linker 6, linker 8, or propargyl linker. The polyvalent molecules were labeled with AF647 or CF570 fluorophores. Two different concentrations (20 nM and 80 nM) of polyvalent molecules were tested. The graphs show trap time in seconds (x-axis) and P90 signal intensity (y-axis). [Figure 59] Three graphs are presented showing the results of a real-time imaging trapping kinetic assay comparing the signal intensity of fluorescently labeled polyvalent molecules carrying nucleotide arms containing one of linker 6 or 10–16. Polyvalent molecules at three different concentrations (15, 7.5, and 2.5 nM) were tested. The graphs show trap time (x-axis) and signal intensity (y-axis) in seconds. [Figure 60] This graph shows the results of a binding kinetics study of a fluorescently labeled polyvalent molecule carrying a nucleotide arm containing one of linkers 6 or 10-16. The graph shows the concentration (x axis, nM) and velocity (y axis) of the polyvalent molecule. The legend shown in Figure 60 is also applicable to Figure 59. [Figure 61] This bar graph shows the determined binding constants (K) for fluorescently labeled polyvalent molecules carrying nucleotide arms containing linker 6 or one of 10-16. [Figure 62] Several examples of systems combining avidity-based sequencing are schematically shown. [Figure 63]This describes a computer system programmed or otherwise configured to implement the methods provided herein. [Figure 64A] This specification describes exemplary embodiments of a multi-surface sample support structure or multi-surface flow cell in which multiple surfaces are axially displaced relative to each other, according to specific embodiments of this specification. [Figure 64B] This specification describes exemplary embodiments of a multi-surface sample support structure or multi-surface flow cell in which multiple surfaces are axially displaced relative to each other, according to specific embodiments of this specification. [Figure 64C] This specification describes exemplary embodiments of a multi-surface sample support structure or multi-surface flow cell in which multiple surfaces are axially displaced relative to each other, according to specific embodiments of this specification. [Figure 64D] This specification describes exemplary embodiments of a multi-surface sample support structure or multi-surface flow cell in which multiple surfaces are axially displaced relative to each other, according to specific embodiments of this specification. [Figure 64E] This specification describes exemplary embodiments of a multi-surface sample support structure or multi-surface flow cell in which multiple surfaces are axially displaced relative to each other, according to specific embodiments of this specification. [Figure 64F] This specification describes exemplary embodiments of a multi-surface sample support structure or multi-surface flow cell in which multiple surfaces are axially displaced relative to each other, according to specific embodiments of this specification. [Figure 65] A non-limiting example of a lighting system for an optical assembly described herein, including a lighting subsystem and a light beam delivery subsystem, is shown. [Figure 66] This specification provides a non-limiting example of a lighting subsystem. [Figure 67A] Figure 65 of this specification illustrates the uniformity of illumination power density provided by the illumination system. Exemplary images of the illumination field and associated illumination intensity are shown. [Figure 67B] Figure 65 of this specification shows the uniformity of the illumination power density provided by the illumination system. Figure 67A shows the line trace of illumination intensity along the long axis. [Figure 67C] Figure 65 of this specification shows the uniformity of the lighting power density provided by the lighting system. Figure 67A shows the line trace of the lighting intensity along the short axis. [Figure 68] This provides non-limiting examples of illumination subsystems and light beam delivery systems for optical assemblies. [Figure 69] This shows a non-restrictive example of a lighting subsystem in an optical assembly. [Figure 70] This shows the relative positions of the despeckra and optical beam delivery subsystems with respect to the collimator. [Figure 71] This shows non-limiting examples of optical fiber and optical beam delivery subsystems. [Figure 72] This shows a non-limiting example of a liquid light guide and light beam delivery subsystem. [Figure 73A] A non-limiting example of despeckling is shown. This example shows a mechanical vibration source loosely or fixedly attached to at least one portion of an optical fiber. A winding portion of the optical fiber is shown according to several embodiments. [Figure 73B] A non-limiting example of despeckling is shown. This example shows a mechanical vibration source loosely or fixedly attached to at least one portion of an optical fiber. A portion of the optical fiber wrapped around the vibration source is shown according to several embodiments. [Figure 73C] A non-limiting example of despeckling is shown. This example shows a mechanical vibration source loosely or fixedly attached to at least one portion of an optical fiber. A portion of an optical fiber wrapped around a fan vibration source is shown according to several embodiments. [Figure 73D] A non-limiting example of despeckling is shown. This example shows a mechanical vibration source loosely or fixedly attached to at least one portion of an optical fiber. A portion of an optical fiber wrapped around a fan vibration source is shown according to several embodiments. [Figure 74] A table is shown showing different despeckle configurations for optical fibers and their corresponding speckle noise levels. [Figure 75] The following are block diagrams of sequencing systems for imaging DNA samples(s) during a DNA sequencing reaction, according to several embodiments. [Figure 76] These are schematic diagrams of various exemplary configurations of polyvalent molecules. Left (Class I): Schematic diagram of a polyvalent molecule having a "starburst" or "helter-skelter" configuration. Center (Class II): Schematic diagram of a polyvalent molecule having a dendrimer configuration. Right (Class III): Schematic diagram of multiple polyvalent molecules formed by reacting streptavidin with 4-armed or 8-armed PEG-NHS and dNTPs containing biotin. Nucleotide units are represented as "N", biotin as "B", and streptavidin as "SA". [Figure 77] This is a schematic diagram of an example of a polyvalent molecule containing a typical core attached to multiple nucleotide arms. [Figure 78] This is a schematic diagram of an example of a polyvalent molecule containing a dendrimer core attached to multiple nucleotide arms. [Figure 79] A schematic diagram shows an example of a polyvalent molecule containing a core attached to multiple nucleotide arms. The nucleotide arms include biotin, spacers, linkers, and nucleotide units. [Figure 80] This is a schematic diagram of an example of a nucleotide arm, including a core attachment portion, spacer, linker, and nucleotide units. [Figure 81] The chemical structure of an example spacer (top) and the chemical structures of various exemplary linkers, including 11-atom linkers, 16-atom linkers, 23-atom linkers, and N3 linkers (bottom), are shown. [Figure 82] The chemical structures of various linker examples, including linkers 1-9, are shown. [Figure 83] The chemical structures of various examples of linkers linked to / attached to nucleotide units are shown. [Figure 84] The chemical structures of various examples of linkers linked to / attached to nucleotide units are shown. [Figure 85] The chemical structures of various examples of linkers linked to / attached to nucleotide units are shown. [Figure 86] The chemical structures of various examples of linkers linked to / attached to nucleotide units are shown. [Figure 87] The chemical structure of an example of a biotinylated nucleotide arm is shown. In this example, the nucleotide units are linked to the linker via a propargylamine bond at position 5 of the pyrimidine base or position 7 of the purine base. [Figure 88] A flowchart of a method for analyzing biomolecules, according to several embodiments, is shown. [Figure 89] A flowchart of a method for analyzing biological samples according to several embodiments is shown. [Figure 90] A perspective view of a non-definitive example of an imaging module or optical assembly is shown. [Figure 91] A cross-sectional view of a non-limiting example of an imaging module or optical assembly is shown. [Figure 92] Figures 90-91 show cross-sectional views of non-limiting examples of single-channel time-series color imaging modules or optical assemblies. [Figure 93] Figures A and B show examples of external actuator coupling according to several embodiments. Figure A shows a detailed view of the external actuator and optical assembly according to several embodiments. Figure B shows a long view of the external actuator and optical assembly according to several embodiments. [Figure 94] Examples of optical elements and associated focal paths of optical assemblies according to several embodiments are shown. [Figure 95] Examples of optical elements and associated focal paths of optical assemblies according to several embodiments are shown. [Figure 96A] The diffraction-modulated transfer function (MTF) of an optical system is provided for several embodiments. An exemplary MTF of an objective lens-based optical system is shown. [Figure 96B] The diffraction-modulated transfer function (MTF) of an optical system is provided for several embodiments. An exemplary MTF of an optical system without an objective lens is shown. [Figure 97A] Wavefront analysis calculations of the optical systems of this disclosure are shown in several embodiments. An exemplary wavefront analysis calculation at position 1 is shown. [Figure 97B] Wavefront analysis calculations of the optical systems of this disclosure are shown in several embodiments. An exemplary wavefront analysis calculation at position 2 is shown. [Figure 98] The optical performance curves of the upper surface according to several embodiments are shown. [Figure 99] The optical performance curves of the lower surface according to several embodiments are shown. [Figure 100] The MTF plots of optical systems according to several embodiments are shown. [Figure 101] The cumulative probability of achieving a given wavefront error in several embodiments is plotted. [Figure 102] This is a schematic diagram of a rotating stage used to move a sample(s) relative to the objective lens of an optical system for imaging a sequencing reaction. [Figure 103A] This specification shows specific exemplary images of morphological targets of in situ cells using the systems and methods disclosed herein. [Figure 103B] This specification shows specific exemplary images of morphological targets of in situ cells using the systems and methods disclosed herein. [Figure 103C] This specification shows specific exemplary images of morphological targets of in situ cells using the systems and methods disclosed herein. [Figure 103D] This specification shows specific exemplary images of morphological targets of in situ cells using the systems and methods disclosed herein. [Figure 104] This section shows specific exemplary images of RNA targets in situ cells using the systems and methods disclosed herein. [Figure 105] Images A–F show specific exemplary images of protein and / or phosphorylated protein targets in situ cells using the systems and methods disclosed herein. [Figure 106A] Specific exemplary images of protein targets in situ cells using the systems and methods disclosed herein (B) compared with protein localization using immunofluorescence (A) are shown. [Figure 106B] Specific exemplary images of protein targets in situ cells using the systems and methods disclosed herein (B) compared with protein localization using immunofluorescence (A) are shown. [Figure 106C] Specific exemplary images of protein targets in situ cells using the systems and methods disclosed herein (B) compared with protein localization using immunofluorescence (A) are shown. [Figure 107] This is a schematic diagram of a guanine quadruplex (for example, a G quadruplex). [Figure 108] This is a schematic diagram of an exemplary intramolecular G quadruple chain structure. [Figure 109A] This is a schematic diagram showing an embodiment of a crosslinked cyclic complex (1600) containing a cyclic barcoded oligonucleotide (1400) hybridized to a linear crosslinked oligonucleotide (1500). [Figure 109B] This is a schematic diagram showing an embodiment of an analyte detection complex that includes an antibody-crosslinked cyclic complex (1700) containing a primary antibody attached to a crosslinked cyclic complex (1600) as shown in Figure 109A. [Figure 110A] This is a schematic diagram showing an embodiment of a crosslinked cyclic complex (1600) containing a cyclic barcoded oligonucleotide (1400) hybridized to a linear crosslinked oligonucleotide (1500). [Figure 110B] Figure 110A is a schematic diagram showing an embodiment of an analyte detection complex containing an antibody-crosslinked cyclic complex (1700) that includes a primary antibody attached to a crosslinked cyclic complex (1600) shown in Figure 110A. [Figure 111A] This is a schematic diagram showing an embodiment of a crosslinked cyclic complex (1600) containing a cyclic barcoded oligonucleotide (1400) hybridized to a linear crosslinked oligonucleotide (1500). [Figure 111B] This is a schematic diagram showing an embodiment of an analyte detection complex that includes an antibody-crosslinked cyclic complex (1700) containing a primary antibody attached to a crosslinked cyclic complex (1600) as shown in Figure 111A. [Figure 112A]This is a schematic diagram showing an embodiment of a crosslinked cyclic complex (1600) containing a cyclic barcoded oligonucleotide (1400) hybridized to a linear crosslinked oligonucleotide (1500). [Figure 112B] This is a schematic diagram showing an embodiment of an analyte detection complex that includes an antibody-crosslinked cyclic complex (1700) containing a primary antibody attached to a crosslinked cyclic complex (1600) as shown in Figure 112A. [Figure 113A] This is a schematic diagram showing an embodiment of a crosslinked cyclic complex (1600) containing a cyclic barcoded oligonucleotide (1400) hybridized to a linear crosslinked oligonucleotide (1500). [Figure 113B] This is a schematic diagram showing an embodiment of an analyte detection complex that includes an antibody-crosslinked cyclic complex (1700) containing a primary antibody attached to a crosslinked cyclic complex (1600) as shown in Figure 113A. [Figure 114A] This is a schematic diagram showing an embodiment of a target analyte containing the first and second epitopes. [Figure 114B] This is a schematic diagram showing an embodiment of an antibody-crosslinked cyclic complex (1700) directly bound to a target analyte. In some embodiments, the antibody-crosslinked cyclic complex (1700) comprises an antibody having an antigen-binding site that binds to a first epitope of a first target analyte. [Figure 115A] This is a schematic diagram showing an embodiment of the first antibody-crosslinked cyclic complex (1700-1) directly bound to the first target analyte. In some embodiments, the first antibody-crosslinked cyclic complex (1700-1) comprises a first primary antibody having an antigen-binding site that binds to the epitope of the first target analyte. In some embodiments, the first antibody-crosslinked cyclic complex (1700-1) comprises a first crosslinked cyclic complex (1600-1) attached to the first primary antibody. [Figure 115B] This is a schematic diagram showing an embodiment of a second antibody-crosslinked cyclic complex (1700-2) directly bound to a second target analyte. In some embodiments, the second antibody-crosslinked cyclic complex (1700-2) comprises a second primary antibody having an antigen-binding site that binds to the epitope of the second target analyte. [Figure 116]This is a schematic diagram showing embodiments of a first antibody-crosslinked cyclic complex (1700-1) and a second antibody-crosslinked cyclic complex (1700-2) bound to different epitopes of the same target analyte. [Figure 117] This is a schematic diagram showing an embodiment of an analyte detection complex, which includes a bipartite complex (1800) comprising a secondary antibody attached to a crosslinked cyclic complex (1600) and a primary antibody bound to the secondary antibody. [Figure 118] This is a schematic diagram showing an embodiment of an analyte detection complex, which includes a bipartite complex (1800) comprising a secondary antibody attached to a crosslinked cyclic complex (1600) and a primary antibody bound to the secondary antibody. [Figure 119] This is a schematic diagram showing an embodiment of an analyte detection complex, which includes a bipartite complex (1800) comprising a secondary antibody attached to a crosslinked cyclic complex (1600) and a primary antibody bound to the secondary antibody. [Figure 120A] This is a schematic diagram showing an embodiment of an analyte detection complex, comprising a first bipartite complex (1800-1) which includes a first secondary antibody attached to a first cross-linked cyclic complex (1600-1) and a first primary antibody bound to the first secondary antibody. [Figure 120B] This is a schematic diagram illustrating an embodiment of an analyte detection complex comprising a second bipartite complex (1800-2), which includes a second secondary antibody attached to a second cross-linked cyclic complex (1600-2) and a second primary antibody conjugated to the second secondary antibody. In some embodiments, the second primary antibody can be conjugated to a second target analyte. [Figure 121] This table shows several embodiments of target barcode sequences that can be used to simultaneously detect and identify two or more cell target analytes (e.g., cell structures) by performing a single sequencing cycle and using multicolor imaging. In some embodiments, the target barcode sequences listed in the table in Figure 121 can be used for cell painting. [Figure 122]Images of the fluorescent signals emitted from sequencing of the barcoded region of concatemers in cell samples are shown. Here, the concatemers were generated from the bipartite complex (1800). Cells were permeabilized, fixed, and reacted with the barcoded bipartite complex under conditions suitable for the bipartite complex to bind to their congeneral target analytes (e.g., C-myc or tubulin). The analyte-bipartite complexes were subjected to rolling circle amplification to generate barcoded concatemers corresponding to tubulin or c-myc. The barcoded concatemers were sequenced using a two-step sequencing workflow with sequencing primers specific to the tubulin or c-myc concatemer, as well as labeled polyvalent molecules and unlabeled nucleotide analogs. The images shown in Figure 27 represent five consecutive sequencing cycles of the same cell and the same field of view using sequencing primers specific to the tubulin barcoded concatemer. The images are rendered in false color. The fluorescent signals released during five sequencing cycles detect and identify intracellular tubulin structures. The target barcode sequences are listed in the table. [Figure 123A]The image shows the fluorescence signals emitted from sequencing of the barcoded regions of concatemers in cell samples. Here, the concatemers were generated from bipartite complexes. Cells were permeabilized, fixed, and reacted with barcoded bipartite complexes under conditions suitable for the bipartite complexes to bind to their congeneral target analytes (e.g., histone or tubulin). The analyte-bipartite complexes were subjected to rolling circle amplification to generate barcoded concatemers corresponding to histone or tubulin. The histone and tubulin concatemers carried sequencing primer binding sites with different sequences. Top: The barcoded concatemers were simultaneously sequenced using a two-step sequencing workflow with a mixture of sequencing primers specific to the histone or tubulin concatemer and labeled polyvalent molecules and unlabeled nucleotide analogs. The top image shows the fluorescence signals emitted from a single sequencing cycle, with the tubulin barcode emitting a green signal and the histone barcode emitting a red signal. The top image shows intracellular histone (red) and tubulin (green) structures. The top image is not an overlay image. Bottom: Sequence read products generated from sequencing of tubulin and histone concatemers were removed from the concatemers by thorough washing. Histone concatemers were sequenced using a two-step sequencing workflow with histone concatemer-specific sequencing primers and labeled polyvalent molecules. The bottom image shows the fluorescence signal emitted from a single sequencing cycle where the histone barcode emits a red signal. The bottom image shows intracellular histone (red) structures. The top and bottom images represent the same cell and the same field of view. [Figure 123B]The upper part is the same fluorescence image as shown in the upper part of Figure 123A. Figure 123B lower part: Sequence reading products (see upper part of Figure 123A) generated from the sequencing of tubulin concatemers and histone concatemers were removed from the concatemers by thorough washing. The tubulin concatemers were sequenced using a two-step sequencing workflow with tubulin concatemer-specific sequencing primers, as well as labeled polyvalent molecules and unlabeled nucleotide analogs. The lower image shows the fluorescence signal emitted from a single sequencing cycle in which the tubulin barcode emits a green signal. The lower image shows intracellular tubulin (green) structures. The upper and lower images represent the same cell and the same field of view. [Figure 124] Images of dividing cells are shown. The images were generated by fluorescence signals emitted from sequencing of the barcode regions of concatemers within the cell sample. Here, the concatemers were generated from bipartite complexes. Cells were permeabilized, fixed, and reacted with barcoded bipartite complexes under conditions suitable for the bipartite complexes to bind to their congeneral target analytes (e.g., histone or tubulin). The analytes-bipartite complexes were subjected to rolling circle amplification to generate barcoded concatemers corresponding to histone or tubulin. The histone and tubulin concatemers carried sequencing primer binding sites with different sequences. Sequencing was performed using a two-step sequencing workflow with labeled polyvalent molecules and unlabeled nucleotide analogs. The images shown in Figure 124 are not superimposed images. [Modes for carrying out the invention]

[0015] There is a need for increased throughput and flexibility in next-generation sequencing (NGS) analysis systems. Disclosed herein are flow cell devices and sequencing systems, including optical system designs, that may offer one or more of the following advantages: higher system throughput for fluorescence imaging-based genomics applications; compatibility with conventional flow cell devices and / or optical systems; flexibility in sample analysis or comparison (e.g., larger sample volumes and / or increased sample varieties); improved optical resolution (including high-performance optical resolution); wide field of view with uniform illumination (e.g., less than 10% variation in excitation energy); simultaneous sequencing and identification of various targets within cells or tissues; and improved image quality. The disclosed optical illumination and imaging system designs may offer one or more of the following advantages: improved dichroic filter performance, improved uniformity of dichroic filter frequency response, improved excitation beam filtering, larger field of view, increased spatial resolution, improved modulation transmission, contrast-to-noise ratio, and image quality, higher spatial sampling frequency, faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., of different fields of view), improved imaging system duty cycle, and higher throughput image acquisition and analysis.

[0016] Optical systems In some embodiments, the optical system 4500 described herein is shown in the non-limiting schematic diagram of Figure 45, which eliminates the need for dichroic or corrective optical systems such as tube lenses for multifaceted imaging of the flow cell. The multifaceted imaging can be two, three, four, or more. The optical system 4500 disclosed herein can be used as a component of a system designed for various chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. As shown in Figure 45, the optical system includes, in some embodiments, a plurality of imaging sensors 4501-4504 configured to image the flow cell 4521. In some embodiments, the imaging sensors 4501-4504 may be CCD imaging sensors. In some embodiments, the imaging sensors 4501-4504 may be CMOS imaging sensors. In some embodiments, pixel shifters 4505-4508 are used to move the object being imaged relative to the corresponding imaging sensors 4505-4508. In some embodiments, the optical system includes a multiband bandpass filter 4509. In some embodiments, the multiband bandpass filter is a multiband fluorescence bandpass filter. In some embodiments, the multiband bandpass filter is a three-band fluorescence bandpass filter. In some embodiments, the three-band fluorescence bandpass filter is referred to as a three-band notch filter. In some embodiments, imaging optics 4510-4513 are positioned between the imaging sensors 4501-4504 and the flow cell 4521. In some embodiments, one imaging optics 4505-4508, also referred to as the imaging optical assembly, focuses the light emitted from the flow cell 4521 onto one of the imaging sensors, for example, 4501, 4502, 4503, or 4504. In some embodiments, the optical system includes an integrated field flattening assembly. In some embodiments, the optical system includes aberration correction. In some embodiments, the optical system lacks a bandpass filter. In some embodiments, the optical system lacks a cutoff filter.In some embodiments, the optical system lacks a dichroic mirror. In some embodiments, the liquid handling system 4514 dispenses the sample 4515 into the flow cell 4521. In some embodiments, the liquid handling system 4514 dispenses the liquid sample into a hydrophobic pad 4516 attached to the flow cell 4521. In some embodiments, the liquid handling system 4514 is a droplet dispensing system. In some embodiments, the droplet dispensing system 4514 delivers the sample 4515 as 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 tensile force. In some embodiments, the tensile force is initiated by a vacuum pump 4518. In some embodiments, the flow cell 4521 includes an internal channel 4517 surrounded by a bottom plate 4519 and a top plate 4520. In some embodiments, the top plate 4520 and the bottom plate 4519 are transparent. In some embodiments, the top plate includes a front inner surface 4528. In some embodiments, the bottom plate includes a rear inner surface 4529. In some embodiments, the sample present in the internal 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 emits a different color or spectrum of light, 4525, 4526, and 4527, respectively. In some embodiments, the optical system 4500 includes a heater.

[0017] Although the flow cell 4521 is shown in Figure 45 having two inner surfaces, in other embodiments the flow cell 4521 may include two or more axially displaced channels and three or more axially displaced inner surfaces.

[0018] In some embodiments, a notch filter refers to a bandstop filter. In some embodiments, a notch filter refers to a bandstop filter. In some embodiments, a notch in the filter refers to a bandstop or stopband. In some embodiments, a notch in the filter refers to a bandpass or passband. In some embodiments, a multiband notch filter refers to a multiband bandpass filter. In some embodiments, a multiband notch filter refers to a multiband stop filter.

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

[0020] This specification describes various methods for various chemical, biochemical, nucleic acid, cell, or tissue analysis applications. Figure 46 provides a schematic diagram of an imaging method 4601 that utilizes the optical system 4500 shown in Figure 45 to image a sample 4515 contained within a flow cell 4521, according to some embodiments of this specification. In some embodiments, the imaging method may be configured for nucleic acid sequencing. In some embodiments, the sample 4515 is contained within or flows through an internal channel 4517 of the flow cell 4521, as shown in Figure 45. In some embodiments, the sample comprises a biopolymer. In some embodiments, the biopolymer comprises units. In some embodiments, the fluorophores are complementary to the units of the biopolymer. In some embodiments, the fluorophores are attached to nucleotides complementary to the units of the biopolymer. In some embodiments, two or more detectably distinct fluorophores are attached to nucleotides complementary to the units of the biopolymer. In some embodiments, the biopolymer is a nucleic acid sequence. In some embodiments, the units are nucleotides complementary to the fluorophor-labeled nucleotides. In some embodiments, multiple light sources emit light that passes through the sample.

[0021] This specification describes various methods for sequencing biopolymers (e.g., nucleic acid molecules). Non-limiting schematic diagrams of sequencing methods and apparatus 4601 and base calling methods 4602 are shown in Figure 46. In some embodiments, the method involves illuminating a sample 4515 using an optical system 4500 including a first light source 4522 among a plurality of light sources, wherein the first light source 4522 emits a first wavelength range 4525 that excites a first fluorophore of the sample 4515, and acquiring a first image of the sample 4515, wherein the optical system 4500 includes a plurality of imaging sensors 4501-4504, and further, the sample 4515 is illuminated by a plurality of light sources 4522-4524 and a plurality of imaging sensors 4501- The optical path between 4504 and 4515 is arranged to acquire and illuminate the sample 4515 using a plurality of second light sources 4523, wherein the second light sources 4523 emit a second wavelength range 4526 that excites a second fluorophore of the sample 4515, acquire a second image of the sample 4515, and illuminate the sample 4515 using a plurality of third light sources 4524, wherein the third light sources 4524 emit a third wavelength range 4527 that excites a third fluorophore of the sample. This includes: obtaining a third image of sample 4515; combining the first, second, and third images into a composite image; identifying the presence of a first nucleotide via a first signal emitted by a first fluorophore, wherein the first signal is extracted and identified from a first region of interest (ROI) in the composite image; identifying the presence of a second nucleotide via a second signal emitted by a second fluorophore, wherein the second signal is extracted and identified from a second ROI in the composite image; identifying the presence of a third nucleotide via a third signal emitted by a third fluorophore, wherein the third signal is extracted and identified from a third ROI in the composite image; and identifying the presence of a fourth nucleotide via first and third signals emitted by first and third fluorophores, respectively, wherein the first and third signals are extracted and identified from a fourth ROI in the composite image.In some embodiments, the optical system 4500 further includes a flow cell 4521, and 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 includes at least one pixel shifter 4505 - 4508. In some embodiments, the optical system 4500 further includes a multi - band band - pass filter 4509 disposed in the optical path between the plurality of imaging sensors 4401 - 4504 and the flow cell 4521. In some embodiments, the method further includes imaging optics 4510 - 4513 disposed in the optical path between the multi - band band - pass filter 4509 and the flow cell 4521. In some embodiments, the optical system 4500 has a reduction ratio of 1×. In some embodiments, the optical system is 1 mm. 2 Greater than, 2 mm 2 Greater than, 4 mm 2 Greater than, 10 mm 2 Greater than, 20 mm 2 Greater than, 36 mm 2 Greater than, 40 mm 2 Greater than, 60 mm 2 Greater than, 80 mm 2 Greater than, or 100 mm 2 Has a field of view (FOV) greater than. In some embodiments, the optical system has a numerical aperture (NA) less than 0.6. In some embodiments, the NA is 0.25. In some embodiments, the FOV is captured by the plurality of image sensors 4501 - 4504.

[0022] In some embodiments, sequencing is avidity sequencing. Further consideration of avidity sequencing is contained in U.S. Patent No. 10,768,173, filed September 23, 2019 (the entire patent is incorporated herein by reference). In some embodiments, a first fluorophore is associated with a first nucleotide conjugate. In some embodiments, a second fluorophore is associated with a second nucleotide conjugate. In some embodiments, a third fluorophore is associated with a nucleotide conjugate. In some embodiments, the first and third fluorophores are associated with a fourth nucleotide conjugate. In some embodiments, the nucleotide conjugate may include a polymer nucleotide conjugate. In some embodiments, the nucleotide conjugate may include a particle-nucleotide conjugate.

[0023] In some embodiments, fluorophores that can function as a first fluorophore, a second fluorophore, and / or a third fluorophore include fluorescein and fluorescein derivatives (e.g., carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidefluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein), rhodamine and rhodamine derivatives (e.g., TRITC, TMR, Lisamin Rhodamine, Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, Lisamin Rhodamine B sulfonyl chloride, Lisamin Rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide), coumarin and coumarin derivatives (e.g., AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide), BODIPY and derivatives (e.g., 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 (e.g., Cascade Blue acetylazide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue Blue hydrazide), Lucifer Yellow and derivatives (e.g., Lucifer Yellow iodoacetamide, Lucifer Yellow CH), cyanines and derivatives (e.g., indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3,Examples include, but are not limited to, Cy5), lanthanide chelates and derivatives (e.g., BCPDA, TBP, TMT, BHHCT, BCOT), europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes, and other known in the art (e.g., 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 their derivatives), or any combination thereof. Cyanine pigments may exist in either sulfonated or unsulfonated forms and contain two indolenin, benzoindlium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine crosslink between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3(this is 1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indole-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indlium, and It may contain 1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indole-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indrolium-5-sulfonate), Cy5 (this is,1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium, or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate), and Cy7 (1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-triene-1-yl]-3H-indolium, or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-triene-1-yl]-3H-indolium-5-sulfonate), where "Cy" represents "cyanine" and the first digit identifies the number of carbon atoms between the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and benzoderivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule. In some embodiments, the reporter moieties can be a FRET pair, whereby multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET can include Förster (excitation exchange) transfer or Dexter (electron exchange) transfer.,

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

[0025] Described herein is an optical system 4700 for imaging a sample in a flow cell for the analysis of biopolymers (e.g., nucleic acid sequencing). In some embodiments, such systems 4700, shown in Figures 47A–47B, are more compact and have higher throughput than previous optical systems. Table 1 and Figures 48A–48B provide non-limiting examples comparing the sequencing cycle times of a standard flow cell and optical system to the optical system described herein. Table 1 provides the cycle and run times, as well as their respective calculated values, for a standard flow cell with 424 individual tiles (e.g., active region, region of interest, etc.) shown in Figure 48A, compared to a flow cell with fewer than 40 individual tiles optimized for imaging with the optical system described herein, shown in Figure 48B. In some embodiments, one image is equivalent to one tile in the region. In some embodiments, when the flow cell 4521 (also shown in Figure 48B) is imaged by the optical system, each tile is exposed to three consecutive light pulses from three separate LED light sources, each emitting a different wavelength. In some embodiments, each different wavelength is matched to the excitation spectrum of different fluorophores described herein. In some embodiments, the imaging sensor of the optical system 4500 is synchronized with each excitation pulse to generate an image. One image is an entire area of ​​one tile, and furthermore, each pixel of the image represents the amount of fluorescence emitted by the fluorophore. In some embodiments, the optical system 4500 images two distinct surfaces on one tile. In some embodiments, a total of eight images with a total exposure time of 0.3 seconds are acquired by the optical systems 4500, 4700, which include eight imaging modules (e.g., optical subsystems). The row highlighted in blue in Table 1, titled "Current (Conventional)," indicates that in a typical flow cell shown in Figure 48A, the total time over 322 cycles when imaged with the IDEX optical system shown in Figure 47B is 36.17 hours.For comparison, the row titled "Sleq" shows that when imaged with the optical system 4700 shown in Figures 47A-47B, the total time for a Sleq cell (see Figure 48B) is 13.63-14.28 hours. The bottom row of Table 1 shows that when only 25 cycles are performed, the total time is 1.11 hours. The reduction in sequencing time demonstrates the advantage of enabling a larger field of view (FOV) with the optical system 4700 described herein.

[0026] [Table 1]

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

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

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

[0030] In some embodiments, the filter 4910 is positioned between the image sensor 4912 and the flow cell 4905. In some embodiments, the imaging optical system 4909, also referred to as the imaging optical assembly, focuses the light emitted from the flow cell 4909 onto the image sensor 4912. In some embodiments, the optical system 4900 includes an integrated image plane flattening assembly. In some embodiments, the optical system includes an aberration correction module. In some embodiments, the optical system includes a wedge block 4916 configured to adjust the path length of the optical system. In some embodiments, the wedge block 4916 includes a first wedge piece 4907, a second wedge piece 4906, or a combination thereof. In some embodiments, the system includes a piezo drive 4908 configured to move the positions of the first wedge piece 4907 and the second wedge piece 4906 relative to each other, and thus adjust the optical path length of the optical system. In some embodiments, the flow cell 4905 is configured for multi-plane imaging (DSI). In some embodiments, the flow cell 4905 includes a front inner surface 4904, a rear inner surface 4905, or a combination thereof. In some embodiments, the front inner surface 4904 and / or the rear inner surface 4903 include the sample area 4902. In some embodiments, the optical system includes an optical axis 4913. In some embodiments, the optical system includes an optimal imaging volume 4915. In certain embodiments, the optimal imaging volume 4915 includes the field of view (FOV), illumination area, acquisition area, focal plane, depth of focus, area and / or volume in which the sample area 4902 emits brightness above an acceptable level, or a combination thereof. Typically, in the field of microscopy, the brightness of an object at the center of the FOV may be maximum at the center and decrease towards the corners and / or edges.

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

[0032] In some cases, imaging with multiple tiles can introduce registration errors (e.g., errors in overlapping image tiles). The amount of registration error can be a measure of the fidelity of the imaging system (for example, the difference in registration between two images can be a measure of the fidelity of the optical system to those two images). The methods and systems of this disclosure can achieve fidelity of at most about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01 micrometers or less. For example, the difference in positioning between multiple images of a sample can be at most about 0.1 micrometers.

[0033] Wedge Block Assembly Various embodiments of optical systems are described herein. In some embodiments, the optical system is configured for fluorescence readout of a sample. In some embodiments, the optical system includes a wedge block assembly 4916, as shown in Figures 49A-49B. In certain embodiments, the wedge block assembly 4916 includes a first wedge piece 4907 and a second wedge piece 4906. In some embodiments, the wedge block assembly 4916 includes an adjustable optical path length. In some embodiments, the first wedge piece 4907 is configured to move relative to the second wedge piece 4906. In some embodiments, the relative movement of the first wedge piece 4907 relative to the second wedge piece 4906 changes the optical path length of the wedge block assembly 4916 due to a change in the physical thickness of the wedge block assembly 4916, as shown in Figures 49A-49B. In some embodiments, the wedge block assembly 4916 includes a gap separating the first wedge piece 4907 from the second wedge piece 4906. In some embodiments, the gap maintains a constant distance regardless of the relative positions of the first wedge piece 4907 and the second wedge piece 4906. In some embodiments, the first wedge piece 4907 and the second wedge piece 4906 are made of fused silica. In some embodiments, the first wedge piece 4907 and the second wedge piece 4906 are made 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 includes a housing. In some embodiments, the wedge block assembly 4916 and the piezo drive 4908 are contained within the housing. In some embodiments, the wedge block assembly 4916 and the piezo drive 4908 include a wedge block-piezo drive assembly. In some embodiments, the second wedge piece 4906 of the wedge block assembly 4916 contacts the housing.In some embodiments, the second wedge piece 4906 of the wedge block assembly 4916 contacts the flow cell 4905.

[0034] In some embodiments, the position of the first wedge piece 4907 relative to the second wedge piece 4906 determines the position of the focal plane along the optical axis 4913 (e.g., the z-axis). In some embodiments, the upper wedge piece 4907 is aligned with the lower wedge piece 4906, as shown in Figure 49A. In such embodiments, the physical distance of the wedge block assembly 4916 results in alignment of the focal plane with the rear inner surface. In this case, the focus is on the sample portion 4902 on the rear inner surface. In some embodiments, the piezo drive 4908 moves the position of the upper wedge piece 4907 relative to the lower wedge piece 4906, as shown in Figure 49B, such that the physical thickness of the wedge block 4916 in the optical path is greater than the alignment shown in Figure 49A. In such embodiments, the focal plane is shifted to align with the front inner surface. In this case, the focus is on the sample portion 4902 on the front inner surface.

[0035] Further examples of flow cells can be found in International Patent Application PCT / US2024 / 010760 (which is incorporated herein by reference in its entirety).

[0036] stage Various embodiments of an optical system including a stage are described herein. The stage may be a tilting stage. The stage may be a tip tilting stage. The stage may be capable of rotation. The stage may be configured to move simultaneously along three different axes (all axes are perpendicular to each other). The stage may be configured to move simultaneously along three different axes (all axes are perpendicular to each other) and to rotate about the axis. The stage may move multiple optical subsystems 5001 relative to a flow cell 4905, as shown in Figure 50. The stage may move the flow cell 4905 relative to multiple optical subsystems 5001, as shown in Figure 50. The stage may move a single optical subsystem 4914. The stage may rotate multiple optical subsystems 5001 about the x-axis of a capillary flow cell 5201, as shown in Figures 53A-53B. As shown in Figures 52A-53B, the stage can move multiple optical subsystems 5001 along the x-axis, coinciding with the long axis of the capillary flow cell 5201.

[0037] Pixel shifter Various embodiments of optical systems comprising a pixel shifter 4911 are described herein. In some embodiments, the pixel shifter 4911 enables sub-pixel resolution imaging. In certain embodiments, the resolution of the optical system can be increased without increasing the actual resolution of the optical system by using the pixel shifter 4911. In some embodiments, the pixel shifter 4911 effectively doubles the resolution of the imaging sensor 4912. In some embodiments, a piezoelectric actuator is configured to define a lateral pixel shift that coincides with the image plane (e.g., in the xy plane). In some embodiments, the piezoelectric actuator is configured for a pixel shift along the optical axis 4913 (e.g., the z-axis or z-axis including the plane). In some embodiments, a tilting stage is configured for a pixel shift in the XZ or YZ or XYZ directions. In some cases, the tilting stage is configured for a two-dimensional pixel shift. In some embodiments, the optical system comprising the pixel shifter is configured to image a 3D sample object. In some cases, the optical system comprising the pixel shifter is configured to image a 2D sample object.

[0038] In some embodiments, the 3D object may include a sample site 4902. In some embodiments, the sample site 4902 is an amplified nucleic acid. In some embodiments, the sample site may include a polony or multiple polonys.

[0039] The pixel shifter 4911 may utilize polarization.

[0040] Autofocus element Various embodiments of optical systems including an autofocusing element are described herein.

[0041] Figures 51A - 51B provide non - limiting cross - sectional views of a focusing lens assembly. The focusing lens assembly is configured to maintain a fixed position within an optical path (e.g., the optical axis) and to enable relative movement between at least a first lens and a second lens included within the lens housing of the focusing lens assembly.

[0042] In some embodiments, the autofocus element is configured for initial focusing. In some embodiments, the autofocus element is included within the lens barrel. In some embodiments, the autofocus element is incorporated into and / or integrated with the lens barrel. In some embodiments, the autofocus element is included within the lens barrel of the lens assembly. In some embodiments, the autofocus element is configured to improve reliability and reduce the mechanical footprint of the optical system. In some embodiments, the autofocus element includes a wedge - block assembly, a piezo - drive, a wedge - block - piezo - drive assembly, or a combination thereof.

[0043] Multiple imaging subsystems In some embodiments, the optical system shown in Figure 50 includes a plurality of optical subsystems 4914. In some embodiments, each optical subsystem 4914 of the plurality of optical subsystems 5001 includes an image sensor 4912, a pixel shifter 4911, a filter 4910, an imaging optical system 4909, a piezo-driven wedge block assembly, a light source 4901, or a combination thereof. In some embodiments, the image sensor 4912 is a mobile phone camera. In some embodiments, the plurality of optical subsystems 5001 includes an array of optical subsystems. In some embodiments, the array of optical subsystems may be configured for multiple depths of focus, multiple wavelengths, or a combination thereof. In some embodiments, each optical subsystem 4914 of the plurality of optical subsystems 5001 is configured for a depth of focus, and the depths of focus of at least two of the optical subsystems are different. In some embodiments, each optical subsystem of the plurality of optical subsystems is configured to detect wavelengths, and the wavelengths detected by at least two of the optical subsystems are different. In some embodiments, the image sensor 4912 of each optical subsystem 4914 of a plurality of optical subsystems 5001 includes an array of image sensors 4912. In some embodiments, a high-resolution, low-cost camera is configured to provide aberration-compensated imaging by software. In some embodiments, the optical system includes one optical subsystem 4914, which includes one optimal imaging volume as shown in Figures 49A-49B. In Figures 49A-49B, the range of the optimal imaging volume 4915 along the x-axis is limited. Certain factors may affect the width of the optimal imaging volume in the xy plane (e.g., the focal plane). The xy plane, or focal plane, includes the cross-section of the optimal imaging volume and may be referred to as the illumination area, acquisition area, or a combination thereof. Surfaces including sample portions 4902 that extend beyond the optimal FOV are not optimally illuminated by the light source, not optimally captured by the image sensor, not resolved by the optical system, or a combination thereof.Such suboptimal areas of the surface exhibit non-uniform brightness and non-uniform resolution, as can be observed at the edges and / or corners of the image in Figure 38. In Figure 38, the sample area becomes darker and the resolution decreases from the center of the image towards the edges and / or corners. Figure 50 shows an embodiment in which the surface covered by the sample area 4902 extends beyond the optimal imaging volume 4915 of one optical subsystem 4916, and the overlapping optimal imaging volumes 4915 overlap to provide a composite optimal imaging volume.

[0044] In some embodiments, the optical system has an optimized FOV of 6 mm × 6 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm to about 9 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm to about 1 mm, about 0.5 mm to about 3 mm, about 0.5 mm to about 6 mm, about 0.5 mm to about 9 mm, about 1 mm to about 3 mm, about 1 mm to about 6 mm, about 1 mm to about 9 mm, about 3 mm to about 6 mm, about 3 mm to about 9 mm, or about 6 mm to about 9 mm. In some embodiments, the system has an optimized FOV of about 0.5 mm, about 1 mm, about 3 mm, about 6 mm, or about 9 mm. In some embodiments, the system has an optimized FOV of at least about 0.5 mm, about 1 mm, about 3 mm, or about 6 mm. In some embodiments, the system has an optimized FOV of at most about 1 mm, about 3 mm, about 6 mm, or about 9 mm.

[0045] In some embodiments, the optical system has an optimized illumination area of ​​6 mm × 6 mm. In some embodiments, the system has an optimized illumination area of ​​about 0.5 mm to about 9 mm. In some embodiments, the system has an optimized illumination area of ​​about 0.5 mm to about 1 mm, about 0.5 mm to about 3 mm, about 0.5 mm to about 6 mm, about 0.5 mm to about 9 mm, about 1 mm to about 3 mm, about 1 mm to about 6 mm, about 1 mm to about 9 mm, about 3 mm to about 6 mm, about 3 mm to about 9 mm, or about 6 mm to about 9 mm. In some embodiments, the system has an optimized illumination area of ​​about 0.5 mm, about 1 mm, about 3 mm, about 6 mm, or about 9 mm. In some embodiments, the system has an optimized illumination area of ​​at least about 0.5 mm, about 1 mm, about 3 mm, or about 6 mm. In some embodiments, the system has an optimized illumination area of ​​at most about 1 mm, about 3 mm, about 6 mm, or about 9 mm.

[0046] In some embodiments, the optical system is configured for rapid imaging of a surface. In some embodiments, the optical system is configured for rapid imaging of the surface of a flow cell. In some embodiments, the optical system is configured for rapid imaging of a first surface and a second surface of a flow cell. In some embodiments, the entire active region (e.g., region of interest, ROI) of surface 4903 or 4904 of the flow cell 4905 is imaged in five imaging steps. In some embodiments, the active region (e.g., region of interest) of surface 4903 or 4904 is imaged in about one to about ten imaging steps. In some embodiments, the active region of the surface (e.g., region of interest) is defined as approximately 1 to 2 imaging steps, approximately 1 to 3 imaging steps, approximately 1 to 4 imaging steps, approximately 1 to 5 imaging steps, approximately 1 to 6 imaging steps, approximately 1 to 10 imaging steps, approximately 2 to 3 imaging steps, approximately 2 to 4 imaging steps, approximately 2 to 5 imaging steps, approximately 2 to 6 imaging steps, The image is captured in approximately 2 to 10 imaging steps, approximately 3 to 4 imaging steps, approximately 3 to 5 imaging steps, approximately 3 to 6 imaging steps, approximately 3 to 10 imaging steps, approximately 4 to 5 imaging steps, approximately 4 to 6 imaging steps, approximately 4 to 10 imaging steps, approximately 5 to 6 imaging steps, approximately 5 to 10 imaging steps, or approximately 6 to 10 imaging steps. In some embodiments, the active region of the surface (e.g., region of interest) is captured in approximately 1, 2, 3, 4, 5, 6, or 10 imaging steps.In some embodiments, the active region of the surface (e.g., region of interest) is imaged in at least about 1 imaging step, about 2 imaging steps, about 3 imaging steps, about 4 imaging steps, about 5 imaging steps, or about 6 imaging steps. In some embodiments, the active region of the surface (e.g., region of interest) is imaged in at most about 2 imaging steps, about 3 imaging steps, about 4 imaging steps, about 5 imaging steps, about 6 imaging steps, or about 10 imaging steps.

[0047] In some embodiments, each imaging step includes imaging the entire active region or at least a portion of the ROI. In some embodiments, each imaging step includes imaging at least an overlapping portion of the ROI, and the overlapping portion of the ROI may also be imaged in different imaging steps. In some embodiments, the complete ROI may be imaged in multiple imaging steps, one portion at a time (with or without some overlapping regions).

[0048] In some embodiments, the images acquired using the optical systems herein are flow cell images. Flow cell images may include a field of view (FOV) that covers the entire active area or at least a portion of the ROI on the surface of the flow cell or otherwise different sample support structure. Flow cell images can be aligned with each other to cover the entire ROI on the surface of the flow cell.

[0049] How to use the optical system This specification describes various methods for imaging biopolymers, the method providing an optical system comprising a plurality of optical subsystems, each optical subsystem comprising a light source configured to emit a first wavelength and a second wavelength separately, wherein the first wavelength is different from the second wavelength, a multiband filter configured to reject each of the first and second wavelengths, and an imaging sensor configured to image one or more biopolymers arranged in an optical path between each light source and each imaging sensor, the method providing, under conditions sufficient to bond a first biopolymer of the one or more biopolymers to a first fluorophore of the plurality of fluorophores, and a second biopolymer of the one or more biopolymers to a second fluorophore of the plurality of fluorophores, The imaging method comprises contacting one or more biopolymers with a plurality of fluorophores, wherein the first fluorophores are different from the second fluorophores; imaging the first biopolymer with each imaging sensor, wherein the imaging includes (i) illuminating the first biopolymer with a first wavelength to excite the first fluorophores, and (ii) acquiring a first image; and imaging the second biopolymer with each imaging sensor, wherein the imaging includes (i) illuminating the second biopolymer with a second wavelength to excite the second fluorophores, and (ii) acquiring a second image, wherein the one or more biopolymers are arranged on a curved surface, and the optical axis of each optical subsystem of the plurality of optical subsystems is perpendicular to the curved surface. In some embodiments, the method further includes imaging a third biopolymer among the one or more biopolymers, comprising (i) illuminating the third biopolymer at a third wavelength to excite a third fluorophore among the plurality of fluorophores, and (ii) acquiring a third image. In some embodiments, the method further includes combining the first image and the second image into a composite image.In some embodiments, the method further includes identifying units of the first biopolymer to which the first fluorophore is bound, including analyzing a first region of interest (ROI) of the composite image to detect a first signal emitted by the first fluorophore. In some embodiments, the method further includes identifying units of the second biopolymer to which the second fluorophore is bound, including analyzing a second ROI of the composite image to detect a second signal emitted by the second fluorophore. In some embodiments, the method further includes identifying first units of the first biopolymer to which the first fluorophore is bound, including analyzing a first ROI of the composite image to detect a first signal emitted by the first fluorophore, and identifying second units of the second biopolymer to which the second fluorophore is bound, including analyzing a second ROI of the composite image to detect a second signal emitted by the first fluorophore. In some embodiments, the method further includes combining the first image, the second image, and the third image into a composite image. In some embodiments, the method further includes identifying a third unit of the third biopolymer to which the third fluorophore is bound, including analyzing the third ROI of the composite image to detect a third signal emitted by the third fluorophore.In some embodiments, the method further includes identifying a first unit of the first biopolymer to which a first fluorophore is bound, including analyzing a first region of interest (ROI) of the composite image to detect a first signal emitted by the first fluorophore; identifying a second unit of the second biopolymer to which a second fluorophore is bound, including analyzing a second ROI of the composite image to detect a second signal emitted by the first fluorophore; identifying a third unit of the third biopolymer to which a third fluorophore is bound, including analyzing a third ROI of the composite image to detect a third signal emitted by the third fluorophore; and identifying a third unit of the third biopolymer to which a third fluorophore is bound, including analyzing a third ROI of the composite image to detect a third signal emitted by the third fluorophore.

[0050] This specification describes various methods for super-resolution imaging using the optical systems described herein. In some embodiments, the method further includes providing a surface comprising at least one sample site containing a plurality of immobilized clone-amplified sample nucleic acid molecules, wherein the plurality of immobilized clone-amplified sample nucleic acid molecules are located at a distance of less than λ / (2*NA), where λ is the central wavelength of the light source and NA is the numerical aperture of the imaging system; and simultaneously applying stochastic photo-switching chemistry to the clone-amplified sample nucleic acid molecules so that the plurality of clone-amplified sample nucleic acid molecules fluoresce in up to four different color on and off events by stochastic photo-switching; and detecting the on and off events in real time in each color channel when on and off events occur for the plurality of clone-amplified sample nucleic acid molecules to determine the identity of the nucleotides in the clone-amplified sample nucleic acid molecules. The stochastic photo-switching may include using the dark state of an emitting fluorophore to randomly switch the fluorophore on and off. This can enable imaging of individual fluorophores, which can then be localized to provide super-resolution images. In some cases, stochastic optical switching may include the use of techniques such as stimulated emission depletion (STED) and stochastic optical reconstruction (STORM).

[0051] In some cases, super-resolution imaging may include imaging at resolutions of approximately 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 nanometers or less. In some cases, the resolution of super-resolution imaging may be controlled by the numerical aperture of the imaging system. In some cases, the resolution of the optical system may be the sub-pixel resolution. The sub-pixel resolution can be higher than the resolution achievable given the size of the pixels used for imaging (e.g., by computer processing of the image).

[0052] light source In some embodiments, the light source 4901 shown in Figures 49A-53B is a solid-state light source. In some embodiments, the solid-state light source is a light-emitting diode (LED). In some embodiments, the light source 4901 is configured to emit multiple wavelengths. In some embodiments, the light source includes multiple light sources. In some embodiments, each light source of the multiple light sources is configured to emit light of a different wavelength. In some embodiments, the light source 4901 is configured to emit light of a first wavelength in a first time, light of a second wavelength in a second time, and light of a third wavelength in a third time. In some embodiments, light of a first wavelength in a first time, light of a second wavelength in a second time, and light of a third wavelength in a third time are emitted sequentially. In some embodiments, the multiple light sources are configured to be delivered in a timing pulse sequence that switches colors sequentially. In some embodiments, the multiple optical subsystems 5001 are configured to increase the detection speed. In some embodiments, the solid-state light source is not a laser. In some applications, the light source includes a filter to narrow the spectrum of light emitted by the light source. In some embodiments, the light source is referred to as the excitation source. In some embodiments, the light emitted by the light source is referred to as the excitation light.

[0053] light delivery component In some embodiments, the optical system includes an optical delivery component. In some embodiments, the optical delivery component is a waveguide. In some embodiments, the optical delivery component is a light pipe 4702 shown in Figure 47. In some embodiments, the optical delivery component is an optical fiber. In some embodiments, a light source delivers light to a flow cell by an optical delivery component. In some embodiments, a light source delivers light to a flow cell by a light pipe. In some embodiments, an optical delivery component is located between a light source 4901 and a flow cell 4905. In some embodiments, a second optical delivery component is located between a flow cell and an image sensor.

[0054] Imaging channel The optical systems described herein may be configured to image one or more fluorophores. In some embodiments, the optical system is configured to clearly image two, three, or more different fluorophores. In certain embodiments, the optical system includes one or more imaging channels. In some embodiments, a first imaging channel of the one or more imaging channels is configured to image a first fluorophore of 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 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 one or more fluorophores. In some embodiments, the imaging channel includes at least one of a light source 4901, a filter 4910, an imaging sensor 4912, or a combination thereof.

[0055] heater Typically, the assay requires heating. In some cases, the flow cell 4905 further includes a heater. In some embodiments, the heater is integrated with the flow cell. In some embodiments, the heater is integrated with the multi-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 IR heater. In some embodiments, the transparent heater fits onto the surface of the flow cell. In some embodiments, the transparent heater fits onto and completely surrounds a flow cell having a non-rectangular cross-section. In some embodiments, the transparent heater fits onto and completely surrounds a flow cell having a circular cross-section. In some embodiments, the transparent heater fits onto and completely surrounds the capillary flow cell 5201. In some embodiments, the transparent heater is transparent to all image channels of one or more image channels of the optical system.

[0056] Aberration correction In some embodiments, aberration correction methods may be applied to enable imaging through bubbles that may appear within the flow cell. In some embodiments, a non-flat flow cell surface allows for right-angle or off-axis illumination. In some embodiments, the optical system described herein may include magnetic positioning of various elements. In some embodiments, the optical system may be configured to image a flow cell having a circular rim.

[0057] Integrated image plane flattener Typically, the illumination area and / or field of view (FOV) of a standard fluorescence microscope imaging system is limited by the size of the single lens system and / or single imaging sensor present. Typically, the system's ability to systematically capture luminance across the FOV can be referred to as the system's field of view uniformity. Non-uniformity of luminance and resolution across the FOV is observed in some cases from the center to the edge of the FOV. In some cases, illumination non-uniformity is caused by the non-uniform field curvature effect of the system's lenses, which are usually a single lens system. Systems, devices, and methods designed to improve field of view uniformity may be referred to as field flatteners or field planarizers, respectively. The optical systems described herein may include field flatteners. In some cases, a field flattener comprises multiple optical subsystems 5001 designed to provide overlapping coverage of the “active area” of the flow cell surface. When an image from one of the individual optical subsystems 4914 of the multiple optical subsystems 5001 begins to become non-uniform (e.g., increased blurring, loss of intensity at corners and edges), the optimal imaging volume 4915 of the second optical subsystem 4914 may overlap. In some cases, the optimal imaging volume 4915 of the first optical subsystem overlaps with that of the second and third optical subsystems.

[0058] In some embodiments, the surface 5101 of the flow cell containing the sample area 4902 is not flat, as shown in Figure 52. In certain embodiments, each optical subsystem 4914 of the multiple optical subsystems 5001 is positioned to conform to the contour of the active area of ​​the flow cell shown in Figure 52.

[0059] Super-resolution optical system When imaging very small sample site features present at high surface density, such as nucleic acid polony (e.g., spots containing amplified target nucleic acids), the super-resolution imaging techniques described herein may be used. In some embodiments, the probabilistic optical switching techniques described herein may be used to improve image resolution. Alternatively, the structured illumination techniques described herein may be used to improve image resolution in optical systems. In some cases, the super-resolution imaging techniques may include structured illumination.

[0060] In some cases, such as in multi-faceted (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or coverslip) thickness >700 μm) and fluid channels (e.g., fluid channel height or thickness 50–200 μm), improved imaging performance can be achieved by using a novel objective lens design that corrects the optical aberrations introduced by imaging the surface opposite the thick coverslip and / or fluid channel as viewed from the objective lens.

[0061] In some cases, for example, in multi-faceted (flow cell) imaging applications involving the use of thick flow cell walls (e.g., wall (or coverslip) thickness >700 μm) and fluid channels (e.g., fluid channel height or thickness 50-200 μm), improved imaging performance can be achieved even with commercially available objective lenses by using a novel tube lens design that corrects optical aberrations induced by the thick flow cell walls and / or intervening fluid layer in combination with the objective lens, unlike conventional microscope tube lenses that simply form images on intermediate image planes.

[0062] In some cases, for example, in multi-channel (e.g., two-color or four-color) imaging applications, improved imaging performance can be achieved by using multiple tube lenses (one for each imaging channel), with each tube lens design optimized for the specific wavelength range used in that imaging channel.

[0063] In some cases, for example in multi-face (flow cell) imaging applications, improved imaging performance can be achieved by using an electro-optical phase plate in combination with an objective lens to compensate for optical aberrations induced by the fluid layer separating the upper (near) and lower (far) inner surfaces of the flow cell. In some cases, this design approach can also compensate for vibrations introduced by a motion-actuated compensator that moves in and out of the optical path depending on which surface of the flow cell is being imaged.

[0064] Various multi-channel fluorescence imaging module designs are disclosed, which may include illumination and imaging optical paths, including a foldable optical path (e.g., including one or more beam splitters or beam combiners, such as a dichroic beam splitter or combiner) that guides an excitation light beam to an objective lens and the emitted light transmitted through the objective lens to multiple detection channels. Some particularly advantageous features of the fluorescence imaging modules described herein include the specification of the dichroic filter incidence angle, which results in a sharper and / or more uniform transition between the passband wavelength region and the stopband wavelength region of the dichroic filter. Such filters may be contained within a foldable optical system and may include a dichroic beam splitter or combiner. Further advantageous features of the disclosed imaging optical system designs may include the position and orientation of one or more excitation light sources and one or more detection optical paths relative to the objective lens, as well as the position and orientation of the dichroic filters that receive the excitation beam. The excitation beam may also be linearly polarized, and the orientation of the linear polarization may be such that the s-polarization is incident on the dichroic reflecting surface of the dichroic filter. Such features may potentially improve excitation beam filtering and / or reduce wavefront errors introduced into the emitted light beam due to surface deformation of the dichroic filter. The fluorescence imaging modules described herein may or may not include any of these features, and may or may not include any of these advantages.

[0065] Also described herein are devices and systems configured to analyze a number of different nucleic acid sequences, for example, by imaging arrays of immobilized nucleic acid molecules or amplified nucleic acid clusters formed on a flow cell surface. The devices and systems described herein may also be useful, for example, for performing comparative genomics sequencing, tracking gene expression, performing microRNA sequencing analysis, performing epigenomics, aptamer and phage display library characterization, and performing other sequencing applications. The devices and systems disclosed herein include various combinations of optical, mechanical, fluid, thermal, electrical, and computational devices / configurations. The advantages provided by the disclosed flow cell devices, cartridges, and systems include, but are not limited to, (i) reduced complexity and cost of manufacturing the devices and systems, (ii) significantly reduced cost of consumables (e.g., compared to those of currently available nucleic acid sequencing systems), (iii) compatibility with typical flow cell surface functionalization methods, (iv) flexibility in flow control when combined with microfluidic components (e.g., syringe pumps and diaphragm valves), and (v) flexibility in system throughput.

[0066] Disclosed herein are capillary flow cell devices and capillary flow cell cartridges, which are constructed from off-the-shelf, disposable single-lumen (e.g., single fluid flow channel) or multi-lumen capillaries, and may also include fluid adapters, cartridge chassis, one or more integrated fluid flow control components, or any combination thereof. Also disclosed herein are capillary flow cell-based systems, which may include one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell cartridges (or microfluidic cartridges), a fluid flow controller module, a temperature control module, an imaging module, or any combination thereof.

[0067] Some of the disclosed design features of capillary flow cell devices, cartridges, and systems include, but are not limited to, (i) a single flow channel structure, (ii) sealed, reliable, and iterative switching between reagent flows that can be implemented with a simple load / unload mechanism (which ensures a sealed fluid interface between the system and the capillary, thereby facilitating capillary replacement and system reuse, and enabling precise control of reaction conditions such as reagent concentration, pH, and temperature), (iii) interchangeability of single fluid flow channel devices or capillary flow cell cartridges containing multiple flow channels (which can be used to provide flexibility in system throughput), and (iv) compatibility with a wide variety of detection methods, such as fluorescence imaging.

[0068] 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, but various embodiments of the disclosed devices and systems may be applicable not only to nucleic acid sequencing but also to any other type of chemical analysis, biochemical analysis, nucleic acid analysis, cell analysis, or tissue analysis applications. It should be understood that the different embodiments of the disclosed methods, devices, and systems may be understood individually, collectively, or in combination with each other. Many of the disclosed optical design approaches and features are considered primarily in the context of fluorescence imaging (including, for example, fluorescence microscopy imaging, fluorescence confocal imaging, and two-photon fluorescence), but it will be understood by those skilled in the art that they are applicable to other imaging modes (e.g., bright-field imaging, dark-field imaging, phase-contrast imaging, etc.).

[0069] Fluorescence imaging as an information pipeline A useful abstraction of the role a fluorescence imaging system plays in typical genome assay techniques (including nucleic acid sequencing applications) is its role as an information pipeline. Here, a photon signal is input at one end of the pipeline (e.g., the objective lens used for imaging), and location-specific information about the fluorescence signal is output at the other end of the pipeline (e.g., the location of the image sensor). As more information is transmitted through this pipeline, some content is inevitably lost during this transmission process and cannot be recovered. An example of this is when labeled molecules (or clusters of cloned molecules) are excessively present in a small area of ​​the substrate surface and are not clearly resolved in the image. At the location of the image sensor, it becomes difficult to distinguish photon signals originating from adjacent molecular clusters, thus increasing the likelihood of attributing the signal to the wrong cluster and leading to detection errors. In some cases, the clusters are Polony clusters.

[0070] Design of an optical imaging module Therefore, the objective of designing the optical imaging module is to maximize the flow of information content through this detection pipeline and minimize detection errors. The design process must address several key design elements: 1) Match the density of physical features on the surface of the substrate being imaged to the overall image quality of the optical imaging system and the pixel sampling frequency of the image sensor used. Failure to match these parameters can lead to loss of information or, at times, the generation of erroneous information. For example, spatial aliasing can occur if the pixel sampling frequency is less than twice the optical resolution limit. 2) The size of the area to be imaged should match the overall image quality and focus quality across the entire field of view of the optical imaging system. 3) While considering background signal and system noise characteristics, the optical acquisition efficiency, modulation transfer function, and image sensor performance characteristics of the optical system design should be matched to the expected fluorescence photon flux and dye efficiency (related to the extinction coefficient and fluorescence quantum yield of the dye) of the input excitation photon flux. 4) Maximize the separation of spectral content to reduce crosstalk between fluorescence imaging channels. 5) The image acquisition step is effectively synchronized with the repositioning of the sample or optical system between image captures of different fields of view to minimize downtime of the imaging system (or maximize the duty cycle), and thus maximize the overall throughput of the image capture process.

[0071] This disclosure addresses each of the design elements outlined above and describes a systematic method for creating component-level specifications for an imaging system.

[0072] Improved optical resolution and image quality to improve or maximize information transfer and throughput. One non-restrictive design approach is to start with the optical resolution required to distinguish two adjacent features (specified in terms of the number of line pairs per millimeter (lp / mm)X) and translate this into a corresponding numerical aperture (NA) requirement. The resulting effect on the modulation transfer function and image contrast can then be evaluated using the NA requirement.

[0073] The standard modulation transfer function (MTF) represents the spatial frequency response to image contrast (modulation) transmitted through an optical system, where image contrast decreases as a function of spatial frequency and increases with increasing numerical aperture (NA). This function limits the contrast / modulation that can be achieved for a given NA. Furthermore, wavefront errors can adversely affect the MTF, and therefore it is desirable to improve or optimize the optical system design using the true system MTF instead of the one predicted by the diffraction-limited optics. Note that, as used herein, MTF refers to the entire system MTF (including the complete optical path from the coverslip to the image sensor). However, in design methodologies, the MTF of the objective lens may be considered primarily.

[0074] In genomic testing applications, if the target being imaged is a high-density array of "spots" on a surface (either randomly distributed or patterned), it is possible to determine the minimum modulation transfer value required by downstream analysis to resolve two adjacent spots and distinguish between four possible states (e.g., on-off, on-on, off-on, and off-off). For example, assume the spots are sufficiently small and approximated as point sources. The detection task is to determine whether two adjacent spots separated by a distance d are on or off (in other words, bright or dark), and the contrast-to-noise ratio (CNR) of the fluorescence signal emanating from the spots on the sample surface (or object plane) is C 試料 Assuming this is the case, under ideal conditions, the image sensor surface (C 画像 The CNR of the readout signals of two adjacent spots in ) is C 画像 =C 試料 *MTF(1 / d) (where MTF(1 / d) is the MTF value at spatial frequency = (1 / d)) can be used as a rough approximation.

[0075] In a typical design, the value of C may be at least 4 so that a simple threshold method can be used to avoid misclassification of the fluorescence signal. Assuming a Gaussian distribution of fluorescence signal intensity around the mean, C 画像 >4, the expected error in correctly classifying the fluorescence signal (e.g., whether it is on or off) is <0.035%. Using proprietary high CNR sequencing and surface chemistry as described in U.S. Patent Application No. 16 / 363,842, when measured on a dilute image plane (e.g., with low surface density of clusters or spots) with an MTF close to 100%, for clusters of clone-amplified labeled oligonucleotide molecules tethered to the substrate surface, the sample surface CNR (C) is greater than 12 (or much higher). 試料 ) This makes it possible to achieve the value C 試料 Assuming a sample surface CNR value of >12, the classification error rate is <0.1% (therefore, C 画像With goal >4), in some implementations, the minimum value of M(1 / d) can be determined as M(1 / d) = 4 / 12 ~ 33%. Therefore, by using a modulation transfer function threshold of at least 33%, the information content of the transmitted image can be preserved.

[0076] In the design method, the minimum separation distance (d) between two features or spots can be related to the optical resolution requirement (specified in terms of X (lp / mm) as above) as d = (1 mm) / X (for example, d is the minimum separation distance between two features or spots that can be fully resolved by the optical system). In some designs disclosed herein, if the objective of the design analysis is to increase or maximize the relevant information transfer, this design criterion may be relaxed to d = (1 mm) / X / A (where 2 > A > 1). For the same optical resolution of X (lp / mm), the value of d (the minimum resolvable spot separation distance on the sample surface) becomes smaller, thereby allowing the use of a higher feature density.

[0077] The design method uses the Nyquist criterion to determine the minimum spatial sampling frequency at the sample plane, where the spatial sampling frequency is S ≥ 2*X (where X is the optical resolution of the imaging system specified in terms of X (lp / mm) as above). When the spatial sampling frequency of the system is close to the Nyquist criterion (as is often the case), aliasing occurs when the resolution of the imaging system exceeds S because higher frequency information resolved by the optical system is not sufficiently sampled by the image sensor.

[0078] In some of the designs disclosed herein, an oversampling scheme may be used to further improve the information transfer capability of the imaging system, based on the relationship S = B * Y (where B ≥ 2 and Y is the MTF limit of the true optical system). As described above, X (lp / mm) corresponds to a practically non-zero (>33%) minimum modulation transfer value, while Y (lp / mm) is the limit of optical resolution, so the modulation at Y (lp / mm) is 0. Therefore, in the disclosed designs, Y (lp / mm) can favorably be significantly larger than X. For a value of B ≥ 2, the disclosed designs are oversampling with respect to the frequency X of the sample object (e.g., S ≥ B * Y > 2 * X).

[0079] The above relationship can be used to determine the system's magnification and may provide an upper limit for the image sensor's pixel size. The selection of the image sensor's pixel size, as well as the system's optical quality, should be aligned with the spatial sampling frequency required to reduce aliasing. The lower limit for the image sensor's pixel size can be determined based on photon throughput, as smaller pixels contribute more to the relative noise.

[0080] However, other design approaches are possible. For example, reducing the NA to less than 0.6 (e.g., 0.5 or less) can provide an increase in depth of field. Such an increase in depth of field may enable imaging of multiple surfaces, allowing simultaneous imaging of two surfaces at different depths, with or without refocusing. As discussed above, reducing the NA can reduce optical resolution. In some implementations, the use of a higher excitation beam power (e.g., 1 watt or more) can be used to generate a strong signal. Also, using inherently high-contrast samples (e.g., sample surfaces exhibiting a strong foreground signal and dramatically reduced background signal) can facilitate the acquisition of images with a high contrast-to-noise ratio (CNR) (e.g., with a CNR value of >20). This provides improved signal identification of base calling, for example, in nucleic acid sequencing applications. In some optical system designs disclosed herein, sample support structures, such as flow cells with hydrophilic surfaces, are used to reduce background noise.

[0081] In various implementations, the disclosed optical systems provide a large field of view (FOV). For example, some optical imaging systems, including objective and tube lenses, may provide an FOV of, for example, more than 2 mm or 3 mm. In some cases, the optical imaging system provides reduced magnification (e.g., less than 10x). Such reduced magnification may facilitate the design of a large FOV in some implementations. Despite the reduced magnification, the optical resolution of such systems may still be sufficient because detector arrays with small pixel sizes or pitches may be used. In some implementations, to satisfy the Nyquist theorem, an image sensor with a pixel size less than twice the optical resolution provided by the optical imaging system (e.g., objective and tube lenses) may be used.

[0082] Further designs are also possible. In some optical designs configured to provide multi-surface imaging that can simultaneously image two surfaces at different depths, the optical imaging system (e.g., objective lens and / or tube lens) is configured to reduce the optical aberration of imaging of the two surfaces (e.g., two planes) at each of those two depths compared to other locations at the other depth (e.g., other planes). Furthermore, the optical imaging system may be configured to reduce the aberration of imaging of the two surfaces (e.g., two planes) at each of those two depths by passing through a transmission layer (e.g., a layer of glass (e.g., a coverslip)) on the sample support structure, and through a solution containing the sample (e.g., an aqueous solution), or by contacting the sample on at least one of the two surfaces.

[0083] Multichannel fluorescence imaging module and system In some cases, the imaging modules or systems disclosed herein may include fluorescence imaging modules or systems. In some cases, the fluorescence imaging systems disclosed herein may include a single fluorescence excitation light source (for providing excitation light within a single wavelength or a single excitation wavelength range) and an optical path configured to deliver the excitation light to a sample (e.g., a fluorescence-tagged nucleic acid molecule or cluster thereof placed on a substrate surface). In some cases, the fluorescence imaging systems disclosed herein may include a single fluorescence emission imaging and detection channel (e.g., an optical path configured to collect fluorescence emitted by a sample and deliver an image of the sample (e.g., an image of the substrate surface on which the fluorescence-tagged nucleic acid molecule or cluster thereof is placed) to an image sensor or other photodetector). In some cases, the fluorescence imaging systems may include two, three, four, or more fluorescence excitation light sources and / or an optical path configured to deliver excitation light within two, three, four, or more excitation wavelengths (or within a range of two, three, four, or more excitation wavelengths). In some cases, the fluorescence imaging systems disclosed herein may include two, three, four, or more fluorescence emission imaging systems and detection channels configured to collect fluorescence at two, three, four, or more emission wavelengths (or within a range of two, three, four, or more emission wavelengths) emitted by a sample, and to deliver an image of the sample (e.g., an image of the substrate surface on which fluorescently tagged nucleic acid molecules or clusters thereof are located) to two, three, four, or more image sensors or other photodetectors.

[0084] Multi-surface imaging In some cases, 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 cases, 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, or more surfaces of a flow cell). Multiple surfaces of a sample support structure or flow cell device can be displaced axially from one another along the axial or z-direction. Multiple surfaces of a sample support structure or flow cell device may be inner surfaces facing a fluid channel(s) disclosed herein. Fluid channels or capillaries of a sample support structure or flow cell can be displaced axially from one another along the axial or z-direction.

[0085] In some cases, the high-resolution images provided by the disclosed imaging system can be used to monitor reactions occurring on two or more surfaces of a flow cell (e.g., nucleic acid hybridization, amplification, and / or sequencing reactions) as various reagents flow through or around the flow cell substrate. Figures 1A and 1B provide schematic diagrams of multi-surface support structures. Figures 64A–64F provide schematic diagrams of four-surface support structures as flow cells.

[0086] Figure 1A shows a multi-surface support structure, such as a flow cell, which includes internal flow channels through which an analyte or reagent can flow. The flow channels may be formed between first and second layers, upper and lower layers, and / or front and rear layers (e.g., first and second plates, upper and lower plates, and / or front and rear plates), as shown. One or more of the plates may include a glass plate, such as a coverslip. In some implementations, the layers include borosilicate glass, quartz, or plastic. The inner surfaces of these upper and lower layers provide walls for the flow channels, which help restrict the flow of analyte or reagent through the flow channels of the flow cell. In some designs, these inner surfaces are planar. Similarly, the upper and lower layers may be planar. In some designs, at least one additional layer (not shown) is placed between the upper and lower layers. This additional layer may have one or more pathways cut into it that help define one or more flow channels and control the flow of analyte or reagent through the flow channels. Further considerations regarding sample support structures (e.g., flow cells) can be found below.

[0087] Figure 1A schematically shows multiple fluorescent sample sites on the first and second inner surfaces, the top and bottom inner surfaces, and / or the front and rear inner surfaces of the flow cell. In some implementations, reactions may occur at these sites, causing the sample to bind and resulting in fluorescence emission from these sites (note that Figure 1A is schematic and not drawn to scale; for example, the size and spacing of the fluorescent sample sites may be smaller than shown).

[0088] Figure 1B shows another multi-surface support structure having two surfaces containing the fluorescent sample sites to be imaged. The sample support structure includes a substrate having first and second outer surfaces, top and bottom outer surfaces, and / or front and rear outer surfaces. In some designs, these outer surfaces are planar. In various implementations, the analyte or reagent flows across these first and second outer surfaces. Figure 1B schematically shows multiple fluorescent sample sites on the first and second outer surfaces, top and bottom outer surfaces, and / or front and rear outer surfaces of the sample support structure. In some implementations, reactions may occur at these sites, causing the sample to bind and resulting in fluorescence emission from these sites (note that Figure 1B is schematic and not drawn to scale; for example, the size and spacing of the fluorescent sample sites may be smaller than shown). For example, the support structures with one or more surfaces in Figures 64A-64E may have a sample site distribution on each surface similar to that shown in Figure 1A or Figure 1B.

[0089] In some cases, the fluorescence imaging modules and systems described herein may be configured to image fluorescent sample sites on each of a plurality of surfaces located at different distances from the objective lens. In some designs, only one of the plurality of surfaces is in focus at a time. Thus, in such designs, one of the surfaces is imaged in a first time and the other surfaces in a second time. Because images of multiple surfaces cannot be in focus simultaneously, the focus of the fluorescence imaging module may be changed after imaging one of the surfaces in order to image the next surface with equivalent optical resolution. In some designs, an optical compensation element may be introduced in 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 large enough to include two or more of the plurality of surfaces. In some implementations of the fluorescence imaging modules described herein, two or more surfaces may be imaged at the same time, for example, simultaneously. For example, the fluorescence imaging module may have a depth of field large enough to include two or more surfaces. In some cases, this increased depth of field can be achieved, for example, by reducing the numerical aperture of the objective lens (or microscope objective lens), as will be discussed in more detail below.

[0090] As shown in Figures 1A and 1B, the imaging optical system (e.g., objective lens) is positioned at a suitable distance from the surface (e.g., a distance corresponding to the working distance) to form a focused image of the surface on the image sensor of the detection channel. The first surface (e.g., 6418 in Figure 64C) may be between the objective lens and the second surface (e.g., 6419). For example, as shown in Figures 1A and 1B, the objective lens is positioned above the multiple surfaces, and the first surface is positioned above the second surface. As shown in Figure 64C, the second surface 6419 is above the third surface 6420, and the third surface is above the fourth surface 6421. The multiple surfaces may be at different depths. The surfaces are at different distances from one or more of the following: the fluorescence imaging module, the illumination and imaging module, the imaging optical system, or the objective lens. The multiple surfaces are separated from each other along the z-direction. The surfaces may be planar and separated from each other along a direction perpendicular to the plane. 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 surfaces may correspond to axial distances such as along the optical path of the excitation beam passing through the fluorescence imaging module and / or the objective lens, and / or along the optical axis. Thus, these surfaces may be separated from each other by distance in the axial (Z) direction, which may also be along the direction of the central axis of the excitation beam, and / or the optical axis of the objective lens, and / or the optical axis of the fluorescence imaging module. This separation may, for example, in some implementations, correspond to a flow channel in a flow cell between a first surface and a second surface, or between a third surface and a fourth surface. This separation may, for example, in some implementations, correspond to an interposer substrate in a flow cell between a second surface and a third surface.

[0091] In various designs, the objective lens (and possibly in combination with another optical system, e.g., a tube lens) has a depth of field and / or depth of focus that is at least equal to the axial (Z-direction) separation between two adjacent surfaces of a plurality of surfaces. In some embodiments, the depth of field and / or depth of focus is at least equal to the axial (Z-direction) separation between a first surface and a last surface (e.g., a fourth surface) along the optical path from the objective lens. Thus, the objective lens, alone or in combination with additional optical components, can simultaneously form focused images of at least two adjacent surfaces on an image sensor with one or more detection channels, such that these images have equivalent optical resolution. In some implementations, the imaging module may or may not need to refocus to capture images of at least two adjacent surfaces with equivalent optical resolution. In some implementations, the adaptive optics do not need to move in and out of the optical path of the imaging module to form focused images of the surfaces. Similarly, in some implementations, one or more optical elements (e.g., lens elements) in the imaging module (e.g., objective lens and / or tube lens) do not need to be moved axially, for example, along the first and / or second optical path (e.g., along the optical axis of the imaging optical system) to form a focused image of one of the surfaces (e.g., the first surface) compared to the position of the one or more optical elements used to form a focused image of another of the surfaces (e.g., the second, third, or fourth surface). However, in some implementations, the imaging module includes an autofocusing system configured to provide at least two adjacent surfaces that are in focus simultaneously. In various implementations, the sample is in focus so as to sufficiently resolve sample parts that are located close to each other in the lateral direction (e.g., the X and Y directions).Therefore, in various implementations, no optical elements enter the optical path between the sample support structure (e.g., the moving stage supporting the sample support structure) and the image sensor (or photodetector array) in at least one detection channel to form a focused image of the fluorescent sample portion on one surface of the sample support structure and on two other surfaces of the sample support structure. In various implementations, optical compensation is not used to form a focused image of the fluorescent sample portion on one surface of the sample support structure (e.g., the first surface) on the image sensor or photodetector array, but this is not the same as the optical compensation used to form a focused image of the fluorescent sample portion on another surface of the sample support structure (e.g., the second, third, or fourth surface) on the image sensor or photodetector array. Furthermore, in certain implementations, the optical elements in the optical path between the sample support structure (e.g., a moving stage supporting the sample support structure) and the image sensor in at least one detection channel are not tuned differently to form a focused image of a fluorescent sample area on one surface of the sample support structure (e.g., the first surface) and to form a focused image of a fluorescent sample area on another surface of the sample support structure (e.g., the second, third, or fourth surface). Similarly, in several different implementations, the optical elements in the optical path between the sample support structure (e.g., a moving stage supporting the sample support structure) and the image sensor in at least one detection channel are not moved by different amounts or in different directions to form a focused image of a fluorescent sample area on one surface of the sample support structure (e.g., the first surface) on the image sensor and to form a focused image of a fluorescent sample area on another surface of the sample support structure (e.g., the second, third, or fourth surface) on the image sensor. Any combination of the features described herein may be possible. For example, in some implementations, focused images of the first and second inner surfaces of the flow cell can be acquired without moving the optical compensator in and out of the optical path between the flow cell and at least one image sensor, and without moving one or more optical elements of the imaging system (e.g., objective lenses and / or tube lenses) along the optical path between them (e.g., the optical axis).For example, focused images of the first inner surface and the second and third inner surfaces of a flow cell can be obtained without moving one or more optical elements of the tube lens in or out of the optical path, or without moving one or more optical elements of the tube lens along the optical path between them (e.g., the optical axis).

[0092] One or more of the fluorescence imaging module, illumination optical path, imaging optical path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at multiple locations, such as planes corresponding to multiple surfaces on a flow cell or other sample support structure where a fluorescent sample portion is located. One or more of the fluorescence imaging module, illumination optical path, imaging optical path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at a selected location or plane relative to other locations or planes, such as the surface containing the fluorescent sample portion on a flow cell. For example, one or more of the fluorescence imaging module, illumination optical path, imaging optical path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at two depths or planes located at different distances from the objective lens compared to aberrations associated with other depths or planes at other distances from the objective lens. For example, optical aberrations may be smaller when imaging a surface in the range of approximately 1 to approximately 10 mm from the objective lens than at other locations. Furthermore, one or more of the fluorescence imaging module, illumination optical path, imaging optical path, objective lens, or tube lens may, in some cases, be configured to compensate for optical aberrations induced by the transmission of emitted light through one or more parts of the sample support structure, such as a layer including one of the surfaces to which the sample adheres, and possibly a solution in contact with the sample. This layer (e.g., a coverslip or flow cell wall) may include, for example, glass, quartz, plastic, or other transparent material having a refractive index that introduces optical aberrations.

[0093] Therefore, imaging performance may be substantially the same when imaging multiple surfaces (e.g., three or four surfaces). For example, the optical transfer function (OTF) and / or modulation transfer function (MTF) may be substantially the same when imaging multiple surfaces. Either or both of these transfer functions may be within, for example, 20%, 15%, 10%, 5%, 2.5%, or 1% of each other, or within any range formed by any of these values ​​when averaged over or at one or more specified spatial frequencies. Thus, the imaging performance metric may be substantially the same when imaging each of multiple surfaces of a flow cell without moving the optical compensator in and out of the optical path between the flow cell and at least one image sensor, and without moving one or more optical elements of the imaging system (e.g., objective lens and / or tube lens) along the optical path between them (e.g., optical axis). For example, the imaging performance metric may be substantially the same when imaging the first, second, third, and fourth surfaces of a flow cell without moving one or more optical elements of the tube lens in or out of the optical path, or without moving one or more optical elements of the tube lens along the optical path between them. In some embodiments, the optical path is the optical axis. Further consideration of the MTF is contained below and in U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020 (the entire application of which is incorporated herein by reference).

[0094] It will be understood by those skilled in the art that the disclosed imaging modules or systems may, in some cases, be standalone optical systems designed to image a sample or substrate surface. In some cases, they may include one or more processors or computers. In some cases, they may include one or more software packages that provide instrument control and / or image processing functions. In some cases, in addition to optical components such as light sources (e.g., solid-state lasers, dye lasers, diode lasers, arc lamps, 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 opticmechanical components such as XY moving stages, XYZ moving stages, piezoelectric focusing mechanisms, and electro-optic phase plates. In some cases, they may function as modules, components, subassemblies, or subsystems of larger systems designed for genomics applications (e.g., genetic testing and / or nucleic acid sequencing applications). For example, in some cases, they may function as modules, components, subassemblies, or subsystems of a larger system, further including light-shielding and / or other environmentally controlled housings, temperature control modules, flow cells and cartridges, fluid 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 genomic applications) will be discussed in more detail below.

[0095] Figures 2A and 2B show non-limiting examples of illumination and imaging modules 100 for multi-channel fluorescence imaging. The illumination and imaging module 100 includes an objective lens 110, an illumination source 115, multiple detection channels 120, and a first dichroic filter 130 (which may include a dichroic reflector or beam splitter). Some designs may include an autofocusing system (which may include an autofocusing laser 102) that projects a spot and monitors its size to determine if the imaging system is in focus. Some or all components of the illumination and imaging module 100 may be coupled to a base plate 105.

[0096] The illumination source or light source 115 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 emitting light within one or more excitation wavelength ranges (or bands). The light source may be a narrowband source emitting light within one or more narrower wavelength ranges. In some cases, the light source may produce a single isolated wavelength (or line) or multiple isolated wavelengths (or lines) corresponding to the desired excitation wavelength. In some cases, the lines may have several very narrow bandwidths. Exemplary light sources that may be suitable for use in the illumination source 115 include, but are not limited to, incandescent filaments, xenon arc lamps, mercury vapor lamps, light-emitting diodes, laser diodes or solid-state lasers, or other types of light sources. In some designs, as will be discussed below, the light source may include a polarization source, such as a linearly polarized light source. In some cases, 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 reflectors of one or more dichroic filters.

[0097] The illumination source 115 may further include one or more additional optical components, such as lenses, filters, optical fibers, or any other suitable transmissive or reflective optical systems, as appropriate, to output an excitation light beam having suitable characteristics toward the first dichroic filter 130. For example, a beam shaping optical system may be included, for example, to receive light from an optical emitter in the light source, generate a beam, and / or provide desired beam characteristics. Such an optical system may include, for example, a collimating lens configured to reduce light divergence and / or increase collimation and / or collimate light.

[0098] In some implementations, multiple light sources are included in the illumination and imaging module 100. In some such implementations, different light sources may produce light with different spectral characteristics, for example, light that excites different fluorescent dyes. In some implementations, the light produced by different light sources may be aligned and directed to form a combined excitation light beam. This combined excitation light beam may consist of excitation light beams from each of the light sources. The combined excitation light beam has more optical power than the individual beams that overlap to form the combined beam. For example, in some implementations, in embodiments including two light sources that produce two excitation light beams, the combined excitation light beam formed from the two individual excitation light beams may have an optical power that is the sum of the optical powers of the individual beams. Similarly, in some implementations, three, four, five, or more light sources may be included, and each of these light sources may output an excitation light beam that together forms a combined beam having an optical power that is the sum of the optical powers of the individual beams.

[0099] In some implementations, the light source 115 outputs a sufficient amount of light to produce sufficiently strong fluorescence emission. Stronger fluorescence emission can increase the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the image acquired by the fluorescence imaging module. In some implementations, the output of the light source and / or the excitation light beam derived therefrom (including the composite excitation light beam) may range in power from about 0.5 watts (W) to about 5.0 watts or more (as will be discussed in more detail below).

[0100] Referring again to Figures 2A and 2B, the first dichroic filter 130 is positioned relative to the light source to receive light from it. The first dichroic filter may include 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, for example, one or more spectral bands within the wavelength range of ultraviolet and blue. Similarly, the second spectral region may include one or more spectral bands, for example, one or more spectral bands extending from green to red and infrared wavelengths. Other spectral regions or wavelength ranges are also possible.

[0101] In some implementations, the first dichroic filter may be configured to transmit light from a light source to a sample support structure, such as a microscope slide, capillary, flow cell, microfluidic chip, or other substrate or support structure. The sample support structure supports and positions the sample, for example, a composition containing fluorescently labeled nucleic acid molecules or their complements, to the illumination and imaging module 100. Thus, the 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 placed or to which the sample is bound. In some cases, the sample may be placed in or bound to different localized regions or sites on at least one surface of the sample support structure.

[0102] In some cases, the support structure may include two, three, four, or more surfaces located at different distances (e.g., different positions or depths along the optical axis of the objective lens 110) from the objective lens 110 in which the sample is placed. As will be considered below, for example, the flow cell may include a fluid channel at least partially formed by first and second (e.g., upper and lower) inner surfaces, and the sample may be placed on the first inner surface, the second inner surface, or a localized area on both inner surfaces. The first and second surfaces may be separated by regions corresponding to the fluid channel through which the solution flows, and thus at different distances or depths from the objective lens 110 of the illumination and imaging module 100. The flow cell may include a second fluid channel axially displaced from the first channel, at least partially formed by third and fourth (e.g., upper and lower) inner surfaces, and the sample may be placed on the third inner surface, the fourth inner surface, or a localized area on both. The third and fourth surfaces may be separated by regions corresponding to second fluid channels through which the solution flows, and are therefore at different distances or depths from the objective lens 110 of the illumination and imaging module 100. The first and second fluid channels may be separated axially by an interposer substrate placed between them.

[0103] The objective lens 110 may be included in a first optical path between the first dichroic filter and the sample. This objective lens may be configured to have, for example, a focal length and working distance, and / or be positioned to focus light from a light source(s) onto the sample, for example, a microscope slide, capillary, flow cell, microfluidic tip, or the surface of another substrate or support structure. Similarly, the objective lens 110 may be positioned to have a suitable focal length and working distance, and / or 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).

[0104] In some implementations, the objective lens 110 may include a microscope objective lens, such as a commercially available objective lens. In some implementations, the objective lens 110 may include a custom objective lens. Examples of custom objective lenses and / or combinations of custom objective lenses with tube lenses are described below and in U.S. Provisional Application No. 62 / 962,723, filed January 17, 2020 (which is incorporated herein by reference in its entirety). The objective lens 110 may be designed to reduce or minimize optical aberrations at two, three, four, or more locations. For example, such locations may include planes corresponding to multiple surfaces of a flow cell. The objective lens 110 may be designed to reduce optical aberrations at selected locations or planes (e.g., the first and second surfaces of a multi-surface flow cell, or the first, second, third, and fourth surfaces of a tetra-surface flow cell) relative to other locations or planes in the optical path. For example, the objective lens 110 may be designed to reduce optical aberrations at two, three, or four depths or planes located at different distances from the objective lens compared to optical aberrations associated with other depths or planes at other distances from the objective lens. For example, in some cases, optical aberrations may be less when imaging the surface of a flow cell than those exhibited elsewhere in the region extending 1 to 10 mm from the front of the objective lens. Furthermore, the custom objective lens 110 may be configured to compensate for optical aberrations induced by the transmission of fluorescence emission light through one or more parts of the sample support structure, such as a layer containing one or more of the flow cell surfaces on which the sample is placed, or a layer containing a solution filling the fluid channels of the flow cell. These layers may include, for example, glass, quartz, plastic, or other transparent materials that have a refractive index and can cause optical aberrations.

[0105] In some implementations, the objective lens 110 may have a numerical aperture (NA) of 0.6 or greater (as will be discussed in more detail below). Such a numerical aperture can provide a reduction in depth of field and / or depth of focus, improved background discrimination, and increased image resolution.

[0106] In some implementations, the objective lens 110 may have a numerical aperture (NA) of 0.6 or less (discussed in more detail below). Such a numerical aperture may provide an increase in depth of field. Such an increase in depth of field may increase the ability to image planes at a certain distance (e.g., the first and second surfaces, the second and third surfaces, or the third and fourth surfaces). In some implementations, the objective lens 110 may have a numerical aperture (NA) of 0.5 or less (e.g., 0.4). Such a numerical aperture may reduce the optical aberrations that need to be compensated for in the optical system compared to when the NA value is higher. Such a numerical aperture may provide the ability to focus on and image additional image planes (e.g., the plane of the third or fourth surface of the flow cell) with minimal modification to the optical system design configured to image one or more surfaces of the flow cell (e.g., without needing to add or remove a compensator from the optical path from the objective lens to the imaged flow cell).

[0107] As discussed above, the flow cell may include first and second layers, each comprising first and second inner surfaces separated by a fluid channel through which the analyte or reagent can flow. The flow cell may also include third and fourth layers, each comprising third and fourth inner surfaces separated by a second fluid channel through which the analyte or reagent can flow. In some implementations, the objective lens 110 and / or illumination and imaging module 100 may be configured to provide a depth of field and / or depth of focus large enough to image at least two adjacent surfaces of the flow cell by sequentially refocusing the imaging module while imaging at least two surfaces with equivalent optical resolution, or by simultaneously ensuring a sufficiently large depth of field and / or depth of focus. In some cases, the depth of field and / or depth of focus may be at least the same as, or greater than, the distance separating the two adjacent surfaces of the flow cell being imaged. In some cases, two adjacent surfaces (e.g., the first and second inner surfaces of a two-surface flow cell or the third and fourth surfaces of a four-surface flow cell) may be separated by a distance ranging from approximately 10 μm to approximately 700 μm or more (as will be discussed in more detail below). In some cases, the depth of field and / or depth of focus may therefore range from approximately 10 μm to approximately 700 μm or more (as will be discussed in more detail below).

[0108] In some designs, the adaptive optics (e.g., “optical compensator” or “compensator”) may be moved in and out of the optical path within the imaging module (e.g., the optical path through which light collected by the objective lens 110 is delivered to the image sensor), enabling the imaging module to image the surface of the flow cell. The imaging module may be configured to image one surface (e.g., the first surface) if, for example, the first adaptive optics is included in the optical path between the objective lens and an image sensor or photodetector array configured to capture an image of the first surface. The imaging module may be configured to image another surface (e.g., the second surface) if, for example, the second adaptive optics is included in the optical path between the objective lens and an image sensor or photodetector array configured to capture an image of the second surface. In such designs, the imaging module may be configured to image yet another surface (e.g., the third surface) if the first and second adaptive optics are removed from or not included in the optical path between the objective lens 110 and an image sensor or photodetector array configured to capture an image of the third surface. The need for an optical compensator may be more pronounced when using an objective lens 110 with a high numerical aperture (NA) value, for example, for an aperture value of at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, or higher. In some implementations, the optical adaptive optics system (e.g., optical compensator or compensator) includes refractive optical elements such as lenses, plates of optically transparent material such as glass, plates of optically transparent material such as glass, or, in the case of polarized beams, quadradic plates or half-wave plates. Other configurations may be employed to allow surfaces to be imaged at different times. For example, one or more lenses or optical elements may be configured to translate in and out of the optical path between the objective lens 110 and the image sensor, or along the optical path.

[0109] In certain designs, the objective lens 110 is configured to be adjustable to change the numerical aperture (NA) of the optical system. In some embodiments, the objective lens 110 can be adjusted so that the NA of the optical system is within the range of 0.25 to 0.6. In some embodiments, the objective lens 110 can be adjusted so that the NA of the optical system is within the range of 0.35 to 0.55. In some embodiments, the objective lens 110 can be adjusted so that the NA of the optical system is 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 110. In some embodiments, the NA of the optical system can be adjusted by changing only the objective lens 110 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 the optical elements within the objective lens 110 without removing an existing objective lens and adding a new objective lens.

[0110] In some embodiments, the objective lens 110 may include an aperture stop, and adjusting the size of the aperture stop may result in a change in the numerical aperture (NA) of the optical system. In some embodiments, changing the size of the aperture stop does not involve moving the objective lens or any other optical element in, out of, or along the optical path between the objective lens and the image sensor. As a non-limiting example, changing the size of the aperture stop may involve rotating an optical element or a portion of the objective lens about the axial or longitudinal axis of the objective lens. As another non-limiting example, changing the size of the aperture stop may involve moving an optical element or a portion of the objective lens so as to be perpendicular to the axial or longitudinal axis of the objective lens.

[0111] In some embodiments, the numerical aperture (NA) of an optical system can be adjusted without changing the size of the aperture diaphragm. Alternatively, the objective lens and / or tube lens can be redesigned to change the NA of the optical system from an optical system having a first NA (e.g., NA of 0.5) to a predetermined different value (e.g., 0.4). The redesign may include, but is not limited to, changing one or more properties of the objective lens and / or tube lens, including diameter, size, magnification, length, cover thickness, working distance, and lens design that functions to correct aberrations.

[0112] In some embodiments, the NA of the optical system may be adjusted from the NA used for imaging conventional single-surface or double-surface flow cells (e.g., NA of 0.5 to NA of 0.4) to image a multi-surface flow cell having three or more axially displaced surfaces at a predetermined image quality. In some embodiments, the NA of the optical system may not need to be changed from the NA used for imaging conventional single-surface or double-surface flow cells (e.g., NA of 0.4) to image a multi-surface flow cell having three or more axially displaced surfaces at a predetermined image quality.

[0113] The optical system herein can be adjusted to change the numerical aperture (NA) (e.g., within the range of 0.5 to 0.4 or 0.4 to 0.5). The optical system can also be used to image flow cell devices whose total thickness falls within a range that matches the NA (e.g., within the range of approximately 220 μm to approximately 360 μm) using the same NA (e.g., NA=0.4). Thus, the optical system herein can provide flexibility and compatibility for imaging both (1) conventional flow cells having one or two surfaces and (2) multi-surface flow cells herein (e.g., having three, four, or more axially displaced surfaces) with sufficient image quality (e.g., a CNR of at least 5, 10, 15, or 20).

[0114] However, in certain designs, the objective lens 110 is configured to provide a sufficiently large depth of field and / or depth of field to allow the surface to be imaged with equivalent optical resolution without such adaptive optics entering and / or leaving the optical path within the imaging module (e.g., the optical path between the objective lens and the image sensor or photodetector array). In various designs, the objective lens 110 is configured to provide a sufficiently large depth of field and / or depth of field to allow the surface to be imaged with equivalent optical resolution without moving optics such as one or more lenses or other optical components that move along the optical path within the imaging module (e.g., the optical path between the objective lens and the image sensor or photodetector array). Examples of such objective lenses are described in more detail below.

[0115] In some implementations, the objective lens (or microscope objective lens) 110 may be configured to have a reduced magnification. For example, the objective lens 110 may be configured such that the fluorescence imaging module has a magnification of less than 2x to less than 10x (as will be discussed in more detail below). Such a reduced magnification may allow the design constraints to be modified so that other design parameters can be achieved. For example, the objective lens 110 may also be configured such that the fluorescence imaging module has a large field of view (FOV) in the range of approximately 1.0 mm to approximately 5.0 mm (e.g., in diameter, width, length, or longest dimension), as will be discussed in more detail below.

[0116] In some implementations, the objective lens 110 may be configured to provide the fluorescence imaging module with the above-described field of view such that the FOV has diffraction-limited performance, for example, an aberration of less than 0.15 wave over at least 60%, 70%, 80%, 90%, or 95% of the field of view, as will be discussed in more detail below.

[0117] In some implementations, the objective lens 110 may be configured to provide the fluorescence imaging module with the above-described field of view such that the FOV has diffraction-limited performance, for example, a Strehr ratio greater than 0.8 over at least 60%, 70%, 80%, 90%, or 95% of the field of view, as will be discussed in more detail below.

[0118] Referring again to Figures 2A and 2B, the first dichroic beam splitter or beam combiner is positioned in a first optical path between the light source and the sample to illuminate the sample with one or more excitation beams. This first dichroic beam splitter or combiner is also in one or more second optical paths to different optical channels used to detect fluorescence emission from the sample. Thus, the first dichroic filter 130 couples the first optical path of the excitation beam emitted by the illumination source 115 and the second optical path of the emitted light emitted by the sample into various optical channels, to which the light is directed to their respective image sensors or photodetector arrays to capture an image of the sample.

[0119] In various implementations, the first dichroic filter 130, for example, the first dichroic reflector or beam splitter or beam combiner, has a passband selected to transmit light from the illumination source 115 only within a specific wavelength band, or possibly multiple wavelength bands, that include the desired excitation wavelength(s). For example, the first dichroic beam splitter 130 includes a reflective surface, for example, a dichroic reflector having a spectral transmittance response configured to transmit light having at least some of the wavelengths output by the light source that form part of the excitation beam. The spectral transmittance response may be configured not to transmit (e.g., reflect instead) light of one or more other wavelengths, for example, one or more other fluorescence emission wavelengths. In some implementations, the spectral transmittance response may also be configured not to transmit (e.g., reflect instead) light of one or more other wavelengths output by the light source. Thus, the first dichroic filter 130 can be used to select which wavelength(s) of light output by the light source reach the sample. Conversely, the dichroic reflector in the first dichroic beam splitter 130 has a spectral reflectance response that reflects light having one or more wavelengths corresponding to desired fluorescence emission from the sample, and optionally reflects light having one or more wavelengths emitted from a light source not intended to reach the sample. Thus, in some implementations, the dichroic reflector has a spectral transmittance that includes one or more passbands that transmit light incident on the sample, and one or more stopbands that reflect light outside the passbands, e.g., one or more emission wavelengths not intended to reach the sample and optionally one or more wavelengths emitted by a light source. Similarly, in some implementations, the dichroic reflector has a spectral reflectance that includes one or more spectral regions configured to reflect one or more emission wavelengths, and optionally one or more wavelengths emitted by a light source not intended to reach the sample, and one or more regions that transmit light outside these reflective regions.The dichroic reflector included in the first dichroic filter 130 may include a reflective filter, such as an interference filter (e.g., a quadruple stack), configured to provide a suitable spectral transmission and reflection distribution. Figures 2A and 2B also show a dichroic filter 105 which may include a dichroic beam splitter or beam combiner, which may be used, for example, to direct an autofocusing laser 102 through an objective lens to a sample support structure.

[0120] In some embodiments, the dichroic filters 105, 130, and 530 may include one or more spectral passbands. This increases the intensity and SNR of the optical signal(s) passed through the filter. Thus, the dichroic filters(s) described herein can provide increased frequency response uniformity compared to conventional dichroic filters(s) with narrower spectral passbands.

[0121] The imaging module 100 shown in Figures 2A and 2B and discussed above is configured such that the excitation beam is transmitted to the objective lens 110 by a first dichroic filter 130. However, in some designs, the illumination source 115 is positioned relative to the first dichroic filter 130, and / or the first dichroic filter is configured (e.g., oriented) such that the excitation beam is reflected to the objective lens 110 by the first dichroic filter 130. Similarly, in some such designs, the first dichroic filter 130 is configured to transmit fluorescence emission from the sample, and possibly light of one or more wavelengths emitted from a light source not intended to reach the sample. As discussed below, designs in which fluorescence emission is transmitted instead of reflected may potentially reduce wavefront errors in the detected emission and / or alternatively may have other advantages. In all cases, in various implementations, the first dichroic reflector 130 is positioned in the second optical path to receive fluorescence emission from the sample, with at least some of them following the detection channel 120.

[0122] Figures 3A and 3B show the optical paths within the multi-channel fluorescence imaging module of Figures 2A and 2B. In the examples shown in Figures 2A and 3A, the detection channel 120 is configured to receive fluorescence emission from a sample that is transmitted by the objective lens 110 and reflected by the first dichroic filter 130. As mentioned above and further described below, in some designs, the detection channel 120 may be configured to receive a portion of the emitted light that is transmitted rather than reflected by the first dichroic filter. In any case, the detection channel 120 may include an optical system for receiving at least a portion of the emitted light. For example, the detection channel 120 may include one or more lenses, such as a tube lens, and one or more image sensors or detectors, such as a photodetector array (e.g., a CCD or CMOS sensor array), for imaging or otherwise generating a signal based on the received light. The tube lens may include one or more lens elements configured to form an image of the sample on the sensor or photodetector array in order to capture its image. Further consideration of detection channels is included in U.S. Provisional Application No. 62 / 962,723, filed on January 17, 2020 (which is incorporated herein by reference in its entirety). In some cases, improved optical resolution can be achieved using image sensors with relatively high sensitivity, small pixels, and a high pixel count, in conjunction with a suitable sampling scheme which may include oversampling or undersampling.

[0123] Figures 3A and 3B are ray tracing diagrams showing the optical paths of the illumination and imaging module 100 shown in Figures 2A and 2B. Figure 3A corresponds to a top view of the illumination and imaging module 100. Figure 3B corresponds to a side view of the illumination and imaging module 100. The illumination and imaging module 100 shown in these figures includes four detection channels 120. However, it will be understood that the disclosed illumination and imaging module can equally be implemented in systems including more or fewer than four detection channels 120. For example, the multi-channel systems disclosed herein can be implemented with as few as one detection channel 120, or up to two, three, four, five, six, seven, eight, or more than eight detection channels 120, without departing from the spirit or scope of this disclosure.

[0124] Non-limiting examples of the imaging module 100 shown in Figures 3A and 3B include four detection channels 120, a first dichroic filter 130 that reflects the emitted beam 150, a second dichroic filter (e.g., a dichroic beam splitter) 135 that splits the beam 150 into a transmitted and a reflective portion, and two channel-specific dichroic filters (e.g., dichroic beam splitters) 140 that further split the transmitted and reflective portions of the beam 150 between the individual detection channels 120. The dichroic reflectors in the dichroic beam splitters 135 and 140 for splitting the beam 150 between the detection channels are shown to be positioned at 45 degrees with respect to the central beam axis of the beam 150 or the optical axis of the imaging module. However, angles less than 45 degrees may be used, as will be discussed below, and may offer advantages such as a sharper transition from the passband to the stopband.

[0125] The different detection channels 120 include an imaging device 124, which may include an image sensor or a photodetector array (e.g., a CCD or CMOS detector array). The different detection channels 120 further include an optical system 126, such as lenses (e.g., one or more tube lenses, each containing one or more lens elements), positioned to focus on a portion of the emitted light entering the detection channel 120 at a focal plane coinciding with the plane of the photodetector array 124. The optical system 126 (e.g., tube lenses) in combination with the objective lens 110 is configured to capture an image of the sample (e.g., an image of the surface on a flow cell or other sample support structure after the sample has been bonded to its surface) by forming an image of the sample on the photodetector array 124. Thus, such an image of the sample may include multiple fluorescence-emitting speckles or regions over a spatial range of the sample support structure where the sample emits fluorescence light. The objective lens 110, together with the optical system 126 (e.g., tube lenses), may provide a field of view (FOV) that includes a portion or the entire sample. Similarly, the photodetector array 124 with different detection channels 120 may be configured to capture an image of the entire field of view (FOV) provided by the objective lens and the tube lens, or a portion thereof. In some implementations, some or all of the photodetector array 124 with detection channels 120 may detect emitted light emitted by a sample placed on or on the surface of a sample support structure, e.g., a flow cell, and record electronic data representing the image. In some implementations, some or all of the photodetector array 124 with detection channels 120 may detect characteristics of emitted light emitted by a sample without capturing and / or storing an image of the sample placed on the flow cell surface and / or an image of the entire field of view (FOV) provided by the objective lens and optics 126 and / or 122 (e.g., elements of the tube lens). In some implementations, the FOV of the disclosed imaging module (e.g., provided by the combination of the objective lens 110 and optics 126 and / or 122) may be in the range of about 1 mm to 5 mm (e.g., in diameter, width, length, or longest dimension), as considered below, for example.The field of view (FOV) can be selected, for example, to provide a balance between the magnification and resolution of the imaging module, and / or based on one or more characteristics of the image sensor and / or objective lens. For example, a relatively small FOV may be provided in conjunction with a smaller, faster imaging sensor to achieve high throughput.

[0126] Referring again to Figures 3A and 3B, in some implementations, the optical system 126 (e.g., a tube lens) in the detection channel may be configured to reduce optical aberrations in the image acquired using the optical system 126 in combination with the objective lens 110. In some implementations, there may be multiple detection channels for imaging at different emission wavelengths, and the optical system 126 (e.g., a tube lens) for each detection channel may have a different design to reduce aberrations at each emission wavelength to which that particular channel is configured to image. In some implementations, the optical system 126 (e.g., a tube lens) may be configured to reduce aberrations when imaging a particular surface (e.g., a plane, an object plane, etc.) on a sample support structure containing a fluorescent sample portion placed thereon, compared to other locations (e.g., other planes in object space). In some implementations, the optical system 126 (e.g., a tube lens) may be configured to reduce aberrations when imaging multiple surfaces (e.g., first and second planes, first and second object planes, etc.) on a sample support structure (e.g., a two- or four-surface flow cell) having a fluorescent sample portion placed thereon, compared to other locations (e.g., other planes in object space). For example, the optical system 126 in the detection channel (e.g., a tube lens) may be designed to reduce aberrations at two, three, or more depths or planes located at different distances from the objective lens compared to aberrations associated with other depths or planes at other distances from the objective lens. For example, optical aberrations may be less in the region about 1 to about 10 mm from the objective lens when imaging multiple surfaces than elsewhere. Furthermore, in some embodiments, the custom optical system 126 in the detection channel (e.g., a tube lens) may be configured to compensate for aberrations induced by the transmission of emitted light through one or more portions of the sample support structure, such as a layer containing one of the surfaces on which the sample is placed, and possibly adjacent to and in contact with the surface on which the sample is placed, such as a solution. The layer containing one of the surfaces on which the sample is placed may include, for example, glass, quartz, plastic, or other transparent material that has a refractive index and introduces optical aberrations.The custom optical system 126 within the detection channel (e.g., a tube lens) may be configured, for example in some implementations, to compensate for optical aberrations induced by the sample support structure, e.g., a coverslip or flow cell wall, or other sample support structure components, and optionally by a solution adjacent to and in contact with the surface on which the sample is placed.

[0127] In some implementations, the optical system 126 (e.g., a tube lens) in the detection channel is configured to have a reduced magnification. The optical system 126 (e.g., a tube lens) in the detection channel may be configured such that the fluorescence imaging module has a magnification of, for example, less than 10x, as will be discussed further below. Such a reduced magnification allows for modification of design constraints to achieve other design parameters. For example, the optical system 126 (e.g., a tube lens) may also be configured such that the fluorescence imaging module has a large field of view (FOV) of, for example, at least 1.0 mm (e.g., in diameter, width, length, or longest dimension), as will be discussed further below.

[0128] In some implementations, the optical system 126 (e.g., a tube lens) may be configured to provide the above-described field of view to the fluorescence imaging module such that the FOV has an aberration of less than 0.15 wave over at least 60%, 70%, 80%, 90%, or 95% of the field of view, as will be further discussed below.

[0129] Referring again to Figures 3A and 3B, in various implementations, the sample is positioned at or near the focal point 112 of the objective lens 110. As described above with reference to Figures 2A and 2B, a light source, such as a laser light source, provides an excitation beam to the sample to induce fluorescence. At least a portion of the fluorescence emission is collected as emitted light by the objective lens 110. The objective lens 110 transmits the emitted light toward a first dichroic filter 130, which reflects some or all of the emitted light toward different detection channels as a beam 150 incident on a second dichroic filter 135, each of which includes a photodetector array 124 (for example, an optical system 126 on the photodetector array that forms an image of the sample (e.g., multiple fluorescent sample areas on the surface of the sample support structure)).

[0130] As discussed above, in some implementations, the sample support structure includes a flow cell, such as a flow cell having multiple surfaces (e.g., two or more inner surfaces) containing sample areas that emit fluorescence. These surfaces may be separated from each other by distance in the longitudinal (Z) direction along the central axis of the excitation beam and / or the optical axis of the objective lens. This separation may correspond, for example, to one or more flow channels within the flow cell. The analyte or reagent may flow through the flow channel(s) and come into contact with the surface of the flow cell, thereby coming into contact with the binding composition such that fluorescence emission is emitted from multiple sites on the surface. The imaging optical system (e.g., objective lens 110) is positioned at a suitable distance from the sample (e.g., a distance corresponding to the working distance) to form a focused image of the sample on one or more detector arrays 124. As discussed above, in various designs, the objective lens 110 (and possibly in combination with the optical system 126) may have a depth of field and / or depth of focus that is at least equal to the longitudinal separation between two adjacent surfaces or between any two surfaces among a plurality of surfaces. Thus, the objective lens 110 and the optical system 126 (for each detection channel) can simultaneously form images of multiple surfaces on the photodetector array 124, and these images of the surfaces can be in focus and have equivalent optical resolution (or can be focused by slightly refocusing on the object to obtain an image with equivalent optical resolution). In various implementations, the adaptive optics do not need to move in and out of the optical path of the imaging module (e.g., in and out of the first and / or second optical path) to form a focused image of the surface (which has equivalent optical resolution). Similarly, in various implementations, when one or more optical elements (e.g., lens elements) within an imaging module (e.g., objective lens 110 or optical system 126) are used to form a focused image of a second surface, it is not necessary to move them longitudinally, for example, along the first and / or second optical paths to form a focused image of the first surface compared to the location of the one or more optical elements.In some implementations, the imaging module includes an autofocusing system configured to rapidly and sequentially refocus the imaging module onto one or more surfaces of a plurality of surfaces so that the image has equivalent optical resolution. In some implementations, the objective lens 110 and / or optical system 126 are configured to simultaneously focus on at least two of a plurality of surfaces (e.g., two adjacent surfaces) with equivalent optical resolution without moving the optical compensator in and out of a first and / or second optical path, and without moving one or more lens elements (e.g., the objective lens 110 and / or optical system 126 (e.g., a tube lens)) longitudinally along the first and / or second optical path. In some implementations, using novel objective lens and / or tube lens designs disclosed herein, images of surfaces acquired sequentially (e.g., with refocusing between surfaces) or simultaneously (e.g., without refocusing between surfaces) may be further processed using a suitable image processing algorithm to improve the effective optical resolution of the image so that the image has equivalent optical resolution. In various implementation configurations, the sample surface is sufficiently focused to resolve sample portions on the flow cell surface where the sample portions are located in close proximity in the lateral direction (e.g., X and Y directions).

[0131] As discussed above, dichroic filters may include 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 specific thicknesses. Therefore, the spectral response (e.g., transmission and / or reflection spectra) of dichroic filters implemented in a multi-channel fluorescence imaging module may depend at least partially on the angle of incidence or range of angles of incidence at which the excitation and / or emission beam light enters the dichroic filter. Such effects may be particularly important with respect to dichroic filters in the detection optical path (e.g., dichroic filters 135 and 140 in Figures 3A and 3B).

[0132] Figure 4 is a graph showing the relationship between dichroic filter performance and beam incidence angle (AOI). Specifically, the graph in Figure 4 shows the effect of the incidence angle on the dichroic filter's transition width or spectral span, corresponding to the wavelength range in which the spectral response (e.g., transmission spectrum and / or reflection spectrum) transitions between the passband and stopband regions of the dichroic filter. Therefore, a transmission edge (or reflection edge) with a relatively small spectral span (e.g., a small delta λ value in the graph in Figure 4) corresponds to a sharper transition between the passband and stopband regions, or between the transmission and reflection regions (or vice versa), while a transmission edge (or reflection edge) with a relatively large spectral span (e.g., a large delta λ value in the graph in Figure 4) corresponds to a less sharp transition between the passband and stopband regions. In various implementations, a sharper transition between the passband and stopband regions is generally desirable. Furthermore, it may also be desirable to have a consistent or relatively consistent increase in transition width across all or most of the field of view and / or beam region.

[0133] Therefore, a fluorescence imaging module in which the dichroic mirror is positioned at a 45-degree angle to the central beam axis of the emitted light or the optical axis of the optical path (e.g., the objective lens and / or tube lens) can have a transition width of approximately 50 nm in an exemplary dichroic filter, as shown in Figure 4. Since the emitted light beam is not parallelized and has some degree of divergence, the fluorescence imaging module can have an incidence angle range of approximately 5 degrees between the two sides of the beam. Thus, as shown in Figure 4, different portions of the emitted light beam can be incident on the channel-splitting dichroic filter at various incidence angles from 40 to 50 degrees. This relatively large range of incidence angles corresponds to a transition width range of approximately 40 nm to approximately 62 nm. This range of relatively large incidence angles thereby results in an increase in the transition width of the dichroic filter in the imaging module. Therefore, the performance of a multi-channel fluorescence imaging module can be improved by providing smaller incidence angles across the entire beam, thereby sharpening the transmission edge and enabling better discrimination between different fluorescence emission bands.

[0134] Figure 5 is a graph showing the relationship between the beam footprint size (DBS) on a dichroic filter and the beam incidence angle (DBS angle). In some cases, a smaller beam footprint may be desirable. For example, a smaller beam footprint allows the use of smaller dichroic filters to split the beam into different wavelength ranges. Using smaller dichroic filters then reduces manufacturing costs and facilitates the production of suitable, flat dichroic filters. As shown in Figure 5, any incidence angle greater than 0 degrees (e.g., perpendicular to the surface of the dichroic filter) results in an elliptical beam footprint with an area larger than the beam's cross-sectional area. An incidence angle of 45 degrees results in a large beam footprint on the dichroic reflector, which is more than 1.4 times the beam's cross-sectional area when incident at zero degrees.

[0135] Figures 6A and 6B schematically illustrate non-limiting exemplary configurations of dichroic filters and detection channels in a multi-channel fluorescence imaging module, where the dichroic mirrors are positioned at an angle of less than 45 degrees with respect to the central beam axis of the emitted light or the optical axis of the optical path (e.g., objective lens and / or tube lens). Figure 6A shows an imaging module 500 including multiple detection channels 520a, 520b, 520c, and 520d. Figure 6B is a detailed view of a portion of the imaging module 500 within the circle 5B shown in Figure 6A. As will be described in more detail, the configurations shown in Figures 6A and 6B include several embodiments that may result in significant improvements over conventional multi-channel fluorescence imaging module designs. In some cases, however, the fluorescence imaging modules and systems of this disclosure may be implemented with respect to one or a subset of the features described with respect to Figures 6A and 6B without departing from the spirit or scope of this disclosure.

[0136] The imaging module 500 shown in Figure 6A includes an objective lens 510 and four detection channels 520a, 520b, 520c, and 520d arranged to receive and / or image the emitted light transmitted by the objective lens 510. A first dichroic filter 530 is provided to combine the excitation optical path and the detection optical path. In contrast to the designs shown in Figures 2A and 2B and Figures 3A and 3B, the first dichroic filter 530 (e.g., a dichroic beam splitter or combiner) is configured to reflect light from the light source to the objective lens 510 and the sample, and to transmit fluorescence emission from the sample to the detection channels 520a, 520b, 520c, and 520d. A second dichroic filter 535 splits the beam of emitted light between at least two detection channels 520a, 520b by transmitting through a first portion 550a and reflecting through a second portion 550b. Additional dichroic filters 540a and 540b are provided to further split the emitted light. Dichroic filter 540a transmits at least a portion of the first portion 550a of the emitted light and reflects portion 550c to the third detection channel 520c. Dichroic filter 540b transmits at least a portion of the second portion 550b of the emitted light and reflects portion 550d to the fourth detection channel 520d. Although the imaging module 500 is shown with four detection channels, in various embodiments the imaging module 500 may include more or fewer detection channels, each having more or fewer dichroic filters as needed to provide a portion of the emitted light to each detection channel. For example, in some embodiments, the features of the imaging module 500 can be implemented with similar advantageous effects in an imaging module that includes only two detection channels 520a and 520b and omits the additional dichroic filters 540a and 540b. In some implementations, only one detection channel may be included. Alternatively, three or more detection channels may be used.

[0137] The detection channels 520a, 520b, 520c, and 520d shown in Figure 6A may include some or all of the same or similar components as those of the detection channel 120 shown in Figures 2A to 3B. For example, different detection channels 520a, 520b, 520c, and 520d may include one or more image sensors or photodetector arrays and may include one or more transmissive and / or reflective optical systems, 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.

[0138] The objective lens 510 is positioned to receive the emitted light emitted by fluorescence from the specimen. In particular, the first dichroic filter 530 is positioned to receive the emitted light collected and transmitted by the objective lens 510. As discussed above, and as shown in Figure 6A, in some designs, an illumination source such as a laser light source (e.g., illumination source 115 in Figures 2A and 2B) is positioned to provide an excitation beam incident on the first dichroic filter 530 such that the first dichroic filter 530 reflects the excitation beam back to the same objective lens 510 that transmits the emitted light in the epifluorescence configuration. In some other designs, the illumination source may be directed to the specimen by other optical components along different optical paths that do not include the same objective lens 510. In such configurations, the first dichroic filter 530 may be omitted.

[0139] Similarly, as discussed above and as shown in Figure 6A, the detection optics (including, for example, 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 positioned on the transmission path of the first dichroic filter 530 rather than on the reflection path of the first dichroic filter 530. In one example implementation, the objective lens 510 and the detection optics are positioned such that the objective lens 510 directly transmits the beam of emitted light 550 toward the second dichroic filter 535. The wavefront quality of the emitted light may be somewhat reduced by the presence of the first dichroic filter 530 along the path of the beam of emitted light 550 (for example, by imparting some wavefront error to the beam 550). However, the wavefront error introduced by the beam transmitted through the dichroic reflector of the dichroic beam splitter is generally significantly smaller (e.g., an order of magnitude smaller) than the wavefront error of the beam reflected from the dichroic reflector of the dichroic beam splitter. Therefore, the wavefront quality of the emitted light and the subsequent imaging quality in a multi-channel fluorescence imaging module can be substantially improved by positioning the detection optics along the transmitted beam path of the first dichroic filter 530 rather than along the reflected beam path.

[0140] Referring further to Figure 6A, within the detection optics of the imaging module 500, dichroic filters 535, 540a, and 540b are provided to split the beam 550 of emitted light between detection channels 520a, 520b, 520c, and 520d. For example, the dichroic filters 535, 540a, and 540b split the beam 550 based on wavelength such that a first detection channel 520a can receive a first wavelength or wavelength band of emitted light, a second detection channel 520b can receive a second wavelength or wavelength band of emitted light, a third detection channel 520c can receive a third wavelength or wavelength band of emitted light, and a fourth detection channel 520d can receive a fourth wavelength or wavelength band of emitted light. In some implementations, the detection channels can receive multiple separated wavelengths or wavelength bands.

[0141] In contrast to the multi-channel fluorescence imaging module designs shown in Figures 2A and 2B, and Figures 3A and 3B, the imaging module 500 has dichroic filters 535, 540a, and 540b positioned at an incident angle of less than 45 degrees with respect to the central beam axis of the incident beam. As shown in Figure 6B, the different beams 550, 550a, and 550b have their respective central beam axes 552, 552a, and 552b. In various implementations, the central beam axes 552, 552a, and 552b are located at the center of the beam cross-section perpendicular to the beam propagation direction. These central beam axes 552, 552a, and 552b may correspond to the optical axes of objective lenses and / or optical systems in separate channels, e.g., the optical axes of their respective tube lenses. Figure 6B shows additional rays 554, 554a, and 554b for each beam 550, 550a, and 550b to illustrate the diameters of each beam 550, 550a, and 550b. The beam diameter can be defined, for example, as the full width at half maximum, D4σ (e.g., 4×σ, where σ is the standard deviation of the horizontal or vertical peripheral distribution of the beam, respectively), or the second moment width, or any other preferred definition of the beam diameter.

[0142] The central beam axis 552 of the emitted beam 550 can serve as a reference point for defining the angle of incidence of the beam 550 to the second dichroic filter 535. Thus, the “angle of incidence” (AOI) of the beam 550 may be the angle between the central beam axis 552 of the incident beam 550 and a line N perpendicular to the surface on which the beam is incident, for example, the dichroic reflecting surface. When the emitted beam 550 is incident on the dichroic reflecting surface of the second dichroic filter 535 at the angle of incidence AOI, the second dichroic filter 535 transmits a first portion 550a of the emitted light (e.g., the portion having wavelengths within the passband region of the second dichroic filter 535) and reflects a second portion 550b of the emitted light (e.g., the portion having wavelengths within the stopband region of the second dichroic filter 535). The first part 550a and the second part 550b can be similarly described with respect to the central beam axes 552a and 552b, respectively. As described above, the optical axis can be used alternatively or additionally.

[0143] In the exemplary configurations of Figures 6A and 6B, the second dichroic filter 535 is positioned so that the central beam axis 552 of the beam 550 is incident at an incident angle of 30 degrees. Similarly, the additional dichroic filters 540a and 540b are positioned so that the central beam axes 552a and 552b of the first and second portions 550a and 550b of the beam 550 are also incident at an incident angle of 30 degrees. However, in various implementations, these incident angles may be other angles smaller than 45 degrees. In some cases, for example, the incident angles may be in the range of about 20 degrees to about 45 degrees, as will be further considered below. Furthermore, the incident angles of each of the dichroic filters 535, 540a, and 540b do not necessarily have to be the same. In some embodiments, some or all of the dichroic filters 535, 540a, and 540b may be positioned so that their incident beams 550, 550a, and 550b have different incident angles. As described above, the angle of incidence can be relative to the optical system within the imaging module, for example, the objective lens and / or optical system (e.g., tube lens) in the detection channel, as well as the dichroic reflector in each dichroic beam splitter. When specifying the AOI using the optical axis, the same range and values ​​of the angle of incidence apply.

[0144] In a fluorescence imaging module system, the emitted beams 550, 550a, and 550b are typically divergent beams. As described above, the emitted beam can have a beam divergence large enough that the region of the beam within the beam diameter is incident on the dichroic filter at an incident angle that differs by up to 5 degrees or more with respect to the incident angle of the central beam axis and / or optical axis of the optical system. In some designs, the objective lens 510 may be configured to have an F-number or numerical aperture selected, for example, to produce a smaller beam diameter with respect to a given field of view of the microscope. In one example, the F-number or numerical aperture of the objective lens 510 may be selected so that the entire diameter of the beams 550, 550a, and 550b is incident on the dichroic filters 535, 540a, and 540b at an incident angle of, 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 or less with respect to the incident angle of the central beam axis 552, 552a, and 552b.

[0145] In some implementations, the focal length of the objective lens suitable for generating such a narrow beam diameter may be longer than that typically used in fluorescence microscopes or imaging systems. For example, in some implementations, the focal length of the objective lens may be in the range of 20 mm to 40 mm, as will be further discussed below. In one example, an objective lens 510 having a focal length of 36 mm can generate a beam 550 characterized by a divergence small enough that light across the entire diameter of the beam 550 enters the second dichroic filter 535 at an angle of incidence of 2.5 degrees or less of the central beam axis.

[0146] Figures 7 and 8 provide graphs illustrating improved dichroic filter performance according to embodiments of the imaging module configurations of Figures 6A and 6B (or any of the imaging module configurations disclosed herein). The graph in Figure 7 is similar to the graph in Figure 4 and shows the effect of the incident angle on the dichroic filter transition width (e.g., spectral span at the transmission edge). Figure 7 shows an example where the orientation of the dichroic filter (e.g., dichroic filters 535, 540a, and 540b) and the dichroic reflecting surface within it are such that the incident beam has an incident angle of 30 degrees instead of 45 degrees. Figure 7 shows how this reduced incident angle significantly improves the sharpness and uniformity of the transition width across the entire beam diameter. For example, with an incident angle of 45 degrees at the central beam axis, the transition width range is approximately 40 nm to approximately 62 nm, while with an incident angle of 30 degrees at the central beam axis, the transition width range is approximately 16 nm to approximately 30 nm. In this example, the average transition width decreases from approximately 51 nm to approximately 23 nm, showing a sharper transition between the passband and stopband. Furthermore, the variation in transition width across the beam diameter decreases by almost 40%, from the 22 nm range to the 14 nm range, showing a more uniform and sharper transition across the beam region.

[0147] Figure 8 illustrates additional advantages that can be achieved in any of the imaging module configurations disclosed herein by selecting an appropriate F-number or numerical aperture for the objective lens to reduce beam divergence. In some implementations, longer focal lengths are used. In the example in Figure 8, the objective lens 510 has a focal length of 36 mm. This reduces the range of incident angles in beam 550 from 30 degrees ± 5 degrees to 30 degrees ± 2.5 degrees, with an appropriate numerical aperture (e.g., less than 5). In this design, the transition width range can be reduced to approximately 19 nm to approximately 26 nm. Compared to the improved system in Figure 7, the average transition width is substantially the same (e.g., a spectral span of approximately 23 nm), but the variation in transition width across the beam diameter is further reduced to a range of 7 nm. This represents a reduction of almost 70% compared to the transition width range shown in Figure 4.

[0148] Referring again to Figure 5, reducing the incidence angle at the central beam axis from 45 degrees to 30 degrees is even more advantageous because it reduces the beam spot size on the dichroic filter. As shown in Figure 5, an incidence angle of 45 degrees results in a beam footprint on the dichroic filter that has an area exceeding 1.4 times the cross-sectional area of ​​the beam. However, an incidence angle of 30 degrees results in a beam footprint on the dichroic filter that has an area of ​​only about 1.15 times the cross-sectional area of ​​the beam. Therefore, reducing the incidence angle on dichroic filters 535, 540a, and 540b from 45 degrees to 30 degrees results in an approximately 18% reduction in the beam footprint area on dichroic filters 535, 540a, and 540b. This reduction in beam footprint area makes it possible to use smaller dichroic filters.

[0149] Referring together to Figures 9A and 9B, the reduction of the incident angle from 45 degrees to 30 degrees can also provide improved performance with respect to surface deformation caused by the dichroic filter in any of the imaging module configurations disclosed herein, as shown by the improvement in the modulation transfer function. Generally, the larger the area of ​​the optical element, the greater the amount of surface deformation. As the area of ​​the dichroic filter increases, the amount of surface deformation increases, thereby introducing more wavefront errors into the beam. Figure 9A shows the effect of the bending angle on image quality degradation induced by applying one wave of peak-versus-valley (PV) spherical power to the final mirror. Figure 9B shows the effect of the bending angle on image quality degradation induced by applying 0.1 waves of PV spherical power to the final mirror. As shown in Figures 9A and 9B, the reduction of the incident angle to 30 degrees significantly reduces the effect of surface deformation in order to achieve performance close to the diffraction-limited performance of the detection optical system.

[0150] In some implementations of the imaging module disclosed herein, the polarization state of the excitation beam may be utilized to further improve the performance of the multichannel fluorescence imaging module disclosed herein. Referring back to Figures 2A, 2B, and 6A, for example, some implementations of the multichannel fluorescence imaging module disclosed herein have an epifluorescence configuration in which a first dichroic filter 130 or 530 merges the optical paths of the excitation light beam and the emitted light beam so that both the excitation light and the emitted light are transmitted through the objective lenses 110, 510. As discussed above, the illumination source 115 may include a light source such as a laser or other source and provide light that forms the excitation beam. In some designs, the light source may include a linearly polarized source and the excitation beam may be linearly polarized. In some designs, a polarization optical system is included to polarize the light and / or rotate the polarization of the light. For example, a polarizer such as a linear polarizer may be included in the optical path of the excitation beam to polarize the excitation beam. Some designs may include retarders to rotate linearly polarized light, such as half-wavelength retarders, multiple quarter-wavelength retarders, or retarders with other amounts of retardation.

[0151] A linearly polarized excitation beam, when incident on any dichroic filter or other planar interface, may be p-polarized (e.g., having an electric field component parallel to the incident plane), s-polarized (e.g., having an electric field component perpendicular to the incident plane), or have a combination of p- and s-polarized states in the beam. The p- or s-polarized state of the excitation beam can be selected and / or modified by selecting the orientation of the illumination source 115 and / or one or more of its components with respect to the first dichroic filters 130, 530 and / or any other surface with which the excitation beam interacts. In some implementations where the light source outputs linear polarization, the light source may be configured to provide s-polarization. For example, the light source may include an emitter such as a solid-state laser or laser diode, which can be rotated about its optical axis or beam axis to orient the linear polarization it outputs. Alternatively, or in addition to this, a retarder may be used to rotate the linear polarization about the optical axis or beam axis. As discussed above, in some implementations, for example, if the light source does not output polarization, a polarizer placed in the optical path of the excitation beam can polarize the excitation beam. In some designs, for example, a linear polarizer is placed in the optical path of the excitation beam. This polarizer may be rotated to provide the appropriate orientation of linear polarization to provide s-polarization.

[0152] In some designs, linearly polarized light is rotated around the optical axis or the central axis of the beam so that s-polarized light is incident on the dichroic reflector of a dichroic beam splitter. When s-polarized light is incident on the dichroic reflector of a dichroic beam splitter, the transition between the passband and stopband is sharper, in contrast to when p-polarized light is incident on the dichroic reflector of a dichroic beam splitter.

[0153] As shown in Figures 10A and 10B, the use of p-polarized or s-polarized states of the excitation beam can significantly affect the narrowband performance of any excitation filter, such as the first dichroic filters 130, 530. Figure 10A shows the transmission spectra of exemplary bandpass dichroic filters from 610 nm to 670 nm at incidence angles of 40 and 45 degrees. Here, the incident beam is linearly polarized with respect to the plane of the dichroic filter and is p-polarized. As shown in Figure 10B, changing the orientation of the light source relative to the dichroic filter so that the incident beam is s-polarized with respect to the plane of the dichroic filter significantly sharpens the edge between the passband and stopband of the dichroic filter. Therefore, the illumination and imaging modules 100, 500 disclosed herein may have an illumination source 115 advantageously oriented with respect to the first dichroic filters 130, 530 so that the excitation beam is s-polarized with respect to the plane of the first dichroic filters 130, 530. As discussed above, in some implementations, a polarizer, such as a linear polarizer, can be used to polarize the excitation beam. This polarizer can be rotated to provide the orientation of linear polarization corresponding to s-polarization. Also, as discussed above, other approaches can be used in some implementations to rotate linear polarization. For example, the orientation of polarization can be rotated using an optical retarder, such as a half-wavelength retarder or multiple quarter-wavelength retarders. Different configurations are also possible.

[0154] As discussed elsewhere in this specification, reducing the numerical aperture (NA) of the fluorescence imaging module and / or objective lens can increase the depth of field, enabling equivalent imaging of multiple surfaces (e.g., three, four, or more surfaces). Figures 11A–16B show that the MTFs of a first and second surface separated by a 1 mm glass are more similar when the numerical aperture is smaller than when the numerical aperture is larger.

[0155] Figures 11A and 11B show the MTF at the first surface (Figure 11A) and the second surface (Figure 11B) when NA is 0.3.

[0156] Figures 12A and 12B show the MTF at the first surface (Figure 12A) and the second surface (Figure 12B) when NA is 0.4.

[0157] Figures 13A and 13B show the MTF at the first surface (Figure 13A) and the second surface (Figure 13B) when NA is 0.5.

[0158] Figures 14A and 14B show the MTF at the first surface (Figure 14A) and the second surface (Figure 14B) when NA is 0.6.

[0159] Figures 15A and 15B show the MTF at the first surface (Figure 15A) and the second surface (Figure 15B) when NA is 0.7.

[0160] Figures 16A and 16B show the MTF at the first surface (Figure 16A) and the second surface (Figure 16B) when the NA is 0.8. The first and second surfaces in each of these figures correspond, for example, to the top and bottom surfaces of the flow cell.

[0161] Figures 17A and 17B provide plots of calculated Strehl ratios (e.g., the ratio of the peak light intensity focused or collected by the optical system to the peak light intensity focused or collected by an ideal optical system and a point light source) for imaging a second flow cell surface through a first flow cell surface. Figure 17A shows plots of the Strehl ratio as a function of the thickness of the intervening fluid layer (fluid channel height) when imaging a second flow cell surface through a first flow cell surface for different numerical apertures of objective lenses and / or optical systems. In some embodiments, the Strehl ratio decreases with increasing separation between two adjacent surfaces (e.g., the first and second surfaces). Therefore, one of the surfaces may have degraded image quality as the separation between the two surfaces increases. The decrease in surface imaging performance with increasing separation distance between two adjacent surfaces is reduced in imaging systems with smaller numerical apertures (e.g., NA of 0.5 or 0.4) compared to imaging systems with larger numerical apertures. Figure 17B shows a plot of the Strehr ratio as a function of numerical aperture when imaging the first flow cell surface and the second flow cell surface through an intervening aqueous layer having a thickness of 0.1 mm. The loss of imaging performance at higher numerical apertures may be due to increased fluid-induced optical aberrations when imaging the second surface. As the NA increases, the optical aberrations introduced by the fluid when imaging the second, third, or fourth surface increase, which can significantly degrade image quality. However, generally, reducing the numerical aperture of an optical system reduces the achievable resolution. This loss of image quality can be at least partially offset by providing an increased contrast-to-noise ratio on the sample surface (or object plane), for example by using chemicals that enhance the fluorescence emission of labeled nucleic acid clusters and / or reduce background fluorescence emission for nucleic acid sequencing applications. In some cases, sample support structures including, for example, hydrophilic substrate materials and / or hydrophilic coatings may be used. In some cases, such hydrophilic substrates and / or hydrophilic coatings can reduce background noise.For example, further considerations of sample support structures, hydrophilic surfaces and coatings, and methods for improving the contrast-to-noise ratio in nucleic acid sequencing applications can be found below.

[0162] In some implementations, one or more of the fluorescence imaging system, illumination and imaging module 100, imaging optical system (e.g., optical system 126), objective lens, and / or tube lens are configured to have a reduced magnification, such as less than 10x, as will be further discussed below. Such reduced magnification may allow for adjustment of design constraints and the achievement of other design parameters. For example, one or more of the fluorescence microscope, illumination and imaging module 100, imaging optical system (e.g., optical system 126), objective lens, or tube lens may also be configured so that the fluorescence imaging module has a large field of view (FOV), for example, at least 3.0 mm (e.g., in diameter, width, height, or maximum dimension), as will be further discussed below. One or more of the fluorescence imaging system, illumination and imaging module 100, imaging optical system (e.g., optical system 126), objective lens, and / or tube lens may be configured to provide the fluorescence microscope with such a field of view, such that the FOV has an aberration of less than 0.1 wave over at least 80% of the field of view. Similarly, one or more of the fluorescence imaging system, illumination and imaging module 100, imaging optical system (e.g., optical system 126), objective lens, and / or tube lens may be configured such that the fluorescence imaging module has such an FOV and is diffraction-limited, or is diffraction-limited over such an FOV.

[0163] As discussed above, in various implementations, the disclosed optical system provides a large field of view (FOV). In some implementations, an increased FOV is easily obtained, in part, by using a larger image sensor or photodetector array. For example, the photodetector array may have an active area with a diagonal length of at least 15 mm, as will be discussed further below. As discussed above, in some implementations, the disclosed optical imaging system provides a reduced magnification, for example, less than 10x, which can facilitate the design of a large FOV. Despite the reduced magnification, the optical resolution of the imaging module may still be sufficient, since a detector array with a small pixel size or pitch may be used. The pixel size and / or pitch may be about 5 μm or less, for example, as will be discussed in more detail below. In some implementations, the pixel size is less than twice the optical resolution provided by the optical imaging system (e.g., objective lens and tube lens) in order to satisfy the Nyquist theorem. Therefore, the pixel dimensions and / or pitch of the image sensor(s) may be such that the spatial sampling frequency of the imaging module is at least twice the optical resolution of the imaging module. For example, the spatial sampling frequency of the photodetector array may be at least twice, at least 2.5 times, at least 3 times, at least 4 times, or at least 5 times, or any spatial sampling frequency within the range of any of these values, of the optical resolution of the fluorescence imaging module (e.g., illumination and imaging module, objective lens and tube lens, object lens and optical system 126 in the detection channel, sample support structure configured to support the sample support stage, or imaging optical system between the stage and the photodetector array).

[0164] A wide range of features relating to fluorescence imaging modules are discussed herein, but any of the features and designs described herein may be applied to other types of optical imaging systems, including but not limited to bright-field and dark-field imaging, and may also be applied to emission imaging or phosphorescence imaging.

[0165] Dual-wavelength excitation / four-channel imaging system Figure 18 shows a dual-excitation wavelength / 4-channel imaging system for two- or four-plane imaging applications. This system includes a combination of objective and tube lenses scanned perpendicular to the optical axis, providing wide-area imaging by, for example, tiling several images to produce a composite image with a much larger field of view (FOV) than each individual image. The system includes two excitation sources operating at different wavelengths, e.g., lasers or laser diodes, and an autofocusing laser. The two excitation light beams and the autofocusing laser beam are combined using a series of mirrors and / or dichroic reflectors and delivered to the surface of the flow cell through the objective lens. Fluorescence emitted by a labeled oligonucleotide (or other biomolecule) tethered to one of the flow cell surfaces is collected by the objective lens, transmitted through the tube lens, and directed to one of four imaging sensors according to the wavelength of the light emitted by the series of intermediate dichroic reflectors. Autofocus laser light reflected from the flow cell surface is collected by an objective lens, transmitted through a tube lens, and directed to an autofocus sensor by a series of intermediate dichroic reflectors. The system allows for precise focus to be maintained while the objective lens / tube lens combination is scanned perpendicular to the optical axis of the objective lens (by adjusting the relative distance between the flow cell surface and the objective lens using, for example, a precision linear actuator, moving stage, or focusing mechanism mounted on a microscope turret, thereby reducing or minimizing the reflected light spot size on the autofocus image sensor). When dual-wavelength excitation is used in combination with four-channel (e.g., four wavelength) imaging capabilities, high-throughput imaging of multiple surfaces of the flow cell is provided.

[0166] Multiplexed optical reading head In some cases, a miniaturized version of any of the imaging modules described herein may be assembled to generate a multiplexing readout head. This may be moved horizontally to the sample surface (e.g., the inner surface of a flow cell) in one or more directions to simultaneously image several sections of the surface. A non-limiting example of a multiplexing readout head is described recently in U.S. Published Patent Application No. 2020 / 0139375A1.

[0167] In some cases, for example, a miniaturized imaging module may include a “microfluorometer,” which includes an illumination or excitation light source such as an LED or laser diode (or the end of an optical fiber connected to an external light source), one or more lenses for parallelizing or focusing the illumination or excitation light, one or more dichroic reflectors, one or more optical filters, one or more mirrors, beam splitters, prisms, apertures, one or more objective lenses, one or more custom tube lenses for enabling imaging of multiple surfaces with minimal focusing adjustment as described elsewhere in this specification, one or more image sensors as described elsewhere in this specification, or any combination thereof. In some cases, the miniaturized imaging module (e.g., “microfluorometer”) may further include an autofocusing mechanism, a microprocessor, power and data transfer connectors, a light-shielding housing, etc. Thus, the resulting miniaturized imaging module may include an integrated imaging package or unit having a small form factor. In some cases, the minimum dimensions (e.g., width or diameter) of a miniaturized imaging module may be less than 5 cm, less than 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, less than 1.2 cm, less than 1 cm, less than 0.8 cm, or less than 0.6 cm. In some cases, the maximum dimensions (e.g., height or length) of a miniaturized imaging module may be less than 16 cm, less than 14 cm, less than 12 cm, less than 10 cm, less than 9 cm, less than 8 cm, less than 7 cm, less than 5 cm, less than 5 cm, less than 4.5 cm, less than 4 cm, less than 3.5 cm, less than 3 cm, less than 2.5 cm, less than 2 cm, less than 1.8 cm, less than 1.6 cm, less than 1.4 cm, less than 1.2 cm, or less than 1 cm. In some cases, one or more individual miniaturized imaging modules within a multiplexed readout head may include an autofocus mechanism.

[0168] In some cases, the multiplexing read heads described herein may hold assemblies of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more than twelve miniaturized imaging modules or microfluorometers in fixed positions relative to one another. In some cases, the optical design specifications and performance characteristics of individual miniaturized imaging modules or microfluorometers, for example, numerical aperture, field of view, depth of field, and image resolution, may be the same as those described elsewhere in this specification for other versions of the disclosed imaging modules. In some cases, the multiple individual miniaturized imaging modules may be arranged in a linear configuration including one, two, three, four, or more rows and / or columns. In some cases, the multiple individual miniaturized imaging modules may be arranged, for example, in a hexagonal close-packed configuration. In some cases, the multiple individual miniaturized imaging modules may be arranged in a circular or helical configuration, a random distribution configuration, or any other configuration known to those skilled in the art.

[0169] Figures 43A and 43B provide non-limiting schematic diagrams of multiplexed read heads disclosed herein. Figure 43A shows a side view of a multiplexed read head in which two rows (viewed from the end face side) of individual microfluorometers having common optical design specifications, e.g., numerical aperture, field of view, working distance, etc., are configured to image a common surface (e.g., the first inner surface of a flow cell). Figure 43B shows a top view of the same multiplexed read head, showing the overlapping imaging paths acquired by the individual microfluorometers of the multiplexed read head as the read head moves relative to the flow cell (and vice versa). In some cases, the individual fields of view of the individual microfluorometers may overlap, as shown in Figure 43B. In some cases, they may not overlap. In some cases, the multiplexed read head may be designed to align with and image a predetermined feature in the flow cell (e.g., individual fluid channels).

[0170] Figures 44A and 44B provide non-limiting schematic diagrams of a multiplexed readout head, in which a first subset of multiple individual miniaturized imaging modules is configured to image a first sample surface (e.g., the first inner surface of a flow cell), and a second subset of multiple individual miniaturized imaging modules is configured to image a second sample surface (e.g., the second inner surface of a flow cell) simultaneously or sequentially. Figure 44A shows a side view of the multiplexed readout head, in which a first subset of individual microfluorometers is configured to image, for example, the first or upper inner surface of a flow cell, and a second subset is configured to image a second surface (e.g., the second or lower inner surface of a flow cell). Figure 44B shows a top view of the multiplexed readout head of Figure 44A, showing the imaging paths acquired by the individual microfluorometers of the multiplexed readout head. In some cases, the individual fields of view of the individual microfluorometers in a given subset may overlap. In some cases, they may not overlap. In some cases, the multiplexed readout head may be designed so that individual miniaturized imaging modules of a first and second subset align with and image predetermined features (e.g., individual fluid channels) within the flow cell.

[0171] In some embodiments, the multiplexing read head may further include a third subset of a plurality of individual miniaturized imaging modules configured to image a third sample surface (e.g., a third inner surface of a flow cell), and a fourth subset of a plurality of individual miniaturized imaging modules configured to image a fourth sample surface (e.g., a fourth inner surface of a flow cell) simultaneously or sequentially.

[0172] Improved or optimized objective and / or tube lenses for use with thicker coverslips Existing design techniques include designing objective lenses to optimize image quality when acquiring images through thin (e.g., less than 200 μm thick) microscope coverslips, and / or using commonly available off-the-shelf microscope objective lenses. When used to image both sides of a fluid channel or flow cell, the extra height of the gap between the two surfaces (e.g., the height of the fluid channel; typically around 50 μm to 200 μm) introduces optical aberrations into the image captured on the non-optimal side of the fluid channel, thereby causing a decrease in optical resolution. This is mainly because the additional gap height is significantly larger compared to the thickness of the optimal coverslip (typical fluid channel or gap height is 50 to 200 μm, while the coverslip thickness is less than 200 μm). Another common design technique is to utilize an additional “compensator” lens in the optical path when imaging is performed on the non-optimal side of the fluid channel or flow cell. This “compensator” lens and the mechanism required to move it in and out of the optical path ensure that all surfaces of the flow cell are imaged. Compensators further increase system complexity and imaging system downtime, and can degrade image quality due to vibration or movement.

[0173] In this disclosure, the imaging system is designed for compatibility with flow cell consumables including thicker coverslips or flow cell walls (thickness ≥ 700 μm). The objective lens design may be improved or optimized for coverslips equal to half the effective gap thickness (e.g., 700 μm + 1 / 2 * fluid channel (gap) height) in addition to the true coverslip thickness. This design significantly reduces the impact of gap height on the image quality of multiple surfaces of the fluid channel, as the gap height is small relative to the total thickness of the coverslip and therefore has a reduced impact on optical quality, thus allowing for a balance in the optical quality of the surface images.

[0174] Further advantages of using thicker coverslips include improved control over manufacturing thickness tolerances, as well as a reduced likelihood of the coverslip deforming due to heat and mounting-induced stresses. Thickness errors and deformation of the coverslip can negatively impact the imaging quality of all surfaces of the flow cell.

[0175] To further improve imaging quality for sequencing applications, our optical system design focuses on improving or optimizing the MTF in the mid-to-high spatial frequency range, which is best suited for imaging and resolving small spots or clusters (e.g., through improvements or optimizations to the objective lens and / or tube lens design).

[0176] Improved or optimized tube lens design for use with commercially available off-the-shelf objective lenses. For low-cost sequencer design, the use of commercially available off-the-shelf objective lenses may be preferable because they are relatively inexpensive. However, as mentioned above, low-cost off-the-shelf objective lenses are mostly optimized for use with thin coverslips of about 170 μm in thickness. In some cases, the disclosed optical systems can utilize tube lens designs that compensate for thicker flow cell coverslips while enabling high image quality of the flow cell surface in multi-surface imaging applications. In some cases, the tube lens designs disclosed herein enable high-quality imaging of multiple surfaces of a flow cell without moving the optical compensator in and out of the optical path between the flow cell and the image sensor, without moving one or more optical elements or components of the tube lens along the optical path, and without moving one or more optical elements or components of the tube lens in and out of the optical path.

[0177] Figure 19 provides a ray trace diagram of a low-light objective lens design improved or optimized for imaging the opposite surface of a 0.17 mm thick coverslip. The modulation transfer function plot of this objective lens, shown in Figure 20, demonstrates near-diffraction-limited imaging performance when used with the designed 0.17 mm thick coverslip.

[0178] Figure 21 provides a plot of the modulation transfer function as a function of spatial frequency when imaging the opposite surface of a 0.3 mm thick coverslip using the same objective lens shown in Figure 19. The relatively small deviation of the MTF value over a spatial frequency range of approximately 100 to 800 lines / mm (or cycles / mm) indicates that the resulting image quality is still good even when using a 0.3 mm thick coverslip.

[0179] Figure 22 provides a plot of the modulation transfer function as a function of spatial frequency when imaging a surface separated by a 0.1 mm thick aqueous fluid layer from the opposite surface of a 0.3 mm thick coverslip (for example, under conditions encountered when imaging a distant surface in multi-face imaging of a flow cell), using the same objective lens shown in Figure 19. As can be seen from the plot in Figure 22, imaging performance degrades, as indicated by the deviation of the MTF curve for the ideal diffraction-limited case over a spatial frequency range of approximately 50 lp / mm to approximately 900 lp / mm.

[0180] Figures 23 and 24 provide plots of the modulation transfer function as a function of spatial frequency for the upper (or proximal) inner surface (Figure 23) and the lower (or distal) inner surface (Figure 24) of the flow cell when imaging is performed through a 1.0 mm thick coverslip using the objective lens shown in Figure 19, and when the upper and lower inner surfaces are separated by a 0.1 mm thick aqueous fluid layer. It can be seen that the imaging performance is significantly reduced for both surfaces.

[0181] Figure 25 provides a ray tracing diagram for a tube lens design that provides improved multi-plane imaging through a 1 mm thick coverslip when used in conjunction with the objective lens shown in Figure 19. The optical design 700, including the composite objective lens (lens elements 702, 703, 704, 705, 706, 707, 708, 709, and 710) and the tube lens (lens elements 711, 712, 713, and 714), has been improved or optimized for use in a flow cell containing a thick coverslip (or wall) (e.g., a coverslip greater than 700 μm thick and a fluid channel at least 50 μm thick), transmitting the internal image from the flow cell 701 to the image sensor 715 with dramatically improved optical image quality and a higher CNR.

[0182] In some cases, a tube lens (or tube lens assembly) may include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more optical lens elements, and the number of optical lens elements, the surface shape of each element, and the order in which they are arranged in the assembly may be improved or optimized to compensate for optical aberrations induced by the thick walls of the flow cell, and in some cases, allow the use of commercially available off-the-shelf objective lenses while still maintaining high-quality multi-plane imaging capabilities.

[0183] In some cases, as shown in Figure 25, the tube lens assembly may include, in order, a first asymmetrical biconvex lens 711, a second plano-convex lens 712, a third asymmetrical biconcave lens 713, and a fourth asymmetrical convex-concave lens 714.

[0184] Figures 26 and 27 provide plots of the modulation transfer function as a function of spatial frequency for the upper (or proximal) inner surface (Figure 26) and lower (or distal) inner surface (Figure 27) of the flow cell when imaging is performed through a 1.0 mm thick coverslip using the objective lens (corrected for a 0.17 mm coverslip) and tube lens combination shown in Figure 25, and when the upper and lower inner surfaces are separated by a 0.1 mm thick aqueous fluid layer. The achieved imaging performance is close to what would be expected from a diffraction-limited optical design.

[0185] Figure 28 provides a ray trace diagram of the tube lens design of this disclosure (left) as improved or optimized to provide high-quality multi-plane imaging performance. Since the tube lens is no longer infinity corrected, a well-designed null lens (right) may be used in combination with the tube lens for manufacturing and testing purposes to compensate for the non-infinity corrected tube lens.

[0186] Adaptation or optimization of imaging channel-specific tube lenses In the design of an imaging system, it is possible to improve or optimize both the objective lens and the tube lens for all imaging channels in the same wavelength range. Typically, the same objective lens is shared by all imaging channels (see, for example, Figure 18), and each imaging channel uses the same tube lens or has tube lenses that share the same design.

[0187] In some cases, the imaging systems disclosed herein may further include tube lenses for each imaging channel, where the tube lenses are independently modified or optimized for a particular imaging channel to reduce or minimize distortion and field curvature, for example, and to improve the depth of field (DOF) performance of each channel. Because the wavelength range (or bandpass) of each particular imaging channel is much narrower than the combined wavelength range of all channels, wavelength or channel-specific adaptations or optimizations of the tube lenses used in the disclosed systems result in significant improvements in imaging quality and performance. These channel-specific adaptations or optimizations result in improved image quality of multiple surfaces of a flow cell in multi-surface imaging applications.

[0188] Multi-surface imaging in a flow cell without fluid present Optimal imaging performance of multiple surfaces of a flow cell typically requires a motion-driven compensator to correct optical aberrations induced by the fluid within the flow cell (typically involving a fluid layer thickness of approximately 50–200 μm). In some cases of the disclosed optical system designs, a first inner surface of the flow cell can be imaged while the fluid is present within the flow cell. Once the sequencing chemical cycle is complete, the fluid can be extracted from the flow cell to image the surface below the first surface. Similarly, the fluid can be extracted from the flow cell from the first and second surfaces to image a third surface. Thus, in some cases, the image quality of the underside can be maintained without the use of a compensator.

[0189] Compensation for optical aberrations and / or vibrations using electro-optical phase plates In some cases, using an electro-optic phase plate (or other corrective lens) in combination with the objective lens can improve image quality without requiring the removal of fluid from the flow cell by canceling out optical aberrations induced by the presence of fluid. In some cases, the use of an electro-optic phase plate (or lens) can be used to eliminate the effects of vibrations resulting from the mechanical operation of motion-driven compensators, which can provide faster image acquisition and sequencing cycle times for genome sequencing applications.

[0190] Improvements to contrast-to-noise ratio (CNR), field of view (FOV), spectral separation, and timing design to increase or maximize information transfer and throughput. Another way to increase or maximize information transfer in imaging systems designed for genomics applications is to increase the size of the field of view (FOV) and reduce the time required to image a particular FOV. In a typical large-scale NA optical imaging system, the area is 1 mm 2 While it is common to acquire images within a field of view of approximately 2mm, the design of the imaging system in this disclosure employs an objective lens with a large FOV and a long working distance, thereby achieving a field of view of 2mm. 2 This makes it possible to image the above regions.

[0191] In some cases, the disclosed imaging systems are designed to be used in combination with proprietary low-binding substrate surfaces and DNA amplification processes. This reduces fluorescence background resulting from various confounding signals, including but not limited to nonspecific adsorption of fluorescent dyes to the substrate surface, nonspecific nucleic acid amplification products (e.g., nucleic acid amplification products that result in substrate surfaces between spots or features corresponding to clone-amplified clusters of nucleic acid molecules (e.g., specifically amplified colonies)), nonspecific nucleic acid amplification products that may occur within amplified colonies, and phased and pre-phased nucleic acid strands. When used in combination with the disclosed optical imaging systems, the low-binding substrate surfaces and DNA amplification processes that reduce fluorescence background can significantly reduce the time required to image each field of view (FOV).

[0192] In the system design of this disclosure, the required acquisition time can be further reduced through improvements or optimization of the acquisition sequence. Here, multiple channels of the fluorescence image are acquired simultaneously or at overlapping timings, and the spectral separation of the fluorescence signal is designed to reduce crosstalk between fluorescence detection channels and crosstalk between excitation light and the fluorescence signal(s).

[0193] The system design of this disclosure can further reduce the required imaging time through improvements or optimizations to the scanning motion sequence. In a typical approach, an XY moving stage is used to move the target FOV to a position below the objective lens, an autofocus step is performed to determine the optimal focal position, the objective lens is moved in the Z direction to the determined focal position, and an image is acquired. A series of fluorescence images are acquired by cycling through a series of target FOV positions. In terms of the information transfer duty cycle, information is transferred only during the fluorescence image acquisition portion of the cycle. In the imaging system design of this disclosure, an autofocus step is used to check for focal position errors by performing a single-step operation in which all axes (XYZ) are simultaneously repositioned. Additional Z movement is commanded only if the focal position error (e.g., the difference between the focal plane position and the sample plane position) exceeds a certain limit (e.g., a specified error threshold). Coupled with high-speed XY movement, this approach increases the system's duty cycle and therefore increases the imaging throughput per unit time.

[0194] In some embodiments, for example, the system execution time for completing the sequence determination analysis includes the imaging time and motion time of the target FOV relative to the objective lens.

[0195] In some embodiments, the imaging time for scanning the target FOV can be doubled by imaging multiple surfaces instead of a single surface, and the imaging time can be further doubled by imaging four surfaces instead of multiple surfaces. The operating time for moving the target FOV relative to the objective lens along the x, y, and / or z directions increases when imaging four surfaces instead of multiple surfaces, but the increase is much less than a doubling because the operating time in the z direction does not increase, and only the operating time in the x and y directions increases. Thus, the optical systems herein can increase the duty cycle of the system and increase the imaging throughput per unit time. For example, information is transferred only during the fluorescence image acquisition portion of the cycle. The amount of information is doubled by imaging four surfaces of the flow cell compared to two surfaces, but the execution time is much less than a doubling because only the operating time in the z direction increases. The system throughput per unit time for imaging a four-surface flow cell can be more than twice that of imaging a two-surface flow cell.

[0196] Furthermore, by matching the optical acquisition efficiency, modulation transfer function, and image sensor performance characteristics of the design to the expected fluorescence photon flux and dye efficiency (related to the dye extinction coefficient and fluorescence quantum yield) of the input excitation photon flux, the time required to acquire high-quality images (high contrast-to-noise ratio (CNR) images) can be reduced or minimized while considering background signal and system noise characteristics.

[0197] The combination of efficient image acquisition and improved or optimized step and settling times for the moving stage results in faster acquisition times (e.g., overall time required per field of view) and higher throughput imaging system performance.

[0198] The disclosed design may also include specifying image plane flatness, chromatic aberration focusing performance between fluorescence detection channels, sensor flatness, image distortion, and focus quality specifications, along with a large FOV and a fast image acquisition duty cycle.

[0199] Chromatic aberration focusing performance is further improved by individually aligning the image sensors for different fluorescence detection channels so that the best focal planes of each detection channel overlap. The design goal is to ensure that images are acquired within ±100 nm (or less) of the best focal plane of each channel over more than 90% of the field of view, and thus increase or maximize the transmission of individual spot intensity signals. In some cases, the disclosed designs further ensure that images over 99% of the field of view are acquired within ±150 nm (or less) of the best focal plane of each channel, and images over a more overall field of view are acquired within ±200 nm (or less) of the best focal plane of each imaging channel.

[0200] Illumination optical path design Another factor for improving the signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and / or throughput is increasing the illumination power density to the sample. In some cases, the disclosed imaging system may include an illumination path design that utilizes a high-power laser or laser diode coupled with a liquid light guide. The liquid light guide eliminates the optical speckle inherent in coherent light sources such as lasers and laser diodes. Furthermore, the coupled optics are designed to underfill the entrance of the liquid light guide. Underfilling the entrance of the liquid light guide reduces the effective numerical aperture of the illumination beam incident on the objective lens, and therefore improves the efficiency of light delivery to the sample surface through the objective lens. This design innovation makes it possible to achieve up to three times the illumination power density of conventional designs for large fields of view (FOV).

[0201] By utilizing angle-dependent discrimination of s and p polarization, in some cases the polarization of the illumination beam can be oriented to reduce the amount of backscattered and backreflected illumination light reaching the imaging sensor.

[0202] Lighting system In some cases, the disclosed imaging modules and systems may include structured illumination optics designs. This improves the effective spatial resolution of the imaging system, and therefore allows for the use of clone-amplified target nucleic acid sequences (clusters) on the flow cell surface at a higher surface density, thereby improving sequencing throughput. Structured illumination microscopy (SIM) utilizes a spatially structured (e.g., periodic) pattern of light for illumination of the sample surface and relies on the generation of interference patterns known as moiré fringes. Some images are acquired under slightly different illumination conditions, for example, by shifting and / or rotating the pattern of structured illumination to generate moiré fringes. The mathematical deconvolution of the resulting interference signals enables the reconstruction of super-resolution images with up to approximately twice the spatial resolution compared to those achieved using diffraction-limited imaging optics [Lutz (2011), “Biological Imaging by Superresolution Light Microscopy”, Comprehensive Biotechnology (Second Ed.), vol.1, pages 579-589, Elsevier, Feiner-Gracia, et al. (2018), “15-Advanced Optical Microscopy Techniques for the Investigation of Cell-Nanoparticle Interactions”, Smart Nanoparticles for Biomedicine: Micro and Nano Technologies, pages 219-236, Elsevier, Nylk, et al. (2019), “Light-Sheet Fluorescence Microscopy With Structured Light”, Neurophotonics and Biomedical Spectroscopy, pages 477-501, Elsevier]. An example of a structured illumination microscope imaging system is recently described in U.S. Patent Application Publication No. 2020 / 0218052 by Hong.

[0203] Figure 41 provides a non-limiting schematic diagram of an imaging system 4100 including a branched structured illumination optical design disclosed herein. The first branch (or arm) of the illumination optical path of system 4100 includes, for example, a light source (optical emitter) 4110A, an optical collimator 4120A for aligning the light emitted by light source 4110A, a diffraction grating 4130A oriented in a first direction with respect to the optical axis, a rotating window 4140A, and a lens 4150A. The second branch of the illumination optical path of system 4100 includes, for example, a light source 4110B, an optical collimator 4120B for aligning the light emitted by light source 4110B, a diffraction grating 4130B oriented in a second direction with respect to the optical axis, a rotating window 4140B, and a lens 4150B. The diffraction gratings 4130A and 4130B enable the projection of a pattern of light fringes onto the sample surface.

[0204] In some cases, light sources 4110A and 4110B may be non-coherent light sources (e.g., including one or more light-emitting diodes (LEDs)) or coherent light sources (e.g., including one or more lasers or laser diodes). In some cases, light sources 4110A and 4110B may include, for example, optical fibers coupled to LEDs, lasers, or laser diodes, which then output light beams parallelized by their respective collimator lenses 4120A and 4120B. In some cases, light sources 4110A and 4110B may output light of the same wavelength. In some cases, light sources 4110A and 4110B may output light of different wavelengths. Either light source 4110A or 4110B may be configured to output light of any wavelength and / or wavelength range described elsewhere in this specification. During imaging, light sources 4110A and 4110B may be switched on or off, for example, using a fast shutter (not shown) placed in the optical path, or by pulse-driving the light sources at a predetermined frequency.

[0205] In the example shown in Figure 41, the first illumination arm of system 4100 includes a fixed vertical grating 4130A used to project a first orientation grating pattern (e.g., a vertical fringe pattern) onto the sample surface (e.g., the first inner surface 4188 of the flow cell 4187), and the second illumination arm includes a fixed horizontal grating 4130B for projecting a second orientation grating pattern (e.g., a horizontal fringe pattern) onto the sample surface 4188. Advantageously, the diffraction gratings of the imaging system 4100 do not need to be mechanically rotated or moved during imaging in this non-limiting example, which can provide improved imaging speed, system reliability, and system repeatability. In some cases, the diffraction gratings 4130A and / or 4130B may be rotatable about their respective optical axes so that the angles between the fringe patterns projected onto the sample surface are adjustable.

[0206] As shown in Figure 41, in some cases the diffraction gratings 4130A and 4130B may be transmission-type diffraction gratings comprising multiple diffracting elements (e.g., parallel slits or grooves) formed on a glass substrate or other suitable surface. In some cases the grating may be implemented as a phase grating that provides periodic fluctuations in the refractive index of the grating material. In some cases the spacing of the grooves or features may be selected to diffract light at a suitable angle and / or adjusted to the minimum resolvable feature size of the imaged sample for the operation of the imaging system 4100. In other cases the diffraction grating may be a reflection-type diffraction grating.

[0207] In the example shown in Figure 41, the orientations of the vertical and horizontal fringe patterns are offset by approximately 90 degrees. In other cases, a diffraction grating with a different orientation may be used to produce an offset of approximately 90 degrees. For example, the diffraction grating may be oriented to project a fringe pattern offset by ±45 degrees from the x-axis or y-axis of the sample surface (e.g., the inner surface of the first flow cell) 4188. The configuration of the imaging system 4100 shown in Figure 41 may be particularly advantageous for a sample support surface (e.g., the inner surface 4188 of the flow cell 4187) containing regularly patterned features laid out on a rectangular grid, since the improvement in image resolution using a structured illumination approach can be achieved using only two vertical grating orientations (e.g., the vertical grating orientation and the horizontal grating orientation). The flow cell 4187 is not limited to having only two inner surfaces as shown in Figure 41. In some embodiments, the flow cell 4187 may have one or more surfaces as shown in Figures 64A–64F.

[0208] In the example of system 4100, diffraction gratings 4130A and 4130B may be configured to diffract the input illumination light beam to a series of intensity maxima resulting from constructive interference, according to the following relationship: m=order=d sin(θ) / λ

[0209] In the equation, d = distance between slits or grooves in the diffraction grating, θ = angle of incidence of illumination light with respect to the normal of the surface of the diffraction grating, λ = wavelength of illumination light, and m = integer value corresponding to the intensity maxima of the diffracted light (e.g., m = 0, ±1, ±2, etc.). In some cases, a specific order of diffracted illumination light, e.g., first-order (m = ±1) light, can be projected onto the sample surface (e.g., the inner surface 4188 of the flow cell). In some cases, for example, a vertical grating 4130A can diffract a parallelized light beam into first-order diffracted beams (±1st order), which can be focused onto the sample surface in a first orientation, and a horizontal grating 4130B can diffract a parallelized light beam into first-order diffracted beams, which can be focused onto the sample surface in a second orientation. In some cases, the zero-order beam and / or all other higher-order beams (e.g., m = ±2 or higher) can be blocked (e.g., filtered out) from the illumination pattern projected onto the sample surface 4188 using beam-blocking elements (not shown), such as order filters that can be inserted into the optical path after the diffraction grating.

[0210] In example 4100, each branch of the illumination system includes optical phase modulators or phase shifters 4140A and 4140B to phase shift the diffracted light transmitted or reflected by diffraction gratings 4130A and 4130B, respectively. During structured imaging, the optical phase of each diffracted beam can be shifted by a certain percentage (e.g., 1 / 2, 1 / 2, 1 / 4, etc.) of the pitch (X) of each fringe in the structured pattern. In the example of Figure 41, the phase modulators 4140A and 4140B are implemented as rotating optical phase plates driven, for example, by a rotary actuator or other actuator mechanism, to rotate and modulate the optical path length of each diffracted beam. For example, optical phase plate 4140A may be rotated around a vertical axis to shift the image projected by the vertical grating 4130A on the sample surface 4188 to the left or right, and optical phase plate 4140B may be rotated around a horizontal axis to shift the image projected by the horizontal grating 4130B on the sample surface 4188 vertically.

[0211] In other implementations, other types of phase modulators that change the optical path length of the diffracted light (e.g., optical wedges mounted on a linear moving stage, etc.) may be used. Furthermore, although the optical phase modulators 4140A and 4140B are shown to be positioned after the diffraction gratings 4130A and 4130B, in other implementations they may be positioned at other locations in the illumination optical path. In some cases, a single optical phase modulator may operate in two different directions to produce different light fringe patterns, or the position of a single optical phase modulator may be adjusted using a single operation to simultaneously adjust the optical path lengths of both arms of the illumination optical path.

[0212] In the example shown in Figure 41, the optical component 4160 may be used to combine light from two illumination optical paths. The optical component 4160 may include, for example, a half-mirror (partially-silvered mirror), a dichroic mirror (depending on the wavelength of light output by light sources 4110A and 4110B), a mirror with a perforated pattern or patterned reflective coating (resulting in the combination of light from the two arms of the illumination system in a lossless or near-lossless manner (e.g., no significant loss of optical power other than a small amount of absorption by the reflective coating)), a polarizing beam splitter (if light sources 4110A and 4110B are configured to generate polarization), and so on. The optical component 4160 may be arranged such that light of a desired order of diffraction, reflected or transmitted by each of the diffraction gratings, is spatially resolved, and light of an undesirable order is blocked. In some cases, the optical component 4160 may allow primary light output by the first illumination optical path to pass through and reflect primary light output by the second illumination optical path. In some cases, the structured illumination pattern on the sample surface 4188 can be switched from vertical (e.g., using diffraction grating 4130A) to horizontal (e.g., using diffraction grating 4130B) by turning each light source on or off, or by opening and closing optical shutters in the optical path of the light sources. In other cases, the structured illumination pattern may be changed by using optical switches to alter the illumination optical path used to illuminate the sample surface.

[0213] Referring again to Figure 41, the lens 4170, a semi-reflection mirror or dichroic mirror 4180, and the objective lens 4185 may be used to focus the structured illumination light onto the sample surface 4188 (e.g., the first inner surface of the flow cell 4187). The light emitted, reflected, or scattered by the sample surface 4188 is then collected by the objective lens 4185, transmitted through the mirror 4180, and imaged by the image sensor or camera 4195. As described above, the mirror 4180 may be a dichroic mirror. The dichroic mirror reflects the structured illumination light received from each branch of the illumination optical path to the objective lens 4185, projecting it onto the sample surface 4188 and allowing the light emitted by the sample surface 4188 (e.g., fluorescence emitted at a different wavelength than the excitation light) to pass through for imaging onto the image sensor 4195.

[0214] In some cases, system 4100 may optionally include a custom tube lens 4190 as described elsewhere in this specification. This allows the focus of the imaging system to be shifted with minimal adjustment from the first inner surface 4188 to the second inner surface 4189, or to the third or fourth inner surface (not shown) of the flow cell 4187. In some cases, lens 4170 may include a custom tube lens as described elsewhere in this specification. This allows the focus of the illumination optical path to be shifted with minimal adjustment from the first inner surface 4188 to the second inner surface 4189, or to the third or fourth surface (not shown) of the flow cell 4187. In some cases, lens 4170 may be implemented to move along the optical axis to adjust the focus of the structured illumination pattern on the sample surface. In some cases, system 4100 may include an autofocusing mechanism (not shown) for adjusting the focus of the illumination light and / or the focus of the image on the plane of the image sensor 4195. In some cases, the system 4100 shown in Figure 41 can provide high optical efficiency because there is no polarizer in the optical path. The use of unpolarized light may or may not significantly affect the contrast of the illumination pattern, depending on the numerical aperture of the objective lens 4185.

[0215] For the sake of brevity, some optical systems of the imaging system 4100 may be omitted from Figure 41 and the preceding description. In this non-limiting example, system 4100 is shown as a single-channel detection system, but in other cases it may be implemented as a multi-channel detection system (e.g., using two different image sensors and appropriate optical systems, as well as light sources emitting at two different wavelengths). Furthermore, in this non-limiting example the illumination optical path of system 4100 is shown to include two branches, but in some cases it may be implemented to include, for example, three branches, four branches, or more than four branches, each of which includes a diffraction grating in a fixed or adjustable relative orientation.

[0216] In some cases, alternative illumination path optical designs may be used to generate structured illumination. For example, in some cases, a single large rotating optical phase modulator may be positioned behind optical component 4160 and used in place of optical phase modulators 4140A and 4140B to modulate the phase of both the vertical and horizontal diffraction gratings 4130A and 4130B. In some cases, instead of being parallel to the optical axis of one of the diffraction gratings, the rotation axis of a single rotating optical compensator may be offset by 45 degrees (or another angular offset) from the respective optical axes of the vertical and horizontal diffraction gratings to allow for phase shifts along both illumination directions. In some cases, a single rotating optical phase modulator may be replaced, for example, by a wedge-type optical component that rotates around the nominal beam axis.

[0217] In an alternative illumination optical path design, the diffraction gratings 4130A and 4130B may be mounted on their respective linear moving stages so that they can be moved to alter the optical path length (and therefore phase) of the light reflected or transmitted by the diffraction gratings 4130A and 4130B. The operating axis of the linear moving stage may be perpendicular to the orientation of each diffraction grating on the linear moving stage, or otherwise offset from it, in order to provide movement of the fringe pattern of the diffraction grating along the sample surface 4188. Suitable moving stages may include, for example, cross-roller bearing stages, linear motors, high-precision linear encoders, and / or other linear actuator techniques to provide precise linear movement of the diffraction grating.

[0218] Figure 42 provides a non-limiting example of a workflow for acquiring and processing images using structured illumination to improve the spatial resolution of an imaging system. In some cases, the workflow illustrated in Figure 42 may be performed to image an entire sample surface (e.g., image tiling the inner surface of a flow cell) or to image a single region of a larger sample surface. The vertical diffraction gratings 4130A and horizontal diffraction gratings 4130B of system 4100 shown in Figure 41 may be used to project illumination fringe patterns onto a sample surface having different known orientations and / or different known phase shifts. For example, the imaging system 4100 may use the vertical gratings 4130A and horizontal gratings 4130B to generate horizontal and vertical illumination patterns, respectively, while the optical phase modulators 4140A and 4140B may be set to three different positions to generate three phase shifts, each indicated for a given orientation.

[0219] During operation, a first illumination condition (e.g., setting a specific orientation and phase shift of the diffraction grating) may be used to project a grating fringe pattern onto the sample surface (e.g., the flow cell surface). After capturing an image using the first illumination condition, one or more phase-shift illumination patterns may be acquired (e.g., one, two, three, four, five, six, or seven or more additional images may be acquired using one, two, three, four, five, six, or seven or more phase-shift illumination patterns). If the imaging system includes a second branch of the illumination optical path, the image acquisition process may be repeated using a second illumination condition (e.g., setting a second specific orientation and phase shift of the diffraction grating) as a starting point. In some cases, images may be acquired for at least three different orientations of the diffraction grating (e.g., spaced 60 degrees apart from each other) using at least five different phase-shift fringe patterns. If no further images are acquired using different orientations of the diffraction grating or phase-shift illumination fringe patterns, an image reconstruction algorithm may be used to process the acquired images and generate a reconstructed super-resolution image. In some cases, images may be obtained for at least one, two, three, four, five, six, or seven different orientations of the diffraction grating using at least one, two, three, four, five, six, or seven or more different phase-shifted fringe patterns in each orientation.

[0220] Potential drawbacks of acquiring multiple images for use in reconstructing a single super-resolution image include the time required to adjust the orientation and / or relative phase shift of the projected light fringe pattern, as well as the exposure time required to acquire each image, and downstream image processing. Therefore, an optical design that minimizes the time required to change the orientation and relative phase of the diffraction grating is preferred, along with a highly efficient image reconstruction algorithm. In some cases, fewer images may be required than those typically required to reconstruct a high-resolution image of a conventional sample (e.g., a stained tissue sample), or fewer images than those typically required to reconstruct a super-resolution image of a flow cell surface, for example, which includes clusters of separate fluorescently labeled amplification target nucleic acid sequences tethered to a low nonspecific binding surface as described elsewhere herein.

[0221] Referring again to Figure 42, the aforementioned cycle may be repeated for different regions of a given flow cell surface, for example, when the image is tiled to produce a high-resolution image of the entire flow cell surface. In some cases, for example, when a second, third, or fourth flow cell surface is imaged, the aforementioned cycle may be repeated after adjusting the focus of the imaging system.

[0222] Other Super-Resolution Imaging Techniques In some cases, the disclosed imaging systems may include the use of alternative superresolution imaging techniques, e.g., photoactivated localization microscopy (PALM), fluorescence-activated localization microscopy (FPALM), and / or stochastic optical reconstruction microscopy (STORM) [see, e.g., Lutz, et al. (2011), “Biological Imaging by Superresolution Light Microscopy”, Comprehensive Biotechnology (Second Ed.), vol.1, pages 579-589, Elsevier]. These are based on statistically curvilinearly fitting the intensity distribution observed in the point spread function (PSF) image of a single molecule to a Gaussian distribution function. The Gaussian distribution function is then used to define the position of the molecule on the sample surface with much higher precision than allowed by the classical resolution limits. Using the same approach, small dispersed subsets of fluorescently labeled molecules can be imaged, for example, clonally amplified clusters of target nucleic acid sequences tethered to a low nonspecific binding surface on a sample support or to the inner surface of a flow cell.

[0223] The spatial accuracy or resolution achieved using these methods depends on the number of photons collected from the molecule before photobleaching and the level of background noise [Lutz, et al. (2011), ibid.]. Positional accuracy of 1–2 nm has been demonstrated when background noise is negligible and at least 10,000 photons can be collected per molecule. In some cases, nucleotide conjugates containing multiple fluorescent labels (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 labels per conjugate) to ensure a high number of photons, for example using avidity sequencing approaches described elsewhere herein, can optionally be used in combination with low nonspecific binding surfaces disclosed elsewhere herein to ensure very low background signal, facilitating the use of these super-resolution imaging techniques for genetic testing and sequencing applications. Spatial accuracy or resolution decreases as the number of collected photons decreases, but positional accuracy or resolution of 20 nm is possible even with moderate numbers of collected photons. In some cases, improvements of more than 10 times in lateral spatial resolution can be achieved. In some cases, image resolutions better than those of 500nm, 400nm, 300nm, 200nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm, or 10nm can be achieved.

[0224] The second fundamental principle underlying this class of imaging is that a small number of spatially separated fluorescent molecules within the sample are imaged at any given time.

[0225] In some cases, the ability to control the fluorescence emission of small, dispersed subsets of fluorescent molecules on the sample surface is key to facilitating super-resolution imaging. For example, in fluorescence-activated localization microscopy (FPALM) and photo-activated localization microscopy (PALM), the use of photoactivatable green fluorescent protein (PA-GFP) as a label allows for the controlled induction of fluorescent subsets in the sample using short pulses of 405 nm light, converting PA-GFP from a dark, non-fluorescent state to an excitable fluorescent state at 488 nm, thereby resulting in a spatially separated subset of fluorescent molecules that can be imaged [Lutz, et al. (2011), ibid.]. In stochastic optical reconstruction microscopy (STORM), for example, the photo-switching properties of cyanine dyes versus Cy5-Cy3 can be used in a similar manner to enable the stochastic induction of Cy5 fluorescence from small subsets of molecules in the sample (e.g., small subsets of molecules spatially separated by at least some resolution units) at any given time point. In some cases, when combined with avidity sequencing approaches as described elsewhere in this specification, for example, the nucleotide conjugate may contain a photoactivatable green fluorescent protein (PA-GFP) or a subdomain or portion thereof. In some cases, the nucleotide conjugate may include a mixture of conjugates in which the first portion is labeled, for example, with Cy3 labeling and the second portion is labeled, for example, with Cy5 labeling. In some cases, the nucleotide conjugate may contain a mixture of Cy3-labeled and Cy5-labeled portions within the same conjugate.

[0226] The super-resolution image is reconstructed from a sum of Gaussian fits from all molecules or features (e.g., clusters of labeled nucleic acids) imaged in a time stack of acquired images [Lutz, et al. (2011), ibid.], where the intensity corresponds to the positional uncertainty of the position of each molecule or subset of molecules. Specific to this type of dataset is the ability to render images with different positional accuracies or resolutions. In some cases, imaging modules, including total internal reflection fluorescence (TIRF) optical imaging designs, may be advantageous for implementing the use of these super-resolution imaging techniques because the evanescent waves used to excite fluorescence are limited to less than 200 nm in the axial dimension from the sample support or flow cell surface, thus suppressing background fluorescence signals. In some cases, the imaging system may include objective lenses with higher numerical apertures than those used in other imaging module designs disclosed herein. Using higher numerical aperture objective lenses may facilitate the implementation of evanescent wave excitation and the highly efficient capture of photons from the fluorescent probe. In some cases, wide-field imaging using single-photon-sensitive EM-CCD cameras or other types of image sensors can enable simultaneous imaging of many molecules or subsets of molecules (e.g., nucleic acid sequence clusters) per frame, thereby improving image acquisition throughput.

[0227] In some cases, the data acquisition time required to obtain images sufficient for proper feature definition and resolution can be reduced by improving the sensitivity and speed of the imaging system through the use of avidity sequencing reagents and low nonspecific binding surfaces disclosed herein, as well as improved image reconstruction algorithms, to increase the signal while reducing or eliminating background noise.

[0228] Image quality evaluation For any embodiment of the optical imaging design disclosed herein, imaging performance or quality may be evaluated using any of the various performance metrics known to those skilled in the art. Examples include measurement of the modulation transfer function (MTF) at one or more specified spatial frequencies, focus blur, spherical aberration, chromatic aberration, coma aberration, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof.

[0229] In some cases, the disclosed optical designs for two- or four-surface imaging (e.g., disclosed objective lens designs, tube lens designs, and the use of electro-optical phase plates in combination with objective lenses, either individually or in combination) can result in a significant improvement in image quality across all inner surfaces of the flow cell. Therefore, the difference in imaging performance metrics for imaging multiple surfaces (e.g., the first, second, third, or fourth surfaces of the flow cell) will be less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% for any of the above imaging performance metrics, either individually or in combination.

[0230] In some cases, the disclosed optical designs for multi-plane imaging (including, for example, the disclosed tube lens designs and the use of electro-optical phase plates in combination with objective lenses) can result in a significant improvement in image quality. In some embodiments, the image quality performance metrics for multi-plane imaging provide, for example, an improvement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% in the case of multi-plane imaging, individually or in combination, compared to two-plane imaging of a conventional system including an objective lens, a motion-driven compensator (which moves in and out of the optical path when imaging the proximal surface (e.g., the first surface) or distal inner surface (e.g., the second surface) of a flow cell), and an image sensor. In some cases, a fluorescence imaging system including one or more of the disclosed tube lens designs provides at least an equivalent improvement in the imaging performance metrics for multi-plane imaging compared to the imaging performance metrics of a conventional system including an objective lens, a motion-driven compensator, and an image sensor. In some cases, fluorescence imaging systems including one or more of the disclosed tube lens designs provide an improvement of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in the imaging performance metric for multi-plane imaging compared to the imaging performance metric of a conventional system including an objective lens, motion-driven compensator, and image sensor.

[0231] Specifications of the imaging module Excitation light wavelength(s): In either the disclosed optical imaging module design or optical system design, the light source(s) of the disclosed imaging module may generate visible light such as green and / or red light. The light source(s) of the disclosed imaging module may generate visible light such as blue light. In some cases, the light source(s), alone or in combination with one or more optical components (e.g., an excitation optical filter and / or a dichroic beam splitter), may generate excitation light of approximately 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. Those skilled in the art will recognize that the excitation wavelength may have any value within this range (e.g., about 620 nm). In some cases, the light source(s) alone or in combination with one or more optical components (e.g., an excitation optical filter and / or a dichroic beam splitter) may generate excitation light having a single wavelength in the range of 300 nm to 600 nm, 400 to 500 nm, 450 to 500 nm, 420 to 520 nm, or 350 to 850 nm. In some cases, a light source(s), either alone or in combination with one or more optical components (e.g., an excitation optical filter and / or a dichroic beam splitter), can generate excitation light having a single wavelength in the range of 300nm–600nm, 400–500nm, 450–500nm, 420–520nm, or 350–850nm, with wavelength bandwidths of ±2nm, ±5nm, ±10nm, ±20nm, ±40nm, ±80nm, or greater. For example, a light source(s) may include blue light having a wavelength of 460nm in a wavelength range of ±5nm.

[0232] Excitation light bandwidth: In either of the disclosed optical imaging module designs or optical system designs, the light source(s) may, alone or in combination with one or more optical components (e.g., an excitation optical filter and / or a dichroic beam splitter), generate light at a specified excitation wavelength within a bandwidth of ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or greater. It will be recognized by those skilled in the art that the excitation bandwidth may have any value within this range (e.g., about ±18 nm).

[0233] Light Source Power Output: In any of the disclosed optical imaging module designs, the output of the light source(s) and / or the excitation light beam(s) derived therefrom (including composite excitation light beams) may range from approximately 0.5 watts to approximately 5.0 watts or more (as will be discussed in more detail below). In some cases, the power of the light source(s) and / or the excitation light beam(s) derived therefrom may be at least 0.5 watts, at least 0.6 watts, at least 0.7 watts, at least 0.8 watts, at least 1 watt, at least 1.1 watts, at least 1.2 watts, at least 1.3 watts, at least 1.4 watts, at least 1.5 watts, at least 1.6 watts, at least 1.8 watts, at least 2.0 watts, at least 2.2 watts, at least 2.4 watts, at least 2.6 watts, at least 2.8 watts, at least 3.0 watts, at least 3.5 watts, at least 4.0 watts, at least 4.5 watts, or at least 5.0 watts. In some implementations, the power of the light source output and / or the excitation light beam derived therefrom (including composite excitation light beams) may be as high as 5.0 watts, 4.5 watts, 4.0 watts, 3.5 watts, 3.0 watts, 2.8 watts, 2.6 watts, 2.4 watts, 2.2 watts, 2.0 watts, 1.8 watts, 1.6 watts, 1.5 watts, 1.4 watts, 1.3 watts, 1.2 watts, 1.1 watts, 1 watt, 0.8 watts, 0.7 watts, 0.6 watts, or 0.5 watts. Any of the lower and upper limits set out in this paragraph may be combined to form a range included in this disclosure, for example, in some cases the output power of the light source and / or the power of the excitation light beam (including a composite excitation light beam) derived therefrom may be in the range of about 0.8 watts to about 2.4 watts. It will be recognized by those skilled in the art that the output power of the light source and / or the power of the excitation light beam (including a composite excitation light beam) derived therefrom may have any value within this range (e.g., about 1.28 watts).

[0234] Light Source Output Power and CNR: In some implementations of the disclosed optical imaging module design, the output power of the light source(s) and / or the power of the excitation light beam(s) (including composite excitation light beams) derived therefrom are sufficient, in combination with a suitable sample, to provide a contrast-to-noise ratio (CNR) of the image acquired by the illumination and imaging module at least 5, at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, or at least 50 or more, or any CNR within any range formed by any of these values.

[0235] In some embodiments, the light source described herein provides a wide field of view (e.g., 60 mm) with uniform illumination power (e.g., variation or difference of less than 10% across the illuminated area). 2 The system may include a lighting system capable of illuminating the area. An exemplary lighting system and corresponding lighting uniformity are disclosed in PCT application PCT / US24 / 12802 (which is incorporated herein by reference in its entirety).

[0236] Fluorescence Emission Bands: In some cases, the disclosed fluorescence optics imaging module may be configured to detect fluorescence emission produced by any of the various fluorophores known to those skilled in the art. Examples of suitable fluorescent dyes for use in genotyping and nucleic acid sequencing applications (e.g., by conjugation to nucleotides, oligonucleotides, or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, cyanine, and their derivatives (including, but not limited to, cyanine derivatives such as cyanine dye 3 (Cy3), cyanine dye 5 (Cy5), cyanine dye 7 (Cy7)).

[0237] Fluorescence Emission Wavelength: In either of the disclosed optical imaging module designs or optical system designs, the detection channel or imaging channel of the disclosed optical system may include one or more optical components (e.g., emission optical filters and / or dichroic beam splitters) configured to collect emitted light at approximately 350 nanometers (nm), 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. In some embodiments, the emitted light may be in the range of 500 nm to 750 nm, 400 to 1200 nm, or 450 to 850 nm. Those skilled in the art will recognize that the emission wavelength may have any value within this range (for example, about 825 nm).

[0238] Fluorescence Emission Bandwidth: In either of the disclosed optical imaging module designs or optical system designs, the detection channel or imaging channel may include one or more optical components (e.g., emission optical filters and / or dichroic beam splitters) configured to collect light at a specified emission wavelength within a bandwidth of ±2 nm, ±5 nm, ±10 nm, ±20 nm, ±40 nm, ±80 nm, or greater. It will be recognized by those skilled in the art that the excitation bandwidth may have any value within this range (e.g., about ±18 nm).

[0239] Numerical Aperture: In some cases, the numerical aperture of the objective lens and / or optical imaging module (e.g., including the objective lens and / or tube lens) in any of the disclosed optical system designs may range from about 0.1 to about 1.4. In some cases, the numerical aperture may be at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, or at least 1.4. In some cases, the numerical aperture may be at most 1.4, at most 1.3, at most 1.2, at most 1.1, at most 1.0, at most 0.9, at most 0.8, at most 0.7, at most 0.6, at most 0.5, at most 0.4, at most 0.3, at most 0.2, or at most 0.1. Any of the lower and upper limits set out in this paragraph may be combined to form a range included in this disclosure, for example, in some cases the numerical aperture may be in the range of about 0.1 to about 0.6. It will be recognized by those skilled in the art that the numerical aperture may have any value within this range (e.g., about 0.55).

[0240] Optical Resolution: In some cases, depending on the numerical aperture of the objective lens and / or optical system (e.g., including the objective lens and / or tube lens), the separation distance of the minimum resolvable spot (or feature) on the sample surface achieved by any of the disclosed optical system designs may range from about 0.5 μm to about 2 μm. In some cases, the separation distance of the minimum resolvable spot may be at least 0.5 μm, at least 0.6 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, at least 1.2 μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or at least 1.0 μm. In some cases, the separation distance of the minimum resolvable spot may be at most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most 0.7 μm, at most 0.6 μm, or at most 0.5 μm. Any of the lower and upper limits described in this paragraph may be combined to form a range included in this disclosure, for example, in some cases the separation distance of the minimum resolvable spot may be in the range of about 0.8 μm to about 1.6 μm. It will be recognized by those skilled in the art that the separation distance of the minimum resolvable spot may have any value within this range (e.g., about 0.95 μm).

[0241] In some cases, the use of any of the novel illumination systems and other designs of optical systems disclosed herein in any of the optical modules or systems disclosed herein may impart equivalent optical resolution to multiple surfaces (e.g., the first, second, third, and / or fourth inner surfaces of a flow cell), regardless of whether refocusing is required while acquiring images of the surfaces. In some cases, the optical resolution of the images of surfaces thus obtained may be within 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, or 1% of each other, or within any of these ranges.

[0242] Magnification: In some cases, the magnification of the objective lens and / or tube lens in any of the disclosed optical configurations, and / or the optical system (e.g., including the objective lens and / or tube lens), may range from about 2x to about 20x. In some cases, the magnification of the optical system may be at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least 10x, at least 15x, or at least 20x. In some cases, the magnification of the optical system may be at most 20x, at most 15x, at most 10x, at most 9x, at most 8x, at most 7x, at most 6x, at most 5x, at most 4x, at most 3x, or at most 2x. Any of the lower and upper limits set out in this paragraph may be combined to form a range included in this disclosure, for example, in some cases the magnification of the optical system may range from about 3x to about 10x. Those skilled in the art will recognize that the magnification of the optical system may have any value within this range (for example, about 7.5x).

[0243] Focal Length of Objective Lens: In some implementations of the disclosed optical design, the focal length of the objective lens may be in the range of 20 mm to 40 mm. In some cases, the focal length of the objective lens may be at least 20 mm, at least 25 mm, at least 30 mm, at least 35 ...

Claims

1. A system for in situ biomolecular analysis, wherein the system is Including an imaging system, the imaging system is A flow cell configured to hold cells or tissues immobilized thereon, wherein the cells or tissues contain a plurality of analytes of different types, A light source configured to illuminate the cells or tissue and thereby generate a plurality of signals corresponding to the plurality of analytes, A detector configured to image the plurality of signals, One or more processors communicably coupled to the imaging system, wherein the one or more processors (a) Using the light source, illuminate the cells or tissue, thereby generating the plurality of signals corresponding to the plurality of analytes, (b) Using the detector, detect the plurality of signals, and (c) A system comprising one or more computer processors, each individually or collectively programmed to determine the identity or arrangement of the plurality of analytes using the plurality of signals.

2. The system according to claim 1, wherein the cells or tissue are in situ cells or a tissue sample.

3. The light source emits a peak-to-valley energy or power fluctuation of at most about 5% over approximately 20 square millimeters (mm) of the flow cell and the cells or tissue. 2 The system according to claim 1, configured to illuminate the superluminal field.

4. The light source has a maximum RMS wavefront error of approximately 0.09λ, and the flow cell and the cells or tissue are approximately mm in diameter. 2 The system according to claim 1, configured to illuminate a superluminescent element.

5. The imaging system according to claim 1, wherein the imaging system has a combined root mean square error of less than approximately 0.

05.

6. The system according to claim 1, wherein the cells or tissue are whole cells or whole tissue.

7. The imaging system according to claim 1, wherein the imaging system does not include an objective lens located in the optical path of the light source or the detector.

8. The imaging system according to claim 7, wherein the imaging system does not include an objective lens.

9. The imaging system according to claim 1, wherein the imaging system does not include a tube lens.

10. The system according to claim 1, wherein the lighting has an illuminance of at least about 40 milliwatts / square meter.

11. The system according to claim 1, wherein the cells or tissue are permeabilized.

12. The system according to claim 1, wherein the plurality of signals are a plurality of fluorescence signals.

13. The system according to claim 1, wherein the plurality of signals are detected with a Q score of at least 30, 40, or 50.

14. The system according to claim 1, wherein the cells or tissue are illuminated by 10 illumination fields in one or more planes perpendicular to the optical axis of the imaging system.

15. The field of view of the detector is at least about 10 mm 2 The system according to claim 1.

16. The system according to claim 1, wherein the cells or tissue are imaged with a resolution of at least about 1 micrometer.

17. The system according to claim 1, wherein the flow cell is configured to allow one or more reagent flows to come into contact with the cells or tissue.

18. The system according to claim 1, wherein the cells or tissue are cultured cells or cultured tissue.

19. The system according to claim 1, wherein the cells or tissue are isolated cells or isolated tissue.

20. The system according to claim 1, wherein the image fidelity of the cells or tissue is at least about 0.1 micrometers.

21. The system according to claim 1, wherein the plurality of analytes include nucleic acid molecules.

22. The system according to claim 21, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.

23. The system according to claim 21, wherein the nucleic acid molecule is a ribonucleic acid molecule.

24. The system according to claim 1, wherein the plurality of analytes include proteins.

25. The system according to claim 1, wherein the plurality of analytes include carbohydrates.

26. A method for imaging an in situ sample, (a) To provide the in situ sample containing multiple different types of analytes, (b) Illuminating the plurality of different types of analytes to generate a plurality of signals related to the plurality of analytes, (c) A method comprising imaging the plurality of signals.

27. The method according to claim 26, wherein the illumination of the plurality of different types of analytes is sequential illumination of the plurality of different types of analytes.

28. The method according to claim 26, wherein illuminating the plurality of different types of analytes is simultaneous illumination of the plurality of different types of analytes.

29. The method according to claim 26, wherein the plurality of different types of analytes are selected from the group consisting of deoxyribonucleic acid molecules, ribonucleic acid molecules, proteins, morphological features, and phosphorylated proteins.

30. The method according to claim 26, further comprising applying one or more sequencing reagents to the in situ sample configured to sequence the plurality of different types of analytes.

31. The aforementioned illumination is approximately 20 square millimeters (mm) 2 The method according to claim 26, wherein the flow cell region exceeding ) has a peak-to-valley variation of at most about 5%.

32. The illumination described above has a maximum RMS wavefront error of approximately 0.09λ, and the flow cell has at least approximately 1 mm 2 The method according to claim 26, relating to the present invention.

33. The aforementioned illumination is approximately 20 square millimeters (mm) 2 The method according to claim 26, wherein the flow cell region exceeding ) has a peak-to-valley variation of at most about 5%.

34. (d) The method according to claim 26, further comprising analyzing the plurality of signals using a computer processor operably coupled to the detector.

35. The method according to claim 34, wherein the analysis of the plurality of signals includes determining the sequence of nucleic acid molecules in the in situ sample.

36. The method according to claim 35, wherein the sequence of the nucleic acid molecule is determined with at least about 95% accuracy, sensitivity, or specificity.

37. The method according to claim 36, wherein the sequence of the nucleic acid molecule is determined without changing the spatial relationship of the nucleic acid in the in situ sample.

38. The method according to claim 26, wherein the in situ sample has a length, width, or height of at least about 10 micrometers.

39. The method according to claim 26, wherein the in situ sample includes tissue.

40. The method according to claim 26, wherein the in situ sample comprises a plurality of cultured cells.

41. The method according to claim 26, wherein the in situ sample comprises a plurality of isolated cells.

42. The method according to claim 26, wherein the in situ sample is imaged in about 10 images in a plane perpendicular to the optical axis of the optical assembly.

43. The method according to claim 26, wherein the plurality of signals are a plurality of fluorescence signals.

44. The method according to claim 26, wherein the plurality of signals are detected with a Q score of at least 30, 40, or 50.

45. The method according to claim 26, wherein the in situ sample contains nucleic acid molecules.

46. The method according to claim 45, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.

47. The method according to claim 45, wherein the nucleic acid molecule is a ribonucleic acid molecule.

48. The field of view of the optical assembly is at least about 10 mm 2 The method according to claim 26.

49. The method according to claim 26, wherein the in situ sample is imaged with a resolution of at least about 1 micrometer.

50. The method according to claim 26, wherein the in situ sample is imaged within a maximum of approximately 24 hours.

51. The method according to claim 26, wherein the fidelity for capturing multiple images of the in situ sample is at least about 0.1 micrometers.

52. An optical assembly for in situ imaging, A flow cell configured to contain an in-situ sample, A light source configured to illuminate the in-situ sample in the flow cell and thereby generate a signal related to the characteristics of the in-situ sample, An optical assembly including a detector configured to image the aforementioned signal.

53. Approximately 20 square millimeters (mm) 2 The optical assembly according to claim 52, wherein the illumination over the region of the flow cell exceeding ) has a peak-to-valley energy or power variation of at most about 5%.

54. The illumination has a root mean square (RMS) wavefront error of at most about 0.09λ over an area of at least about 1 square millimeter (mm 2 ), the optical assembly according to claim 52.

55. The optical assembly according to claim 52, further comprising a processor configured to analyze the signal and determine the characteristics of the in situ sample.

56. The optical assembly according to claim 52, wherein the in situ sample has a length, width, or height of at least about 10 micrometers.

57. The optical assembly according to claim 52, wherein the optical assembly does not include an objective lens.

58. The optical assembly according to claim 57, wherein the system does not include an objective lens.

59. The optical assembly according to claim 52, wherein the optical assembly does not include a tube lens.

60. The optical assembly according to claim 59, wherein the system does not include an objective lens.

61. The optical assembly according to claim 52, wherein the in situ sample includes tissue.

62. The optical assembly according to claim 52, wherein the in situ sample comprises a plurality of cultured cells.

63. The optical assembly according to claim 52, wherein the in situ sample comprises a plurality of isolated cells.

64. The optical assembly according to claim 52, wherein the in situ sample is imaged in a plane perpendicular to the optical axis of the optical assembly in a maximum of about 10 images.

65. The optical assembly according to claim 52, wherein the signal is a fluorescence signal.

66. The optical assembly according to claim 52, wherein the signal is detected with a Q score of at least about 30.

67. The optical assembly according to claim 52, wherein the in situ sample contains nucleic acid molecules.

68. The optical assembly according to claim 67, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.

69. The optical assembly according to claim 67, wherein the nucleic acid molecule is a ribonucleic acid molecule.

70. The field of view of the optical assembly is at least about 10 mm 2 The optical assembly according to claim 52.

71. The optical assembly according to claim 52, wherein the in situ sample is imaged with a resolution of at least about 1 micrometer.

72. The optical assembly according to claim 52, wherein the in situ sample is imaged within a maximum of approximately 24 hours.

73. The optical assembly according to claim 52, wherein the fidelity for capturing multiple images of the in situ sample is at least about 0.1 micrometers.

74. A method for imaging an in situ sample, (a) Providing the in situ sample to a flow cell included in a system including an optical assembly including a light source and a detector, (b) Illuminating the in situ sample and generating a signal related to the analyte of the in situ sample, (c) A method comprising imaging the signal using the detector.

75. The aforementioned illumination is approximately 20 square millimeters (mm) 2 The method according to claim 74, wherein the flow cell region exceeding ) has a peak-to-valley variation of at most about 5%.

76. The illumination described above has a maximum RMS wavefront error of approximately 0.09λ, and the flow cell has at least approximately 1 mm 2 The method according to claim 74, relating to the present invention.

77. (d) The method of claim 74, further comprising analyzing the signal using a computer processor operably coupled to the detector.

78. The method according to claim 77, wherein analyzing the signal includes determining the sequence of nucleic acid molecules in the in situ sample.

79. The method according to claim 78, wherein the sequence of the nucleic acid molecule is determined with at least about 95% accuracy, sensitivity, or specificity.

80. The method according to claim 79, wherein the sequence of the nucleic acid molecule is determined without destroying the in situ sample.

81. The method according to claim 74, wherein the in situ sample has a length, width, or height of at least about 10 micrometers.

82. The method according to claim 74, wherein the optical assembly does not include an objective lens.

83. The method according to claim 82, wherein the system does not include an objective lens.

84. The method according to claim 74, wherein the optical assembly does not include a tube lens.

85. The method according to claim 84, wherein the system does not include an objective lens.

86. The method according to claim 74, wherein the in situ sample includes tissue.

87. The method according to claim 74, wherein the in situ sample comprises a plurality of cultured cells.

88. The method according to claim 74, wherein the in situ sample comprises a plurality of isolated cells.

89. The method according to claim 74, wherein the in situ sample is imaged in a plane perpendicular to the optical axis of the optical assembly in a maximum of about 10 images.

90. The method according to claim 74, wherein the signal is a fluorescence signal.

91. The method according to claim 74, wherein the signal is detected with a Q score of at least about 30.

92. The method according to claim 74, wherein the in situ sample contains nucleic acid molecules.

93. The method according to claim 92, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.

94. The method according to claim 92, wherein the nucleic acid molecule is a ribonucleic acid molecule.

95. The field of view of the optical assembly is at least about 10 mm 2 The method according to claim 74.

96. The method according to claim 74, wherein the in situ sample is imaged with a resolution of at least about 1 micrometer.

97. The method according to claim 74, wherein the in situ sample is imaged within a maximum of approximately 24 hours.

98. The method according to claim 74, wherein the fidelity for capturing multiple images of the in situ sample is at least about 0.1 micrometers.

Images