Illumination system for nucleic acid sequencing

Abstract

The present disclosure describes illumination methods and systems for illumination and sequencing applications, which can be used, for example, for microscopes and sequencing platforms. The methods and systems of the present disclosure may provide large area, uniform illumination, which may reduce errors and improve system throughput.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Application No. 63 / 481,583, filed January 25, 2023, and U.S. Provisional Application No. 63 / 489,150, filed March 8, 2023, the entire contents of which are incorporated herein by reference. Summary of the Invention

[0003] This paper describes an optical system for an imaging module used to sequence nucleic acids from DNA samples. The optical system and method described herein illuminate multiple axially displaced surfaces of a flow cell with a relatively uniform illumination power density. This illumination field is wider than that provided by some illumination systems, thus facilitating increased sequencing throughput within the set system runtime. Speckle noise in the illumination system can be advantageously reduced using cost-effective and easily implemented speckle eliminaters. Therefore, the illumination system and method described herein can increase the effectiveness and efficiency of sequencing analyses, including next-generation sequencing (NGS).

[0004] In certain situations, the optical systems and assemblies of this disclosure can provide wide-range temporal color imaging. This imaging can enhance sequencing or other imaging performance, where wide-area high-resolution imaging can increase throughput and reduce the time required for surface imaging. The optical systems and assemblies of this disclosure can provide a reduced volume, which can reduce the coverage area and enable new system architectures. The optical systems and assemblies of this disclosure can be used to reduce the number of optical components (e.g., lenses), thereby reducing the number of elements to be aligned and the number of system failure points, extending uptime, and reducing manufacturing burden. The optical systems and assemblies of this disclosure can eliminate stage movement in the system's z-direction (e.g., along the optical axis, relative to the optical assembly), which can simplify setup and enhance the reliability of the optical system.

[0005] In some embodiments, the aspects disclosed herein provide an optical system comprising: a stage configured to hold a solid support; a light source configured to illuminate the solid support; and an optical assembly at least partially disposed within an optical path from the stage to the light source, wherein the optical assembly is configured to be positioned on the solid support at a depth greater than about 20 square millimeters (mm²). 2Illumination is provided over an area where the peak-to-valley variation is at most about 5%. In some embodiments, the optical assembly does not include an objective lens. In some embodiments, the optical system does not include the objective lens. In some embodiments, the optical assembly does not include a tube lens. In some embodiments, the optical system does not include the tube lens. In some embodiments, the stage is not adjusted along the optical axis of the system. In some embodiments, the irradiance of the illumination is at least about 40 milliwatts per square millimeter. In some embodiments, the optical assembly is configured to receive emitted light from the solid support. In some embodiments, the numerical aperture (NA) of the optical assembly is at least about 0.3. In some embodiments, the wavelength of the emitted light is from about 500 nanometers to about 750 nanometers. In some embodiments, the working distance of the optical assembly is at least about 1 mm to 25 mm. In some embodiments, the optical system further includes a motion coil housed within the optical assembly, the motion coil being configured to move a focusing element within the optical path of the optical system. In some embodiments, a motor located outside the optical system is configured to move the focusing element along the optical axis in one or both directions. In some embodiments, the motor is directly coupled to a portion of the first, second, or third housing of the optical assembly, and a portion of the first, second, or third housing of the optical assembly is directly coupled to the focusing element. In some embodiments, the light source is a pulsed light source. In some embodiments, the composite root mean square error of the optical system is less than about 0.05. In some embodiments, the illumination efficiency of the optical assembly is at least about 90%. In some embodiments, the area is greater than 30 mm. 2 In some embodiments, the region is greater than 50 mm. 2 Or 60mm 2In some embodiments, the optical system further includes the solid support within the stage. In some embodiments, the solid support includes two or more surfaces having one or more samples imaged by the optical system fixed thereon. In some embodiments, the solid support includes three or more surfaces having one or more samples imaged by the optical system fixed thereon. In some embodiments, the three or more surfaces are axially displaced from each other at least along the optical axis of the optical system. In some embodiments, the solid support includes a probe configured to bind nucleic acid molecules. In some embodiments, the probe binds to a surface of the solid support. In some embodiments, the light source is a laser light source. In some embodiments, the optical assembly includes a dichroic filter configured to transmit the illumination. In some embodiments, the optical assembly includes: a first segment including a first housing including a first plurality of lenses; a second segment including a second housing; and a third segment including a third housing including a second plurality of lenses. In some embodiments, the first segment and the third segment are optically aligned. In some embodiments, the first segment is positioned between the third segment and the stage. In some embodiments, the third segment is positioned between the first segment and an image sensor of the optical system. In some embodiments, the first plurality of lenses are movable along the optical path ranging from about 0 to about 2 mm. In some embodiments, the first plurality of lenses include asymmetric biconvex lenses. In some embodiments, the second plurality of lenses include asymmetric biconcave lenses. In some embodiments, the asymmetric biconcave lens is an aspherical asymmetric biconcave lens. In some embodiments, the optical system is configured to acquire an image of the solid support without moving the optical compensator into the optical path between the solid support and the detector of the optical system. In some embodiments, the optical system is configured to acquire an image of the solid support without removing the optical compensator from the optical path between the sample and the detector of the optical system. In some embodiments, the solid support is a flow cell. In some embodiments, the optical component is configured to generate one or more spatial contractions transverse to the optical path of light traveling through it. In some embodiments, the optical component is configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it. In some embodiments, the optical component is configured to generate at least one field curvature correction transverse to the optical path of light traveling through it in a first, second, or third segment.

[0006] On the other hand, this disclosure provides a method for analyzing biomolecules, comprising: (a) providing a solid support comprising the biomolecules containing a label; and (b) illuminating the biomolecules containing the label using an optical system including a light source to generate signal light or a change thereof, wherein the illumination is provided over an area of ​​the solid support greater than about 20 square millimeters (mm²). 2(a) wherein the peak-to-valley variation is at most about 5%; (c) detecting the signal light or the said variation therein using a detector of the optical system; and (d) processing the signal light or the said variation therein at least partially to analyze the biomolecule. In some embodiments, the biomolecule is a nucleic acid molecule, a protein, or a polypeptide. In some embodiments, the biomolecule is a nucleic acid. In some embodiments, the method further includes, prior to (a), binding the biomolecule to a probe bound to the solid support and conjugating the label to the biomolecule. In some embodiments, the label is conjugated to the biomolecule by hybridization. In some embodiments, the optical system does not include an objective lens. In some embodiments, the solid support does not move along the optical axis of the optical system. In some embodiments, multiple images of the solid support are acquired without moving the solid support along the optical axis. In some embodiments, the irradiance of the illumination is at least about 40 milliwatts per square millimeter. In some embodiments, the wavelength of the signal light is from about 500 nanometers to about 750 nanometers. In some embodiments, the detection in (c) is performed using an optical element with a numerical aperture of at least about 0.3. In some embodiments, the method further includes, in (b), using a motion coil within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focal point of the optical system on the solid support. In some embodiments, the light source is a pulsed light source. In some embodiments, the illumination is provided with an efficiency of at least about 90%. In some embodiments, the method further includes repeating (b)-(d) on additional biomolecules coupled to another surface of the solid support. In some embodiments, the method further includes, after (c), removing the marker from the biomolecule. In some embodiments, the method further includes repeating (a)-(d) on additional markers bound to another portion of the biomolecule. In some embodiments, the optical component is configured to generate one or more spatial contractions transverse to the optical path of light traveling through it. In some embodiments, the optical component is configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it. In some embodiments, the optical component is configured to generate at least one field curvature correction transverse to the optical path of light traveling through it in a first, second, or third segment. In some embodiments, (d) includes at least partially processing the signal light or its variations to generate one or more solid support images, and analyzing the one or more solid support images to generate a base interpretation of the sample. In some embodiments, each solid support image comprises more than 20 square millimeters (mm²). 2 The field of view (FOV) of the solid support. In some embodiments, the solid support is a flow cell.

[0007] In some embodiments, the aspects disclosed herein provide an optical system comprising: a stage configured to hold a solid support; a light source configured to illuminate the solid support; and a speckle eliminator optically coupled to the light source and disposed within an optical path from the light source to the stage. In some embodiments, the speckle eliminator is configured to reduce speckle noise introduced between the light source and the stage. In some embodiments, the optical system further includes an additional light source optically coupled to the speckle eliminator. In some embodiments, light from the additional light source is configured to illuminate the solid support with light of a different wavelength than that of the light source. In some embodiments, at least about four light sources are coupled to the speckle eliminator. In some embodiments, the speckle eliminator is a vibratory speckle eliminator. In some embodiments, the speckle eliminator is a passive speckle eliminator. In some embodiments, the passive speckle eliminator includes a diffuser plate. In some embodiments, the speckle eliminator is a tension speckle eliminator. In some embodiments, the speckle eliminator is configured to reduce speckle noise to at most about 5%. In some embodiments, the solid support is a flow cell.

[0008] In some embodiments, aspects disclosed herein provide a method for analyzing biomolecules, the method comprising: (a) providing a solid support comprising a biological sample containing a marker; (b) illuminating the biological sample containing the marker using an optical system including a light source to generate signal light or a variation thereof, wherein the illumination is provided via a speckle eliminator in the optical path of the optical system; (c) detecting the signal light or the said variation thereof using a detector of the optical system; and (d) processing the signal light or the said variation thereof at least partially to analyze the biomolecules.

[0009] In some embodiments, the method further includes repeating steps (b)–(d) with additional biological samples coupled to a further surface of the solid support. In some embodiments, the method further includes removing the marker from the biological sample after (c). In some embodiments, the method further includes repeating (a)–(d) with additional markers bound to another portion of the biological sample. In some embodiments, the speckle eliminator uses vibration to perform speckle elimination on the illumination. In some embodiments, the method further includes illuminating the solid support with an additional light source. In some embodiments, the additional light source provides light of a different wavelength to the solid support. In some embodiments, the additional light source is optically coupled to the speckle eliminator. In some embodiments, the biological sample includes nucleic acid molecules, proteins, or peptides. In some embodiments, the biological sample includes nucleic acids. In some embodiments, optical components are at least partially disposed within the optical path from the stage to the detector of the optical system. In some embodiments, the illumination system of the optical components is disposed within the optical path from the stage to the detector of the optical system. In another aspect, the present invention provides a sample stage for holding a DNA sample for DNA sequencing reaction and imaging, the sample stage comprising: a stage including a top surface, wherein the stage is rotatable about a z-axis relative to the optical system of the sequencing system; one or more top stages positioned on the top surface of the stage, wherein each of the one or more top stages is configured to receive and fix one or more flow cell devices thereon, and wherein each of the one or more top stages is movable relative to the stage.

[0010] A first motor is configured to actuate a stage to rotate at a first resolution. In some embodiments, the top surface is circular. In some embodiments, the first resolution is an angular resolution and is less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees, or 50 degrees. In some embodiments, each flow cell device in the flow cell apparatus includes one or more samples to be sequenced fixed thereon. In some embodiments, at least one of the flow cell devices includes an in-situ sample fixed thereon. In some embodiments, the sample stage further includes one or more second motors configured to actuate one or more top stages individually relative to the stage at a second resolution. In some embodiments, the sample stage further includes second motors configured to actuate one or more top stages simultaneously relative to the stage at a second resolution. In some embodiments, the second resolution is less than 0.01 mm, 0.015 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.2 mm, or 1 mm. In some embodiments, the sequencing system includes a fluid control device in fluid communication with a flow cell assembly positioned on a sample stage. In some embodiments, each of the one or more top stages is movable relative to the stage in a sample plane. In some embodiments, a first top stage of the one or more top stages is movable independently relative to a second top stage of the one or more top stages. In some embodiments, a first top stage of the one or more top stages is movable simultaneously relative to the stage and a second top stage of the one or more top stages. In some embodiments, each of the one or more top stages is movable relative to the stage along a radius of the top surface of the stage. In some embodiments, each of the one or more top stages is movable relative to the stage with a radius orthogonal to the top surface of the stage.

[0011] In some embodiments, the aspects disclosed herein provide a method for sequencing multiple DNA samples positioned on a rotating sample stage, the method comprising: obtaining a sample stage including a base and one or more top stages positioned on a top surface of the base, wherein the base is rotatable about a z-axis relative to an optical system of a sequencing system; positioning and fixing a first flow cell device relative to a first top stage of the one or more top stages; positioning and fixing a second flow cell device relative to a second top stage of the one or more top stages; dispensing one or more sequencing reagents into the first flow cell device using a first fluid control device; imaging a first sample region of the first flow cell device using the optical system of the sequencing system; moving the first top stage relative to the optical system in the xy plane while preventing movement of the second flow cell device relative to the optical system; imaging a second sample region of the first flow cell device using the optical system of the sequencing system; rotating the sample stage at a predetermined angular resolution to position the second flow cell device relative to the optical system at a predetermined location; and imaging the first sample region of the second flow cell device using the optical system of the sequencing system. In some embodiments, moving the first top stage relative to the optical system in the xy plane while preventing the second flow cell device from moving relative to the optical system includes: independently moving the first top stage relative to the optical system at a predetermined distance along the radius of the top surface of the base while preventing the second flow cell device from moving relative to the optical system. In some embodiments, moving the first top stage relative to the optical system in the xy plane while preventing the second flow cell device from moving relative to the optical system includes: independently moving the first top stage relative to the optical system at a predetermined distance along a direction orthogonal to the radius of the top surface of the base while preventing the second flow cell device from moving relative to the optical system. In some embodiments, the method further includes: moving a first fluid control device or a second fluid control device to position the second flow cell device relative to the first fluid control device or the second fluid control device at a predetermined position. In some embodiments, the first sample area or the second sample area includes a block. In some embodiments, the range of movement of each of the one or more top stages along a radius or orthogonal to the top surface of the base is greater than 15 mm and less than 80 mm. In some embodiments, the range of motion of each of the one or more top platforms along the radius of the top surface of the base or orthogonal to the top surface of the base is greater than 25 mm and less than 100 mm. Attached Figure Description

[0012] The novel features of the inventive concept are set forth in detail in the appended claims. The features and advantages of the inventive concept will be better understood by referring to the following detailed description, which illustrates illustrative embodiments employing the principles of the inventive concept, and the accompanying drawings:

[0013] Figure 1A non-limiting example of an illumination system for the optical components described herein is shown, which includes an illumination subsystem and a beam transmission subsystem.

[0014] Figure 2 A non-limiting example of the illumination subsystem described in this paper is shown.

[0015] Figures 3A to 3C This article shows Figure 1 The uniformity of the lighting power density of the lighting system shown. Figure 3A Example images showing the illumination field and its associated illumination intensity are provided. Figure 3B It shows along Figure 3A The linear trajectory of the illumination intensity along the medium-long axis. Figure 3C It shows along Figure 3A The linear trajectory of the illumination intensity along the middle and short axes.

[0016] Figure 4 Non-limiting examples of the illumination subsystem and beam transmission system of the optical components are shown.

[0017] Figure 5 A non-limiting example of an illumination subsystem of an optical component is shown.

[0018] Figure 6 The speckle canceller and its relative position to the collimator of the beam transmission subsystem are shown.

[0019] Figure 7 A non-limiting example of an optical fiber and beam transmission subsystem is shown.

[0020] Figure 8 A non-limiting example of a liquid-core optical guide and a beam transmission subsystem is shown.

[0021] Figures 9A to 9D A non-limiting example of a speckle canceller is shown, in which the speckle canceller is a mechanical vibration source loosely or fixedly attached to at least a portion of an optical fiber. Figure 9A A wound portion of an optical fiber according to some embodiments is shown. Figure 9B A portion of an optical fiber around a vibration source is shown according to some embodiments. Figure 9C A portion of an optical fiber surrounding a fan vibration source is shown according to some embodiments. Figure 9D A portion of an optical fiber surrounding a fan vibration source is shown according to some embodiments.

[0022] Figure 10 A table showing the corresponding speckle noise levels for different speckle canceller configurations associated with optical fibers is provided.

[0023] Figure 11 A block diagram of a sequencing system for imaging a DNA sample during a DNA sequencing reaction, according to some embodiments, is shown.

[0024] Figure 12 These are schematic diagrams illustrating various examples of multivalent molecular configurations. Left (Class I): Schematic diagram of multivalent molecules with a "starburst" or "helix-skelter" configuration. Middle (Class II): Schematic diagram of multivalent molecules with a dendritic macromolecular configuration. Right (Class III): Schematic diagram of multiple multivalent molecules formed by the reaction of streptavidin with a 4-arm or 8-arm PEG-NHS containing biotin and dNTPs. Nucleotide units are designated 'N', biotin is designated 'B', and streptavidin is designated 'SA'.

[0025] Figure 13 This is a schematic diagram of an example of a multivalent molecule containing a universal core attached to multiple nucleotide arms.

[0026] Figure 14 This is a schematic diagram of an example of a multivalent molecule containing a dendritic macromolecular core attached to multiple nucleotide arms.

[0027] Figure 15 A schematic diagram of an example of a multivalent molecule comprising a core attached to multiple nucleotide arms is shown, wherein the nucleotide arms comprise biotin, spacers, linkers, and nucleotide units.

[0028] Figure 16 This is a schematic diagram of an example of a nucleotide arm that includes a core attachment portion, spacers, linkers, and nucleotide units.

[0029] Figure 17 The chemical structures of spacer examples are shown (top), as well as the chemical structures of various connector examples, including 11-atom connectors, 16-atom connectors, 23-atom connectors, and N3 connectors (bottom).

[0030] Figure 18 The chemical structures of various connector examples are shown, including connectors 1 through 9.

[0031] Figure 19 The chemical structures of various examples of linkers that connect to / attach to nucleotide units are shown.

[0032] Figure 20 The chemical structures of various examples of linkers that connect to / attach to nucleotide units are shown.

[0033] Figure 21 The chemical structures of various examples of linkers that connect to / attach to nucleotide units are shown.

[0034] Figure 22 The chemical structures of various examples of linkers that connect to / attach to nucleotide units are shown.

[0035] Figure 23 The chemical structure of an example of a biotinylated nucleotide arm is shown. In this example, the nucleotide unit is linked to the linker via a propargylamine attachor located at the 5-position of a pyrimidine base or the 7-position of a purine base.

[0036] Figure 24 A flowchart of a method for analyzing biomolecules according to some embodiments is shown.

[0037] Figure 25 A flowchart of a method for analyzing biological samples according to some embodiments is shown.

[0038] Figure 26 A perspective view showing a non-limiting example of an imaging module or optical component is shown.

[0039] Figure 27 A cross-sectional view of a non-limiting example of an imaging module or optical component is shown.

[0040] Figure 28 It shows Figures 26 to 27 A cross-sectional view of a non-limiting example of a single-channel temporal color imaging module.

[0041] Figures 29A to 29B An example of external actuator coupling according to some embodiments is shown. Figure 29A Detailed views of an external actuator and optical components according to some embodiments are shown. Figure 29B A remote view of an external actuator and optical components according to some embodiments is shown.

[0042] Figure 30 Examples of optical elements and associated focusing paths of optical assemblies according to some embodiments are shown.

[0043] Figure 31 Examples of optical elements and associated focusing paths of optical assemblies according to some embodiments are shown.

[0044] Figures 32A to 32B A diffraction modulation transfer function (MTF) for an optical system according to some embodiments is provided. Figure 32A An example MTF of an objective-based optical system is shown. Figure 32B An example MTF of an optical system excluding the objective lens is shown.

[0045] Figures 33A to 33B Wavefront analysis calculations of an optical system according to some embodiments of the present disclosure are shown. Figure 33A Example wavefront analysis calculations at location 1 are shown. Figure 33B Example wavefront analysis calculations at location 2 are shown.

[0046] Figure 34 The optical performance curves of the top surface according to some embodiments are shown.

[0047] Figure 35 The bottom surface optical performance curves according to some embodiments are shown.

[0048] Figure 36 A diagram of the MTF of an optical system according to some embodiments is shown.

[0049] Figure 37 A cumulative probability plot of a given wavefront error is shown according to some embodiments.

[0050] Figure 38 This is a schematic diagram of a rotating stage used to move a sample relative to the objective lens of the optical system for imaging the sequencing reaction. Detailed Implementation

[0051] Next-generation sequencing (NGS) analysis systems require higher throughput and flexibility. This paper discloses an optical system, its design, and its usage, which can provide one or more of the following advantages: wide field of view, uniform illumination power; cost-effective and easy-to-implement methods to reduce speckle noise; higher system throughput for fluorescence imaging-based genomics applications; compatibility with conventional flow cell devices and / or optical systems; flexibility in sample analysis or comparison (e.g., larger sample volumes and / or increased sample types); reduced system size; reduced complexity and other requirements for optical components (e.g., simpler optical setup); larger field of view; and improved illumination power uniformity.

[0052] This document discloses imaging modules configured for multi-channel fluorescence imaging. Each optical system may include multiple imaging modules, or equivalently, multiple optical components, such as one imaging module per color channel, and one or more imaging modules may include the illumination system and image acquisition system disclosed herein, the image acquisition system being configured to acquire flow cell images of one or more samples fixed on a sample stage and positioned at a sample plane. The illumination system and / or image acquisition system may operate independently for each respective imaging module or may be shared among multiple imaging modules. As disclosed herein, according to some embodiments, the imaging modules may be used interchangeably as optical components.

[0053] Image acquisition systems and methods

[0054] In some embodiments, the image acquisition system includes one or more image sensors and one or more objectives. In some embodiments, the optical system for imaging next-generation sequencing (NGS) responses (e.g., Figure 11The imager 116 may include one or more multi-channel fluorescence imaging modules, each corresponding to a different color channel. Each imaging module may include an image acquisition system having an image sensor and an objective lens corresponding to that color channel. The objective lens may be shared among multiple imaging modules. In some embodiments, each imaging module may include its own sensor and may not include any objective lens. In some embodiments, each imaging module is configured to generate flow cell images without using any objective lens.

[0055] In some embodiments, the imaging module includes three distinct segments. These segments can be optically aligned independently of each other and can be coupled together to form the imaging module. Having multiple segments advantageously allows each segment to be manufactured and optically aligned independently. Each segment may include its own separate housing. Alternatively, the imaging module may have a housing that houses all three distinct segments. In some embodiments, a first segment houses a first set of lens elements therein. Some of these lens elements are movable relative to the housing. For example, Figure 28 Lenses 1 to 3 and 5 to 8 are lens elements housed in the first segment. In some embodiments, the third segment partially houses a second set of lens elements, G2. For example, Figure 28 9 to 13 are in the third segment. Various methods can be used to control the centering and angular alignment of optical elements between multiple segments or within a single segment. For example, alignment flipping techniques can be used for control, and angular alignment can be based on sub-units, utilizing alignment flipping techniques to control centering and angular alignment. The second segment can house an excitation dichroic beam splitter, for example... Figures 26 to 27 As shown in Figure 2770. In some embodiments, the two orthogonal lens groups can be actively aligned at a nominal 45° angle, which can control the pointing difference between the first lens element G1 and the second lens element group G2. In some embodiments, the active alignment of the two orthogonal lens groups can reduce the alignment error to a satisfactory range. The satisfactory range can be customized according to different applications. For example, the alignment error may include one or more of the following: eccentricity, tilt, lens spacing error, and the need for defocus to meet the error budget.

[0056] As disclosed herein, focusing of the imaging module is advantageously internalized. The imaging module enables the movement of a single lens element, or elements therein, relative to the housing of the imaging module to achieve focusing at least along the z-axis, rather than moving multiple lens elements, such as all objectives (one or more optical compensators), relative to the sample for focusing. In some embodiments, a single lens element can be moved relative to the housing to achieve focusing along the z-axis. In some embodiments, two lens elements can be moved together or separately relative to the housing to achieve focusing along the z-axis. Figures 29A to 29BAs shown, the lens element can be mounted on a linear bearing driven by an external actuator, allowing the lens element to be moved automatically a predetermined distance in a controlled manner. The lens element can move along the optical axis of the optical assembly. The optical axis of the optical assembly between the detector and the stage (e.g., Figure 26 The optical axis of the optical component (2790 in A) can be along the z-axis of the segment closest to the sample stage (e.g., the first segment). The optical axis of the optical component can be along an axis orthogonal to the z-axis of the segment closest to the image sensor (e.g., the third segment). The optical axis of the optical component between the light source and the stage (e.g., Figure 26 2791 in A) can be along the z-axis of the segment closest to the sample stage (e.g., the first segment). The optical axis of the optical component can be along an axis orthogonal to the z-axis to the segment closest to the image sensor (e.g., the third segment).

[0057] In some embodiments, the range of motion of the lens element along the z-axis for focusing can be customized based on the size and dimensions of the flow cell. For example, when the lens element moves toward or away from the imaging sensor, the z-motion range for imaging from the top surface to the bottom surface of the flow cell can be approximately 810 μm. In some embodiments, the housing may include a rigid travel limiter for limiting the range of motion of the lens element during focusing. For example, Figure 28 The element number 8 is movable for focusing the imaging module. As another example, Figure 28 5 in E7 can be a movable element for focusing the imaging module. The travel range is sufficient to allow focusing on multiple surfaces without interfering with, or contacting, other lens elements. For example, the lens element used for focusing can move approximately 0.1 to 5 mm toward the sensor and approximately 0.1 to 4.0 mm away from the sensor. In some embodiments, a travel range may be included to assist in handling decoupling or placement errors of the flow cell surface (e.g., the top surface) relative to the apex of the lens element. In some embodiments, the lens element actively aligns E7 with E1 through E6. Sample stage and usage instructions

[0058] In some embodiments, the optical systems described herein (e.g., those for imaging samples or for sequencing reactions) may include a sample stage configured to hold samples and / or their respective supports (e.g., flow cell devices with solid supports) in a predetermined position relative to the optical system. In some embodiments, the sample stage may include a base and one or more top stages positioned thereon. Figure 38 An exemplary sample stage 3800 with a base 3810 and a top stage 3820 is shown.

[0059] base (e.g., Figure 38The stage 3810 (as shown in the image) may include a thickness along the z-axis and a top surface. The thickness of the stage can be customized to various values, for example, in the range of 1 mm to 5 cm. A top stage (e.g., 3820) can be positioned on the top surface 3811 of the stage. The top surface can be planar. The top surface can be of various geometries. In some embodiments, the top surface of the stage can be, but is not limited to, circular, annular, elliptical, square, rectangular, or rhomboid. The dimensions of the top surface of the stage are sufficient to position one or more top stages thereon for sequencing purposes. For example, the top surface is sufficient to position 5, 10, 20, 30, or more top stages thereon.

[0060] The stage can be configured to move relative to the optical system (116), such as relative to the focal plane of the objective lens or the focal plane of the optical system herein, to allow a sample positioned on the stage to be focused for imaging. The stage can be configured to move in one or more directions in 3D space. For example, the stage can be configured to move along the x, y, and / or z axes relative to the focal plane of the optical system. As another example, the stage can be configured to rotate about an axis (e.g., the z-axis) to focus different areas of the stage's top surface, thereby focusing a sample positioned thereon relative to the focal plane of the optical assembly.

[0061] In some embodiments, the sample stage can be of various geometries. In some embodiments, the stage can move relative to the optical axis of the optical system. In some embodiments, the stage can rotate about the optical axis or the z-axis of the optical system.

[0062] One or more top platforms can be of various geometries. For example, such as Figure 38 As shown, the five top platforms are rectangular. In some embodiments, the top platforms may be, but are not limited to, circular, annular, elliptical, square, rectangular, or rhomboid. In some embodiments, each top platform may have a shape and size sufficient to hold one or more flow cell devices located thereon.

[0063] In some embodiments, one or more top platforms may be movable relative to the base along the radius of the base's top surface, for example, along the y-axis, such as... Figure 38 As shown. In some embodiments, one or more top platforms may be movable relative to the base with a radius orthogonal to the top surface of the base, for example, along the x-axis, as... Figure 38 As shown. In some embodiments, one or more top platforms can move in various directions within the xy plane.

[0064] In some embodiments, a first top platform of one or more top platforms may move independently relative to at least a second top platform of one or more top platforms. In some embodiments, a first top platform of one or more top platforms may move simultaneously relative to a base platform and at least a second top platform of one or more top platforms.

[0065] In some embodiments, each top platform may have one or more flow cell devices (not shown) fixed thereon. In some embodiments, the flow cell devices are detachably fixed to the respective top platform. In some embodiments, movement of the top platform may cause the one or more flow cell devices fixed thereon to move in the same way. The flow cell devices may be fixed relative to the top platform such that there is no relative movement between the flow cell devices and the respective top platform when the top platform moves. The flow cell devices may be fixed by various fixing or fastening methods, including but not limited to mechanically clamping the flow cell devices, fixing them with magnetic or electromagnetic forces, positioning the flow cell devices in a suitable housing fastened to the top platform, coupling the pins or posts of the top platform to the holes or grooves of the flow devices, and vice versa.

[0066] In some embodiments, each flow cell device has a sample immobilized thereon. The sample may be a 2D DNA sample. The sample may be a 3D volume sample of cells and / or tissues in situ. In some embodiments, the sample may be a multiplexed sample. In some embodiments, the nucleotide diversity of the sample may be balanced or unbalanced.

[0067] Figure 38 A non-limiting example of a sample stage 3800 for holding a sample imaged by the optical system 116 of the sequencing system 110 disclosed herein is shown. In this particular embodiment, the stage 3810 has a circular top surface 3811. The top surface may include one or more top stages 3820 coupled thereto. Figure 38 In the illustrated embodiment, five top platforms are spaced apart on the top surface of the substrate. In some embodiments, there are 1 to 30 top platforms positioned on the sample stage. In some embodiments, the sample stage (e.g., the substrate and the top platforms) can rotate about an optical axis (e.g., the z-axis). When the substrate rotates, the top platforms fixed thereto can also rotate together with the substrate in the same rotational motion.

[0068] In some embodiments, the top stage can be movable relative to the base. This movement can be separate from or simultaneous with the rotational movements of the base and the top stage. For example, rotation of the base relative to the optical system and linear movement of the top stage relative to the base can occur simultaneously to position a predetermined sample region of the flow cell apparatus relative to the optical system for efficient imaging. As another example, rotation of the base relative to the optical system and linear movement of the top stage relative to the base can occur sequentially and can be controlled by the same motor. The movement of the top stage relative to the base can occur in the sample plane (e.g., an xy-plane orthogonal to the z-axis). For each top stage, the x-axis can extend axially from the center of the top surface of the base, and the y-axis can be orthogonal to both the x-axis and z-axis. For example, in Figure 38The top stage can be moved relative to the sample stage along the y-axis and / or x-axis, allowing different regions of the top stage to be moved relative to the optical system (e.g., objectives) to designated positions for imaging. In some embodiments, the y-axis and x-axis corresponding to different top stages can be oriented differently in the xy-plane, such that the y-axis is along the longest dimension of the flow cell assembly (e.g., along the radius of the top surface of the stage), while the x-axis is along the transverse direction of the flow cell assembly (e.g., along the tangential direction of the top surface of the stage).

[0069] Each stage can be configured to hold a sample and its corresponding support thereon. The sample and its corresponding support can be fixed to the stage for movement with the stage. By fixing various samples to different stages, specific sample regions can be rotated by rotating the stage and / or linearly moved by linearly moving the stage to image different samples in a temporal manner, so that the sample region can be positioned relative to the optical system for imaging.

[0070] In some embodiments, the stage includes a range of motion in the xy-plane sufficient to image a predetermined region of the sample. In some embodiments, the range of motion along the x-axis can be 0 to 50 mm, 0 to 40 mm, 0 to 30 mm, or 0 to 20 mm. In some embodiments, the range of motion along the y-axis can be 0 to 50 mm, 0 to 40 mm, 0 to 30 mm, 0 to 20 mm, 0 to 16 mm, or 0 to 10 mm. The resolution of movement along the x-axis or y-axis can be customized based on different samples or sequencing applications. In some embodiments, the resolution of movement along the x-axis or y-axis can be 1 μm to 40 μm, 1 μm to 30 μm, 1 μm to 20 μm, or 1 μm to 10 μm.

[0071] In some embodiments, the range of motion of each of the one or more top platforms along a radius perpendicular to or perpendicular to the top surface of the base is greater than 15 mm and less than 80 mm. In some embodiments, the range of motion of each of the one or more top platforms along a radius perpendicular to or perpendicular to the top surface of the base is greater than 25 mm and less than 100 mm.

[0072] An optical system may include one or more imaging heads, such as one or more optical components disclosed herein. Figure 38Two imaging heads are shown. Having a single imaging head can advantageously reduce the cost, size, and complexity of the optical system, while having multiple imaging heads can advantageously increase imaging throughput and reduce the total imaging time for imaging a certain number of samples, but at the cost of increased system hardware cost, complexity, etc. In some embodiments, the sample stage further includes a first motor configured to actuate the sample stage (e.g., the base and top stage) to rotate at a first resolution. The rotation of the sample stage can be relative to the optical system, for example, the optical axis of the optical system. The first resolution can be an angular resolution less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees, or 50 degrees. The first resolution can also be an angular resolution greater than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees, or 50 degrees. In some embodiments, various actuation mechanisms can be used to enable the sample stage to rotate. For example, a gear mechanism or an induction motor can be used to actuate the movement of the sample stage. In some embodiments, the resolution of the rotational motion can be customized. For example, the resolution can be 0.1 degrees, 0.2 degrees, or 0.5 degrees. In some embodiments, the sample stage can rotate at least 10 to 360 degrees. In some embodiments, the sample stage can rotate any number of full revolutions. In some embodiments, the sample stage can rotate in one or both directions.

[0073] In some embodiments, the same mechanism as the base or a different actuation mechanism may be used to actuate the top stage for movement. In some embodiments, the sample stage further includes one or more second motors configured to independently actuate some of the one or more top stages relative to the base at a second resolution, while the remaining top stages remain stationary. In some embodiments, the sample stage further includes second motors configured to simultaneously actuate one or more top stages relative to the base at a second resolution. In some embodiments, the second resolution is less than 0.01mm, 0.015mm, 0.02mm, 0.025mm, 0.03mm, 0.05mm, 0.08mm, 0.1mm, 0.15mm, 0.2mm, or 0.5mm. In some embodiments, the second resolution is less than 0.01mm, 0.02mm, 0.05mm, 0.1mm, 0.2mm, 0.4mm, 0.8mm, 1mm, 2mm, or 5mm. In some embodiments, the second resolution is greater than 0.01mm, 0.02mm, 0.05mm, 0.1mm, 0.2mm, 0.4mm, 0.8mm, 1mm, 2mm, or 5mm. In some embodiments, the second resolution is greater than 0.01mm, 0.015mm, 0.02mm, 0.025mm, 0.03mm, 0.05mm, 0.08mm, 0.1mm, 0.15mm, 0.2mm, or 0.5mm.

[0074] In some embodiments, the sample stage is coupled to one or more fluid control devices (e.g., Figure 38 The fluid control device is coupled to the sample stage (e.g., a flow cell device) and can be configured to hold, dispense, and collect various fluids used in the sequencing reactions within the flow cell device during a sequencing run. The fluid control device can also be individually fluidized with the sample on its respective stage. Figure 38 Five different fluid control devices are shown, each fluidly connected to a sample on a corresponding top stage. In some embodiments, the fluid control devices may be fixed relative to a base. In some embodiments, the fluid control devices may be fixed relative to a corresponding top stage. In some embodiments, each fluid control device may include a dispenser configured to dispense one or more reagents into the sample. For example, the dispenser may openly dispense reagents into a corresponding inlet of a flow cell. Another example is that the dispenser may be connected to the inlet of a flow cell device via a conduit, and the reagent may pass through the conduit to contact the sample in the flow cell device. In some embodiments, the fluid control devices may include one or more pumps to facilitate the dispensing of fluid into and / or collection of fluid from the sample.

[0075] In some embodiments, compared to sequencing systems using multiple fluid control devices, flow cell devices on multiple first stages can share a single fluid control device to simplify the system, reduce system costs, and minimize sequencing reagent waste. In such embodiments, different tubing can be used to provide fluid communication with different flow cell devices on different stages. These different tubing can be used for different reagents or the same reagent. Alternatively, different dispensing tips can be used to allow fluid application to different flow cell devices on different stages. In some embodiments, different stages can share the same dispensing tip, and reagent dispensing can be performed sequentially over time.

[0076] In some embodiments, the sample may be fixed to a solid support (e.g., a flow cell) for imaging using an optical system. The flow cell may include one or more lanes, each lane corresponding to a microfluidic channel that allows sequencing reagents or other fluids (e.g., wash buffer) to flow through during a sequencing run. In some flow cells with two lanes, the lanes are positioned parallel to each other. In some embodiments, the sample stage described herein may utilize a flow cell with a lane orientation different from that of these flow cells. In some embodiments, the flow cell described herein may include multiple lanes, and each pair of lanes may be positioned at an acute angle between their longitudinal directions such that they are not parallel to each other. For example, multiple lanes may be positioned along different radial axes of the sample stage (e.g., a top stage with a predetermined angle between each pair of adjacent lanes). In such embodiments, movement of the top stage relative to the stage along the y-axis of the top stage can be eliminated. Instead, the stage may be rotated at a predetermined angle to move to the next lane of the sample flow cell. Such embodiments with non-parallel lanes can advantageously eliminate the need to move the top stage along its respective x-axis, thereby simplifying the movement of the top stage relative to the base in the xy plane.

[0077] In some embodiments, the top platform here may include a manifold that can securely hold one or more flow cell devices therein. The manifold may include an open state in which the flow cell devices can be removed or installed within the manifold. The manifold may also include a closed state in which the flow cell devices are secured within the manifold, forming a sealed fluid communication between the flow cell devices (e.g., cleaning outlets) and the manifold. Further, in the closed state, the relative position of the flow cell devices with respect to the manifold is fixed. In some embodiments, a sealed fluid communication is formed between the manifold and a fluid control device.

[0078] In some embodiments, the fluid control device includes one or more sealed fluid passages leading to a manifold and a flow pool device. In some embodiments, some of the sealed fluid passages are configured to seal the application of fluid to the flow pool device. In some embodiments, some or all of the sealed fluid passages are configured to collect sealed fluid (e.g., clean fluid residue from the flow pool device inlet) from the flow pool device.

[0079] In some embodiments, the sample stage, optical system, and optical assembly described herein advantageously eliminate the need for movement of the sample stage relative to the optical assembly or system described herein along the z-axis. Therefore, the problems and complexities that may arise from moving the sample stage and sample in the z-direction are also eliminated. Achieving z-axis movement of the focused sample can be done by moving its individual lens element (e.g., a single lens element) relative to the housing of the imaging module, which is simpler, more convenient, and more accurate than some optical systems.

[0080] In some embodiments, this document discloses methods for sequencing multiple DNA samples positioned on a rotating sample stage for DNA sequencing using various sequencing methods, including but not limited to sequencing by synthesis, affinity sequencing, and binding sequencing. During a sequencing run, such methods can be repeated in one or more sequencing cycles.

[0081] In some embodiments, a method for sequencing multiple DNA samples positioned on a rotating sample stage for DNA sequencing includes obtaining a sample stage comprising a stage and one or more top stages positioned on the top surface of the stage, wherein the stage is rotatable about a z-axis relative to the optical system or imaging module of the sequencing system.

[0082] In some embodiments, the method includes positioning and fixing a first flow pool device relative to one or more top platforms.

[0083] Flow cell devices can have 2D or 3D samples embossed thereon. Flow cell devices can have different numbers of microfluidic channels with channel surfaces on which samples can be fixed. The flow cell device described herein can have two, three, four, or more channel surfaces. Multiple channel surfaces can be displaced relative to each other along the z-axis such that at least two, three, or more channel surfaces are located at two, three, or more different z-positions relative to the optical system. For example, a flow cell device can have two channels in the z-direction, thus having four surfaces at different z-positions.

[0084] The flow cell device can be fixed relative to the top platform such that there is no relative movement between the flow cell device and the corresponding top platform when the top platform moves. The flow cell device can be fixed by various fixing or fastening methods, including, but not limited to, mechanically clamping the flow cell device, fastening with magnetic or electromagnetic force, positioning the flow cell device in a suitable housing (e.g., a manifold) fastened to the top platform, and connecting the pins or posts of the top platform to holes or slots in the flow cell device. For example, the flow cell device can be fixed in its corresponding manifold in the closed state, and a sealed fluid communication can be established between the flow cell device and the manifold in the closed state.

[0085] In some embodiments, the method further includes positioning and securing the second flow cell device relative to a second top platform among one or more top platforms. The second top platform may have the same or different securing or fastening method as the first top platform.

[0086] In some embodiments, the method further includes dispensing one or more sequencing reagents to a first flow cell device positioned on a first top stage via a first fluid control device to enable sequencing of the sample. Such dispensing of sequencing reagents can be performed openly, for example, by dispensing a tip into an open area leading to a channel in the flow cell device. Alternatively, such dispensing of sequencing reagents can be performed via a closed conduit.

[0087] In some embodiments, the method further includes imaging a first sample region of the first flow cell apparatus using the optical system of the sequencing system. The first sample region may include at least a portion of a first patch of the flow cell apparatus. Such imaging operations may include collecting emitted light signals from the sample via an image sensor of the imaging module. Such operations may also include automatically focusing the imaging module onto the sample using various autofocusing methods. Such imaging operations may further include generating excitation light propagating to the sample.

[0088] After imaging the first sample region of the first flow cell apparatus, the method may include moving the first stage in the xy-plane relative to the optical system while preventing the second flow cell apparatus from moving relative to the optical system. This operation allows the second sample region (e.g., at least a portion of a second patch) to be correctly positioned for imaging. This movement may occur in the sample plane (e.g., the xy-plane) along the x, y, or any other direction. During this movement of the first stage, the stage may remain stationary relative to the optical system.

[0089] In some embodiments, the method further includes moving a first fluid control device to keep the first fluid device (e.g., its dispensing tip) stationary relative to the first flow cell device while the first flow cell device moves relative to the optical system. In some embodiments, for example, the first fluid control device does not need to move when a closed conduit contacts the first flow cell device while the first flow cell device moves relative to the optical system. In some embodiments, the first top stage is actuated by a first motor configured to actuate a base. In some embodiments, the first top stage is actuated by a second motor configured to actuate one or more top stages independently of actuation of the base.

[0090] In some embodiments, the operation of moving the first top stage relative to the optical system in the xy plane while preventing the second flow cell device from moving relative to the optical system includes: moving the first top stage relative to the optical system by a predetermined distance along the radius of the top surface of the stage while preventing the second flow cell device from moving independently relative to the optical system.

[0091] In some embodiments, the operation of moving the first top stage relative to the optical system in the xy plane while preventing the second flow cell device from moving relative to the optical system includes: moving the first top stage independently relative to the optical system by a predetermined distance along a direction orthogonal to the top surface of the base while preventing the second flow cell device from moving relative to the optical system.

[0092] In some embodiments, the method further includes imaging a second sample region of the first flow cell device using the optical system of the sequencing system.

[0093] After imaging the second sample region of the first flow cell apparatus, the method may further include moving the first stage and imaging other sample regions of the first flow cell apparatus until all desired sample regions of the first flow cell apparatus have been imaged.

[0094] In some embodiments, the method may further include rotating the sample stage at a predetermined angular resolution to position the second flow cell device at a predetermined location relative to the optical system. This operation may occur in the same sequencing cycle as the operation of imaging a sample region of the first flow cell device. Alternatively, this operation may occur in a different sequencing cycle than the operation of imaging a sample region of the first flow cell device. The angular resolution may be the first resolution disclosed herein.

[0095] After rotating the sample stage at a predetermined angular resolution to position the second flow cell device relative to the optical system, in some embodiments, the method may further include dispensing one or more sequencing reagents to the second flow cell device via a first or second fluid control device such that the sample fixed on the second flow cell device can be subjected to a sequencing reaction. The one or more sequencing reagents dispensed to the second flow cell device may be different from those dispensed to the first flow cell device, for example, for different sequencing chemistry or applications. Dispensing sequencing reagents to the second flow cell device may be optional, for example, if the same sequencing reagents are used and dispensed simultaneously to both the first and second flow cell devices before imaging the first flow cell device.

[0096] In some embodiments, the method further includes: moving a first fluid control device or a second fluid control device to position the second fluid pool device at a predetermined position relative to the first fluid control device or the second fluid control device.

[0097] In some embodiments, after rotating the sample stage at a predetermined angular resolution to position the second flow cell device relative to the optical system at a predetermined location, the method may further include imaging a first sample region of the second flow cell device (e.g., at least a portion of a first patch of the second flow cell device) using the optical system of the sequencing system. The imaging operation of the first sample region of the second flow cell device may be the same as the imaging operation of the sample region in the first flow cell device. For example, when the first flow cell device contains a 2D sample and the second flow cell device contains a 3D sample such that the imaging operation may include imaging the 3D sample at different z-levels and the 2D sample at a single z-level, the imaging operation may differ from the imaging operation of the first sample region of the second flow cell device.

[0098] In some embodiments, the rotating sample stage and the method of sequencing samples using the rotating sample stage can advantageously improve sequencing capacity and system throughput by allowing sequencing and imaging of multiple flow cell devices using a single sample stage, and such multiple flow cell devices can contain different samples for different sequencing reactions. Furthermore, in some embodiments, the rotating sample stage and the sequencing method using the rotating sample stage can advantageously improve sequencing efficiency by allowing imaging of the first flow cell device while sequencing reagents are dispensed and flowed into the second flow cell device.

[0099] In some cases, the optical system may include a stage. This stage may be as described elsewhere herein (e.g., it may be configured to hold a flow cell device, a glass slide, or other similar solid support and on which a sample is placed). The stage may lack movement along the optical axis of the system. Such movement may be relative to a non-movable housing of the optical assembly (e.g., the housing of the first or third element). For example, in a system configured to illuminate and image a sample (e.g., an in situ sample of cells or tissue) at multiple different z-levels along the z-axis, the stage may not move along the z-axis relative to the non-movable housing of the optical system (e.g., the housing of the first or third element of the optical assembly) during sample imaging. In another instance, the stage and the optical assembly may not move relative to each other along the z-axis, yet still be able to focus the sample such that it lies within the focal plane of the optical assembly along the z-axis before or during imaging. The optical systems described herein may employ a method of focusing by moving only one or more lens elements within the optical assembly (such as moving the stage or optical assembly to move the sample into the focal plane of the optical system). One or more lens elements of the optical assembly may not include objectives. In some embodiments, the optical system described herein may employ the focusing method disclosed herein, which eliminates the need for a z-stage that responds to movement of a sample relative to an objective along the z-axis in another optical system.

[0100] Solid supports (e.g., flow cells) may include probes configured to bind nucleic acid molecules, proteins, peptides, etc. For example, probes complementary to nucleic acid molecules may be immobilized (e.g., bound) on the surface of the solid support.

[0101] A solid support (e.g., a flow cell) may include one or more samples fixed thereon. For example, a solid support may include one or more probe molecules configured to bind one or more samples. A solid support may include two or more surfaces on which one or more samples are fixed. For example, a solid support may include a first surface with a first sample fixed thereon and a second surface with a second sample fixed thereon. In some cases, a solid support includes at least about two, about three, about four, about five, about six, or more surfaces. In some cases, each surface of the solid support has a different sample fixed thereon, and each sample can be illuminated and imaged by an optical system. In some cases, two or more surfaces of the solid support may be axially displaced relative to each other along the optical axis (e.g., the z-axis) of the optical system. For example, the surfaces of the solid support may be stacked relative to the optical axis of the optical system.

[0102] The optical system may include a light source configured to illuminate the flow cell. The illumination may be used for at least a portion of an imaging operation (e.g., a sequencing operation as described elsewhere herein). The irradiance of the illumination is at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, or more milliwatts per square millimeter. In some cases, the light source may be a pulsed light source (e.g., a flash lamp, a pulsed laser, a pulsed light-emitting diode, etc.). In other cases, the light source may be a continuous light source (e.g., an incandescent lamp, a fluorescent lamp, a light-emitting diode, a continuous laser, etc.).

[0103] The optical system may include optical components. These optical components may be disposed within the optical path from the stage to the light source. The optical components may be configured to provide flow cell illumination of approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 square millimeters or more. The optical components can be configured to illuminate the flow cell region with variations of up to about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (e.g., peak-to-valley variation, standard deviation, variance, interquartile range, mean absolute deviation, coefficient of variation, etc.). The optical components can also be configured to illuminate the flow cell region with variations of up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (e.g., peak-to-valley variation, standard deviation, variance, interquartile range, mean absolute deviation, coefficient of variation, etc.). In some embodiments, such illumination variations may be variations in power over the flow cell region. In some embodiments, such illumination variations may be variations in irradiance over the flow cell region.

[0104] In some cases, the optical assembly does not include an objective lens (e.g., an objective lens assembly). For example, the optical assembly may not include an objective lens assembly. In some cases, the optical system does not include an objective lens. For example, the entire optical system may not include an objective lens at any point in the optical system. In another instance, the optical system may not include an objective lens in the optical path of the optical system. The optical assembly may not include a barrel lens. In some cases, the optical system may not include a barrel lens. For example, a barrel lens may not be present in the optical path of the optical system. Despite the absence of a barrel lens and / or an objective lens, the optical assembly or optical system can still achieve the large-area illumination described herein. For example, the optical system can achieve wide-range, uniform illumination without the use of a barrel lens or objective lens.

[0105] Optical components, and more specifically, the illumination system of the optical components (e.g., such as...) Figure 1 (As shown) can be configured to transmit illumination light from a light source to a stage and a sample fixed on the stage. For example, the illumination system can be positioned above an excitation dichroic filter, such as... Figure 27 Above 2770, the excitation dichroic filter of the optical assembly can be configured to transmit illumination from the illumination system to the sample and stage via a first segment. In other words, the excitation dichroic filter and the optical elements of the optical assembly located between the excitation dichroic filter and the stage are situated within the optical path between the light source and the stage, for example, Figure 27 The first segments 2710 and 2770. In some embodiments, the excitation dichroic filter may also be configured to reflect emitted light from the sample into the detector's optical path. In other words, the excitation dichroic filter and the optical components, also located between the excitation dichroic filter and the stage, are situated within the optical path between the stage and the detector, for example, Figure 27 The first paragraphs 2710 and 2770. An optical component may be configured to receive emitted light from a flow cell (e.g., light generated by the interaction of illumination light with a marker in the sample within the flow cell). For example, the optical component may be configured to receive the emitted light and transmit it to a detector. Non-limiting examples of detection include a CCD camera or a CMOS camera. The optical component may have a numerical aperture of at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or greater. The emitted light may have a wavelength of at least about 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000 nanometers or greater. The emitted light may have a wavelength of up to about 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400 nanometers or less. The emitted light may have a wavelength within the range defined by any two of the aforementioned values. The optical component may have an operating distance of at least about 1 mm to about 30 mm. Alternatively, the optical component may have an operating distance of at least about 2 mm to about 25 mm.

[0106] In some cases, the optical assembly may include a motion coil housed within it. The motion coil may be configured to move a focusing element within the optical path of the optical system. For example, the motion coil may be used to move the focusing element to change the focal point of the optical system without moving other parts of the system (e.g., the optical assembly, stage, etc.). Alternatives to the motion coil include, but are not limited to, piezoelectric actuators, motors (e.g., stepper motors, servo motors, etc.), electrostatic actuators, hydraulic actuators, pneumatic actuators, etc., or any combination thereof. In some cases, the motion coil (or other actuator) may be located externally to the optical assembly or optical system and configured to move the focusing element along the optical axis. For example, a motor located outside the optical assembly may be operatively coupled to the focusing element and may adjust the position of the focusing element within the optical assembly. Figure 27 Figure 2760 shows an actuator integrated with optical components. Examples of external actuators can be seen in... Figures 29A to 29B The actuator 2901 is coupled to the focusing element 2903 via a coupling element 2902. In some cases, the actuator is directly coupled to the focusing element or the focusing element housing. For example, the actuator may be coupled without using a coupling element. In some embodiments, the focusing element includes one or more lenses of an optical assembly. Figure 29A and Figure 31 In the specific embodiment shown, the focusing element is a single lens of an optical assembly. The focusing element can be mechanically coupled to a focusing element housing such that movement of the focusing element housing causes movement of the focusing element. The mechanical coupling between the focusing element and the focusing element housing can be achieved using various methods. Such mechanical coupling can be direct, without contact with a third element. Such mechanical coupling can be indirect, contacting a third element. For example, as... Figure 29A As shown, the two ends of the focusing element 2903 are directly clamped to the focusing housing element 2904.

[0107] The optical system may have a wavefront compounding root-mean-square (RMS) error of up to about 0.2, 0.15, 0.14, 0.13, 0.12, 0.11, 0.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less for the light transmitted through the optical system. The optical components or system may have an illumination efficiency of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or greater (e.g., the efficiency of the illumination light transmitted through the optical components or system). The optical system may be configured to image a solid support without moving an optical compensator into, out of, or into and out of the optical path between the solid support and the detector. For example, an image of the sample can be captured without moving the optical compensator into or out of the optical path between the sample and the detector. An optical component may be configured to produce one or more spatial contractions (e.g., one or more double beam waists in the light) transverse to the optical path through which the light travels. These one or more spatial contractions may advantageously: enhance imaging resolution; enhance the depth of focus of the optical component; provide low illumination variation at the sample stage; provide a wide field of view with low illumination variation; or a combination thereof. An optical component may be configured to produce at least one field curvature correction transverse to the optical path through which the light travels. The at least one field curvature correction may similarly improve optical resolution and depth of focus. An optical component may be configured to produce more than two field curvature corrections transverse to the optical path through which the light travels.

[0108] Figure 27Examples of optical components according to some embodiments are shown. A first segment 2710 may include a first housing containing a first plurality of lenses. A second segment 2720 may include a second housing. A third segment 2730 may include a third housing containing a second plurality of lenses. The first and third segments may be optically aligned (e.g., aligned on the same optical axis). The first segment may be positioned between the third segment and a stage (e.g., a solid support 2740). The third segment may be positioned between the first segment and an image sensor (e.g., a detector) of an optical system 2750. One or more lens elements in the first plurality of lenses may be movable along the optical path of the optical system by at least about 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mm or greater. The first plurality of lenses can be moved along the optical path of the optical system by a maximum of approximately 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or 0 mm. One or more lens elements of the first plurality of lenses can be moved along the optical path of the optical system by a distance within the range defined by any two of the aforementioned values. For example, a single lens element in the first plurality of lenses may be movable within a range of approximately 0 to approximately 25 millimeters along the optical path of the optical system in one or both directions along the optical axis. The optical assembly may include a dichroic filter 2770 configured to transmit illumination light from outside the optical assembly to a solid support.

[0109] In some cases, the first plurality of lenses includes one or more asymmetric biconvex lenses. In some cases, the second plurality of lenses includes one or more asymmetric biconcave lenses. The use of both biconvex and biconcave lenses, as well as the adjustment between the lenses, can be used to adjust the focal plane of the optical system (e.g., to move the focal plane to image the individual surfaces of a solid support).

[0110] In some cases, the actuator (e.g., a motion coil) may be coupled to a portion of the optical assembly (e.g., a first, second, or third housing of the optical assembly), and the focusing element may be coupled to the portion of the optical assembly to which the actuator is coupled. In this way, the actuator can adjust the focus of the optical assembly. The optical assembly may be configured to produce at least one field curvature correction transverse to the optical path of the first, second, or third segment.

[0111] Figure 30 Examples of optical elements and associated focusing paths of an optical assembly according to some embodiments are shown. The optical assembly can be configured to focus light onto a solid substrate 3001 and a detector 3002, and between the two. For example, illumination light can pass through a dichroic filter 3003 and be focused onto the solid substrate 3001. Once the illumination light interacts with a sample on the solid support, the resulting sample light can be refocused onto the optical assembly, reflected from the dichroic filter, and passed through an internal focusing group 3004. The internal focusing group can be configured to adjust the focus of the optical assembly, thereby allowing imaging of multiple surfaces of the solid support. The internal focusing group can be coupled to an actuator as described elsewhere herein. Signal light can pass through a multi-bandpass filter 3005. The multi-bandpass filter can have, for example... Figure 30 The optical density diagram is shown in the inset. A multi-bandpass filter can be configured to transmit sample light from the marker in the sample while suppressing other light to reduce noise. Passing through field leveler 3006, the signal light can be detected by detector 3002 and further analyzed, as described elsewhere herein.

[0112] Similarly, Figure 31 An alternative lens configuration for illuminating and collecting light from a solid support (e.g., a flow cell, a glass slide, etc.) 3101 is shown. Lens group 3102 may be configured to focus incident illumination light (e.g., illumination light transmitted through a dichroic filter 3103) onto a solid substrate, while also focusing signal light from the solid substrate through a notch filter 3104 and through a focusing element 3105 (part of lens group 3106). The focusing element may be as described elsewhere herein. Lens group 3017 may be configured to focus the signal light onto a detector 3108 for processing, as described elsewhere herein.

[0113] Figure 24A flowchart of a method 2400 for analyzing biomolecules according to some embodiments is shown. In operation 2410, method 2400 may include providing a solid support containing biomolecules. The solid support may be a flow cell. The biomolecules may include markers. The biomolecules may include, for example, nucleic acid molecules, proteins, peptides, carbohydrates, lipids, etc. The markers may vary depending on the characteristics of the biomolecules. For example, markers for nucleic acids and proteins may be different so that they can bind to different molecules. In another example, for nucleic acids, the markers may hybridize with the nucleic acids.

[0114] The label can be an optical label (e.g., a fluorescent label, a luminescent label, a Raman label, a scattering label, a plasma label, etc.), a magnetic label, etc. In some cases, the probe is indispensable to the biomolecule. For example, a protein containing green fluorescent protein can be both a biomolecule and a label. In some cases, prior to operation 2410, method 2400 may include binding the biomolecule to a probe in a solid support and / or coupling the label to the biomolecule. For example, the biomolecule may flow into a flow cell containing the probe, bind to the probe, and then the fluorescent label may be coupled to the biomolecule after binding to the probe. In another example, the biomolecule containing the probe may flow into the flow cell and bind to the probe. As yet another example, the sample to be sequenced and imaged may be immobilized on a flow cell device, and the label or probe may flow into the flow cell during various sequencing reactions. Examples of sequencing samples will be described in detail below.

[0115] In another operation 2420, method 2400 may include providing illumination to biomolecules containing the marker using an optical system including a light source, thereby generating signal light or a change thereof. Illumination may be provided over an area of ​​a flow cell larger than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 square millimeters or larger. The lighting may have variations of up to about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less (e.g., peak-to-valley variation, standard deviation, variance, interquartile range, mean absolute deviation, coefficient of variation, etc.).

[0116] The optical system may be as described elsewhere herein. For example, the optical system may not include an objective lens or a tube lens. The solid support may not move along the optical axis of the optical system. For example, the flow cell may be fixed along the z-axis of the flow cell (e.g., the optical axis of the optical system) while being movable along the x- and y-axis of the flow cell. In some cases, multiple images of the flow cell can be acquired without moving the flow cell along the optical axis. For example, a first image at a first depth of focal length can be acquired, and a second image at a second depth of focal length can be acquired without moving the solid support.

[0117] Motion coils or other actuators, as described elsewhere herein, can be used within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focal point of the optical system on a solid support. For example, the actuator can move the focusing element along the optical path to change the parameters of the optical system, thereby changing the focal point of the optical system from one side of the flow cell to the other. The light source can be a light source as described elsewhere herein (e.g., a pulsed light source). The optical system can have an illumination efficiency (e.g., the efficiency of the illumination light transmitted through the optical system) of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or greater.

[0118] In another operation 2430, method 2400 may include detecting a signal light or a change thereof using a detector of an optical system. The signal light may have a wavelength of at least about 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000 nanometers or greater. The signal light may have a wavelength of at most about 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400 nanometers or less. The signal light may have a wavelength within the range defined by any two of the aforementioned values. The optical system may have a numerical aperture of at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or greater. In some cases, the label can be removed from the biomolecule. For example, a hybridization label can be dehybridized from a nucleic acid molecule.

[0119] In another operation 2440, method 2400 may include at least partially processing the signal light or variations thereof to analyze biomolecules. Processing may be as described elsewhere herein (e.g., using properties of a marker to determine a portion of the biomolecule, etc.). Images of the solid support may include a field of view greater than about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 square millimeters or larger. Processing may include at least partially processing the signal light or variations thereof to generate one or more images of the solid support, and analyzing one or more images of the solid support to generate a base interpretation of the sample.

[0120] In some cases, method 2400 may include repeated operations 2420 to 2440 on additional biomolecules coupled to a further surface of a solid support. For example, multiple biomolecules may be coupled to a solid support having multiple markers, each of which is attached to a multiple biomolecule, and an optical system may image each of the markers. In some cases, method 2400 may include repeated operations 2410 to 2440 on additional markers bound to another portion of a biomolecule. For example, a first marker may identify a first nucleotide of a nucleic acid, which may be removed from the nucleic acid, and a second marker may hybridize to a second nucleotide of the nucleic acid molecule. In this example, the second marker may be identified in a similar manner to the first marker, providing information related to a second nucleotide of the nucleic acid molecule.

[0121] Lighting system

[0122] The illumination system described herein can advantageously provide at least 10 mm of illumination at the sample plane. 2 20mm 2 30mm 2 40mm 2 Or 50mm 2 The illumination system provides an ultra-wide illumination field. In some embodiments, the illumination system may advantageously provide an illumination field that is 2, 4, 5, 6, 8, 10, or 15 times larger than the illumination field produced by other illumination systems in the NGS optical system. The illumination system may advantageously provide a minimum of 30, 40, 50, 60, or 70 milliwatts / mm² at the sample plane. 2 The illumination power density. In some embodiments, the illumination system is configured to generate an illumination power density greater than 20, 30, 40, or 50 mm at the sample stage. 2 The lighting field has a power density variance or standard deviation of less than ±2%, 5%, 8%, 10%, or 12%. In some embodiments, the variance or standard deviation is measured as a percentage of the average power density, maximum power density, or median power density.

[0123] The illumination system advantageously enables imaging of a wide field of view (FOV) 2, 4, 5, 6, 8, 10, or 15 times larger than the maximum FOV offered by existing optical systems for NGS applications, thereby increasing system throughput and flexibility in NGS applications. Because it can illuminate a wide field of view and image part or all of the wide illumination field, the illumination system and imaging module of this paper advantageously eliminate the photon bleaching problem in unimaged areas associated with some optical systems.

[0124] In some embodiments, the lighting system may provide a power efficiency of not less than 65%, 70%, 75%, or 80%. In other words, the power loss within the lighting system may be less than 20%, 25%, 30%, or 35%. In some embodiments, the lighting subsystem, the beam transmission subsystem, or both may each provide a power efficiency of not less than 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, one or more optical elements in the lighting system may each provide a power efficiency of not less than 65%, 70%, 75%, 80%, 85%, or 90%, such as the power efficiency of optical fibers, lens arrays, etc. In some embodiments, power efficiency may be determined as the ratio or percentage of power leaving the optical element to power entering the optical element.

[0125] The sample plane in this paper can be the location where the sample is positioned, and it can be orthogonal to the z-axis or optical axis of the imaging module. In some embodiments, the sample plane overlaps with the focal plane of the objective lens of the imaging module.

[0126] In some embodiments, the lighting system includes a lighting subsystem and a beam transmission subsystem optically coupled to the lighting system.

[0127] Lighting subsystem

[0128] The lighting subsystem may include a light source, which may be used alone or in combination with a speckle eliminator for the light source.

[0129] The light source may include one or more lasers. Lasers can be of various types. Some non-limiting examples of lasers include: gas lasers, solid-state lasers, fiber lasers, dye lasers, and semiconductor lasers (laser diodes). The one or more lasers may include one or more laser diodes. The one or more lasers may emit light of multiple wavelengths. In some embodiments, each laser or laser diode may emit light of a predetermined color (e.g., red, green, or blue). In some embodiments, each laser or laser diode may emit light within a wavelength range corresponding to the predetermined color (e.g., red, green, or blue). The wavelength range of the predetermined color may be less than 0.1 Hz, 1 Hz, 10 Hz, 20 Hz, 50 Hz, or more. In some embodiments, the one or more lasers or laser diodes may emit light of multiple colors or wavelength ranges. In some embodiments, each laser or laser diode may emit light of multiple colors, such as white light.

[0130] In some embodiments, the light source comprises one or more multicolor laser arrays. Each multicolor laser array may include lasers arranged in an array in any direction in the xy-plane, such as a 2D array. The lasers in the array may have different colors, such that each laser may have a different color from the lasers immediately adjacent to it in the array. In some embodiments, the multicolor laser array comprises an array of laser diodes that emit light of 2, 3, 4, 5, or 6 wavelengths or wavelength ranges of 2, 3, 4, 5, or 6 wavelengths. Each wavelength or wavelength range may correspond to a different color. In some embodiments, the multicolor laser array comprises lasers that emit light of 2, 3, or 4 color wavelengths or wavelength ranges in at least a direction orthogonal to the z-axis. Figure 2 An example of an embodiment of a multicolor laser array is shown, wherein the laser diodes produce at least three different colors, such as blue, red, and green.

[0131] In some embodiments, the illumination subsystem may further include one or more optical fibers that can be coupled to a light source to transmit light from that source. In some embodiments, a single optical fiber is coupled to a corresponding laser or laser diode (multicolor or monochromatic). The optical fibers may have various fiber lengths. For example, the length of one or more optical fibers may be from 0.5 m to 5 m. In some embodiments, one or more optical fibers may include a core. The core may have a maximum dimension (e.g., diameter) of 50 μm to 2000 μm in its cross-section. The cross-section may be orthogonal to the longitudinal axis extending along the length of the optical fiber. In some embodiments, the cross-section of the core may be circular or substantially circular. Figure 1 Figure 3 to Figure 4 and Figure 7 An example of an embodiment of a laser diode and optical fiber coupling to the laser diode is shown.

[0132] In some embodiments, the lighting subsystem further includes, for example, Figure 2 The single optical fiber shown may be a multimode fiber. This multimode fiber is configured to transmit light of different colors, wavelengths, or wavelength ranges within the same fiber. The single optical fiber may include a core having a maximum dimension (e.g., diameter) of 400 μm to 2000 μm in a cross-section orthogonal to the fiber's longitudinal axis. The single optical fiber may include a core having a maximum dimension (e.g., diameter) of 600 μm to 1600 μm. The single optical fiber may include a core having a maximum dimension (e.g., diameter) of 800 μm to 1300 μm.

[0133] In some embodiments, the illumination subsystem may include multiple optical fibers. Each optical fiber may be optically coupled to one or more corresponding lasers of the light source. The one or more corresponding lasers may emit light of the same wavelength or wavelength range. The one or more corresponding lasers may emit light of the same color. In some embodiments, the illumination subsystem further includes one or more dichroic filters, optical lens elements, or both.

[0134] Figure 3 illustrates an example of one embodiment where the light source comprises an array of red, green, and blue laser diodes. A single optical fiber is coupled to the laser, and various optical elements, such as dichroic filters and lens elements, can then be used to combine light of different colors from the different optical fibers.

[0135] In some embodiments, the light source includes one or more beam combiners. Each beam combiner is configured to combine two distinct beams (e.g., beams with different polarizations or other beam characteristics) into a combined beam. In some embodiments, the beam combiner may be a polarization beam combiner. In some embodiments, the light source may include two or more lasers that emit light of the same wavelength or the same wavelength range. Each beam combiner may combine light emitted by such two or more lasers of the same wavelength or the same wavelength range into a combined beam. In some embodiments, each beam combiner may combine light emitted by two or more lasers that emit light of the same color into a combined beam. In some embodiments, the beam combiner is configured to increase the power coupled into an optical fiber or sample plane by combining two beams into a combined beam. In some embodiments, the power of the combined beam is greater than the power of each individual beam before combination. Figure 5 An example of an embodiment is shown, which combines two beams of the same color from two lasers with a higher success rate than the combined beam of each individual beam.

[0136] In some embodiments, the optical fiber coupled to a monochromatic or multicolor laser may include a core having a non-circular cross-section orthogonal to the z-axis. The non-circular cross-section may be of various shapes, such as oval, triangular, rhomboid, pentagonal, hexagonal, etc. For example, the core may include a rectangular or square cross-section. Figure 7 An example of an embodiment of an optical fiber with a rectangular core is shown, which can advantageously facilitate the transmission of more uniform optical power on a rectangular sample plane, thereby better matching the imaging FOV, which is also rectangular.

[0137] In some embodiments, the light source may be coupled to a liquid-core optical guide with a liquid core. In some embodiments, the one or more liquid-core optical guides are optically coupled to the light source in the absence of an optical fiber. In some embodiments, each laser may be coupled to a liquid-core optical guide. In other embodiments, multiple individual lasers may be coupled to a single liquid-core optical guide. Figure 8An example of one embodiment of a liquid-core optical guide is shown. In some embodiments, the liquid-core optical guide facilitates the transmission of high optical power, wherein homogenization is achieved within a wide illumination field over a sample plane. In some embodiments, the one or more liquid-core optical guides include a liquid core. The liquid core may have a maximum dimension of 0.5 mm to 10 mm in a cross-section orthogonal to the z-axis. In some embodiments, the one or more liquid-core optical guides include a liquid core having a maximum dimension of 0.2 mm to 20 mm in a cross-section orthogonal to the z-axis. In some embodiments, the liquid core includes a circular cross-section. In some embodiments, the liquid core includes a non-circular cross-section. In some embodiments, the liquid core may include a cross-section having various non-circular shapes.

[0138] In some embodiments, the illumination subsystem further includes one or more coupling elements, such as optical lenses. The one or more coupling lenses may be positioned between the light source and the optical fiber, for example, as shown in the image. Figure 2 As shown. The coupling lens can be configured to couple laser light from a light source to an optical fiber, a liquid-core optical guide, or other optical element (e.g., a collimator) for transmitting light. The coupling lens may include one or more of the following: an asymmetric biconvex lens, a convex-plano lens, a concave-plano lens, an asymmetric biconcave lens, and an asymmetric convex-concave lens.

[0139] Figures 26 to 27 Non-limiting examples of the single-channel temporal color imaging module or optical assembly disclosed herein are shown. The single-channel temporal color imaging module of this paper can be advantageously used to image optical signals of different wavelengths, such that it can be configured to image in multiple color channels within a conventional system without the need for additional image sensors and other optical elements (such as associated dichroic beam splitters and excitation notch filters). Compared to systems in which signals are acquired through different channels, images of optical signals of different wavelengths can be acquired sequentially in a single channel. Compared to existing optical systems, the single-channel temporal color imaging module helps to reduce system size, complexity, and cost.

[0140] Figure 26 This is a perspective view of the imaging module, showing the housing of different segments (e.g., 3 segments) of the imaging module. Figure 27 This is a cross-sectional view showing the different lens elements in different segments and their relative positions to each other. The autofocusing beam and the excitation / illumination beam can pass through... Figures 26 to 27 The excitation dichroic filter is injected into the single-channel time-series color imaging module.

[0141] The imaging module can have a "double-waist" design, which includes at least two contractions in the optical path passing through the imaging module, for example, between the sample and the image sensor. Figure 31The diagram illustrates two contractions, one occurring in the first segment and the other potentially occurring in the third segment. These contractions can induce overcorrection or backward bending components in field curvature correction compared to some imaging systems, thereby enabling flat-field imaging of samples at a much wider scale than allowed by those systems. This wider field of view advantageously translates to significantly improved image acquisition time to cover specific sample regions, thus providing the ability to build high-throughput sequencing systems. In this particular embodiment, the sample region or field of view that can be imaged within a single image is increased by 10x, 13x, 15x, or 20x compared to some imaging systems.

[0142] In some embodiments, a three-notch filter or a double-notch filter (not shown) may embed two lens elements (e.g., Figure 28 The collimation space between points 5 and 7 is used. The maximum angle of the optical path (with the optical axis) at this location is controlled to be less than 10, 8, 6, or 5 degrees to achieve OD6 suppression of the excitation wavelength. In some embodiments, a double-notch or triple-notch filter can be added at this location to suppress possible leakage from the excitation wavelength, e.g., leakage from the illumination system to the sample. For example, a triple-notch or double-notch filter can be located... Figure 38 The top of the 9 is located within the housing, which provides an accessible hard aperture stop. This accessible hard aperture stop is accessible from outside the imaging module.

[0143] In some embodiments, the imaging module described herein is capable of autofocusing at least along the optical axis by moving one or more elements along the optical axis. In some embodiments, the lens elements can be moved longitudinally to achieve multi-surface imaging, wherein the z-motion range is 0 to 5 mm, 0 to 3 mm, 0 to 2 mm, 0 to 1 mm, 0 to 0.8 mm, 0 to 0.6 mm, 0 to 0.5 mm, or 0 to 0.4 mm. The movement of the internal lens elements advantageously eliminates the z-stage assembly used for moving the entire objective relative to the sample, and the associated integration problems.

[0144] In some embodiments, the imaging module may include a lens element for aberration correction. This lens element may be aspherical. For example, Figure 28 8 represents a lens element used for aberration correction. In some embodiments, this lens element can achieve spherical aberration correction to help improve both aberration correction and transmittance simultaneously. In some embodiments, this lens element, or any other optical element, can be manufactured using a glass type with low autofluorescence. In some embodiments, the lens element may include a global surface design.

[0145] In some embodiments, the imaging module or optical component described herein includes an illumination system. This illumination system may include an illumination subsystem and a beam transmission system. Figure 1A non-limiting example of an illumination system with excitation / illumination light is shown.

[0146] speckle eliminateer

[0147] Due to the coherent characteristics of lasers, interference can occur between light waves of the same frequency, leading to undesirable laser speckle noise. This speckle noise can cause inhomogeneity in the illumination field, thus resulting in errors in DNA sequencing results. Therefore, there is a need for an effective and easily implemented method to reduce speckle. Commercially available speckle eliminators are expensive, for example, costing $1000 per unit. The speckle eliminator described in this paper provides a low-cost, easy-to-implement, and effective way to reduce speckle noise from a light source.

[0148] In some embodiments, various laser-based illumination sources may be affected by speckle noise, and speckle intensity can be controlled to minimize potential errors in image intensity-based sequencing analysis. Source coherence (e.g., speckle) can be reduced by using predetermined fiber lengths and / or core sizes (e.g., fibers 1 to 3 m in length with a 200 to 800 μm core). In some embodiments, the illumination system may allow mode mixing to reduce speckle to a predetermined level. In some embodiments, time-varying diffuser attenuation methods may be integrated into the optical path to improve source coherence.

[0149] In some embodiments, a single optical fiber can be coupled to a corresponding laser or laser diode. In some embodiments, the characteristics of the optical fiber can be predetermined to reduce laser speckle. Some non-limiting characteristics of the optical fiber may include: fiber length, fiber bending radius, core shape, core size, and attachment of the fiber to the vibration source, etc.

[0150] In some embodiments, the speckle eliminator comprises an optical fiber optically coupled to a light source. In some embodiments, the speckle eliminator is coupled or associated with the optical fiber to mitigate speckle from the light source. In some embodiments, the speckle eliminator is coupled or associated with the optical fiber such that a speckle reduction effect can be generated in a portion or all of the optical fiber.

[0151] In some embodiments, the speckle eliminate includes a mechanical vibration source, such as a vibrating motor. The vibration source can generate vibration at a predetermined frequency or frequency range. In some embodiments, the mechanical vibration source is configured to vibrate at one or more frequencies within an audible sound frequency range, an ultrasonic frequency range, or both. In some embodiments, the mechanical vibration source is configured to vibrate at one or more frequencies between 10 and 500 Hz. For example, the vibration source can vibrate at a single frequency, wherein the standard deviation is less than 1%, 5%, or 10% of that single frequency. As another example, the vibration source can vibrate at randomly selected frequencies within a bandwidth (e.g., 80 to 90 Hz). In some embodiments, the mechanical vibration source is configured to generate one-dimensional, two-dimensional, or three-dimensional vibrational motion. In some embodiments, the mechanical vibration source is configured to generate vibrational motion including two-dimensional or three-dimensional translation, rotation, or both. In some embodiments, the mechanical vibration source is configured, but not limited to, generating linear or nonlinear vibration, random or deterministic vibration, and / or undamped or damped vibration.

[0152] In some embodiments, at least a portion of the optical fiber is wound or coiled one or more times, such as Figures 9A to 9D As shown. The winding or coiling portion may include a radius not less than the minimum bending radius of the optical fiber to avoid damaging the fiber. For example, the winding or coiling portion may include a radius of 60mm, 65mm, 70mm or larger, with a minimum bending radius of 60mm. The winding or coiling portion may be, but does not necessarily have to be, a perfect circle, such as... Figures 9A to 9D As shown. In some embodiments, the winding or coiling portion may include at least 2, 3, 4, 5 or more turns. In some embodiments, the number of turns of the winding or coiling portion may be limited by the fiber length and the minimum bending radius of the fiber to achieve the maximum possible number of turns. In some embodiments, the number of turns of the winding or coiling portion may be maximized based on the fiber length and the minimum bending radius of the fiber. In some embodiments, at least a portion of the fiber is not wound or coiled at or near its ends. The uncoiled portion of the fiber at each end may be less than 2%, 4%, 5%, 8% or 10% of the total fiber length. The uncoiled portion of the fiber at each end may be less than 0.05m, 0.1m, 0.15m, 0.2m or 0.3m.

[0153] In some embodiments, at least a portion of the optical fiber is loosely or fixedly attached to a mechanical vibration source. For example, the optical fiber can be taped to an off-the-shelf fan, whether or not it is wound or coiled one or more times. This off-the-shelf fan is a cooling fan for a CPU or other component of the sequencing system described herein. Figures 9C to 9D As shown, the optical fiber is wound or coiled around an existing fan, or further attached to the fan with tape in some locations. For example... Figure 9B As shown, the optical fiber is coiled around the wheel, and the vibration source is fixedly attached to the center of the wheel at the bottom.

[0154] Mechanical vibration sources can be any type of machinery capable of generating vibrational motion. They can be readily available machines that are more cost-effective than commercially available speckle eliminaters. As non-limiting examples, mechanical vibration sources may include one or more of the following: eccentric rotating mass (ERM) vibratory motors, linear resonant actuators, coin-type vibratory motors, mobile phone vibratory motors, acoustic or ultrasonic vibratory motors (e.g., vibratory motors such as those used in electric toothbrushes), track gears or gear sets, tracked weights, etc.

[0155] In some embodiments, the speckle eliminator is physically isolated from other components of the imaging module, except for the optical fiber, to minimize the impact of speckle eliminator movement on sequencing response and / or imaging quality. For example, the speckle eliminator is not coupled to the optical fiber at or near either end of the fiber (e.g., less than 0.1 m). For example, the speckle eliminator is not positioned within a predetermined threshold distance (e.g., at least 0.1 m, 0.2 m, 0.5 m, or greater) from other components of the imaging module. In some embodiments, the speckle eliminator is physically isolated from the sample stage, objectives, and / or one or more image sensors, such that the mechanical movement of the speckle eliminator is independent of the sample stage, objectives, and one or more image sensors. For example, the speckle eliminator is located at least 0.1 m, 0.2 m, 0.5 m, or greater away from the sample stage, objectives, and / or one or more image sensors. In some embodiments, the speckle eliminator is configured to reduce speckle noise to no more than 4%, 4.5%, 5%, or 5.5%. In some embodiments, the speckle canceller is configured to reduce speckle noise by at least 10%, 15%, 20%, 30%, 35%, 40% or more, such that the reduced speckle noise is less than 40%, 50%, 55%, 60%, 65%, 70% or 70% of the original speckle noise.

[0156] Figure 10 Table 1 illustrates the effects of different embodiments of the speckle canceller described in this paper on speckle noise in optical fibers. Two optical fibers, each coupled to a green and a red light source, were examined. By winding the fiber three times and vibrating at least the wound portion with an off-the-shelf fan, the speckle noise was reduced from 6.53% or 6.32% to less than 4.5%. The speckle reduction effect was more pronounced when the fan was kept upright rather than flat on a table or other horizontal surface. Figures 9C to 9D The image shows a fan in either an upright or horizontal position.

[0157] In some embodiments, various methods for determining the uniformity level of the beam (e.g., the standard deviation of the beam intensity at the sample plane) can be used to calculate speckle noise. For example, the 2D beam profile within the imaging FOV can be divided into multiple regions, the standard deviation of the intensity of each individual region can be determined, and the average standard deviation of all regions can be used as the speckle noise level.

[0158] In some embodiments, the speckle canceller is positioned in the optical path between the collimator and the objective lens of the imaging module, for example, as... Figure 6 As shown. The speckle canceller is configured to generate micro-motion. At such large beam positions, the speckle canceller advantageously avoids high optical power density on the speckle canceller, preventing damage to it. In some embodiments, the speckle canceller described herein may comprise a single speckle canceller. In some embodiments, the speckle canceller described herein may comprise a combination of one or more first speckle cancellers associated with an optical fiber and one or more second large beam speckle cancellers positioned after the collimator in the optical path but before the objective or sample plane. In some embodiments, the large beam speckle canceller is positioned where the maximum dimension (e.g., diameter or diagonal) of the beam in its cross-section (orthogonal to the z-axis) is greater than 1 mm, 2 mm, 5 mm, 8 mm, 10 mm, or 20 mm.

[0159] In one aspect, this disclosure provides an optical system. The optical system may include a stage configured to fix a solid support (e.g., a flow cell). The stage may be as described elsewhere herein. The optical system may include a light source configured to illuminate the solid support (as described elsewhere herein). The optical system may include a speckle eliminator optically coupled to the light source and disposed within the optical path from the light source to the stage.

[0160] The speckle canceller can be as described elsewhere herein. It can be configured to reduce speckle noise, act as a coupler for light sources, or a combination thereof. For example, the speckle canceller can be configured to receive light from multiple light sources and combine the light from the multiple light sources into a single beam. In this example, various excitation wavelengths can be combined into a single optical path and simultaneously speckle. At least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more light sources can be optically coupled to a single speckle canceller. In some cases, each light source can be optically coupled to a different speckle canceller. For example, four light sources can each be coupled to four speckle cancellers, thereby speckling the light from each light source.

[0161] Speckle cancellers may include diffuse speckle cancellers (e.g., speckle cancellers including diffusers such as fixed diffusers, rotating diffusers, etc.), spatial light modulators, phase speckle cancellers (e.g., speckle cancellers configured to change the phase of light), polarization speckle cancellers, vibration speckle cancellers (e.g., speckle cancellers configured to vibrate one or more optical elements such as mirrors, optical fibers, lenses, etc.), tension speckle cancellers (e.g., speckle cancellers configured to adjust the tension of an optical fiber to induce speckle), and any combination thereof. A speckle canceller can be configured to reduce speckle noise in an optical system by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. A speckle canceller can be configured to reduce speckle noise in an optical system to at most about 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. Using a speckle canceller can improve the image quality of an optical system by reducing speckle noise generated in the optical system, enhancing contrast, or a combination thereof. Using a vibration speckle eliminator can provide unexpected benefits to optical systems, which are typically avoided because vibration affects the resolution of the optical system. By using vibration in the opposite way (e.g., vibration already present in the system from fans, motors, etc.), the methods and systems of this disclosure can provide enhanced illumination distribution and imaging.

[0162] Figure 25 A flowchart of a method 2500 for analyzing a biological sample according to some embodiments is shown. In operation 2510, method 2500 may include providing a solid support (e.g., a flow cell) containing the biological sample. The biological sample may include markers as described elsewhere herein. The biological sample may be as described elsewhere herein (e.g., the biological sample may include nucleic acid molecules, proteins, peptides, etc.). The marker may be an optical marker as described elsewhere herein. The biological sample may include a two-dimensional biological sample. The biological sample may include a three-dimensional biological sample.

[0163] In another operation 2520, method 2500 may include illuminating a biological sample containing a marker using an optical system including a light source, thereby generating signal light or a variation thereof. The optical system may be as described elsewhere herein. For example, the optical system may include a speckle eliminator. Illumination may be provided by a speckle eliminator oriented in the optical path of the optical system. This speckle eliminator may be as described elsewhere herein. For example, the speckle eliminator may be a vibrating speckle eliminator. In some cases, an additional light source may be used to illuminate the solid support. The additional light source may provide light of a different wavelength than the light source to the flow cell. For example, the light source may provide a first wavelength configured to excite a first marker, and the additional light source may provide a second wavelength configured to excite a second marker. This additional light source may be optically coupled to the speckle eliminator. For example, both the light source and the additional light source may be optically coupled to the same speckle eliminator, and the output of the speckle eliminator may include light from both light sources.

[0164] In another operation 2530, method 2500 may include detecting a signal light or a change thereof using a detector of an optical system. Detection may be as described elsewhere herein. For example, detection may include using optical components to direct the signal light or a change thereof to the detector. In some cases, detection may include time-gated detection.

[0165] In another operation 2540, method 2500 may include at least partially processing the signal light or variations thereof to analyze a biological sample. Processing may include using one or more computer systems as described elsewhere herein. Processing may include generating one or more base readouts of the sample.

[0166] In some cases, method 2500 may include repeating operations 2520 to 2540 on additional biological samples coupled to additional surfaces of a solid support. For example, multiple biomolecules may be coupled to a solid support having multiple markers, each of which is attached to a multiple biomolecule, and an optical system may image each of the markers. In some cases, method 2500 may include repeating operations 2510 to 2540 on additional markers bound to another portion of a biomolecule. For example, a first marker may identify a first nucleotide of a nucleic acid, which may be removed from the nucleic acid, and a second marker may hybridize to a second nucleotide of the nucleic acid molecule. In this example, the second marker may be identified in a similar manner to the first marker, providing information related to a second nucleotide of the nucleic acid molecule.

[0167] Beam transmission subsystem

[0168] The beam transmission subsystem described herein may include one or more collimators and one or more optical lens elements. In some embodiments, the power in the beam transmission subsystem is greater than 5, 8, 10, 12, or 14 watts for one or more wavelengths or wavelength ranges. In some embodiments, the power at the sample plane is greater than 5, 8, 10, 12, or 14 watts for one or more wavelengths or wavelength ranges. In some embodiments, the power in the beam transmission subsystem is greater than 5, 8, 10, 12, or 14 watts for one or more colors. In some embodiments, the power at the sample plane is greater than 5, 8, 10, 12, or 14 watts for one or more colors.

[0169] In some embodiments, one or more collimators may be spaced apart along the z-axis or in the xy-plane. In some embodiments, the beam transmission system may include a single collimator. Figure 1 and Figure 6 An example of an embodiment of the beam transmission subsystem is shown. For example... Figure 1 As shown, one or more optical lens elements include one or more multi-lens arrays (e.g., MLA1 and MLA2). In some embodiments, each multi-lens array includes one or more of the following: an asymmetric biconvex lens, a convex-plano lens, a concave-plano lens, an asymmetric biconcave lens, and an asymmetric convex-concave lens. Each multi-lens array may include a plurality of lens elements at least in a direction orthogonal to the z-axis. For example, the plurality of lens elements of the array may be distributed along the x-axis or y-axis, or in any direction in the xy-plane orthogonal to the z-axis. Figure 1 In some embodiments, one or more optical lens elements include: an asymmetric biconvex lens, a convex-planar lens, a concave-planar lens, an asymmetric biconcave lens, an asymmetric convex-concave lens, or a combination thereof.

[0170] In some embodiments, one or more optical lens elements include: a first multilens array (MLA1) and a second multilens array (MLA2), which are positioned along the z-axis between the collimator and the entrance pupil of the illumination system, such as... Figure 1 As shown. In this particular embodiment, the illumination system includes a wide-field illumination module with fiber-coupled diode laser inputs interlaced at an angle on adjacent columns of a first multi-element lens array pair MLA1 and MLA2. Multiple images of the light sources are generated externally or in the entrance pupil to form a uniform illumination field at the sample field (e.g., a flow cell). The illumination system may include fiber-coupled laser diodes. The illuminator design is based on generating multiple source images on the entrance pupil plane of the imaging module. The output light from the fiber-coupled laser is connected to a collimator and then split in the pupil space by a plurality of multi-element lens arrays to form these secondary illumination sources. Figure 1Imaging group G3 is an alternative to the imaging module between the illumination system and the sample. Imaging group G3 may be shared by the imaging and illumination optical paths. In some embodiments, the illumination system is configured to generate secondary illumination sources overlapping on the sample stage or the sample positioned thereon, thereby averaging the individual intensities to provide intensity with improved uniformity compared to some methods.

[0171] In some embodiments, various laser-based illumination sources may be affected by speckle, and the speckle intensity may be controlled. Source coherence (e.g., speckle) can be mitigated by using optical fibers of 1 to 3 m in length (with a core of 200 to 800 μm). In some embodiments, the illumination system may allow mode mixing to reduce speckle to a predetermined level. In some embodiments, a time-varying diffuser attenuation method may be integrated into the optical path to improve source coherence. Figures 3A to 3C The intensity distribution at a sample using the lighting system described herein is shown.

[0172] sequencing system

[0173] Figure 11 A block diagram of a system 100 for imaging a sequencing reaction of a sample on a flow cell, according to one embodiment, is shown. System 100 has a sequencing system 110, which may include a flow cell 112, a sequencer 114, an imager (e.g., an optical system) 116, a data storage device 122, and a user interface 124. Sequencing system 110 may be connected to a cloud 130. Sequencing system 110 may include one or more of the following: a dedicated processor 118, a field-programmable gate array (FPGA) 120, and a computer system 126.

[0174] In some embodiments, flow cell 112 is configured to capture DNA fragments and form DNA sequences for base interpretation on the flow cell. Flow cell 112 may include a support as disclosed herein. The support may be a solid support. As disclosed herein, the support may include a surface coating thereon. The surface coating may be a polymer coating as disclosed herein.

[0175] The flow pool 112 may include multiple tiles or other imaging regions, and each tile may be divided into sub-tile grids. Each sub-tile may include multiple clusters or communities. As a non-limiting example, the flow pool may have 424 tiles, and each tile may be divided into a 6x9 grid, thus having 54 sub-tiles.

[0176] The flow cell images described herein are images of samples fixed to a support (e.g., a flow cell). Flow cell images as disclosed herein may be images of signals comprising multiple clusters or communities. A flow cell image may include one or more signal patches or one or more signal subpatches. In some embodiments, a flow cell image may be an image including all patches and approximately all signals on them. Flow cell images may be acquired from channels using imager 116 during an imaging or sequencing cycle. In some embodiments, each patch may include millions of communities or clusters. As a non-limiting example, a patch may include from about one to 10 million clusters or communities. Each community may be a collection of many copies of DNA fragments. Clusters or communities may appear as bright spots ranging from less than one pixel to several pixels.

[0177] Flow cell images can have various sizes or fields of view (FOV). In some embodiments, each flow cell image in the flow cell image includes a size greater than 20 mm. 2 30mm 2 40mm 2 Or 50mm 2 The wide field of view (FOV). In some embodiments, each flow cell image in the flow cell image contains a field of view (FOV) that completely overlaps with or is contained within the illumination field generated by the illumination system on the sample plane. In some embodiments, each flow cell image contains a field of view (FOV) that overlaps with at least 80%, 85%, 90%, or 95% of the illumination field generated by the illumination system on the sample plane. In some embodiments, each flow cell image contains a field of view (FOV) of at least 80%, 85%, 90%, or 95% of the size of the illumination field generated by the illumination system on the sample plane.

[0178] In some embodiments, to capture such a wide FOV, the image sensor may have a wider size than other image sensors used in some NGS sequencing systems. In some embodiments, the image sensor may include a size greater than 20 mm. 2 30mm 2 40mm 2 Or 50mm 2 The sensor size. In some NGS systems, flow cell images are typically 1 mm. 2 Up to 8mm 2 Within this range. Therefore, in these NGS systems, greater than 10 mm 2The illumination field is not preferred to avoid or reduce unwanted photon bleaching in the vicinity of the image's FOV. The illumination system described herein produces an illumination field that is 2, 5, 10, or greater than the illumination fields in these NGS systems. In some embodiments, the imager 116 is capable of producing an FOV of the flow cell image that is comparable in size to the wide illumination field described herein. The FOV and illumination field can be customized such that they overlap or substantially overlap each other. Furthermore, the illumination field can be customized such that its shape matches the shape of the FOV to facilitate such overlap. Through such overlap, photon bleaching in unimaged areas of the sample caused by an illumination field wider than the FOV of the flow cell image can be avoided or minimized.

[0179] The sequencer 114 can be configured to flow a mixture of nucleotides onto a flow cell 112, cleave blocking agents from the nucleotides between flow steps, and perform other steps for forming DNA sequences on the flow cell 112. The nucleotides may have linked fluorescent elements that emit light or energy at wavelengths indicating the type of nucleotide. Each type of fluorescent element may correspond to a specific nucleotide base (e.g., A, G, C, T). The fluorescent elements may emit light at visible wavelengths.

[0180] For example, each nucleotide base can be assigned a color. In some cases, adenine may be red, cytosine may be blue, guanine may be green, and thymine may be yellow. The color or wavelength of the fluorescent element can be selected for each nucleotide, so that the nucleotides can be distinguished from each other based on the wavelength of light emitted by the fluorescent element.

[0181] Imager 116 may be configured to capture an image of flow cell 112 after each flow step. Imager 116 may include one or more imaging modules disclosed herein. For example, the imager may include an optical system comprising four different imaging modules, each for capturing flow cell images from different color channels.

[0182] In one embodiment, imager 116 includes a camera, such as a CMOS or CCD camera, configured to capture digital images. The camera may be configured to capture images of wavelengths of fluorescent elements that bind to nucleotides.

[0183] The resolution of imager 116 controls the level of detail in the flow cell image, including pixel size. This resolution is critical in existing systems because it controls the accuracy of the spot-finding algorithm in identifying cluster centers. One way to improve spot-finding accuracy is to improve the resolution of imager 116 or to improve the processing of the images captured by imager 116. The methods described herein can detect cluster centers in pixels other than those detected by the spot-finding algorithm. These methods allow for improved cluster center detection accuracy without increasing the resolution of imager 116. The imager resolution can even be lower than that of existing systems with comparable performance, which can reduce the cost of sequencing system 110.

[0184] In one embodiment, images of the flow cell may be captured in groups, wherein each image in the group is taken at a wavelength or spectrum that matches or includes only one of the fluorescent elements. In another embodiment, images may be captured as a single image that captures at least a portion (e.g., a portion, all, etc.) of the wavelength of the fluorescent element.

[0185] The sequencing system 100 can be configured to identify cluster locations on flow cell 112 based on flow cell images. Processing for cluster identification can be performed by a dedicated processor 118, one or more FPGAs 120, a computing system 126, or a combination thereof. Identifying or determining cluster locations may involve conventional cluster lookup methods combined with those described more specifically herein.

[0186] General-purpose processors provide interfaces for use in systems such as Windows. TM or Linux TM Various programs run within an operating system. Such operating systems offer users great flexibility.

[0187] In some embodiments, a dedicated processor 118 may be configured to perform operations of the cluster discovery method described herein. The dedicated processor may not be a general-purpose processor, but rather a custom processor with specific hardware or instructions for performing these operations. The dedicated processor runs specific software directly, without an operating system. The lack of an operating system reduces overhead, but at the cost of limited processor flexibility. The dedicated processor may use a custom programming language, which can be designed to operate more efficiently than software running on a general-purpose processor. This increases the speed of operation execution and allows for real-time processing.

[0188] In some embodiments, FPGA 120 can be configured to perform the cluster discovery method described herein. The FPGA is programmed to be hardware that performs only specific tasks. Software operations can be translated into hardware components using a special programming language. Once the FPGA is programmed, the hardware directly processes the digital data provided to it without running software. Instead, the FPGA uses logic gates and registers to process digital data. Because the operating system requires no overhead, FPGAs typically process data faster than general-purpose processors. Similar to dedicated processors, this comes at the cost of flexibility.

[0189] The lack of software overhead also allows FPGAs to operate faster than dedicated processors, although this will depend on the exact processing to be performed and the specific FPGA and dedicated processor.

[0190] A group of FPGAs 120 can be configured to perform these operations in parallel. For example, multiple FPGAs 120 can be configured to perform processing operations on an image, a set of images, or cluster locations within one or more images. Each FPGA 120 can execute its own portion of the processing operation simultaneously, thereby reducing the time required to process data. This allows processing steps to be completed in real time. Further discussion of the use of FPGAs is provided below.

[0191] Compared to methods that may require storing data before processing it (which could require more memory or access to a computer system located in Cloud 130), performing processing steps in real time allows the system to use less memory because the data can be processed as it is received.

[0192] In some embodiments, data storage 122 is used to store information used in identifying cluster locations. This information may include the image itself or information derived from an image captured by imager 116. DNA sequences determined by base interpretation may be stored in data storage 122. Parameters identifying cluster locations may also be stored in data storage 122.

[0193] User interface 124 can be used by users to operate the sequencing system or access data stored in data storage 122 or computer system 126.

[0194] Computer system 126 controls the general operation of the sequencing system and can be coupled to user interface 124. It can also perform operations such as cluster location identification and base reading. In some embodiments, computer system 126 is a computer system. Computer system 126 can store information about the operation of sequencing system 110, such as configuration information, instructions for operating sequencing system 110, or user information. Computer system 126 can be configured to transfer information between sequencing system 110 and cloud 130.

[0195] As described above, the sequencing system 110 may have a dedicated processor 118, an FPGA 120, or a computer system 126. The sequencing system may use one, two, or all of these components to perform the necessary processing described above. In some embodiments, when these components are present together, the processing tasks are separated among them. For example, one or more FPGAs 120 may be used to perform the cluster center finding method described herein, while the computer system 126 may perform other processing functions for the sequencing system 110. Various combinations of these components allow for various system embodiments that balance the efficiency and speed of processing with the cost of the processing components.

[0196] The cloud 130 can be a network, a remote storage device, or some other remote computing system separate from the sequencing system 110. Connection to the cloud 130 allows access to data stored outside the sequencing system 110 or allows updates to the software within the sequencing system 110.

[0197] Support and low nonspecific coating

[0198] In some embodiments, NGS sequencing compositions and methods (e.g., pairwise sequencing) employ a support containing a plurality of oligonucleotide surface primers immobilized thereon. In some embodiments, the support is passivated with a low-nonspecific binding coating. The surface coatings described herein exhibit very low nonspecific binding to reagents commonly used in nucleic acid capture, amplification, and sequencing workflows, such as dyes, nucleotides, enzymes, and nucleic acid primers. The surface coatings exhibit a low background fluorescence signal or high contrast-to-noise (CNR) ratio compared to some other surface coatings.

[0199] Low nonspecific bonding coatings consist of one or more layers ( Figure 11In some embodiments, multiple surface primers are immobilized to a low-nonspecific binding coating. In some embodiments, at least one surface primer is embedded within the low-nonspecific binding coating. The low-nonspecific binding coating enables improved nucleic acid hybridization and amplification performance. Generally, the support comprises a substrate (or support structure), one or more layers of covalently or non-covalently attached low-binding chemically modified layers (such as silane layers or polymer films), and one or more covalently or non-covalently attached surface primers for tethering single-stranded nucleic acid library molecules to the support. In some embodiments, the formulation of the coating (e.g., the chemical composition of one or more layers, the coupling chemistry for crosslinking the one or more layers with the support and / or with each other, and the total number of layers) can be varied such that nonspecific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the coating is minimized or reduced relative to a comparable monolayer. The formulation of the coating described herein can be varied such that nonspecific hybridization on the coating is minimized or reduced relative to a comparable monolayer. The formulation of the coating can be varied such that nonspecific amplification on the coating is minimized or reduced relative to a comparable monolayer. The coating formulation can be varied to maximize the specific amplification rate and / or yield on the coating. In some of the cases disclosed herein, suitable amplification levels for detection are achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 amplification cycles.

[0200] The support structure comprising one or more chemically modified layers (e.g., layers of low nonspecifically binding polymers) may be standalone or integrated into another structure or component. For example, in some embodiments, the support structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure may include one or more surfaces within a microwell plate format (e.g., the bottom surface of a hole in a microwell plate). In some embodiments, the support structure includes the inner surface of a capillary (such as an inner lumen surface). In some embodiments, the support structure includes the inner surface of a capillary etched into a planar chip (such as an inner lumen surface).

[0201] The attachment chemistry used to graft the first chemically modified layer onto the surface of the support will generally depend on both the material from which the surface is made and the chemical properties of the layer. In some embodiments, the first layer may be covalently attached to the surface. In some embodiments, the first layer may be non-covalently attached to the support (e.g., adsorbed onto the support) through non-covalent interactions between the support and the molecular components of the first layer (such as electrostatic interactions, hydrogen bonding, or van der Waals interactions). In either case, the support may be treated prior to the attachment or deposition of the first layer. A variety of surface preparation techniques can be used to clean or treat the surface. For example, glass or silicon surfaces may be acid-washed using a Piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2), alkaline-treated with KOH and NaOH, and / or cleaned using oxygen plasma treatment methods.

[0202] The silane chemistry is a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amine or carboxyl groups), which can then be used to couple connector molecules (e.g., linear hydrocarbon molecules of various lengths (such as C6, C12, or C18 hydrocarbons) or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to produce any of the disclosed low-binding coatings include, but are not limited to: (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), PEG-silanes (e.g., containing molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (e.g., containing free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, etc.

[0203] Various molecules (including, but not limited to, amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof) can be used to generate one or more chemically modified layers on a support, wherein the selection of the components used can be varied to alter one or more properties of the layer, such as the surface density of functional groups and / or tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the layer, or the three-dimensional properties of the layer (e.g., “thickness”). Examples of polymers that can be used to generate one or more layers of low-nonspecific binding material in any of the disclosed low-binding coatings include, but are not limited to: polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyesters, dextran, polylysine and polylysine copolymers, or any combination thereof. Examples of conjugation chemistry that can be used to graft one or more layers (e.g., polymer layers) of a material onto a surface and / or crosslink the layers to each other include, but are not limited to: biotin-streptavidin interactions (or variants thereof), His-tagged Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylic acid ester conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxy resins, azides, hydrazides, alkynes, isocyanates, and silanes.

[0204] Low nonspecificity bonding surface coatings can be uniformly applied to a support. Alternatively, the surface coating can be patterned such that the chemically modified layer is confined to one or more discrete regions of the support. For example, photolithography can be used to pattern the coating to create an ordered array or random pattern of chemically modified regions on the support. Alternatively or in combination, contact printing and / or inkjet printing techniques can be used to pattern the coating. In some embodiments, the ordered array or random pattern of chemically modified regions may include at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions.

[0205] In some embodiments, the low nonspecific binding coating comprises a hydrophilic polymer that is nonspecifically adsorbed or covalently grafted to a support. Passivation can be performed using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyethylene oxide) or other hydrophilic polymers with different molecular weights and end groups chemically linked to the support, such as silanes. End groups remote from the surface may include, but are not limited to, biotin, methoxy ethers, carboxylic esters, amines, NHS esters, maleimides, and bissilanes. In some embodiments, two or more layers of a hydrophilic polymer (e.g., a linear polymer, a branched polymer, or a multibranched polymer) may be deposited on the surface. In some embodiments, the two or more layers may be covalently coupled to each other or internally crosslinked to improve the stability of the resulting coating. In some embodiments, surface primers (or other biomolecules, such as enzymes or antibodies) with different nucleotide sequences and / or base modifications may be tethered to the resulting layers at various surface densities. In some embodiments, for example, both the surface functional group density and the surface primer concentration may be varied to obtain a specified range of surface primer densities. Furthermore, the surface primer density can be controlled by diluting the surface primers with other molecules having the same functional groups. For example, amine-labeled surface primers can be diluted with amine-labeled polyethylene glycol during reactions with NHS-ester-coated surfaces to reduce the final primer density. Surface primers with linkers of varying lengths between the hybridization region and the surface attachment functional groups can also be applied to control surface density. Examples of suitable linkers include poly-T and poly-A chains (e.g., 0 to 20 bases) at the 5' end of the primer, PEG linkers (e.g., 3 to 20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.). To measure primer density, fluorescently labeled primers can be tethered to the surface, and the fluorescence readings can then be compared to the fluorescence readings of a dye solution of known concentration.

[0206] In some embodiments, the low nonspecific binding coating comprises a functionalized polymer coating covalently bound to at least a portion of the support via chemical groups on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM).

[0207] To scale primer surface density and add additional dimensions to hydrophilic or amphoteric coatings, supports comprising multilayer coatings of PEG and other hydrophilic polymers have been developed. Primer loading density on supports can be significantly increased by using hydrophilic and amphoteric surface layering methods (including, but not limited to, polymer / copolymer materials described below). Some PEG coating methods use monolayer primer deposition, which is often reported for single-molecule applications but does not produce high copy numbers for nucleic acid amplification applications. As described herein, “layering” can be achieved using conventional crosslinking methods with any compatible polymer or monomer subunit, allowing for the sequential construction of surfaces comprising two or more highly crosslinked layers. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyesters, dextran, polylysine, and copolymers of polylysine and PEG. In some embodiments, different layers may be attached to each other via any of a variety of conjugation reactions, including but not limited to: biotin-streptavidin binding, azide-alkyne click reaction, amine-NHS ester reaction, thiol-maleimide reaction, and ionic interactions between positively charged polymers and negatively charged polymers. In some embodiments, high primer density materials may be constructed in solution and subsequently layered onto a surface in multiple steps.

[0208] Examples of materials from which support structures can be manufactured include, but are not limited to: glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)) or any combination thereof. Various combinations of both glass and plastic support structures are contemplated.

[0209] The support structure can take on various geometries and sizes and can comprise a variety of materials. For example, the support structure can be partially planar (e.g., including a microscope slide or the surface of a microscope slide). Generally, the support structure can be cylindrical (e.g., including a capillary or the inner surface of a capillary), spherical (e.g., including the outer surface of a non-porous bead), or irregular (e.g., including the outer surface of an irregularly shaped non-porous bead or particle). In some embodiments, the surface of the support structure for nucleic acid hybridization and amplification can be a solid, non-porous surface. In some embodiments, the surface of the support structure for nucleic acid hybridization and amplification can be porous, such that the coating described herein penetrates the porous surface, and the nucleic acid hybridization and amplification reactions performed thereon can occur within the pores.

[0210] The support structure comprising one or more chemically modified layers (e.g., layers of low nonspecifically binding polymers) can be standalone or integrated into another structure or component. For example, the support structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure may include one or more surfaces within a microwell plate format (e.g., the bottom surface of a hole in a microwell plate). In some embodiments, the support structure includes the inner surface of a capillary (such as an inner lumen surface). In some embodiments, the support structure includes the inner surface of a capillary etched into a planar chip (such as an inner lumen surface).

[0211] As noted, the low-nonspecific binding supports of this disclosure exhibit reduced nonspecific binding to proteins, nucleic acids, and other components of hybridization and / or amplification formulations used for solid-phase nucleic acid amplification. The degree of nonspecific binding exhibited on a given support surface can be assessed qualitatively or quantitatively. For example, exposing the surface to a fluorescent dye (e.g., anthocyanins (such as Cy3 or Cy5, etc.), fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein), fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases) under a standardized set of conditions, followed by a specified rinsing protocol and fluorescence imaging, can be used as a qualitative tool for comparing nonspecific binding on supports containing different surface formulations. In some embodiments, exposing a surface to a fluorescent dye, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases) under a standardized set of conditions, followed by a specified rinsing protocol and fluorescence imaging, can be used as a quantitative tool for comparing nonspecific binding on supports containing different surface formulations. Care should be taken to ensure that fluorescence imaging is performed under conditions where the fluorescence signal is linearly correlated (or predictably correlated) with the number of fluorophores on the support surface and using suitable calibration standards (e.g., where signal saturation and / or fluorophore self-quenching are not problematic). In some embodiments, other techniques, such as radioisotope labeling and counting methods, can be used to quantitatively assess the extent of nonspecific binding exhibited by the different support surface formulations of this disclosure.

[0212] Some surfaces disclosed herein exhibit a ratio of specific to nonspecific binding to fluorophores (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the scope of this document. Some surfaces disclosed herein exhibit a ratio of specific to nonspecific fluorescence to fluorophores (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the scope of this document.

[0213] The degree of nonspecific binding exhibited by the disclosed low-binding supports can be evaluated using a standardized protocol that involves contacting the surface with labeled proteins (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc., under a standardized set of incubation and rinsing conditions, followed by detection of the amount of label remaining on the surface and comparison of the resulting signal with an appropriate calibration standard. In some embodiments, the label may include a fluorescent label. In some embodiments, the label may include a radioisotope. In some embodiments, the label may include any other detectable label. In some embodiments, the degree of nonspecific binding exhibited by a given support surface formulation can therefore be evaluated based on the number of nonspecifically bound protein molecules (or nucleic acid molecules or other molecules) per unit area. In some embodiments, the low-binding supports of this disclosure may exhibit less than 0.001 molecules / μm. 2 Less than 0.01 molecules / μm 2 Less than 0.1 molecules / μm 2 Less than 0.25 molecules / μm 2 Less than 0.5 molecules / μm 2 Less than 1 molecule / μm 2 Less than 10 molecules / μm 2 Less than 100 molecules / μm 2 or less than 1,000 molecules / μm 2 Nonspecific binding to proteins (or nonspecific binding to other specified molecules (e.g., anthocyanins such as Cy3 or Cy5, fluorescein, coumarin, rhodamine, or other dyes disclosed herein)). The surface of any given support disclosed herein may exhibit nonspecific binding at any location falling within this range (e.g., less than 86 molecules / μm). 2(Non-specific binding). For example, some of the modified surfaces disclosed herein showed less than 0.5 molecules / μm after contacting with 1 μM Cy3-labeled streptavidin (GE Amersham) solution in phosphate-buffered saline (PBS) buffer for 15 minutes, followed by rinsing three times with deionized water. 2 Non-specific protein binding. Some of the modified surfaces disclosed in this paper exhibited less than 0.25 molecules / μm. 2Nonspecific binding of Cy3 dye molecules was observed. In independent nonspecific binding assays, 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5 SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM 7-propynylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propynylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated in 384-well plates on a low-binding coated support at 37 °C for 15 min. Each well was rinsed 2 to 3 times with 50 μL of deionized RNase-free / DNase-free water, and then 2 to 3 times with 25 mM MACES buffer (pH 7.4). The 384-well plate was imaged on a GE Typhoon instrument using a Cy3, AF555, or Cy5 filter set as specified by the manufacturer (depending on the dye assay performed) at a PMT gain setting of 800 and a resolution of 50 to 100 μm. For higher resolution imaging, images were collected on an Olympus IX83 microscope (e.g., an inverted fluorescence microscope) (Olympus Corp., Center Valley, Pa.) equipped with a total internal reflection fluorescence (TIRF) objective (100×, 1.5NA, Olympus), a CCD camera (e.g., an Olympus EM-CCD monochrome camera, an Olympus XM-10 monochrome camera, or an Olympus DP80 color and monochrome camera), an illumination source (e.g., an Olympus 100W mercury lamp, an Olympus 75W xenon lamp, or an Olympus U-HGLGPS fluorescence source), and an excitation wavelength of 532 nm or 635 nm. Dichroic mirrors, such as 405 nm, 488 nm, 532 nm, or 633 nm dichroic mirrors / beam splitters, were purchased from Semrock (IDEX Health & Science, LLC, Rochester, NY), and bandpass filters were selected to be 532 LP or 645 LP integrated with the appropriate excitation wavelength. Some of the modified surfaces disclosed in this paper exhibit a density of less than 0.25 molecules / μm. 2 This involves the non-specific binding of dye molecules. In some embodiments, the coated support is immersed in a buffer solution (e.g., 25 mMACES, pH 7.4) while images are acquired.

[0214] In some embodiments, the surfaces disclosed herein exhibit a ratio of specific to nonspecific binding to a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the scope herein. In some embodiments, the surfaces disclosed herein exhibit a ratio of specific to nonspecific fluorescence signal for a fluorophore (such as Cy3) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value covered by the scope herein.

[0215] Low-background surfaces conforming to this disclosure may exhibit a ratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 specific dye molecules attached to each nonspecifically adsorbed molecule, with a ratio of specific dye attachment (e.g., Cy3 attachment) to nonspecific dye adsorption (e.g., Cy3 dye adsorption). Similarly, when subjected to excitation energy, low-background surfaces conforming to this disclosure that have attached fluorophores (e.g., Cy3) may exhibit a ratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50:1, with a ratio of specific fluorescence signal (e.g., generated from Cy3-labeled oligonucleotides attached to the surface) to nonspecifically adsorbed dye fluorescence signal.

[0216] In some embodiments, the degree of hydrophilicity (or “wetability” with an aqueous solution) of the disclosed support surface may be assessed, for example, by measuring the water contact angle, wherein a small drop of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some embodiments, a static contact angle may be determined. In some embodiments, a forward or backward contact angle may be determined. In some embodiments, the water contact angles disclosed herein for hydrophilic, low-binding support surfaces may range from about 0 degrees to about 30 degrees. In some embodiments, the water contact angles disclosed herein for hydrophilic, low-binding support surfaces may not exceed 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle does not exceed 40 degrees. A given hydrophilic, low-binding support surface of this disclosure may exhibit a water contact angle with values ​​at any location within this range.

[0217] In some embodiments, the hydrophilic surfaces disclosed herein help reduce washing time for bioassays, typically due to reduced nonspecific binding of biomolecules to low-binding surfaces. In some embodiments, a sufficient washing step can be performed in less than 60, 50, 40, 30, 20, 15, 10 seconds, or less than 10 seconds. For example, a sufficient washing step can be performed in less than 30 seconds.

[0218] Some of the low-binding surfaces disclosed herein exhibit significant improvements in stability or durability under prolonged exposure to solvents and elevated temperatures, or under repeated cycles of solvent exposure or temperature variations. For example, the stability of the disclosed surfaces can be tested by fluorescently labeling functional groups or tethered biomolecules (e.g., oligonucleotide primers) on the surface, and monitoring the fluorescence signal before, during, and after prolonged exposure to solvents and elevated temperatures, or under repeated cycles of solvent exposure or temperature variations. In some embodiments, the degree of fluorescence change used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, or 25% (or any combination of these percentages as measured within these time periods) over a period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours of exposure to solvents and / or elevated temperatures. In some embodiments, the degree of change in fluorescence used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages as measured by the range of cycles experienced) after repeated exposure to solvent variations and / or temperature variations for 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 cycles.

[0219] In some embodiments, the surfaces disclosed herein may exhibit a high ratio of specific signal to non-specific signal or other background. For example, when used for nucleic acid amplification, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 times greater than the signal in adjacent unpopulated regions of the surface. Similarly, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 times greater than the signal in adjacent amplified nucleic acid population regions of the surface.

[0220] In some embodiments, when used for nucleic acid hybridization or amplification applications to generate communities of hybridized or clonally amplified nucleic acid molecules (e.g., directly or indirectly labeled with fluorophores), the disclosed low-background surface fluorescence image exhibits a contrast-to-noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250 or greater than 250.

[0221] One or more types of primers may be attached to or tethered to the surface of a support. In some embodiments, the one or more types of adaptors or primers may include: spacer sequences, adaptor sequences for hybridizing with target library nucleic acid sequences linked to the adaptor, forward amplification primers, reverse amplification primers, sequencing primers and / or molecular barcode sequences, or any combination thereof. In some embodiments, one primer or adaptor sequence may be tethered to at least one layer of the surface. In some embodiments, at least two, three, four, five, six, seven, eight, nine, ten, or more than ten different primer or adaptor sequences may be tethered to at least one layer of the surface.

[0222] In some embodiments, the length of the tethered adaptor and / or primer sequence may range from about 10 nucleotides to about 100 nucleotides. In some embodiments, the length of the tethered adaptor and / or primer sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some embodiments, the length of the tethered adaptor and / or primer sequence may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides. 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 embodiments, the length of the tethered adaptor and / or primer sequence may range from about 20 nucleotides to about 80 nucleotides. The length of the tethered adaptor and / or primer sequence can have any value within this range, for example, about 24 nucleotides.

[0223] In some embodiments, the resulting surface density of primers (e.g., trapping primers) on the low-binding support surface of this disclosure can be approximately 100 primer molecules / μm. 2 Approximately 100,000 primer molecules / μm 2 Within the range. In some embodiments, the resulting surface density of primers on the low-binding support surface of this disclosure can be approximately 1,000 primer molecules / μm. 2 Approximately 1,000,000 primer molecules / μm 2 Within the range. In some embodiments, the surface density of the primers may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules / μm. 2 In some embodiments, the surface density of the primers may be up to 1,000,000, up to 100,000, up to 10,000, or up to 1,000 molecules / μm². Any of the lower and upper limits described in this paragraph may be combined to form a range included within this disclosure; for example, in some embodiments, the surface density of the primers may be about 10,000 molecules / μm². 2 Approximately 100,000 molecules / μm 2 The surface density of primer molecules can have any value within this range, for example, approximately 455,000 molecules / μm. 2In some embodiments, the surface density of the target library nucleic acid sequence initially hybridized with the adaptor or primer sequence on the support surface may be less than or equal to the surface density indicated by the surface density of the tethered primer. In some embodiments, the surface density of the target library nucleic acid sequence cloned and hybridized with the adaptor or primer sequence on the support surface may span the same range as indicated by the surface density of the tethered primer.

[0224] The local density listed above does not exclude variations in density across the surface, such that the surface may include densities with, for example, 500,000 / μm. 2 The region of oligonucleotide density also includes at least a second region with substantially different local densities.

[0225] Contrast-to-noise ratio (CNR)

[0226] In some embodiments, fluorescence imaging techniques can be used to evaluate the performance of nucleic acid hybridization, amplification reactions, or combinations thereof, using the disclosed reaction formulations and low-binding supports, wherein the contrast-to-noise ratio (CNR) of the image provides a key metric for evaluating amplification specificity and non-specific binding on the support. CNR is defined as: CNR = (Signal - Background) / Noise. The background term is considered as the signal measured against the gap region surrounding a specific feature (diffraction-limited spot, DLS) in a specified region of interest (ROI). While signal-to-noise ratio (SNR) is generally considered a benchmark for overall signal quality, it has been demonstrated that in applications requiring rapid image capture (e.g., sequencing applications where cycle time must be minimized), an improved CNR can provide a significant advantage over SNR as a benchmark for signal quality, as illustrated in the examples below. At high CNRs, even slight improvements in CNR can drastically reduce the imaging time required to achieve accurate differentiation (and therefore, accurate base interpretation in the case of sequencing applications). Improved CNR in imaging data regarding imaging integration time provides a method for more accurately detecting features such as clonal amplified nucleic acid colonies on support surfaces.

[0227] In some ensemble-based sequencing methods, background components can be measured as signals associated with "gap" regions. In addition to the "gap" background (B-gap), the "intrastitial" background (B-endoplasm) exists within regions occupied by amplified DNA colonies. The combination of these two background signals determines the achievable CNR and subsequently directly impacts optical instrumentation requirements, architecture costs, reagent costs, run time, cost / genome, and ultimately, accuracy and data quality for cyclic array-based sequencing applications. The B-gap background signal has various sources; some examples include: autofluorescence from consumable flow cells, nonspecific adsorption of detection molecules that produce spurious fluorescence signals (which may mask signals from the ROI), and the presence of nonspecific DNA amplification products (e.g., those from primer dimers). In some next-generation sequencing (NGS) applications, this background signal in the current field of view (FOV) is averaged and subtracted over time. Signals generated from individual DNA colonies (e.g., (signal)-B(gap) in the FOV) produce classifiable, distinguishable features. In some embodiments, the endoplasmic background (B(endoplasm)) can contribute hybrid fluorescence signals that are not specific to the target of interest but exist in the same ROI, thus making averaging and subtraction much more difficult.

[0228] Nucleic acid amplification on the low-binding coated support described herein can reduce the B (gap) background signal by decreasing nonspecific binding, leading to improvements in specific nucleic acid amplification and reductions in nonspecific amplification (which can affect the background signal generated from both the gap and endoplasmic regions). Compared to some methods, the disclosed low-binding coated support (optionally used in combination with the disclosed hybridization and / or amplification reaction formulations) can cause improvements in CNR of 2, 5, 10, 100, 250, 500, or 1000 times. Although described herein in the context of using fluorescence imaging as a readout or detection mode, the same principles apply to the use of the disclosed low-binding coated support and nucleic acid hybridization and amplification formulations for other detection modes, including both optical and non-optical detection modes.

[0229] Methods for sequencing

[0230] This disclosure provides a method for an autofocusing optics system for sequencing immobilized or non-immobilized template nucleotide molecules. In some embodiments, the immobilized template molecule comprises a plurality of nucleic acid template molecules having one copy of a target sequence of interest. In some embodiments, the nucleic acid template molecule having one copy of the target sequence of interest can be generated by bridging amplification using a linear library molecule. In some embodiments, the immobilized template molecule comprises a plurality of nucleic acid template molecules, each having two or more tandem copies (e.g., tandem copies) of the target sequence of interest. In some embodiments, the nucleic acid template molecule comprising tandem copy molecules can be generated by rolling circle amplification of a circularized linear library molecule. In some embodiments, the non-immobilized template molecule comprises a circular molecule. In some embodiments, the method for sequencing employs a soluble (e.g., non-immobilized) sequencing polymerase or a sequencing polymerase immobilized to a support.

[0231] In some embodiments, the sequencing reaction employs a detectably labeled nucleotide analog. In some embodiments, the sequencing reaction employs a two-stage sequencing reaction, including binding a detectably labeled multivalent molecule and incorporating a nucleotide analog. In some embodiments, the sequencing reaction employs an unlabeled nucleotide analog. In some embodiments, the sequencing reaction employs phosphate ester-labeled nucleotides.

[0232] Multivalent molecules

[0233] This disclosure provides a method for using an autofocusing optical system to sequence template nucleic acid molecules. In some embodiments, the sample fixed or otherwise positioned on a support may include at least one multivalent molecule. In some embodiments, the sample used for autofocusing the optical system may include at least one multivalent molecule. In some embodiments, the sequencing method using the optical system for imaging may employ at least one multivalent molecule. In some embodiments, the sequencing method using the optical system for imaging may include autofocusing the optical system prior to imaging one or more surfaces during a sequencing run flow cycle.

[0234] In some embodiments, the multivalent molecule comprises multiple nucleotide arms attached to the nucleus and having any conformation, including starburst, ladder, or bottle-brush conformations (e.g., Figure 12The multivalent molecule comprises: (1) a nucleus; and (2) a plurality of nucleotide arms, the plurality of nucleotide arms comprising: (i) a nucleus attachment portion; (ii) a spacer comprising a PEG portion; (iii) a linker; and (iv) a nucleotide unit, wherein the nucleus is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and at least one phosphate ester group, and the linker is attached to the nucleotide unit via a base. In some embodiments, the linker comprises an aliphatic chain or an oligomeric glycol chain, wherein both linker chains have 2 to 6 subunits. In some embodiments, the linker further comprises an aromatic portion. Figure 16 An example of a nucleotide arm is shown in the figure. Figures 12 to 15 Examples of multivalent molecules are shown in the figure. Figure 17 An example of a spacer is shown (at the top), and Figure 17 (bottom) and Figure 18 An example of a connector is shown in the figure. Figures 19 to 22 An example of a nucleotide attached to a linker is shown in the figure. Figure 23 An example of a biotinylated nucleotide arm is shown in the figure.

[0235] In some embodiments, the multivalent molecule includes a nucleus linked to a plurality of nucleotide arms, wherein the plurality of nucleotide arms have the same type of nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0236] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein each arm comprises a nucleotide unit. The nucleotide unit comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). The plurality of multivalent molecules may comprise a type of multivalent molecule having a type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of multivalent molecules may comprise a mixture of any combination of two or more types of multivalent molecules, wherein the individual multivalent molecules in the mixture comprise nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, dUTP, or combinations thereof.

[0237] In some embodiments, the nucleotide unit comprises a chain of one, two, or three phosphorus atoms, wherein the chain is attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, at least one nucleotide unit is a nucleotide analog having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (including O, S, or BH3). In some embodiments, the chain comprises a phosphate ester group substituted with an analog, which includes phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide groups.

[0238] In some embodiments, the multivalent molecule comprises a core linked to a plurality of nucleotide arms, wherein each nucleotide arm comprises a nucleotide unit that is a nucleotide analog having a chain termination portion (e.g., a blocking portion) at the 2' position, the 3' position, or at both the 2' and 3' positions of the sugar. In some embodiments, the nucleotide unit comprises a chain termination portion (e.g., a blocking portion) at the 2' position, the 3' position, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain termination portion may inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides into the nascent chain during primer extension reactions. In some embodiments, the chain termination portion is attached to the 3' sugar position, wherein the sugar comprises a ribose or deoxyribose portion. In some embodiments, the chain termination portion may be removed / cleaved from the 3' sugar position to produce a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the chain-terminating portion may be cleaved / removed from the nucleotide unit, for example, by reacting the chain-terminating portion with a chemical agent, a pH change, light, or heat. In some embodiments, the alkyl, alkenyl, alkynyl, and allyl groups of the chain-terminating portion may be cleaved with tetrakis(triphenylphosphine)palladium(O) (Pd(PPh3)4), with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl and benzyl groups of the chain-terminating portion may be cleaved with H2 Pd / C. In some embodiments, the amine, amide, ketone, isocyanate, phosphate, sulfur, and disulfide groups of the chain-terminating portion may be cleaved with phosphine or with a thiol group (including β-mercaptoethanol or dithiothreitol (DTT)). In some embodiments, the chain-terminating carbonate portion can be cleaved using potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the chain-terminating urea and silyl groups can be cleaved using tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0239] In some embodiments, the nucleotide unit comprises a chain termination portion (e.g., a blocking portion) at the 2' position of the sugar, at the 3' position of the sugar, or at both the 2' and 3' positions of the sugar. In some embodiments, the chain termination portion comprises an azide, an azide group, or an azide methyl group. In some embodiments, the chain termination portion comprises a 3'-O-azido group or a 3'-O-azido methyl group. In some embodiments, the chain termination portion azide, azide group, and an azide methyl group can be cleaved / removed using a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine portion or a derived triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleavage agent comprises 4-dimethylaminopyridine (4-DMAP).

[0240] In some embodiments, the nucleotide unit comprises a chain termination portion selected from the group consisting of: 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl, and derivatives thereof.

[0241] In some embodiments, the multivalent molecule includes a core attached to a plurality of nucleotide arms, wherein the nucleotide arms include spacers, linkers, and nucleotide units, and wherein the core, linkers, and / or nucleotide units are labeled with a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of a nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of nucleotide bases.

[0242] In some embodiments, at least one nucleotide arm of the multivalent molecule has a nucleotide unit attached to a detectable reporter gene portion. In some embodiments, the detectable reporter gene portion is attached to a nucleotide base. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to the multivalent molecule may correspond to a base of the nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of nucleotide bases.

[0243] In some embodiments, the core of the multivalent molecule comprises an avidin-like or streptavidin-like moiety, and the core-attached portion comprises biotin. In some embodiments, the core comprises a streptavidin-type or avidin-type moiety (containing avidin protein), and any derivative, analog, or other non-natural form of avidin that can bind to at least one biotin moiety. Other forms of the avidin moiety include natural and recombinant avidin and streptavidin, as well as derived molecules, such as non-glycosylated avidin and truncated streptavidin. For example, the avidin moiety includes deglycosylated forms of avidin, bacterial streptavidin produced by *Streptomyces* (e.g., *Streptomyces averdin*), and derived forms such as N-acylavidin, such as N-acetyl, N-phthaloyl, and N-succinylavidin, and commercially available products such as EXTRAVIDIN, CAPTAVIDIN, NEUTRAVIDIN, and NEUTRAALITE AVIDIN.

[0244] In some embodiments, any method for sequencing the nucleic acid molecules described herein may include forming a binding complex comprising (i) a polymerase, a primer-double-helicaled nucleic acid template molecule, and nucleotides, or the binding complex comprising (ii) a polymerase, a primer-double-helicaled nucleic acid template molecule, and nucleotide units of a multivalent molecule. In some embodiments, the binding complex has a residence time greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 second. The binding complex has a residence time of greater than about 0.1 to 0.25 seconds, or about 0.25 to 0.5 seconds, or about 0.5 to 0.75 seconds, or about 0.75 to 1 second, or about 1 to 2 seconds, or about 2 to 3 seconds, or about 3 to 4 seconds, or about 4 to 5 seconds, and / or the method is carried out at or above 15°C, at or above 20°C, at or above 25°C, at or above 35°C, at or above 37°C, at or above 42°C, at or above 55°C, at or above 60°C, or at or above 72°C, or at or above 80°C, or within the range defined by any of the foregoing. The binding complex (e.g., a ternary complex) remains stable before being subjected to conditions that lead to dissociation between the polymerase, template molecule, primer and / or nucleotide unit or any of the nucleotides. For example, dissociation conditions include contacting the binding complex with any or any combination of a detergent, EDTA, and / or water. In some embodiments, this disclosure provides a method in which the binding complex is deposited onto, attached to, or hybridized to a surface exhibiting a contrast-to-noise ratio greater than 20 in a detection step. In some embodiments, this disclosure provides a method in which contact is performed under conditions that stabilize the binding complex when the nucleotide or nucleotide unit is complementary to the next base of the template nucleic acid, and destabilize the binding complex when the nucleotide or nucleotide unit is not complementary to the next base of the template nucleic acid.

[0245] Methods for sequencing using phosphate-labeled nucleotides

[0246] In some embodiments, the methods described herein can be used for autofocusing of an optical system that can be used for sequencing using an immobilized sequencing polymerase bound to a non-immobilized template molecule. This disclosure provides a method for sequencing using an immobilized sequencing polymerase bound to a non-immobilized template molecule, wherein the sequencing reaction is performed with phosphate-labeled nucleotides. In some embodiments, the sequencing method includes the action (a): providing a support to which multiple sequencing polymerases are immobilized. In some embodiments, the sequencing polymerase includes a persistent DNA polymerase. In some embodiments, the sequencing polymerase comprises a wild-type or mutant DNA polymerase, including, for example, Phi29 DNA polymerase. In some embodiments, the support comprises multiple individual compartments, and the sequencing polymerase is immobilized to the bottom of the compartments. In some embodiments, the individual compartments comprise a silica bottom that is permeable to light. In some embodiments, the individual compartments comprise a silica bottom configured with a nanophotonic confinement structure comprising pores in a metal coating (e.g., an aluminum coating). In some embodiments, the pores in the metal coating have a small aperture, for example, approximately 70 nm. In some embodiments, the height of the nanophotonic confinement structure is approximately 100 nm. In some embodiments, the nanophotonic confinement structure comprises a zero-mode waveguide (ZMW). In some embodiments, the nanophotonic confinement structure contains a liquid.

[0247] In some embodiments, the sequencing method further includes operation (b): contacting multiple immobilized sequencing polymerases with multiple single-stranded circular nucleic acid template molecules and multiple oligonucleotide sequencing primers under conditions suitable for binding of individual immobilized sequencing polymerases to single-stranded circular template molecules and suitable for hybridization of individual sequencing primers to individual single-stranded circular template molecules, thereby generating multiple polymerase / template / primer complexes. In some embodiments, individual sequencing primers hybridize to universal sequencing primer binding sites on single-stranded circular template molecules.

[0248] In some embodiments, the sequencing method further includes the action (c): contacting a plurality of polymerase / template / primer complexes with a plurality of phosphate-ester-tagged nucleotides, each phosphate-ester-tagged nucleotide comprising an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and a phosphate ester chain comprising 3 to 20 phosphate groups, wherein the terminal phosphate group is linked to a detectable reporter gene moiety (e.g., a fluorophore). The first, second, and third phosphate groups may be referred to as α, β, and γ phosphate groups. In some embodiments, the specific detectable reporter gene moiety attached to the terminal phosphate group corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of nucleotides. In some embodiments, the plurality of polymerase / template / primer complexes are contacted with the plurality of phosphate-ester-tagged nucleotides under conditions suitable for polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase is capable of binding to a complementary phosphate-ester-tagged nucleotide and incorporating a complementary nucleotide to a nucleotide in the template molecule. In some embodiments, the polymerase-catalyzed nucleotide incorporation reaction involves cleavage between α-phosphate groups and β-phosphate groups, thereby releasing a polyphosphate chain linked to the fluorophore.

[0249] In some embodiments, the sequencing method further includes step (d): detecting a fluorescent signal emitted by a phosphate-labeled nucleotide, which is bound by a sequencing polymerase and incorporated into the end of a sequencing primer. In some embodiments, step (d) further includes identifying the phosphate-labeled nucleotide bound by the sequencing polymerase and incorporated into the end of the sequencing primer.

[0250] In some embodiments, the sequencing method further includes operation (e): repeating steps (c) through (d) at least once. In some embodiments, the sequencing method using phosphate-labeled nucleotides is performed according to the methods described in U.S. Patent Nos. 7,170,050; 7,302,146; and / or 7,405,281, each of which is incorporated herein by reference in its entirety.

[0251] The headings provided herein are not intended to limit the individual aspects of this disclosure, which can be understood by referring to the specification as a whole.

[0252] certain terms

[0253] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. Generally, terms related to the techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in conjunction with the laboratory procedures and techniques described herein is that well-known and commonly used in the art.

[0254] Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. Unless explicitly and definitively limited to a single referent, the singular forms “a / an” and “the” and any other word used in the singular include multiple referents.

[0255] It should be understood that the use of alternative terms (e.g., "or") is considered to refer to one or both of the alternatives or any combination thereof.

[0256] As used herein, the term “and / or” should be considered to refer to a specific disclosure of each of the specified features or components, whether or not they are together with another. For example, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). Similarly, the term “and / or” as used herein, such as “A, B and / or C”, is intended to cover each of the following aspects: “A, B and C”; “A, B or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0257] As used herein and in the appended claims, the terms “comprising,” “including,” “having,” and “containing,” and their grammatical variations, are intended to be non-limiting, such that one or more items in the list do not exclude other items that may be substituted for or added to the listed items. It should be understood that whenever an aspect is described herein with the language “comprising,” other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0258] As used herein, the terms “about,” “approximately,” and “substantially” mean a value or composition within an acceptable range of error for a particular value or composition, as determined by one of ordinary skill in the art, depending in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” or “substantially” may mean within one or more standard deviations. Alternatively, “about” or “approximately” may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, specifically with respect to biological systems or processes, the term may mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about,” “approximately,” or “substantially” should be assumed to be within an acceptable range of error for said particular value or composition. Furthermore, in the case of providing ranges and / or subranges of values, the range and / or subrange may include the endpoints of the range and / or subrange.

[0259] As used herein, the term "polony" refers to a nucleic acid library molecule that can be clonally amplified in solution or on a support to produce an amplicon that can serve as a template molecule for sequencing. In some embodiments, linear library molecules may be circularized to produce circularized library molecules, and circularized library molecules may be clonally amplified in solution or on a support to produce tandem molecules. In some embodiments, tandem molecules can serve as nucleic acid template molecules that can be sequenced. Tandem molecules are sometimes referred to as polony. In some embodiments, a polony comprises nucleotide chains.

[0260] References to "an embodiment," "an example embodiment," "some embodiments," or similar phrases herein indicate that the described embodiments may include specific features, structures, or characteristics, but not every embodiment necessarily includes such specific features, structures, or characteristics. Furthermore, such phrases do not necessarily refer to the same embodiment. Additionally, when a specific feature, structure, or characteristic is described in relation to an embodiment, incorporating such feature, structure, or characteristic into other embodiments (whether expressly mentioned or described herein) will be within the knowledge of those skilled in the art.

[0261] It should be understood that the Detailed Description section, and not any other section, is intended to interpret the claims. The other sections may set forth one or more, but not all, exemplary embodiments contemplated by the inventors, and therefore are not intended to limit this disclosure or the appended claims in any way.

[0262] While this disclosure describes exemplary embodiments of some fields and applications, it should be understood that this disclosure is not limited thereto. Other embodiments and modifications thereof are possible and are within the scope and spirit of this disclosure. For example, and without limiting the generality of this paragraph, the embodiments are not limited to the software, hardware, firmware, and / or entities shown in the figures and / or described herein. Furthermore, the embodiments (whether or not explicitly described herein) have significant utility in fields and applications beyond those described herein.

[0263] This document has described embodiments using functional building blocks that illustrate implementations of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries can be defined as long as the specified functions and their relationships (or equivalents) are appropriately performed. Similarly, alternative embodiments may use a different order of execution of functional blocks, steps, operations, methods, etc., than those described herein.

[0264] Example

[0265] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims presented herein.

[0266] Example 1 – Optical System Efficiency

[0267] Figures 32A to 32B A diffraction modulation transfer function (MTF) for an optical system according to some embodiments is provided. Figure 32A The MTF of the objective-based system at p=480nm is shown, while Figure 32B The MTF of the optical system of this disclosure at p = 500 nm is shown. As can be seen from the figure, the overall effectiveness of the optical system of this disclosure is higher than that of the objective-based system at all spatial frequencies, and the system of this disclosure provides a much larger field of view (9 mm, compared to 2 mm for the objective-based system). Figures 33A to 33B Wavefront analysis calculations of an optical system according to some embodiments of the present disclosure are shown. In both cases, the optical system exhibits low compound root mean square errors of approximately 24 mλ and 26 mλ, respectively, within a wide field of view (e.g., 9 mm).

[0268] Figures 34 to 35Optical performance curves for the top and bottom surfaces according to some embodiments are shown. In both cases, longitudinal spherical aberration, astigmatism curves, and distortion are all low, indicating improved optical (e.g., imaging) performance. Figure 36 A plot of the MTF (Mean Transmission Flow) of an optical system according to some embodiments is shown. The MTF plot can show a wide depth of field (e.g., ±1 micrometer) with low field curvature, which indicates the optical system's ability to provide wide-area, large-depth-of-field imaging, enabling simultaneous imaging of multiple samples on a solid support. In some cases, the wide depth of field enables multi-surface imaging (e.g., simultaneous imaging of multiple surfaces of a solid support). Figure 37 A cumulative probability plot of achieving a given wavefront error according to some embodiments is shown. This plot illustrates data regarding the low wavefront error achievable by the systems of this disclosure.

[0269] Examples of the numbered embodiments of this disclosure

[0270] 1. An optical system, the optical system comprising:

[0271] A stage, configured to hold a solid support;

[0272] A light source, configured to illuminate the solid support; and

[0273] An optical component, at least partially disposed within the optical path from the stage to the light source, wherein the optical component is configured to be larger than approximately 20 square millimeters (mm) on the solid support. 2 Lighting is provided in the area where the peak-to-valley variation is at most about 5%.

[0274] 2. The optical system according to any one of the foregoing embodiments, wherein the optical components do not include an objective lens.

[0275] 3. The optical system according to any one of the foregoing embodiments, wherein the optical system does not include the objective lens.

[0276] 4. The optical system according to any one of the foregoing embodiments, wherein the optical components do not include a barrel lens.

[0277] 5. The optical system according to any one of the foregoing embodiments, wherein the optical system does not include the barrel lens.

[0278] 6. The optical system according to any one of the foregoing embodiments, wherein the stage is not adjusted on the optical axis of the system.

[0279] 7. The optical system according to any one of the foregoing embodiments, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter.

[0280] 8. The optical system according to any one of the foregoing embodiments, wherein the optical component is configured to receive emitted light from the solid support.

[0281] 9. The optical system according to any one of the foregoing embodiments, wherein the numerical aperture (NA) of the optical component is at least about 0.3.

[0282] 10. The optical system according to any one of the foregoing embodiments, wherein the wavelength of the emitted light is about 500 nanometers to about 750 nanometers.

[0283] 11. The optical system according to any one of the foregoing embodiments, wherein the working distance of the optical component is at least about 1 mm to 25 mm.

[0284] 12. The optical system according to any one of the foregoing embodiments, the optical system further comprising a movable coil housed within the optical component, the movable coil being configured to move a focusing element within the optical path of the optical system.

[0285] 13. The optical system according to any one of the foregoing embodiments, wherein a motor located outside the optical system is configured to move the focusing element along the optical axis in one or two directions.

[0286] 14. The optical system according to any one of the foregoing embodiments, wherein the motor is directly coupled to a portion of the first, second, or third housing of the optical component, and a portion of the first, second, or third housing of the optical component is directly coupled to the focusing element.

[0287] 15. The optical system according to any one of the foregoing embodiments, wherein the light source is a pulsed light source.

[0288] 16. The optical system according to any one of the foregoing embodiments, wherein the composite root mean square error of the optical system is less than about 0.05.

[0289] 17. The optical system according to any one of the foregoing embodiments, wherein the illumination efficiency of said optical component is at least about 90%.

[0290] 18. The optical system according to any one of the foregoing embodiments, wherein the area is greater than 30 mm². 2 .

[0291] 19. The optical system according to any one of the foregoing embodiments, wherein the area is greater than 50 mm². 2 Or 60mm 2 .

[0292] 20. The optical system according to any one of the foregoing embodiments, the optical system further comprising the solid support within the stage.

[0293] 21. The optical system according to any one of the foregoing embodiments, wherein the solid support comprises two or more surfaces having one or more samples imaged by the optical system fixed thereon.

[0294] 22. The optical system according to any one of the foregoing embodiments, wherein the solid support comprises three or more surfaces having one or more samples imaged by the optical system fixed thereon.

[0295] 23. The optical system according to any one of the foregoing embodiments, wherein the three or more surfaces are axially displaced from each other at least along the optical axis of the optical system.

[0296] 24. The optical system according to any one of the foregoing embodiments, wherein the solid support includes a probe configured to bind nucleic acid molecules.

[0297] 25. The optical system according to any one of the foregoing embodiments, wherein the probe is attached to the surface of the solid support.

[0298] 26. The optical system according to any one of the foregoing embodiments, wherein the light source is a laser light source.

[0299] 27. The optical system according to any one of the foregoing embodiments, wherein the optical component includes a dichroic filter configured to transmit the illumination.

[0300] 28. The optical system according to any one of the foregoing embodiments, wherein the optical component comprises: a first segment including a first housing, the first housing including a first plurality of lenses; a second segment including a second housing; and a third segment including a third housing, the third housing including a second plurality of lenses.

[0301] 29. The optical system according to any one of the foregoing embodiments, wherein the first segment is optically aligned with the third segment.

[0302] 30. The optical system according to any one of the foregoing embodiments, wherein the first segment is positioned between the third segment and the stage.

[0303] 31. The optical system according to any one of the foregoing embodiments, wherein the third segment is positioned between the first segment and the image sensor of the optical system.

[0304] 32. The optical system according to any one of the foregoing embodiments, wherein the first plurality of lenses are movable along the optical path in a range of about 0 to about 2 millimeters.

[0305] 33. The optical system according to any one of the foregoing embodiments, wherein the first plurality of lenses comprises an asymmetric biconvex lens.

[0306] 34. The optical system according to any one of the foregoing embodiments, wherein the second plurality of lenses comprises an asymmetric biconcave lens.

[0307] 35. The optical system according to any one of the foregoing embodiments, wherein the asymmetric biconcave lens is an aspherical asymmetric biconcave lens.

[0308] 36. The optical system according to any one of the foregoing embodiments, wherein the optical system is configured to acquire an image of the solid support without moving the optical compensator into the optical path between the solid support and the detector of the optical system.

[0309] 37. The optical system according to any one of the foregoing embodiments, wherein the optical system is configured to acquire an image of the solid support without removing the optical compensator from the optical path between the sample and the detector of the optical system.

[0310] 38. The optical system according to any one of the foregoing embodiments, wherein the solid support is a flow cell.

[0311] 39. The optical system according to any one of the foregoing embodiments, wherein the optical components are configured to generate one or more spatial contractions transverse to the optical path of light traveling through it.

[0312] 40. The optical system according to any one of the foregoing embodiments, wherein the optical components are configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it.

[0313] 41. The optical system according to any one of the foregoing embodiments, wherein the optical components are configured to generate at least one field curvature correction in a first, second, or third segment transverse to the optical path of light traveling through it.

[0314] 42. A method for analyzing biomolecules, the method comprising:

[0315] (a) Providing a solid support comprising the biomolecule containing the marker;

[0316] (b) Illuminating the biomolecule containing the marker using an optical system including a light source, thereby generating signal light or a change thereof, wherein the solid support is larger than about 20 square millimeters (mm²). 2 The lighting is provided over the area where the peak-to-valley variation is at most about 5%;

[0317] (c) Detecting the signal light or any changes thereof using the detector of the optical system; and

[0318] (d) Process the signal light or its changes at least partially to analyze the biomolecules.

[0319] 43. The method according to any one of the foregoing embodiments, wherein the biomolecule is a nucleic acid molecule, a protein, or a polypeptide.

[0320] 44. The method according to any one of the foregoing embodiments, wherein the biomolecule is a nucleic acid.

[0321] 45. The method according to any one of the foregoing embodiments, the method further comprising, prior to (a), binding the biomolecule to a probe bound to the solid support and coupling the marker to the biomolecule.

[0322] 46. ​​The method according to any one of the foregoing embodiments, wherein the marker is coupled to the biomolecule via hybridization.

[0323] 47. The method according to any one of the foregoing embodiments, wherein the optical system does not include an objective lens.

[0324] 48. The method according to any one of the foregoing embodiments, wherein the solid support is not moved on the optical axis of the optical system.

[0325] 49. The method according to any one of the foregoing embodiments, wherein multiple images of the solid support are acquired without moving the solid support along the optical axis.

[0326] 50. The method according to any one of the foregoing embodiments, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter.

[0327] 51. The method according to any one of the foregoing embodiments, wherein the wavelength of the signal light is from 500 nanometers to about 750 nanometers.

[0328] 52. The method according to any one of the foregoing embodiments, wherein the detection in (c) is performed using an optical element with a numerical aperture of at least about 0.3.

[0329] 53. The method according to any one of the foregoing embodiments, the method further includes (b) using a motion coil within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focal point of the optical system on the solid support.

[0330] 54. The method according to any one of the foregoing embodiments, wherein the light source is a pulsed light source.

[0331] 55. The method according to any one of the foregoing embodiments, wherein the illumination is provided with an efficiency of at least about 90%.

[0332] 56. The method according to any one of the foregoing embodiments, the method further comprising repeating (b)-(d) additional biomolecules coupled to an additional surface of the solid support.

[0333] 57. The method according to any one of the foregoing embodiments, the method further comprising, after (c), removing the marker from the biomolecule.

[0334] 58. The method according to any one of the foregoing embodiments, the method further comprising repeating (a)-(d) on an additional marker that binds to another portion of the biomolecule.

[0335] 59. The method according to any one of the foregoing embodiments, wherein the optical component is configured to generate one or more spatial contractions transverse to the optical path of light traveling through it.

[0336] 60. The method according to any one of the foregoing embodiments, wherein the optical component is configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it.

[0337] 61. The method according to any one of the foregoing embodiments, wherein the optical component is configured to generate at least one field curvature correction in the first, second, or third segment transverse to the optical path through which the light travels.

[0338] 62. The method according to any one of the foregoing embodiments, wherein (d) includes at least partially processing the signal light or the said variation thereof to generate one or more solid support images, and analyzing the one or more solid support images to generate a base interpretation of the sample.

[0339] 63. The method according to any one of the foregoing embodiments, wherein each solid support image in the solid support images comprises more than 20 square millimeters (mm²). 2 ) field of view (FOV).

[0340] 64. The method according to any one of the foregoing embodiments, wherein the solid support is a flow cell.

[0341] 65. An optical system, the optical system comprising:

[0342] A stage, configured to hold a solid support;

[0343] A light source, configured to illuminate the solid support; and

[0344] A speckle eliminater, which is optically coupled to the light source and disposed within the optical path from the light source to the stage.

[0345] 66. The optical system according to any one of the foregoing embodiments, the optical system further comprising an additional light source optically coupled to the speckle eliminator.

[0346] 67. The optical system according to any one of the foregoing embodiments, wherein light from the additional light source is configured to illuminate the solid support with light of a different wavelength than the light source.

[0347] 68. The optical system according to any one of the foregoing embodiments, wherein at least about four light sources are coupled to the speckle eliminator.

[0348] 69. The optical system according to any one of the foregoing embodiments, wherein the speckle eliminator is a vibration speckle eliminator.

[0349] 70. The optical system according to any one of the foregoing embodiments, wherein the speckle eliminator is a passive speckle eliminator.

[0350] 71. The optical system according to any one of the foregoing embodiments, wherein the passive speckle eliminator comprises a diffuse scattering plate.

[0351] 72. The optical system according to any one of the foregoing embodiments, wherein the speckle eliminator is a tension speckle eliminator.

[0352] 73. The optical system according to any one of the foregoing embodiments, wherein the speckle canceller is configured to reduce speckle noise to at most about 5%.

[0353] 74. The optical system according to any one of the foregoing embodiments, wherein the solid support is a flow cell.

[0354] 75. A method for analyzing biomolecules, the method comprising:

[0355] (a) Providing a solid support, said solid support comprising a biological sample containing a marker;

[0356] (b) Illuminating the biological sample containing the marker using an optical system including a light source to generate signal light or a variation thereof, wherein the illumination is provided by a speckle eliminator in the optical path of the optical system;

[0357] (c) Detecting the signal light or any changes thereof using the detector of the optical system; and

[0358] (d) Process the signal light or its changes at least partially to analyze the biomolecules.

[0359] 76. The method according to any one of the foregoing embodiments, the method further comprising repeating (b)-(d) on additional biological samples coupled to additional surfaces of the solid support.

[0360] 77. The method according to any one of the foregoing embodiments, the method further comprising, after (c), removing the marker from the biological sample.

[0361] 78. The method according to any one of the foregoing embodiments further includes repeating steps (a)-(d) on additional markers bound to the biological sample.

[0362] 79. The method according to any one of the foregoing embodiments, wherein the speckle eliminator uses vibration to eliminate speckle on the illumination.

[0363] 80. The method according to any one of the foregoing embodiments, the method further comprising illuminating the solid support using an additional light source.

[0364] 81. The method according to any one of the foregoing embodiments, wherein the additional light source provides light of a different wavelength to the solid support.

[0365] 82. The method according to any one of the foregoing embodiments, wherein the additional light source is coupled to the speckle eliminator.

[0366] 83. The method according to any one of the foregoing embodiments, wherein the biological sample comprises nucleic acid molecules, proteins or polypeptides.

[0367] 84. The method according to any one of the foregoing embodiments, wherein the biological sample comprises nucleic acid.

[0368] 85. An illumination system for a multi-channel fluorescence imaging module, the illumination system comprising:

[0369] The lighting subsystem includes:

[0370] light source;

[0371] speckle eliminater; and

[0372] A beam delivery subsystem, optically coupled to the illumination system, comprising:

[0373] Collimator; and

[0374] One or more optical lens elements.

[0375] 86. The illumination system according to any one of the foregoing embodiments, wherein the illumination system is configured to provide a light intensity of not less than 10 mm at the sample plane. 2 20mm 2 30mm 2 40mm 2 Or 50mm 2 The lighting field.

[0376] 87. The illumination system according to any one of the foregoing embodiments, wherein the illumination system is configured to provide not less than 40, 50, or 60 milliwatts / mm² at the sample plane. 2 The power density.

[0377] 88. The lighting system according to any one of the foregoing embodiments, wherein the sample plane is orthogonal to the z-axis.

[0378] 89. The lighting system according to any one of the foregoing embodiments, wherein the light source comprises one or more lasers.

[0379] 90. The lighting system according to any one of the foregoing embodiments, wherein the one or more lasers comprise one or more laser diodes.

[0380] 91. The lighting system according to any one of the foregoing embodiments, wherein the one or more lasers emit light of multiple wavelengths.

[0381] 92. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises one or more optical fibers.

[0382] 93. The lighting system according to any one of the foregoing embodiments, wherein at least one of the optical fibers has a length of 0.5m to 5m.

[0383] 94. The lighting system according to any one of the foregoing embodiments, wherein at least one of the optical fibers comprises a core having a maximum cross-sectional dimension of 50 μm to 1500 μm.

[0384] 95. The lighting system according to any one of the foregoing embodiments, wherein the cross-section of the fiber core is circular.

[0385] 96. The lighting system according to any one of the foregoing embodiments, wherein the power efficiency of the lighting system is not less than 65%, 70%, 75% or 80%.

[0386] 97. The lighting system according to any one of the foregoing embodiments, wherein the lighting field is rectangular or square.

[0387] 98. The lighting system according to any one of the foregoing embodiments, wherein the one or more optical lens elements comprise one or more multi-lens arrays.

[0388] 99. The lighting system according to any one of the foregoing embodiments, wherein each of the one or more multi-lens arrays comprises one or more of the following: an asymmetric biconvex lens, a convex plano lens, a concave plano lens, an asymmetric biconcave lens, and an asymmetric convex-concave lens.

[0389] 100. The lighting system according to any one of the foregoing embodiments, wherein each of the one or more multi-lens arrays includes at least a plurality of lens elements in a direction orthogonal to the z-axis.

[0390] 101. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises an optical fiber optically coupled to a laser diode and reducing light source speckle.

[0391] 102. The lighting system according to any one of the foregoing embodiments, wherein the speckle eliminator comprises an optical fiber optically coupled to a laser diode and reducing light source speckle.

[0392] 103. The lighting system according to any one of the foregoing embodiments, wherein the one or more optical lens elements comprise:

[0393] Asymmetric biconvex lenses, convex-plano lenses, concave-plano lenses, asymmetric biconcave lenses, asymmetric convex-concave lenses, or combinations thereof. 104. The lighting system according to any one of the foregoing embodiments, wherein the one or more optical lens elements comprise:

[0394] A first multi-lens array and a second multi-lens array are positioned along the z-axis between the collimator and the incident pupil of the illumination system.

[0395] 105. The lighting system according to any one of the foregoing embodiments, wherein the lighting system is configured at the sample stage.

[0396] Generate more than 50mm 2 The lighting field has a lighting power density variation of less than ±2%, 5%, 8%, 10%, or 12%.

[0397] 106. The lighting system according to any one of the foregoing embodiments, wherein the speckle eliminator includes a mechanical vibration source.

[0398] 107. The lighting system according to any one of the foregoing embodiments, wherein the speckle eliminator includes a vibration source that generates vibrations within a predetermined frequency range.

[0399] 108. The lighting system according to any one of the foregoing embodiments, wherein the mechanical vibration source is configured to vibrate at one or more frequencies in the audible range, the ultrasonic range, or both.

[0400] 109. The lighting system according to any one of the foregoing embodiments, wherein the mechanical vibration source is configured to generate one-dimensional, two-dimensional or three-dimensional vibrational movement.

[0401] 110. The lighting system according to any one of the foregoing embodiments, wherein at least a portion of each of the optical fibers or single optical fibers is wound or coiled one or more times.

[0402] 111. The lighting system according to any one of the foregoing embodiments, wherein at least a portion of each of the optical fibers or single optical fibers is fixedly or loosely attached to the mechanical vibration source.

[0403] 112. The illumination system according to any one of the foregoing embodiments, wherein the speckle eliminator is integrated with the sample stage, objective lens, and...

[0404] The one or more image sensors are physically isolated, so that the mechanical movement of the speckle eliminate is independent of the sample stage, objective lens and the one or more image sensors.

[0405] 113. The lighting system according to any one of the foregoing embodiments, wherein the speckle eliminator is configured to reduce speckle noise to no more than 4%, 4.5%, 5%, or 5.5%.

[0406] 114. The lighting system according to any one of the foregoing embodiments, wherein the light source comprises a multicolor laser array.

[0407] 115. The lighting system according to any one of the foregoing embodiments, wherein the multicolor laser array includes a laser diode.

[0408] The laser diode array emits laser light of 2, 3, 4, 5 or 6 wavelengths, or laser light within a wavelength range of 2, 3, 4, 5 or 6.

[0409] 116. The lighting system according to any one of the foregoing embodiments, wherein the multicolor laser array comprises a laser that emits light of at least two, three, or four color wavelengths or wavelength ranges in a direction orthogonal to the z-axis.

[0410] 117. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further includes one or more coupling lenses.

[0411] 118. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem comprises a single optical fiber.

[0412] 119. The lighting system according to any one of the foregoing embodiments, wherein the single optical fiber includes a core having a maximum cross-sectional dimension of 500 μm to 1500 μm.

[0413] 120. The lighting system according to any one of the foregoing embodiments, wherein the power in the beam transmission subsystem is greater than 5, 8, 10, 12 or 14 watts for one or more wavelengths or wavelength ranges.

[0414] 121. The lighting system according to any one of the foregoing embodiments, wherein the power at the sample plane is greater than 5, 8, 10, 12 or 14 watts for one or more wavelengths or wavelength ranges.

[0415] 122. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises multiple light rays.

[0416] Each optical fiber is optically coupled to one or more corresponding lasers of the light source, wherein the one or more corresponding lasers emit light of the same wavelength or wavelength range as the light source.

[0417] 123. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further includes one or more dichroic filters.

[0418] 124. The lighting system according to any one of the foregoing embodiments, wherein the light source includes a plurality of beam combiners.

[0419] 125. The lighting system according to any one of the foregoing embodiments, wherein the light source includes a plurality of polarized beam combiners.

[0420] 126. The lighting system according to any one of the foregoing embodiments, wherein the light source comprises two or more lasers that emit light of the same wavelength or the same wavelength range.

[0421] 127. The lighting system according to any one of the foregoing embodiments, wherein each polarization beam combiner in the polarization beam combiner is configured to combine light of the same wavelength or the same wavelength range from two or more lasers.

[0422] 128. The illumination system according to any one of the foregoing embodiments, wherein the speckle eliminator is positioned within the optical path between the collimator and the sample plane.

[0423] 129. The lighting system according to any one of the foregoing embodiments, wherein the speckle eliminator is positioned at a position where the beam diameter is greater than 5 mm, 10 mm or 20 mm, and wherein the beam diameter is orthogonal to the z-axis.

[0424] 130. The lighting system according to any one of the foregoing embodiments, wherein the optical fiber comprises a core having a rectangular or square cross-section.

[0425] 131. The lighting system according to any one of the foregoing embodiments, wherein the optical fiber comprises a core having a non-circular cross-section.

[0426] 132. The lighting system according to any one of the foregoing embodiments, wherein the lighting subsystem further comprises one or more liquid light guides.

[0427] 133. The lighting system according to any one of the foregoing embodiments, wherein the one or more liquid light guides are optically coupled to the light source in the absence of optical fibers.

[0428] 134. The lighting system according to any one of the foregoing embodiments, wherein the one or more liquid light guides include a liquid core, the maximum dimension of the cross-section of the liquid core being 0.5 mm to 10 mm, and wherein the cross-section is orthogonal to the z-axis.

[0429] 135. The lighting system according to any one of the foregoing embodiments, wherein the liquid core comprises a circular cross-section.

[0430] 136. The lighting system according to any one of the foregoing embodiments, wherein the liquid core comprises a non-circular cross-section.

[0431] 137. An imaging module for multi-channel fluorescence imaging, the imaging module comprising:

[0432] The lighting system according to any one of the foregoing embodiments; and

[0433] An image acquisition system configured to acquire flow cell images of a sample fixed on a sample stage at the sample plane.

[0434] 138. The imaging module according to any one of the foregoing embodiments, wherein each flow cell image in the flow cell image comprises a portion greater than 20 mm. 2 30mm 2 40mm 2 Or 50mm 2 Field of view (FOV).

[0435] 139. The imaging module according to any one of the foregoing embodiments, wherein each flow cell image in the flow cell image includes a field of view (FOV) that overlaps with the illumination field generated by the illumination system at the sample plane.

[0436] 140. The imaging module according to any one of the foregoing embodiments, wherein each flow cell image in the flow cell image...

[0437] This includes a field of view (FOV) that overlaps with at least 80%, 85%, 90%, or 95% of the illumination field generated by the illumination system at the sample plane.

[0438] 141. The imaging module according to any one of the foregoing embodiments, wherein each flow cell image in the flow cell image...

[0439] The image includes a field of view (FOV) of at least 80%, 85%, 90%, and 95% of the illumination field generated by the illumination system at the sample plane.

[0440] 142. The imaging module according to any one of the foregoing embodiments, wherein the imaging module comprises:

[0441] One or more image sensors; and

[0442] Objective lens.

[0443] 143. The imaging module according to any one of the foregoing embodiments, wherein the numerical aperture (NA) of the imaging module is greater than 0.4, 0.5 or 0.6.

[0444] 144. The imaging module according to any one of the foregoing embodiments, wherein the imaging module includes an optical axis parallel to the z-axis. 145. The imaging module according to any one of the foregoing embodiments, wherein the flow cell image is an image of a sample fixed to one or more surfaces of a solid support.

[0445] 146. The imaging module according to any of the foregoing embodiments, wherein the one or more surfaces comprise at least two or more surfaces that are axially displaced from each other along the z-axis.

[0446] 147. The imaging module according to any one of the foregoing embodiments, wherein the flow cell image is acquired without moving the optical compensator into the optical path between the objective lens and the one or more image sensors.

[0447] 148. The imaging module according to any of the foregoing embodiments, wherein the flow cell image of one or more surfaces is acquired without removing the optical compensator from the optical path between the objective lens and the at least one image sensor.

[0448] 149. The imaging module according to any one of the foregoing embodiments, wherein the flow cell diagram is rendered when the following conditions are met.

[0449] Each flow cell image in the image includes a contrast-to-noise ratio (CNR) of at least 5: nucleic acid polones set on three or more surfaces are labeled with cyanine dye 3 (Cy3); the dichroic mirror and bandpass filter set are optimized for Cy3 emission; and the flow cell images are acquired by an optical system under non-signal saturation conditions while one or more of the surfaces are immersed in 25 mMACES, pH 7.4 buffer.

[0450] 150. The imaging module according to any one of the foregoing embodiments, wherein the imaging module is configured to determine the nucleotides of nucleic acid molecules in a sample.

[0451] 151. The imaging module according to any one of the foregoing embodiments, wherein the imaging module is configured for use in the one

[0452] Sequencing reactions based on affinity, nucleotide base pairing, nucleotide binding, or nucleotide incorporation are performed on at least one of one or more surfaces.

[0453] 152. The imaging module according to any one of the foregoing embodiments, wherein each of the one or more surfaces

[0454] Each includes multiple induced target nucleic acid sequences coupled thereto, wherein the induced target nucleic acid sequence of the multiple induced target nucleic acid sequences has a polymerase intended to bind.

[0455] 153. A method for sequencing nucleic acid molecules, the method comprising:

[0456] Provides a flow pool comprising one or more surfaces, wherein each surface includes:

[0457] At least one hydrophilic polymer coating;

[0458] Multiple oligonucleotide molecules, which are attached to at least one hydrophilic polymer coating; and

[0459] At least one discrete region on each surface, comprising a plurality of cloned and amplified sample nucleic acid molecules immobilized to the plurality of attached oligonucleotide molecules;

[0460] The illumination system causes the multiple cloned and amplified sample nucleic acid molecules in the illumination field to emit different colors of fluorescence during on / off events; and

[0461] When the multiple cloned and amplified sample nucleic acid molecules experience the on and off events, the on and off events are detected in one or more color channels by the one or more image sensors to determine the nucleotide identity of the cloned and amplified sample nucleic acid molecules.

[0462] 154. The method according to any one of the foregoing embodiments, wherein the method further comprises:

[0463] Adjust the NA of the imaging module.

[0464] 155. The method according to any one of the foregoing embodiments, wherein the method further comprises:

[0465] The NA of the imaging module can be varied within the range of 0.4 to 0.6 by changing the adjustable optical aperture size.

[0466] 156. The method according to any one of the foregoing embodiments, wherein the illumination system is configured to provide at least 10 mm of illumination at the sample plane. 2 20mm 2 30mm 2 40mm 2 Or 50mm 2 The lighting field.

[0467] 157. The method according to any one of the foregoing embodiments, wherein the illumination system is configured to provide not less than 40, 50, or 60 milliwatts / mm² at the sample plane. 2 The power density.

[0468] 158. The method according to any one of the foregoing embodiments, wherein the lighting system comprises:

[0469] The lighting subsystem includes:

[0470] light source;

[0471] speckle eliminater; and

[0472] A beam delivery subsystem, optically coupled to the illumination system, comprising:

[0473] Collimator; and

[0474] One or more optical lens elements.

[0475] 159. The method according to any one of the foregoing embodiments, wherein the sample plane is orthogonal to the z-axis.

[0476] 160. The method according to any one of the foregoing embodiments, wherein the light source comprises one or more lasers.

[0477] 161. The method according to any one of the foregoing embodiments, wherein the one or more lasers comprises one or more laser diodes.

[0478] 162. The method according to any one of the foregoing embodiments, wherein the one or more lasers emit light of multiple wavelengths. 163. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further comprises one or more optical fibers.

[0479] 164. The method according to any one of the foregoing embodiments, wherein at least one of the optical fibers has a length of 0.5m to 5m.

[0480] 165. The method according to any one of the foregoing embodiments, wherein at least one of the optical fibers comprises a core having a maximum cross-sectional dimension of 50 μm to 1500 μm.

[0481] 166. The method according to any one of the foregoing embodiments, wherein the cross-section of the core is circular.

[0482] 167. The method according to any one of the foregoing embodiments, wherein the power efficiency of the lighting system is not less than 65%, 70%, 75% or 80%.

[0483] 168. The method according to any one of the foregoing embodiments, wherein the illumination field is rectangular or square.

[0484] 169. The method according to any one of the foregoing embodiments, wherein the one or more optical lens elements comprise one or more multi-lens arrays.

[0485] 170. The method according to any one of the foregoing embodiments, wherein each of the multi-lens arrays comprises one or more of the following: an asymmetric biconvex lens, a convex-planar lens, a concave-planar lens, an asymmetric biconcave lens, and an asymmetric convex-concave lens.

[0486] 171. The method according to any one of the foregoing embodiments, wherein each of the multi-lens arrays comprises a plurality of lens elements at least in a direction orthogonal to the z-axis.

[0487] 172. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further comprises an optical fiber optically coupled to a laser diode and reducing light source speckle.

[0488] 173. The method according to any one of the foregoing embodiments, wherein the speckle eliminater is composed of an optical fiber, the optical fiber being optically coupled to a laser diode and reducing the speckle of the light source.

[0489] 174. The method according to any one of the foregoing embodiments, wherein the one or more optical lens elements comprise: non-

[0490] Symmetrical biconvex lens, convex-plano lens, concave-plano lens, asymmetrical biconcave lens, asymmetrical convex-concave lens, or combinations thereof. 175. The method according to any one of the foregoing embodiments, wherein the one or more optical lens elements comprise: a...

[0491] A first multi-lens array and a second multi-lens array are positioned along the z-axis between the collimator and the entrance pupil of the illumination system.

[0492] 176. The method according to any one of the foregoing embodiments, wherein the illumination system is configured to generate at the sample stage.

[0493] Greater than 50mm 2 The lighting field has a lighting power density difference of less than ±2%, 5%, 8%, 10%, or 12%.

[0494] 177. The method according to any one of the foregoing embodiments, wherein the speckle eliminator includes a mechanical vibration source.

[0495] 178. The method according to any one of the foregoing embodiments, wherein the speckle eliminator includes a vibration source that generates vibrations within a predetermined frequency range.

[0496] 179. The method according to any one of the foregoing embodiments, wherein the mechanical vibration source is configured to vibrate at one or more frequencies in the audible sound range, the ultrasonic range, or both.

[0497] 180. The method according to any one of the foregoing embodiments, wherein the mechanical vibration source is configured to generate vibrational motion in one, two or three dimensions.

[0498] 181. The method according to any one of the foregoing embodiments, wherein at least a portion of each optical fiber or single optical fiber is wound or coiled one or more times.

[0499] 182. The method according to any one of the foregoing embodiments, wherein at least a portion of each of the optical fibers or single optical fibers is fixedly or loosely attached to a mechanical vibration source.

[0500] 183. The method according to any one of the foregoing embodiments, wherein the speckle eliminator is connected to the sample stage and the material

[0501] The objective lens and the one or more image sensors are physically isolated so that the mechanical movement of the speckle eliminator is independent of the sample stage, the objective lens, and the one or more image sensors.

[0502] 184. The method according to any one of the foregoing embodiments, wherein the speckle eliminator is configured to reduce speckle noise to no more than 4%, 4.5%, 5%, or 5.5%.

[0503] 185. The method according to any one of the foregoing embodiments, wherein the light source comprises a multicolor laser array.

[0504] 186. The method according to any one of the foregoing embodiments, wherein the multicolor laser array comprises a laser diode array that emits laser light in the range of 2, 3, 4, 5 or 6 wavelengths or wavelengths of 2, 3, 4, 5 or 6 wavelengths.

[0505] 187. The method according to any one of the foregoing embodiments, wherein the multicolor laser array comprises a laser that emits light of at least two, three, or four color wavelengths or wavelength ranges in a direction orthogonal to the z-axis.

[0506] 188. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further includes one or more coupling lenses.

[0507] 189. The method according to any one of the foregoing embodiments, wherein the illumination subsystem comprises a single optical fiber.

[0508] 190. The method according to any one of the foregoing embodiments, wherein the single optical fiber includes a core, the maximum cross-sectional dimension of the core being 500 μm to 1500 μm.

[0509] 191. The method according to any one of the foregoing embodiments, wherein the power in the beam transmission subsystem is greater than 5, 8, 10, 12 or 14 watts for one or more wavelengths or wavelength ranges.

[0510] 192. The method according to any one of the foregoing embodiments, wherein the power at the sample plane is greater than 5, 8, 10, 12 or 14 watts for one or more wavelengths or wavelength ranges.

[0511] 193. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further comprises a plurality of optical fibers, each optical fiber being optically coupled to one or more corresponding lasers of the light source, the one or more corresponding lasers emitting light of the same wavelength or wavelength range.

[0512] 194. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further comprises one or more dichroic filters.

[0513] 195. The method according to any one of the foregoing embodiments, wherein the light source comprises a plurality of beam combiners.

[0514] 196. The method according to any one of the foregoing embodiments, wherein the light source comprises a plurality of polarizing beam combiners.

[0515] 197. The method according to any one of the foregoing embodiments, wherein the light source comprises two or more lasers that emit light of the same wavelength or the same wavelength range.

[0516] 198. The method according to any one of the foregoing embodiments, wherein each polarized beam in the polarized beam combiner

[0517] The combiner is configured to combine light of the same wavelength or the same wavelength range from two or more lasers.

[0518] 199. The method according to any one of the foregoing embodiments, wherein the speckle eliminator is positioned in the optical path between the collimator and the sample plane.

[0519] 200. The method according to any one of the foregoing embodiments, wherein the speckle canceller is positioned at a location where the beam diameter is greater than 5 mm, 10 mm or 20 mm, and wherein the beam diameter is orthogonal to the z-axis.

[0520] 201. The method according to any one of the foregoing embodiments, wherein the optical fiber comprises a core having a rectangular or square cross-section.

[0521] 202. The method according to any one of the foregoing embodiments, wherein the optical fiber comprises a core having a non-circular cross-section.

[0522] 203. The method according to any one of the foregoing embodiments, wherein the illumination subsystem further comprises one or more liquid light guides.

[0523] 204. The method according to any one of the foregoing embodiments, wherein the one or more liquid light guides are optically coupled to the light source in the absence of an optical fiber.

[0524] 205. The method according to any one of the foregoing embodiments, wherein the one or more liquid optical guides include a liquid fiber core, the maximum dimension of the cross-section of the liquid fiber core being 0.5 mm to 10 mm, and wherein the cross-section is orthogonal to the z-axis.

[0525] 206. The method according to any one of the foregoing embodiments, wherein the liquid fiber core comprises a circular cross-section.

[0526] 207. The method according to any one of the foregoing embodiments, wherein the liquid fiber core comprises a non-circular cross-section.

[0527] 208. A sample stage for holding a DNA sample for DNA sequencing reactions and imaging, the sample stage comprising:

[0528] A platform, including a top surface, wherein the platform is rotatable about the z-axis relative to the optical system of the sequencing system;

[0529] One or more top platforms are positioned on the top surface of the base, wherein each of the one or more top platforms is configured to receive and fix one or more flow cell devices thereon, and wherein each of the one or more top platforms is movable relative to the base.

[0530] A first motor is configured to actuate the base to rotate at a first resolution.

[0531] 209. The sample stage according to any one of the foregoing embodiments, wherein the top surface is circular.

[0532] 210. The sample stage according to any one of the foregoing embodiments, wherein the first resolution is an angular resolution and is less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees or 50 degrees.

[0533] 211. The sample stage according to any one of the foregoing embodiments, wherein each flow cell device in the flow cell device includes one or more samples to be sequenced fixed thereon.

[0534] 212. The sample stage according to any one of the foregoing embodiments, wherein at least one flow cell device in the flow cell device includes an in-situ sample fixed thereon.

[0535] 213. The sample stage according to any one of the foregoing embodiments, wherein the sample stage further comprises one or more first...

[0536] Two motors, the second motor being configured to individually actuate the one or more top stages relative to the base with a second resolution.

[0537] 214. The sample stage according to any one of the foregoing embodiments, wherein the sample stage further includes a second motor configured to simultaneously actuate the one or more top stages relative to the base with a second resolution.

[0538] 215. The sample stage according to any one of the foregoing embodiments, wherein the second resolution is less than 0.01 mm, 0.015 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.2 mm or 1 mm.

[0539] 216. The sample stage according to any one of the foregoing embodiments, wherein the sequencing system includes a fluid control device in fluid communication with the flow cell device positioned on the sample stage.

[0540] 217. The sample stage according to any of the foregoing embodiments, wherein each of the one or more top stages is movable relative to the base stage in the sample plane.

[0541] 218. The sample stage according to any one of the foregoing embodiments, wherein the first top stage of the one or more top stages is movable independently relative to the second top stage of the one or more top stages.

[0542] 219. The sample stage according to any of the foregoing embodiments, wherein the first top stage of the one or more top stages is movable simultaneously with the second top stage of the one or more top stages relative to the base.

[0543] 220. The sample stage according to any one of the foregoing embodiments, wherein each of the one or more top stages is movable relative to the base stage along the radius of the top surface of the base stage.

[0544] 221. The sample stage according to any one of the foregoing embodiments, wherein each of the one or more top stages is movable relative to the base stage by a radius orthogonal to the top surface of the base stage.

[0545] 222. A method for sequencing multiple DNA samples positioned on a rotating sample stage, the method comprising:

[0546] A sample stage is obtained, the sample stage including a base and one or more top stages positioned on the top surface of the base, wherein the base is rotatable about the z-axis relative to the optical system of the sequencing system.

[0547] Positioning and fixing a first flow cell device relative to a first stage of the one or more stagees; positioning and fixing a second flow cell device relative to a second stage of the one or more stagees; dispensing one or more sequencing reagents into the first flow cell device using a first fluid control device; and imaging a first sample region of the first flow cell device using the optical system of the sequencing system.

[0548] While preventing the second flow cell device from moving relative to the optical system, the first top platform is moved in the xy plane relative to the optical system;

[0549] The second sample region of the first flow cell device is imaged using the optical system of the sequencing system.

[0550] Rotate the sample stage with a predetermined angular resolution to position the second flow cell device relative to the optical system at a predetermined position; and

[0551] The first sample region of the second flow cell device is imaged using the optical system of the sequencing system. 223. The method according to any one of the foregoing embodiments, wherein the second flow cell device is prevented from being positioned relative to the...

[0552] Moving the first top platform relative to the optical system in the xy plane while moving the optical system includes:

[0553] While preventing the second flow cell device from moving relative to the optical system, the first top stage is moved independently relative to the optical system at a predetermined distance along the radius of the top surface of the base.

[0554] 224. The method according to any one of the foregoing embodiments, wherein preventing the second flow cell device from being relative to the

[0555] Moving the first top platform relative to the optical system in the xy plane while moving the optical system includes:

[0556] While preventing the second flow cell device from moving relative to the optical system, the first top platform is moved independently relative to the optical system at a predetermined distance in a direction orthogonal to the radius of the top surface of the base.

[0557] 225. The method according to any one of the foregoing embodiments, wherein the method further comprises:

[0558] Move the first fluid control device or the second fluid control device to position the second fluid pool device relative to the first fluid control device or the second fluid control device at a predetermined position.

[0559] 226. The method according to any one of the foregoing embodiments, wherein the first sample region or the second sample region comprises a pattern.

[0560] 227. The method according to any one of the foregoing embodiments, wherein each of the one or more top stages includes

[0561] The range of motion is greater than 15 mm and less than 80 mm along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base.

[0562] 228. The method according to any one of the foregoing embodiments, wherein the range of motion of each of the one or more top platforms along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base is greater than 25 mm and less than 100 mm.

[0563] While preferred embodiments of the inventive concept have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Various changes, modifications, and substitutions will now occur to those skilled in the art without departing from the inventive concept. It should be understood that various alternatives to the embodiments of the inventive concept described herein can be used to practice these inventive concepts. The following claims are intended to define the scope of the inventive concept and cover methods and structures within the scope of these claims and their equivalents.

Claims

1. An optical system, the optical system comprising: A stage, configured to hold a solid support; A light source configured to illuminate the solid support; as well as An optical component, at least partially disposed within the optical path from the stage to the light source, wherein the optical component is configured to be larger than approximately 20 square millimeters (mm) on the solid support. 2 Lighting is provided in the area where the peak-to-valley variation is at most about 5%.

2. The optical system of claim 1, wherein the optical components do not include an objective lens.

3. The optical system of claim 2, wherein the optical system does not include the objective lens.

4. The optical system of claim 1, wherein the optical components do not include a barrel lens.

5. The optical system of claim 4, wherein the optical system does not include the barrel lens.

6. The optical system of claim 1, wherein the stage is not adjusted on the optical axis of the system.

7. The optical system of claim 1, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter.

8. The optical system of claim 1, wherein the optical component is configured to receive emitted light from the solid support.

9. The optical system of claim 8, wherein the numerical aperture (NA) of the optical component is at least about 0.

3.

10. The optical system of claim 8, wherein the wavelength of the emitted light is about 500 nanometers to about 750 nanometers.

11. The optical system of claim 1, wherein the working distance of the optical component is at least about 1 mm to 25 mm.

12. The optical system of claim 1, further comprising a motion coil housed within the optical component, the motion coil being configured to move a focusing element within the optical path of the optical system.

13. The optical system of claim 1, wherein a motor located outside the optical system is configured to move the focusing element along the optical axis in one or both directions.

14. The optical system of claim 13, wherein the motor is directly coupled to a portion of the first, second, or third housing of the optical assembly, and the portion of the first, second, or third housing of the optical assembly is directly coupled to the focusing element.

15. The optical system according to claim 1, wherein the light source is a pulsed light source.

16. The optical system of claim 1, wherein the composite root mean square error of the optical system is less than about 0.

05.

17. The optical system of claim 1, wherein the illumination efficiency of said optical component is at least about 90%.

18. The optical system of claim 1, wherein the region is greater than 30 mm 2 .

19. The optical system of claim 1, wherein the region is greater than 50 mm 2 Or 60mm 2 .

20. The optical system of claim 1, further comprising the solid support within the stage.

21. The optical system of claim 20, wherein the solid support comprises two or more surfaces having one or more samples imaged by the optical system fixed thereon.

22. The optical system of claim 21, wherein the solid support comprises three or more surfaces having one or more samples imaged by the optical system fixed thereon.

23. The optical system of claim 22, wherein the three or more surfaces are axially displaced from each other at least along the optical axis of the optical system.

24. The optical system of claim 20, wherein the solid support comprises a probe configured to bind nucleic acid molecules.

25. The optical system of claim 24, wherein the probe is attached to the surface of the solid support.

26. The optical system according to claim 1, wherein the light source is a laser light source.

27. The optical system of claim 1, wherein the optical component includes a dichroic filter configured to transmit the illumination.

28. The optical system of claim 1, wherein the optical components comprise: The first segment includes a first housing, the first housing including a first plurality of lenses; The second section includes a second housing. And the third segment, which includes a third housing, the third housing including a second plurality of lenses.

29. The optical system of claim 28, wherein the first segment is optically aligned with the third segment.

30. The optical system of claim 28, wherein the first segment is positioned between the third segment and the stage.

31. The optical system of claim 28, wherein the third segment is positioned between the first segment and the image sensor of the optical system.

32. The optical system of claim 28, wherein the first plurality of lenses are movable along the optical path ranging from about 0 to about 2 millimeters.

33. The optical system of claim 28, wherein the first plurality of lenses comprises an asymmetric biconvex lens.

34. The optical system of claim 28, wherein the second plurality of lenses comprises an asymmetric biconcave lens.

35. The optical system according to claim 34, wherein the asymmetric biconcave lens is an aspherical asymmetric biconcave lens.

36. The optical system of claim 1, wherein the optical system is configured to acquire an image of the solid support without moving the optical compensator into the optical path between the solid support and the detector of the optical system.

37. The optical system of claim 1, wherein the optical system is configured to acquire an image of the solid support without removing the optical compensator from the optical path between the sample and the detector of the optical system.

38. The optical system of claim 1, wherein the solid support is a flow cell.

39. The optical system of claim 1, wherein the optical components are configured to produce one or more spatial contractions transverse to the optical path of light traveling through it.

40. The optical system of claim 1, wherein the optical components are configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it.

41. The optical system of claim 1, wherein the optical component is configured to generate at least one field curvature correction in the first, second, or third segment, transverse to the optical path of light traveling through it.

42. A method for analyzing biomolecules, the method comprising: (a) Providing a solid support comprising the biomolecule containing the marker; (b) Illuminating the biomolecule containing the marker using an optical system including a light source, thereby generating signal light or a change thereof, wherein the solid support is larger than about 20 square millimeters (mm²). 2 The lighting is provided over the area where the peak-to-valley variation is at most about 5%; (c) Detecting the signal light or any changes thereof using the detector of the optical system; and (d) Process the signal light or its changes at least partially to analyze the biomolecules.

43. The method according to claim 42, wherein the biomolecule is a nucleic acid molecule, a protein, or a polypeptide.

44. The method according to claim 43, wherein the biomolecule is a nucleic acid.

45. The method of claim 42, further comprising, prior to (a), binding the biomolecule to a probe bound to the solid support and coupling the marker to the biomolecule.

46. ​​The method of claim 42, wherein the marker is coupled to the biomolecule via hybridization.

47. The method of claim 42, wherein the optical system does not include an objective lens.

48. The method of claim 42, wherein the solid support is not moved along the optical axis of the optical system.

49. The method of claim 48, wherein multiple images of the solid support are acquired without moving the solid support along the optical axis.

50. The method of claim 42, wherein the irradiance of the illumination is at least about 40 milliwatts per square millimeter.

51. The method of claim 42, wherein the wavelength of the signal light is about 500 nanometers to about 750 nanometers.

52. The method of claim 42, wherein the detection in (c) is performed using an optical element with a numerical aperture of at least about 0.

3.

53. The method of claim 42, further comprising (b) using a motion coil within the optical system to move a focusing element within the optical path of the optical system, thereby changing the focal point of the optical system on the solid support.

54. The method according to claim 42, wherein the light source is a pulsed light source.

55. The method of claim 42, wherein the illumination is provided with an efficiency of at least about 90%.

56. The method of claim 42, the method further comprising repeating (b)-(d) additional biomolecules coupled to additional surfaces of the solid support.

57. The method of claim 42, further comprising, after (c), removing the marker from the biomolecule.

58. The method of claim 57, further comprising repeating (a)-(d) on an additional marker that binds to another portion of the biomolecule.

59. The method of claim 42, wherein the optical components are configured to produce one or more spatial contractions transverse to the optical path through which light travels.

60. The method of claim 42, wherein the optical components are configured to generate one or more field curvature corrections transverse to the optical path of light traveling through it.

61. The method of claim 42, wherein the optical components are configured to generate at least one field curvature correction in the first, second, or third segment, transverse to the optical path through which the light travels.

62. The method of claim 42, wherein (d) comprises at least partially processing the signal light or the said variation thereof to generate one or more solid support images, and analyzing the one or more solid support images to generate a base interpretation of the sample.

63. The method of claim 62, wherein each solid support image in the solid support images comprises more than 20 square millimeters (mm²). 2 ) field of view (FOV).

64. The method of claim 42, wherein the solid support is a flow cell.

65. An optical system, the optical system comprising: A stage, configured to hold a solid support; A light source configured to illuminate the solid support; as well as A speckle eliminater, which is optically coupled to the light source and disposed within the optical path from the light source to the stage.

66. The optical system of claim 65, further comprising an additional light source optically coupled to the speckle eliminator.

67. The optical system of claim 66, wherein light from the additional light source is configured to illuminate the solid support with light of a different wavelength than that of the light source.

68. The optical system of claim 66, wherein at least about four light sources are coupled to the speckle eliminator.

69. The optical system of claim 65, wherein the speckle eliminator is a vibration speckle eliminator.

70. The optical system of claim 65, wherein the speckle eliminator is a passive speckle eliminator.

71. The optical system of claim 70, wherein the passive speckle eliminator comprises a diffuse scattering plate.

72. The optical system of claim 65, wherein the speckle eliminator is a tension speckle eliminator.

73. The optical system of claim 65, wherein the speckle canceller is configured to reduce speckle noise to at most about 5%.

74. The optical system of claim 65, wherein the solid support is a flow cell.

75. A method for analyzing biomolecules, the method comprising: (a) Providing a solid support, said solid support comprising a biological sample containing a marker; (b) Illuminating the biological sample containing the marker using an optical system including a light source to generate signal light or a variation thereof, wherein the illumination is provided by a speckle eliminator in the optical path of the optical system; (c) Detecting the signal light or the changes thereof using the detector of the optical system; as well as (d) Process the signal light or its changes at least partially to analyze the biomolecules.

76. The method of claim 75, further comprising repeating (b)-(d) on additional biological samples coupled to additional surfaces of the solid support.

77. The method of claim 75, further comprising, after (c), removing the marker from the biological sample.

78. The method of claim 77, further comprising repeating (a)-(d) on an additional marker bound to the biological sample.

79. The method of claim 75, wherein the speckle eliminator uses vibration to eliminate speckle on the illumination.

80. The method of claim 75, further comprising illuminating the solid support using an additional light source.

81. The method of claim 80, wherein the additional light source provides light of a different wavelength to the solid support.

82. The method of claim 80, wherein the additional light source is optically coupled to the speckle eliminator.

83. The method of claim 75, wherein the biological sample comprises nucleic acid molecules, proteins, or polypeptides.

84. The method of claim 83, wherein the biological sample comprises nucleic acid.

85. The optical system of claim 1, wherein the optical components are at least partially disposed within the optical path from the stage to the detector of the optical system.

86. The optical system of claim 1, wherein the illumination system of the optical component is disposed within the optical path from the stage to the detector of the optical system.

87. A sample stage for holding a DNA sample for DNA sequencing reactions and imaging, the sample stage comprising: A platform, including a top surface, wherein the platform is rotatable about the z-axis relative to the optical system of the sequencing system; One or more top platforms are positioned on the top surface of the base, wherein each of the one or more top platforms is configured to receive and fix one or more flow cell devices thereon, and wherein each of the one or more top platforms is movable relative to the base. A first motor is configured to actuate the base to rotate at a first resolution. The sample stage according to any one of the preceding claims, wherein the top surface is circular.

88. The sample stage according to any one of the preceding claims, wherein the first resolution is an angular resolution and is less than 0.1 degrees, 0.2 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 10 degrees, 20 degrees, 30 degrees or 50 degrees.

89. The sample stage according to any one of the preceding claims, wherein each flow cell device in the flow cell device includes one or more samples to be sequenced fixed thereon.

90. The sample stage according to any one of the preceding claims, wherein at least one flow cell device in the flow cell device includes an in-situ sample fixed thereon.

91. The sample stage according to any one of the preceding claims, wherein the sample stage further comprises one or more second motors configured to individually actuate the one or more top stages relative to the base stage with a second resolution.

92. The sample stage according to any one of the preceding claims, wherein the sample stage further comprises a second motor configured to simultaneously actuate the one or more top stages relative to the base stage with a second resolution.

93. The sample stage according to any one of the preceding claims, wherein the second resolution is less than 0.01 mm, 0.015 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.2 mm or 1 mm.

94. The sample stage according to any one of the preceding claims, wherein the sequencing system includes a fluid control device in fluid communication with the flow cell device positioned on the sample stage.

95. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable in the sample plane relative to the base stage.

96. The sample stage according to any one of the preceding claims, wherein the first of the one or more top stages is movable independently relative to the second of the one or more top stages.

97. The sample stage according to any one of the preceding claims, wherein the first of the one or more top stages is movable simultaneously relative to the base stage and the second of the one or more top stages.

98. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable relative to the base stage along the radius of the top surface of the base stage.

99. The sample stage according to any one of the preceding claims, wherein each of the one or more top stages is movable relative to the base with a radius orthogonal to the top surface of the base.

100. A method for sequencing multiple DNA samples positioned on a rotating sample stage, the method comprising: A sample stage is obtained, the sample stage including a base and one or more top stages positioned on the top surface of the base, wherein the base is rotatable about the z-axis relative to the optical system of the sequencing system. The first flow pool device is positioned and fixed relative to the first of the one or more top platforms; The second flow cell device is positioned and fixed relative to the second of the one or more top platforms; One or more sequencing reagents are dispensed into the first flow cell device using a first fluid control device; The first sample region of the first flow cell device is imaged using the optical system of the sequencing system. While preventing the second flow cell device from moving relative to the optical system, the first top platform is moved in the xy plane relative to the optical system; The second sample region of the first flow cell device is imaged using the optical system of the sequencing system. The sample stage is rotated with a predetermined angular resolution to position the second flow cell device relative to the optical system at a predetermined position. as well as The first sample region of the second flow cell device is imaged using the optical system of the sequencing system.

101. The method according to any one of the preceding claims, wherein moving the first top stage relative to the optical system in the xy plane while preventing the second flow cell device from moving relative to the optical system comprises: While preventing the second flow cell device from moving relative to the optical system, the first top stage is moved independently relative to the optical system at a predetermined distance along the radius of the top surface of the base.

102. The method according to any one of the preceding claims, wherein moving the first top stage relative to the optical system in the xy plane while preventing the second flow cell device from moving relative to the optical system comprises: While preventing the second flow cell device from moving relative to the optical system, the first top platform is moved independently relative to the optical system at a predetermined distance in a direction orthogonal to the radius of the top surface of the base.

103. The method according to any one of the preceding claims, wherein the method further comprises: Move the first fluid control device or the second fluid control device to position the second fluid pool device relative to the first fluid control device or the second fluid control device at a predetermined position.

104. The method according to any one of the preceding claims, wherein the first sample region or the second sample region comprises a pattern.

105. The method according to any one of the preceding claims, wherein each of the one or more top platforms includes a range of motion greater than 15 mm and less than 80 mm along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base.

106. The method according to any one of the preceding claims, wherein each of the one or more top platforms includes a range of motion greater than 25 mm and less than 100 mm along the radius of the top surface of the base or orthogonal to the radius of the top surface of the base.

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