Apparatus for reducing signal fluctuations in a sequencing system

Abstract

The device includes a sequencing stage and an illumination assembly. The sequencing stage is configured to receive a flow cell that includes a plurality of reaction sites. Each reaction site is configured to contain a biological sample. The illumination assembly is configured to project light toward the sequencing stage, thereby illuminating the reaction sites. The illumination assembly includes a light source, a first small lens array, a second small lens array, and a diffuser plate. The first small lens array is interposed between the light source and the sequencing stage. The second small lens array is also interposed between the light source and the sequencing stage. The diffuser plate is further interposed between the light source and the sequencing stage.

Description

Technical Field

[0001] Priority This application claims priority to U.S. Provisional Patent Application No. 63 / 357,074, filed Jun. 30, 2022, entitled "Apparatus for Reduction of Signal Variation in Sequencing System", the disclosure of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Subject matter considered in this section should not be assumed to be prior art merely as a result of mention in this section. Similarly, problems mentioned in this section, or problems associated with the subject matter provided as background, should not be assumed to have been previously recognized in the prior art. The subject matter of this section merely represents different approaches, which may in themselves further correspond to implementations of the claimed technology.

[0003] Aspects of the present disclosure generally relate to biological or chemical analysis, and more particularly to systems and methods for using an imaging sensor for biological or chemical analysis.

[0004] Various protocols in biological or chemical research involve performing a number of controlled reactions on a local support surface or within a predefined reaction chamber. Next, the specified reaction can be observed or detected, and subsequent analysis may be useful for identifying or elucidating the properties of the chemical substances involved in the reaction. For example, in some multiplex assays, an unknown analyte having an identifiable label (e.g., a fluorescent label) may be exposed to thousands of well-known probes under controlled conditions. Each well-known probe may be deposited in a corresponding well of a flow cell channel. Observing any chemical reaction that occurs between the well-known probe and the unknown analyte in the well may be useful for identifying or elucidating the properties of the analyte. Other examples of such protocols include well-known DNA sequencing processes such as Sequencing-By-Synthesis (SBS) or circular array sequencing.

[0005] In some conventional fluorescence detection protocols, an optical system is used to direct excitation light onto a fluorescently labeled analyte and further detect the fluorescence signal that may be emitted from the analyte. Such an optical system may include the arrangement of lenses, filters, and light sources. It may be desirable to provide uniformity in the spatial irradiance from the excitation light and thereby obtain spatial uniformity in the collected fluorescence emission signal. However, providing such uniformity in the spatial irradiance from the excitation light and thereby obtaining spatial uniformity in the collected fluorescence emission signal can present challenges.

Brief Description of the Drawings

[0006]

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DETAILED DESCRIPTION OF THE INVENTION

[0007] I. Overview of Systems for Biological or Chemical Analysis Described herein are devices, systems, and methods for providing spatial irradiance uniformity from excitation light emitted within an optical system, thereby obtaining spatial uniformity in fluorescence signals emitted from analytes exposed to the excitation light. The examples described herein may be used in various biological or chemical processes and systems for academic analysis, commercial analysis, or other analysis. More specifically, the examples described herein may be used in various processes and systems where it is desirable to detect events, properties, qualities, or characteristics indicative of a specified reaction.

[0008] A bioassay system as described herein may be configured to perform a plurality of specified reactions that can be detected individually or collectively. Biosensors and bioassay systems may be configured to perform a number of cycles in which a plurality of specified reactions occur in parallel. For example, a bioassay system may be used to sequence a high-density array of DNA features through iterative cycles of enzymatic manipulation and image acquisition. The cartridges and biosensors used in bioassay systems may include one or more microfluidic channels that deliver reagents or other reaction components to the reaction sites. The reaction sites may be randomly distributed over a substantially flat surface or may be patterned over a substantially flat surface in a predetermined pattern. In some variations, the reaction sites are located within reaction chambers that compartmentalize the specified reactions therein.

[0009] Regardless of the form taken by the reaction site, each of the reaction sites may be imaged in order to detect light from the reaction site. In some embodiments, one or more imaging sensors may detect light emitted from the reaction site. The signals indicative of the photons emitted from the reaction site and detected by the individual imaging sensors may be referred to as the illumination values of those sensors. These illumination values may be combined into an image indicative of the photons detected from the reaction site. These images may be further analyzed to identify the composition, reactions, conditions, etc. at each reaction site.

[0010] The following detailed description of specific embodiments will be better understood when read in conjunction with the accompanying drawings. To the extent that the drawings show diagrams of functional blocks of various embodiments, the functional blocks do not necessarily represent a division between hardware components. Thus, for example, one or more of the functional blocks (e.g., a processor or a memory) may be implemented in one piece of hardware (e.g., a general-purpose signal processor or a random access memory, a hard disk, etc.). Similarly, a program may be an independent program, may be incorporated as a subroutine within an operating system, may be a function within an installed software package, etc. It should be understood that the various embodiments are not limited to the arrangements and means shown in the drawings.

[0011] As used herein, an element or step described in the singular and preceded by the word "a" or "an" should be understood not to exclude a plurality of such elements or steps, unless such exclusion is explicitly recited. Further, reference to "one example" is not intended to be construed as excluding the existence of additional examples that also incorporate the recited features. Further, unless explicitly stated to the contrary, embodiments "comprising" or "having" one element or a plurality of elements with a particular characteristic may include additional elements, whether or not they have that characteristic.

[0012] As used herein, a "designated reaction" includes at least one change in a chemical, electrical, physical, or optical property (or quality) of an analyte of interest. In some examples, the designated reaction is a positive binding event (e.g., incorporation of a fluorescently labeled biomolecule with an analyte of interest). More generally, the designated reaction may be a chemical conversion, chemical change, or chemical interaction. In some examples, the designated reaction includes incorporation of a fluorescently labeled molecule into an analyte. The analyte may be an oligonucleotide and the fluorescently labeled molecule may be a nucleotide. The designated reaction may be detected when excitation light is directed at an oligonucleotide having a labeled nucleotide and the fluorophore emits a detectable fluorescent signal. In alternative examples, the detected fluorescence is the result of chemiluminescence or bioluminescence. The designated reaction may further, for example, increase fluorescence (or Forster) resonance energy transfer (FRET) by bringing a donor fluorophore in proximity to an acceptor fluorophore, decrease FRET by separating the donor fluorophore and the acceptor fluorophore, increase fluorescence by separating a quencher from a fluorophore, or decrease fluorescence by co-localizing a quencher and a fluorophore.

[0013] As used herein, a "reaction component" or "reactant" includes any substance that can be used to effect a designated reaction. For example, reaction components include reagents, enzymes, samples, other biomolecules, and buffers. Reaction components may be delivered to a reaction site in solution and / or immobilized at the reaction site. Reaction components may interact directly or indirectly with another substance, such as an analyte of interest.

[0014] As used herein, the term "reaction site" is a localized region where a specified reaction can occur. The reaction site may include a support surface of a substrate on which a substance can be immobilized. For example, the reaction site may include a substantially planar surface within a channel of a flow cell having a colony of nucleic acids thereon. The nucleic acids in the colony may have the same sequence and may be, for example, a cloned copy of a single-stranded template or a double-stranded template. However, in some embodiments, the reaction site may contain only a single nucleic acid molecule, for example, in single-stranded or double-stranded form. Further, a plurality of reaction sites may be randomly distributed along the support surface or arranged in a predetermined pattern (e.g., in a matrix such as in a microarray). The reaction site may further include a reaction chamber that at least partially defines a spatial region or volume configured to compartmentalize a specified reaction. As used herein, the term "reaction chamber" includes a spatial region in fluid communication with a flow channel. The reaction chamber may be at least partially separated from the surrounding environment or other spatial regions. For example, a plurality of reaction chambers may be separated from each other by a shared wall. As a more specific example, the reaction chamber may include a cavity defined by the inner surface of a well and may have an opening or aperture such that the cavity is in fluid communication with the flow channel. The reaction site need not necessarily be provided within the reaction chamber and may instead be provided within any other suitable type of structure or within a structure.

[0015] As used herein, the term "adjacent", when used with respect to two reaction sites, means that there are no other reaction sites between the two reaction sites. The term "adjacent" may have a similar meaning when used with respect to adjacent detection paths and adjacent imaging sensors (e.g., adjacent imaging sensors do not have other imaging sensors between them). In some cases, a reaction site may not be adjacent to another reaction site, but may still be in close proximity to other reaction sites. The first reaction site may be in close proximity to the second reaction site when a fluorescence emission signal from the first reaction site is detected by an imaging sensor associated with the second reaction site. More specifically, the first reaction site may be in close proximity to the second reaction site when an imaging sensor associated with the second reaction site detects, for example, crosstalk from the first reaction site. Adjacent reaction sites may be continuous such that they abut each other, or the adjacent sites may be discontinuous with an intervening space therebetween.

[0016] As used herein, "substance" includes items or solids such as capture beads, as well as biological or chemical substances. As used herein, "biological or chemical substance" includes biomolecules, target samples, target analytes, and other chemical compounds (s). Biological or chemical substances may be used to detect, identify, or analyze other chemical compounds (s), or may function as intermediaries for studying or analyzing other chemical compounds (s). In certain embodiments, biological or chemical substances include biomolecules. As used herein, "biomolecule" includes at least one of biopolymers, nucleotides, nucleic acids, polynucleotides, oligonucleotides, proteins, enzymes, polypeptides, antibodies, antigens, ligands, receptors, polysaccharides, carbohydrates, polyphosphates, cells, tissues, organisms, or fragments thereof, or any other biologically active chemical compounds (s) such as analogs or mimetics of the foregoing species.

[0017] Biomolecules, samples, and biological or chemical substances can be naturally occurring or synthetic and can be suspended in a solution or mixture within a spatial region. The biomolecules, samples, and biological or chemical substances may further be bound to a solid phase or a gel material. The biomolecules, samples, and biological or chemical substances may further include a pharmaceutical composition. In some cases, the biomolecules, samples, and biological or chemical substances of interest may be referred to as targets, probes, or analytes.

[0018] As used herein, when the terms "removably" and "coupled" (or "engaged") are used together to describe the relationship between components, these terms are intended to mean that the connection between the components is readily separable without breaking or damaging the components. Components are readily separable if they can be separated from each other without undue effort or without spending an inordinate amount of time when separating the components. For example, the components may be electrically removably coupled or engaged such that the mating contacts of the components are not broken or damaged. The components may further be removably coupled or engaged in a mechanical manner such that the features holding the components are not broken or damaged. The components may further be removably coupled or engaged in a fluid manner such that the ports of the components are not broken or damaged. Components are not considered broken or damaged if only simple adjustments (e.g., repositioning) or simple replacements (e.g., nozzle replacement) to the components are required.

[0019] As used herein, the terms "fluid communication" or "fluidically coupled" refer to two spatial regions that are connected such that a liquid or gas can flow between the two spatial regions. For example, a microfluidic channel may be in fluid communication with a reaction chamber such that fluid can flow freely from the microfluidic channel into the reaction chamber. The terms "in fluid communication" or "fluidically coupled" mean that two spatial regions are in fluid communication through one or more valves, restrictors, or other fluid components, enabling control or regulation of fluid flow through the system.

[0020] As used herein, the term "immobilized", when used with respect to a biomolecule or biological or chemical substance, includes substantially attaching the biomolecule or biological or chemical substance to a surface at the molecular level. For example, a biomolecule or biological or chemical substance may be immobilized on the surface of a substrate material using adsorption techniques including non-covalent interactions (e.g., electrostatic forces, van der Waals, and dehydration of hydrophobic interfaces), as well as covalent bonding techniques where functional groups or linkers facilitate attachment of the biomolecule to the surface. Immobilizing a biomolecule or biological or chemical substance to the surface of a substrate material may be based on the properties of the substrate surface, the liquid medium carrying the biomolecule or biological or chemical substance, and the properties of the biomolecule or biological or chemical substance itself. In some cases, the substrate surface may be functionalized (e.g., chemically or physically modified) to facilitate immobilizing the biomolecule (or biological or chemical substance) to the substrate surface. The substrate surface may first be modified to have functional groups attached to the surface. The functional groups may then bind to the biomolecule or biological or chemical substance to immobilize them thereon.

[0021] As used herein, the term "magnet" includes permanent magnets and electromagnets.

[0022] In some embodiments, nucleic acids can be attached to a surface and amplified. Examples of such amplification are described in U.S. Patent No. 7,741,463, issued June 22, 2010, entitled "Method of Preparing Libraries of Template Polynucleotides", the disclosure of which is incorporated herein by reference in its entirety. Optionally, iterative rounds of extension (e.g., amplification) using immobilized primers and primers in solution may provide multiple copies of the nucleic acid.

[0023]

[0022] In certain embodiments, the assay protocols performed by the systems and methods described herein include the use of natural nucleotides and enzymes configured to interact with the natural nucleotides. Examples of natural nucleotides include ribonucleotides or deoxyribonucleotides. The natural nucleotides may be in the mono-phosphate, di-phosphate, or tri-phosphate form and may have a base selected from adenine (A), thymine (T), uracil (U), guanine (G), or cytosine (C). However, it will be understood that non-natural nucleotides, modified nucleotides, or analogs of the foregoing nucleotides may be used.

[0024] FIG. 1 shows an example of components of a system (100) that can be used to provide biological or chemical analysis. In some examples, the system (100) can be a workstation similar to a benchtop device. For example, most (or all) of the systems and components for performing a specified reaction may be within a common housing. In certain examples, the system (100) is a nucleic acid sequencing system (or sequencer) configured for various applications including, but not limited to, de novo sequencing, re-sequencing of whole or target genomic regions, and metagenomics. The sequencer may further be used for DNA or RNA analysis. In some variations, the system (100) may further be configured to generate reaction sites within a flow cell (110). For example, the system (100) may be configured to receive a sample and generate surface-attached clusters of clonally amplified nucleic acids derived from the sample. In some implementations, the clusters may include a particular sample that, as a result of one or more other samples present within the cluster, is a distinguishable part of the cluster even if the cluster is polyclonal. The system (100) is further configured to utilize an imaging assembly (120) to capture an image of the reaction sites on the flow cell (110).

[0025] In certain embodiments, system (100) is for performing a number of parallel reactions within flow cell (110). Flow cell (110) includes one or more reaction sites where a designated reaction can occur. The reaction sites may be immobilized, for example, on the solid surface of flow cell (110) or on beads (or other movable substrates) located within corresponding reaction chambers of flow cell (110). The reaction sites may include, for example, clusters of clonally amplified nucleic acids. Flow cell (110) may include one or more flow channels that receive a solution from system (100) and direct the solution towards the reaction sites. Optionally, flow cell (110) may engage a thermal element for transferring thermal energy into or out of the flow channels.

[0026] System (100) may include various components, assemblies, and systems (or subsystems) that interact with each other to perform a given method or assay protocol for biological or chemical analysis. For example, system (100) may include a system controller (195) that can communicate with the various components, assemblies, and subsystems of system (100). Examples of such components are described in more detail below. Controller (195) may include one or more microprocessors, memory devices, and / or any other suitable electrical components configured to cooperate to execute control algorithms, data processing, and the like.

[0027] In this embodiment, the imaging assembly (120) includes a light emitter (150) that emits light reaching a reaction site on the flow cell (110). The light emitter (150) may include an incoherent light emitter (e.g., emitting a light beam output by one or more excitation diodes), or a coherent light emitter such as a light emitter output by one or more lasers or laser diodes. In this embodiment, the light emitter (150) includes an optical fiber (152) for guiding the light beam output through the light emitter. However, other configurations of the light emitter 150 may be used. In some implementations, the optical fiber (152) may be optically coupled to a plurality of different light sources (not shown), each light source emitting light in a different wavelength range. The system (100) is shown as having a single light emitter (150), but in some other implementations, a plurality of light emitters (150) may be included.

[0028] In this embodiment, the light output from the light emitter (150) is collimated by the collimating lens (154). The collimated light is structured (patterned) by the light structuring optical assembly (156) and reaches the projection lens (158). In some variations, the projection lens (158) includes a lens element operable to translate along an axis (i.e., the axis along which the light emitter (150), the collimating lens (154), and the light structuring optical assembly (156) are aligned) to adjust the structured beam shape and path. For example, the projection lens (156) may be translated along this axis taking into account the range of thicknesses of the sample within the flow cell (110) (e.g., different cover glass thicknesses). In other implementations, the projection lens (156) may be fixed and / or omitted, and the movable lens element may be positioned within the imaging lens assembly in the radiation optical path taking into account the focusing on the upper or lower inner surface of the flow cell (110) and / or the spherical aberration introduced by the movement of the objective lens assembly (142). The foregoing illumination components (150, 152, 154, 156, 158) are merely examples. Alternatively, the system (100) may include any other suitable components for providing illumination in addition to, or instead of, any of the above-described illumination components (150, 152, 154, 156, 158). For example, some other variations may omit the light structuring optical assembly (156) and use unstructured illumination and / or any other type of illumination and / or optical arrangement (e.g., epifluorescence microscopy, etc.).

[0029] In this embodiment, light irradiates the sample in the flow cell (110) from the dichroic mirror (160) through the objective lens assembly (142), and the sample in the flow cell (110) is positioned on the moving stage (170). In the case of fluorescence microscopy of the sample, the fluorescent element related to the target sample emits fluorescence in response to the excitation light, and the resulting light is collected by the objective lens assembly (142) and directed to the imaging sensor of the camera system (140) to detect the emitted fluorescence. As described above, in some implementations, an imaging lens assembly may be positioned between the objective lens assembly (142) and the dichroic mirror (160), or between the dichroic mirror (160) and the imaging sensor of the camera system (140). The movable lens element may be translatable along the longitudinal axis of the imaging lens assembly in consideration of focusing on the upper or lower inner surface of the flow cell (110) and / or the spherical aberration introduced by the movement of the objective lens assembly (142). In some embodiments, a filter switching assembly (162) having one or more emission filters may be included, and one or more emission filters may be used to pass a specific emission wavelength range and block (or reflect) other emission wavelength ranges. For example, one or more emission filters may be used to direct different wavelength ranges of the emitted light to different imaging sensors of the camera system (140) of the imaging assembly (120). For example, the emission filter may be implemented as a dichroic mirror that directs emitted light of different wavelengths from the flow cell (110) to different imaging sensors of the camera system (140). In some variations, the projection lens (158) is interposed between the filter switching assembly (162) and the camera system (140) instead of being positioned as shown in FIG. 1. The filter switching assembly (162) may be omitted in some variations.

[0030] In an example of the system (100), the fluid delivery module or device (190) may direct the flow of reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and through) the flow cell (110) and the waste valve (180). The flow cell (110) may include one or more substrates to which the sample is provided. For example, in the case of a system for analyzing a number of different nucleic acid sequences, the flow cell (110) may include one or more substrates to which the nucleic acid to be sequenced binds, attaches, or associates. Substrates may include, for example, any inert substrate or matrix to which nucleic acids can be attached, such as a glass surface, a plastic surface, latex, dextran, a polystyrene surface, a polypropylene surface, a polyacrylamide gel, a gold surface, and a silicon wafer. In some applications, the substrate includes channels formed within the substrate or other areas, in the form of a matrix or array, within the channel or across the flow cell (110). The system (100) may further include a temperature station actuator (130) and a heater / cooler (132) that can optionally adjust the temperature of the fluid conditions within the flow cell (110). In some implementations, the heater / cooler (132) may be fixed to the sample stage (170) on which the flow cell (110) is placed, and / or integrated into the sample stage (170) internally.

[0031] In certain implementations, the flow cell (110) may be implemented as a patterned flow cell that includes a transparent cover plate and a substrate and is configured to contain a liquid therebetween, and the biological sample may be located on the inner surface of the transparent cover plate and / or the inner surface of the substrate. The flow cell may include a large number (e.g., thousands, millions, or billions) of wells (also referred to as nanowells), or regions patterned in a defined array (e.g., hexagonal array, rectangular array, etc.) within the substrate. Such wells may define reaction chambers that provide reaction sites as described above. Each region may form a cluster (e.g., monoclonal cluster, substantially monoclonal cluster, or polyclonal cluster) or multiple clusters of biological samples such as DNA, RNA, or another genomic material that can be sequenced, for example, using sequencing by synthesis. A substantially monoclonal cluster may be one in which a particular sample forms a distinguishable portion of the cluster, even if the cluster itself is polyclonal as a result of the presence of one or more other samples within the cluster. The flow cell may be further divided into some separated lanes (e.g., eight lanes), and each lane includes a hexagonal array of clusters or a linear array of clusters.

[0032] The flow cell (110) may be mounted on the sample stage (170) to provide movement and alignment of the flow cell (110) relative to the objective lens assembly (142). The sample stage (170) may have one or more actuators to enable the sample stage (170) to move in any of three dimensions. For example, with respect to a Cartesian coordinate system, the actuators may enable the sample stage (170) to move in the x, y, and z directions relative to the objective lens assembly (142), to tilt relative to the objective lens assembly (142), and / or to move relative to the objective lens assembly (142) in other ways. Movement of the sample stage (170) may enable one or more sample positions on the flow cell (110) to be optically aligned and positioned with the objective lens assembly (142). Movement of the sample stage (170) relative to the objective lens assembly (142) may be achieved by moving the sample stage (170) itself, by moving the objective lens assembly (142), by moving other components of the imaging assembly (120), by moving other components of the system (100), or by any combination of the foregoing. For example, in some implementations, the sample stage (170) may be operable in the X and Y directions relative to the objective lens assembly (142), and the focus component (175) or Z stage may move the objective lens assembly (142) along the Z direction relative to the sample stage (170). Further implementations may further include moving the imaging assembly (120) over a stationary flow cell (110). Thus, in some variations, the flow cell (110) may be fixed during imaging, and one or more components of the imaging assembly (120) may be moved to capture images in different regions of the flow cell (110).

[0033] In some implementations, the focus component (175) may be included to control the positioning of the objective lens with respect to the flow cell (110) in a focus direction (e.g., along the z-axis or z-dimension). The focus component (175) includes one or more actuators physically coupled to the objective lens assembly (142), an optical stage, a sample stage (170), or a combination thereof, and may move the flow cell (110) on the sample stage (170) relative to the objective lens assembly (142) to provide appropriate focusing for an imaging operation. In some implementations, the focus component (175) detects displacement of the objective lens assembly (142) relative to a portion of the flow cell (110) and may utilize a focus tracking module (not shown) configured to output data indicative of an in-focus position to the focus component (175) to move the objective lens assembly (142) to position the corresponding portion of the flow cell (110) within the focus of the objective lens assembly (142).

[0034] In some implementations, the actuator for the focus component (175) or the sample stage (170) may be physically coupled to the objective lens assembly (142), an optical stage, a sample stage (170), or a combination thereof, for example, by direct or indirect mechanical, magnetic, fluidic, or other attachment or contact to or with the stage or its components. The actuator of the focus component (175) may be configured to move the objective lens assembly (142) in the z-direction while maintaining the sample stage (170) in the same plane (e.g., while maintaining a level or horizontal orientation perpendicular to the optical axis). In some implementations, the sample stage (170) includes an X-direction actuator and a Y-direction actuator to form an X-Y stage. The sample stage (170) may further be configured to include one or more tilt actuators for tilting the sample stage (170) and / or portions thereof, such as a flow cell chuck. This may be done, for example, so that the flow cell (110) can be dynamically leveled considering any tilt of its surface.

[0035] The camera system (140) may include one or more imaging sensors for monitoring and tracking the imaging (e.g., sequencing) of the flow cell (110). The camera system (140) may be implemented, for example, as a CCD or CMOS imaging sensor camera, although other imaging sensor technologies (e.g., active pixel sensors) may be used. As merely a further example, the camera system (140) may include a dual sensor time delay integration (TDI) camera, a single sensor camera, a camera having one or more two-dimensional imaging sensors, and / or other types of camera technologies. The camera system (140) and associated optical components are shown positioned above the flow cell (110) in FIG. 1, although one or more imaging sensors or other camera components may be incorporated into the system (100) in a number of other ways that will be apparent to those skilled in the art in view of the teachings herein. For example, one or more imaging sensors may be positioned below the flow cell (110), such as within or under the sample stage (170), or may even be incorporated into the flow cell (110).

[0036] II. Examples of Features for Modifying the Illumination Footprint in the Flow Cell As described above, it may be desirable to provide uniformity of spatial irradiance from the excitation light radiated from the imaging assembly (120) towards the flow cell (110), thereby obtaining spatial uniformity of the fluorescence emission signal collected by the camera system (140). Otherwise, non-uniformity of spatial irradiance in the illumination footprint illuminating the reaction sites within the flow cell (110) may ultimately result in spatial signal variations of the fluorescence emission signal from the fluorophores at the reaction sites of the flow cell (110) collected via the camera system (140). The spatial signal variations of the fluorescence emission signal collected via the camera system (140) may in turn cause lower accuracy of the nucleotide sequencing information extracted from the imaging process. For example, the fluorescence emission signal collected via the camera system (140) may generally exhibit a dependence on the position within the imaged field of view. Such a dependence may be due to variations in the excitation irradiance across the field of view. That is, when the illumination footprint in the flow cell (110) has a higher luminance amplitude near the center and a lower luminance amplitude at the edges of the footprint, the response emission from the fluorophores at the edges may be similarly lower in luminance compared to those near the center. Some illumination beam shaping features may tend to impart a periodic modulation on the excitation "line" that is mapped onto the field of view imaged on the sensor of the camera system (140).

[0037] The uniformity of spatial irradiance from the excitation light radiated from the imaging assembly (120) may be achieved by reducing the periodic modulation that might otherwise be seen in the excitation light. In other words, it may be desirable to minimize the amplitude of modulation of the excitation light radiated from the imaging assembly (120) towards the flow cell (110). As will be described in more detail below, providing a rectangular excitation illumination footprint may minimize the amplitude of modulation in the excitation light, thereby maximizing the spatial uniformity in the fluorescence emission signal collected by the camera system (140).

[0038] To reduce the adverse effects from the periodic modulation of the excitation light, to the extent that image processing techniques can be applied to the images captured by the camera system (140) through software (e.g., executed via the controller (195)), instead, it may be desirable to provide a hardware-based solution for preventing or at least reducing the periodic modulation of the excitation light. Examples of hardware-based solutions for reducing or at least preventing the periodic modulation in the excitation light are described in more detail below. After these examples of the hardware-based solutions, examples of software-based solutions are described. Although the examples of the hardware-based solutions and the examples of the software-based solutions are described separately, there may be scenarios where one or more aspects of the hardware solution can be used in combination with one or more aspects of the software solution, and thus, the examples of the hardware-based solutions and the examples of the software-based solutions should not necessarily be considered mutually exclusive.

[0039] A. Examples of Different Optical Fiber Configurations FIG. 2 shows an example of an optical fiber (200) that may be included in the imaging assembly (120) as a deformed form of the above-described optical fiber (152). The optical fiber (200) of this example includes a cladding (coating portion) (202) and a core (204). The depiction of the optical fiber (200) in FIG. 2 is simplified, and it should be understood that the optical fiber (200) may include various other layers and components including, but not limited to, coatings, strengthening mechanisms, jackets, and the like. Any suitable material may be used to form the cladding (202), the core (204), and / or other mechanisms of the optical fiber (200). In some variations, a laser diode (not shown) is optically coupled to the optical fiber (200) such that the core (204) is configured to transmit light generated by the laser diode. In some such variations, the laser diode includes a multimode laser diode and the optical fiber (200) is configured as a multimode fiber. As a mere further example, the multimode laser diode used with the optical fiber (200) may include a laser diode that provides multimode light on one axis and single-mode light on another axis. Alternatively, any other suitable type of light source (e.g., incoherent, etc.) may be optically coupled to the optical fiber (200).

[0040] In this example, the core (204) has a square cross-sectional profile such that the height (H) and width (W) of the core (204) are equal to each other. When the core (204) has a square cross-sectional profile, the light directly radiated from the optical fiber (200) may provide a square illumination footprint that includes a substantially top hat (uniform) irradiance profile.

[0041] FIG. 3 shows a graph (210) having a first plot (212) and a second plot (214). The first plot (212) represents an example of the amplitude of modulation of a first wavelength range of excitation light (e.g., light within the green wavelength range) from the optical fiber (200) as a function of frequency, while the second plot (214) represents an example of the amplitude of modulation of a second wavelength range of excitation light (e.g., light within the blue wavelength range) from the optical fiber (200) as a function of frequency. As can be seen from the figure, the plots (212, 214) include non-negligible spikes in the amplitude of modulation, which may indicate an increase in the lack of uniformity of the spatial irradiance from the excitation light emitted from the optical fiber (200). This, in turn, may indicate that the spatial signal variation of the fluorescence emission signal from the fluorophore at the reaction site of the flow cell (110) irradiated by the excitation light from the optical fiber (200) can increase.

[0042] FIG. 4 shows another example of an optical fiber (220) that may be included in the imaging assembly (120) as a deformed form of the above-described optical fiber (152). The optical fiber (220) of this example includes a cladding (222) and a core (224). The depiction of the optical fiber (200) in FIG. 2 is simplified, and it should be understood that the optical fiber (220) may include various other layers and components, including but not limited to coatings, reinforcement mechanisms, jackets, and the like. Any suitable material may be used to form the cladding (222), the core (224), and / or other mechanisms of the optical fiber (220). In some variations, a laser diode (not shown) is optically coupled to the optical fiber (220) such that the core (224) is configured to transmit light generated by the laser diode. In some such variations, the laser diode includes a multimode laser diode, and the optical fiber (220) is configured as a multimode fiber. As merely a further example, the multimode laser diode used with the optical fiber (220) may include a laser diode that provides multimode light on one axis and single-mode light on another axis. Alternatively, any other suitable type of light source (e.g., incoherent, etc.) may be optically coupled to the optical fiber (220).

[0043] In this example, the core (224) has a circular cross-sectional profile. When the core (224) has a circular cross-sectional profile, the light directly radiated from the optical fiber (220) may provide a circular illumination footprint, which includes a somewhat top-hat (uniform) irradiance profile, although this is not substantial.

[0044] FIG. 5 shows a graph (230) having a first plot (232) and a second plot (234). The first plot (232) represents an example of the amplitude of modulation of a first wavelength range of excitation light (e.g., light within the green wavelength range) from the optical fiber (220) as a function of frequency, while the second plot (234) represents an example of the amplitude of modulation of a second wavelength range of excitation light (e.g., light within the blue wavelength range) from the optical fiber (220) as a function of frequency. As can be seen from the figure, the plots (232, 234) include non-negligible spikes in the amplitude of modulation, which may indicate an increase in the lack of uniformity of the spatial irradiance from the excitation light emitted from the optical fiber (220). This, in turn, may indicate that the spatial signal variation of the fluorescence emission signal from the fluorophore at the reaction site of the flow cell (110) irradiated by the excitation light from the optical fiber (220) may increase. As long as the optical fiber (220) provides a smaller amplitude of modulation than the optical fiber (200), it may be desirable to provide an even further reduced amplitude of modulation.

[0045] FIG. 6 shows another embodiment of an optical fiber (240) that may be included in the imaging assembly (120) as a deformed form of the above-described optical fiber (152). The optical fiber (240) of this embodiment includes a cladding (242) and a core (244). The depiction of the optical fiber (240) in FIG. 2 is simplified, and it should be understood that the optical fiber (240) may include various other layers and components including, but not limited to, coatings, strengthening mechanisms, jackets, and the like. Any suitable material may be used to form the cladding (242), the core (244), and / or other mechanisms of the optical fiber (240). In some variations, a laser diode (not shown) is optically coupled to the optical fiber (240) such that the core (244) is configured to transmit light generated by the laser diode. In some such variations, the laser diode includes a multimode laser diode, and the optical fiber (240) is configured as a multimode fiber. As a mere further example, the multimode laser diode used with the optical fiber (240) may include a laser diode that provides multimode light on one axis and single-mode light on another axis. Alternatively, any other suitable type of light source (e.g., incoherent, etc.) may be optically coupled to the optical fiber (240).

[0046] In this embodiment, the core (244) has a rectangular cross-sectional profile such that the height (H) and width (W) of the core (204) are not equal to each other. In some variations, the height (H) and width (W) of the core (204) are configured such that the core (204) has an aspect ratio of 2:1. In some other variations, the height (H) and width (W) of the core (204) are configured such that the core (204) has an aspect ratio of 3:1. In some other variations, the height (H) and width (W) of the core (204) are configured such that the core (204) has an aspect ratio of 4:1. Alternatively, the height (H) and width (W) of the core (204) may be configured to have some other aspect ratio, including but not limited to an aspect ratio greater than 4:1, based on the rectangular shape of the core (204). As a mere further example, the width (W) of the core (204) may be parallel to the pitch direction of the light-structured optical assembly (156). In variations where a microlens (lenslet) array (380, 390) is used as described below, the width (W) of the core (204) may be parallel (or perpendicular) to the pitch direction of the microlens array (380, 390). The cladding (242) of this embodiment also has a rectangular cross-sectional profile. When the core (244) has a rectangular cross-sectional profile, the light radiated directly from the optical fiber (240) may provide a rectangular illumination footprint that includes a substantially top-hat (uniform) irradiance profile.

[0047] FIG. 7 shows a graph (250) having a first plot (252) and a second plot (254). The first plot (252) represents an example of the amplitude of modulation of a first wavelength range of excitation light (e.g., light within the green wavelength range) from the optical fiber (240) as a function of frequency, while the second plot (254) represents an example of the amplitude of modulation of a second wavelength range of excitation light (e.g., light within the blue wavelength range) from the optical fiber (240) as a function of frequency. As can be seen from the figure, the plots (252, 254) include relatively small spikes in the amplitude of modulation, which may indicate substantial uniformity of the spatial irradiance from the excitation light emitted from the optical fiber (240). This, in turn, may indicate that there may be minimal or otherwise acceptable spatial signal variations in the fluorescence emission signal from the fluorophore at the reaction site of the flow cell (110) illuminated by the excitation light from the optical fiber (240).

[0048] Thus, compared to the optical fibers (200, 220), the optical fiber (240) may ultimately provide better performance in the context of the system (100). This may be due, at least in part, to the rectangular cross-sectional profile of the core (244). In particular, the rectangular cross-sectional profile of the core (244) may provide a rectangular illumination footprint on a far-field surface (e.g., on or at other positions of the flow cell (110)) illuminated by the light from the optical fiber (240).

[0049] B. Examples of beam shaping features for converting the illumination footprint As described above, it may be desirable to provide a rectangular illumination footprint in which the excitation light is radiated from the imaging assembly (120) towards the flow cell (110). Further as described above, the rectangular illumination footprint may be achieved by using an optical fiber (152) having a rectangular core (244) as the optical fiber (240) within the imaging assembly (120). Alternatively, one or more beam shaping features positioned along the optical path between the optical fiber (152) and the flow cell (110) may be used to achieve an illumination footprint in the shape of a rectangle. The following describes several embodiments of illumination assemblies (300, 330, 360, 500) configured and arranged to provide a beam shaping feature that provides a rectangular illumination footprint in the flow cell (110).

[0050] It should be understood that the components of the illumination assemblies (300, 330, 360, 500) described below may be incorporated into the imaging assembly (120) instead of the optical fiber (152), light emitter (150), collimating lens (154), light structuring optical assembly (156), and projection lens (158) shown in FIG. 1. Thus, the light from the illumination assemblies (300, 330, 360, 500) described below may be transmitted directly to the dichroic mirror (160) and reach one or more reaction sites of the flow cell (110) through the objective lens assembly (142). Alternatively, any other suitable optical component(s) (or without additional optical components) may be interposed between the illumination assemblies (300, 330, 360, 500) described below and the flow cell (110).

[0051] 1. Example of an illumination assembly having an anamorphic lens assembly for reforming an illumination footprint FIG. 8 shows an embodiment of an illumination assembly (300) that can be incorporated into an imaging assembly (120). The illumination assembly (300) of this embodiment includes a light source (302), an optical fiber (310), a collimator (312), and a lens assembly (314). In some variations, the light source (302) includes a laser diode. In some such variations, the laser diode includes a multimode laser diode, and the optical fiber (310) is configured as a multimode fiber. As a mere further example, the multimode laser diode of the light source (302) may include a laser diode that provides multimode light on one axis and single mode light on another axis. Alternatively, the light source (302) may be configured to provide incoherent light and / or any other type of light to the optical fiber (310).

[0052] In this embodiment, the optical fiber (310) has a circular core such that the optical fiber (310) is configured like the optical fiber (220). Alternatively, the optical fiber (310) may have a square core like the optical fiber (200), a rectangular core like the optical fiber (240), or any other suitable configuration. The light emitted from the optical fiber (310) reaches the collimator (312) in a first plane (P1). In this embodiment, the illumination footprint (320) in the first plane (P1) has a circular shape as shown in FIG. 9. It should be understood that even in a variant configuration where the optical fiber (310) is configured like the optical fiber (200) or like the optical fiber (240), the illumination footprint (320) may not necessarily be purely circular but may still have some rounded aspect. The collimator (312) collimates the light from the optical fiber (310). As a mere example, the collimator (312) may include a rotationally symmetric lens. Alternatively, the collimator (312) may include a non-rotationally symmetric lens, may include any other suitable mechanism(s), and / or may take any other suitable form.

[0053] After passing through the collimator (312), the light reaches the lens assembly (314). By way of mere example, the lens assembly (314) may include an anamorphic lens assembly. Alternatively, the lens assembly (314) may include a non-rotationally symmetric lens, may include any other suitable mechanism(s), and / or may take any other suitable form. The lens assembly (314) is optically configured to convert the illumination footprint into a rectangular shape. In particular, FIG. 8 shows the illuminated surface (316) in the second plane (P2). The illuminated surface (316) may correspond to the surface of the reaction site within the flow cell (110) (e.g., in an arrangement such as that shown in FIG. 1, the output of the lens assembly (314) is directed through the objective lens assembly (142) and reaches the flow cell (110)). FIG. 10 shows the illumination footprint (322) in a second plane (P2) having a rectangular shape such that the height (H) and width (W) of the illumination footprint (322) are not equal to each other. In some variations, the width (W) of the illumination footprint (322) is substantially greater than the height (H) of the illumination footprint (322). In some such variations, the illumination footprint has an aspect ratio of about 10:1 or greater. Alternatively, the illumination footprint (322) may have any other suitable rectangular aspect ratio. Some variations may provide an illumination footprint (322) having an elliptical shape. As described above, the illumination footprint (322) may provide irradiance to the surface of the reaction site within the flow cell (110) (e.g., when the output of the lens assembly (314) is directed through the objective lens assembly (142) and reaches the flow cell (110) in an arrangement such as that of FIG. 1).

[0054] Accordingly, even if the optical fiber (310) has a core of circular, square, or other shape, the illumination assembly (300) may provide substantially the same illumination output as the output from the optical fiber (240) having a rectangular core (244). In some variations, the optical fiber (310) may have a rectangular core (such as the rectangular core (244) of the optical fiber (240)) such that the lens assembly (314) can further broaden the aspect ratio of the illumination footprint. In other words, the rectangular illumination footprint in the second plane (P2) may have an aspect ratio wider than the aspect ratio of the square or rectangular fiber core (204, 244) due to the anamorphic transformation imposed by the lens assembly (314).

[0055] The aforementioned illumination assembly (300) may be incorporated into the system (100) such that the illumination footprint (322) is configured to correspond to the corresponding imaging sensor size and / or a predetermined dimension size while utilizing an optical fiber (310) that does not need to be specially designed for the illumination footprint (322) to illuminate a portion of the flow cell (110).

[0056] 2. Example of an illumination assembly having an anamorphic collimator for reforming an illumination footprint FIG. 11 shows another example of an illumination assembly (330) that can be incorporated into the imaging assembly (120). The illumination assembly (330) of this example includes a light source (332), an optical fiber (340), and a collimator (342). In some variations, the light source (332) includes a laser diode. In some such variations, the laser diode includes a multimode laser diode, and the optical fiber (340) is configured as a multimode fiber. As a mere further example, the multimode laser diode of the light source (332) may include a laser diode that provides multimode light on one axis and single mode light on another axis. Alternatively, the light source (332) may be configured to provide incoherent light and / or any other type of light to the optical fiber (340).

[0057] In this example, the optical fiber (340) has a circular core such that the optical fiber (340) is configured like the optical fiber (220). Alternatively, the optical fiber (340) may be configured like the optical fiber (200), or may have any other suitable configuration. The light emitted from the optical fiber (340) reaches the collimator (342) in a first plane (P1). In this example, the illumination footprint (not shown) in the first plane (P1) has a circular shape similar to the illumination footprint (320) shown in FIG. 9.

[0058] The collimator (342) collimates the light from the optical fiber (310). By way of mere example, the collimator (342) may include an anamorphic collimator, and the collimator (342) is optically configured to convert an illumination footprint (not shown) into a rectangle or an ellipse on the illuminated surface (344) in the second plane (P2). The illuminated surface (344) may correspond to the surface of the reaction site within the flow cell (110). The illumination footprint on the illuminated surface (344) in the second plane (P2) may be similar to the illumination footprint (322) shown in FIG. 10. Thus, even if the optical fiber (340) has a circular, square, or other shaped core, the illumination assembly (330) may provide substantially the same illumination output as the output from the optical fiber (240) having a rectangular core (244).

[0059] In some variations, the optical fiber (340) may have a rectangular core (such as, for example, the rectangular core (244) of the optical fiber (240)) so that the collimator (342) can further broaden the aspect ratio of the illumination footprint. In other words, the rectangular illumination footprint in the second plane (P2) may have an aspect ratio wider than the aspect ratio of the rectangular core (244) due to the anamorphism imposed by the collimator (342). In some variations, the light from the collimator (342) further passes through an anamorphic lens assembly (such as, for example, the lens assembly (314)), whereby the anamorphic lens assembly further emphasizes the anamorphism imposed by the collimator (342), thereby providing an even wider aspect ratio in the illumination footprint on the illuminated surface (344) in the second plane (P2).

[0060] Although not shown in FIG. 8, the illumination assembly (330) may further include other optical components interposed between the collimator (342) and the illuminated surface (344), which may include, but are not limited to, one or more microlens arrays and / or other optical features. Examples of other arrangements including microlens arrays will be described in more detail below.

[0061] The aforementioned illumination assembly (330) may be incorporated into the system (100) while utilizing an optical fiber (340) that does not need to be specially designed for the illumination footprint (344) on the illuminated surface, so that the illumination footprint (344) on the illuminated surface is configured to correspond to the imaging sensor size and / or a predetermined dimensional size corresponding to the portion of the flow cell (110) to be illuminated.

[0062] 3. Example of an illumination assembly having a diffuser plate and a microlens array for reforming an illumination footprint FIG. 12 shows another example of an illumination assembly (360) that can be incorporated into the imaging assembly (120). The illumination assembly (360) of this example includes a light source (362), an optical fiber (370), a collimator (372), a diffuser plate (374), a first microlens array (380), and a second microlens array (390). In some variations, the light source (362) includes a laser diode. In some such variations, the laser diode includes a multimode laser diode, and the optical fiber (370) is configured as a multimode fiber. As a mere further example, the multimode laser diode of the light source (362) may include a laser diode that provides multimode light on one axis and single-mode light on another axis. Alternatively, the light source (362) may be configured to provide incoherent light and / or any other type of light to the optical fiber (370).

[0063] In this embodiment, like the optical fiber (220), the optical fiber (370) has a circular core. Alternatively, the optical fiber (370) may be configured like the optical fiber (200), or may have any other suitable configuration. The light emitted from the optical fiber (370) reaches the collimator (372) in the first plane (P1). In this embodiment, the illumination footprint (not shown) in the first plane (P1) has a circular shape similar to the illumination footprint (320) shown in FIG. 9. The collimator (372) collimates the light from the optical fiber (370). By way of example only, the collimator (372) may include an anamorphic collimator such as the collimator (342) of the illumination assembly (330). As another example, the collimator (372) may include a rotationally symmetric lens such as the collimator (312) of the illumination assembly (300). In some variations where the collimator (372) includes a rotationally symmetric lens, the light may pass through an anamorphic lens assembly such as the lens assembly (314) of the illumination assembly (300) before reaching the diffuser plate (374), as described below. Alternatively, the collimator (372) may include any other suitable feature(s) and / or may take any other suitable form.

[0064] After passing through the collimator (372), the light reaches the diffuser plate (374). The diffuser plate (374) is configured to increase the viewing angle of the light entering the small lens arrays (380, 390). In this embodiment, the diffuser (374) includes a one-dimensional diffuser, although some other variations may include a two-dimensional diffuser. The light exiting the collimator (372) and reaching the diffuser plate (374) may have a residual divergence related to the size of the core of the optical fiber (370) divided by the focal length of the collimator (372). In variations where the diffuser (374) includes a one-dimensional diffuser, the diffuser (374) increases the residual divergence in one direction. In other words, the diffuser plate (374) increases the viewing angle of the light.

[0065] After passing through the diffuser plate (374), the light reaches the first small lens array (380) and then the second small lens array (390). Each small lens array (380, 390) includes a plurality of respective small lenses (382, 392). In some variations, the small lens arrays (380, 390) are configured substantially identically to each other but face in opposite directions. For example, the small lenses (382) of the first small lens array (380) may be configured and arranged substantially identically to the configuration and arrangement of the small lenses (392) of the second small lens array (390). As a mere further example, the small lens arrays (380, 390) may be separated from each other by a distance approximately equal to the focal length of the small lenses (382, 392). The small lens arrays (380, 390) may represent optical components having an array on two surfaces. Some variations include, but are not limited to, variations where the camera system (140) includes a time delay integration (TDI) imaging component. In some variations, each small lens array (380, 390) includes a cylindrical microlens array surface (CuLA). Such a CuLA can spread a light beam in a fan shape in one direction. In such variations, a one-dimensional variation of the diffuser plate (374) may be arranged and configured to increase the fill of the CuLa small lenses in the pitch direction without increasing the fill of the CuLa small lenses in the non-pitch direction. Some variations of the small lens arrays (380, 390) include a cylindrical microlens array, but some other variations of the small lens arrays (380, 390) may include a two-dimensional small lens array. Alternatively, the small lens arrays (380, 390) may have any other suitable configuration.

[0066] When light passes through the first small lens array (380), each small lens (382) of the first small lens array (380) forms an image of the core of the optical fiber (370) close to the active surface (394) of the second small lens array (394). The size of this image of the optical fiber (370) core may be proportional to the apparent size of the core of the optical fiber (370). Similarly, the size of this image of the optical fiber (370) core close to the active surface (394) of the second small lens array (394) may be proportional to the residual divergence of the light. In some cases, as the apparent size of the core of the optical fiber (370) increases, the fill factor of the second small lens array (390) may increase accordingly. This fill factor may be understood as the ratio of the effective refractive area (i.e., the area of the small lenses (392) of the second small lens array (390) that direct light towards the irradiated surface (376)) to the total continuous area occupied by the small lenses (392) of the second small lens array (390). As the fill factor of the small lenses (392) increases, the modulation depth of the light emitted from the second small lens array (390) may decrease.

[0067] The light emitted from the second small lens array (390) provides a rectangular-shaped illumination footprint (not shown) on the illuminated surface (376) in a second plane (P2) that is remote from the second small lens array (390). The illuminated surface (376) may correspond to the surface of the reaction site within the flow cell (110) (e.g., in an arrangement such as that shown in FIG. 1, oriented such that the output of the illumination assembly (360) reaches the flow cell (110) through the objective lens assembly (142)). The illumination footprint on the illuminated surface (376) in the second plane (P2) may be similar to the illumination footprint (322) shown in FIG. 10. The combination of the collimator (372), the diffuser plate (374), and the small lens arrays (380, 390) may effectively produce an illuminated footprint on the second plane (P2) of the illuminated surface (376) that is substantially larger than the footprint in the first plane (P1). In other words, the collimator (372), the diffuser plate (374), and the small lens arrays (380, 390) may provide an illumination output such as that of an illumination output of a light source that is much larger than the optical fiber (370), which may provide a modulation that is substantially reduced compared to the modulation provided in the light transmitted directly from the optical fiber (370) in the absence of the collimator (372), the diffuser plate (374), and the small lens arrays (380, 390). Further, the illumination assembly (360) may provide an illumination output that is substantially the same as the output from the optical fiber (240) having a rectangular core (244), even when the optical fiber (370) has a circular, square, or other shaped core. Some variations of the illumination assembly (360) may further utilize an optical fiber having a rectangular core, such as the rectangular core (244) of the optical fiber (240).

[0068] The foregoing illumination assembly (360) can be configured such that the illumination footprint at the illuminated surface (376) corresponds to a corresponding imaging sensor size and / or a predetermined dimensional size for illuminating a portion of the flow cell (110), and can be incorporated into the system (100) to utilize an optical fiber (370) that need not be specially designed for the illumination footprint at the illuminated surface (376).

[0069] FIG. 13 shows a plot (402) having an example of the modulation amplitude of the excitation light as a function of frequency, from a variant of the illumination assembly (360) in which the optical fiber (200) is used as the optical fiber (370) such that the illumination footprint at the focal point of the small lens (382) of the first small lens array (380) is square. The plot (402) may be understood to represent the modulation of the illumination footprint in a second plane (P2) associated with the reaction site within the flow cell (110). As can be seen from the figure, the plot (402) has relatively small spikes in the amplitude of the modulation. However, this spike is substantially smaller than the spikes seen in the plots (212, 214) of FIG. 3, which may be understood to correspond to the modulation of the illumination footprint in the first plane (P1) when the optical fiber (200) is used as the optical fiber (370) in the illumination assembly (360).

[0070] Compared to the spikes in plots (212, 214), substantially smaller spikes in the modulation amplitude in plot (402) may indicate how the collimator (372), diffuser plate (374), and small lens arrays (380, 390) together ultimately reduce the modulation in the illumination footprint from the optical fiber (200). Thus, the collimator (372), diffuser plate (374), and small lens arrays (380, 390) together may provide better uniformity of the spatial irradiance of the reaction sites within the flow cell (110), which may provide a reduction in the spatial signal variation of the fluorescence emission signal from the fluorophore at the reaction sites of the flow cell (110) illuminated by the excitation light from the illumination assembly (360). Only one plot (402) is shown in FIG. 13 and represents only one channel of the excitation light. However, substantially similar results can be obtained for the other channels of the excitation light from the optical fiber (370) within the illumination assembly (360).

[0071] FIG. 14 shows a graph (420) having a plot (422) representing an example of the amplitude of the modulation of the excitation light as a function of frequency, from a variant of the illumination assembly (360) in which the optical fiber (220) is used as the optical fiber (370) such that the illumination footprint at the focal point of the small lens (382) of the first small lens array (380) is circular. The plot (422) may be understood to represent the modulation of the illumination footprint in a second plane (P2) associated with the reaction sites within the flow cell (110). As can be seen from the figure, the plot (422) has relatively small spikes in the amplitude of the modulation. However, this spike is substantially smaller than the spikes seen in the plots (232, 234) of FIG. 5, which may be understood to correspond to the modulation of the illumination footprint in the first plane (P1) when the optical fiber (220) is used as the optical fiber (370) in the illumination assembly (360).

[0072] Compared to the spikes in plot (232, 234), the substantially smaller spikes in the modulation amplitude in plot (422) may indicate how the collimator (372), diffuser plate (374), and small lens array (380, 390) together ultimately reduce the modulation in the illumination footprint from the optical fiber (220). Thus, the collimator (372), diffuser plate (374), and small lens array (380, 390) together may provide better uniformity of the spatial irradiance of the reaction site within the flow cell (110), which may provide a reduction in the spatial signal variation of the fluorescence emission signal from the fluorophore at the reaction site of the flow cell (110) illuminated by the excitation light from the illumination assembly (360). Only one plot (422) is shown in FIG. 14 and represents only one channel of the excitation light. However, substantially similar results can be obtained for the other channels of the excitation light from the optical fiber (370) within the illumination assembly (360).

[0073] FIG. 15 shows a graph (440) having a plot (442) representing an example of the amplitude of the modulation of the excitation light as a function of frequency from a variant of the illumination assembly (360) in which the optical fiber (240) is used as the optical fiber (370) such that the illumination footprint at the focus of the small lens (382) of the first small lens array (380) is circular. The plot (442) may be understood to represent the modulation of the illumination footprint in a second plane (P2) associated with the reaction site within the flow cell (110). As can be seen from the figure, the plot (442) has relatively small spikes in the amplitude of the modulation. However, this spike is substantially smaller than the spikes seen in the plots (252, 254) of FIG. 7, which may be understood to correspond to the modulation of the illumination footprint in the first plane (P1) when the optical fiber (240) is used as the optical fiber (370) in the illumination assembly (360).

[0074] Compared to the spikes in plots (252, 254), substantially smaller spikes in the modulation amplitude in plot (442) may indicate how the collimator (372), diffuser plate (374), and small lens arrays (380, 390) together ultimately reduce the modulation in the illumination footprint from the optical fiber (240). Thus, the collimator (372), diffuser plate (374), and small lens arrays (380, 390) together may provide better uniformity of the spatial irradiance of the reaction site within the flow cell (110), which may provide a reduction in the spatial signal variation of the fluorescence emission signal from the fluorophore at the reaction site of the flow cell (110) illuminated by the excitation light from the illumination assembly (360). Only one plot (442) is shown in FIG. 15 and represents only one channel of the excitation light. However, substantially similar results can be obtained for the other channels of the excitation light from the optical fiber (370) within the illumination assembly (360).

[0075] FIG. 16 shows another example of an illumination assembly (500) that can be incorporated into the imaging assembly (120). The illumination assembly (500) of this example is a variant of the illumination assembly (360), and the illumination assembly (500) includes the same components as the illumination assembly (360), but includes them in a different arrangement. In particular, the illumination assembly (500) includes a light source (362), an optical fiber (370), a collimator (372), a diffuser plate (374), a first small lens array (380), and a second small lens array (390). However, unlike the arrangement of the imaging assembly (360) where the diffuser plate (374) is positioned between the collimator (372) and the first small lens array (380), the diffuser plate (374) is positioned between the first small lens array (380) and the second small lens array (390) in the illumination assembly (500). This arrangement of the diffuser plate (374) between the first small lens array (380) and the second small lens array (390) in the illumination assembly (500) may result in substantially the same outcome as the different arrangement in the illumination assembly (360). In other words, the illumination assembly (500) may provide a rectangular-shaped illumination footprint on the illuminated surface (376) in the second plane (P2) with a substantially reduced modulation amplitude.

[0076] FIG. 17 shows another embodiment of an illumination assembly (550) that can be incorporated into the imaging assembly (120). The illumination assembly (550) of this embodiment is a variant of the illumination assembly (500), whereby the illumination assembly (500) includes most of the same components as the illumination assembly (500), except that the illumination assembly (500) includes a refractive substrate (560) instead of the diffuser plate (374). Similar to the illumination assembly (500), the illumination assembly (550) of this embodiment further includes a light source (362), an optical fiber (370), a collimator (372), a first small lens array (380), and a second small lens array (390). The refractive substrate (560) of this embodiment includes a first surface (562) and a second surface (564). The first small lens array (380) is disposed on the first surface (562). The second small lens array (390) is disposed on the first surface (564). Thus, the refractive substrate (560) is directly interposed between the small lens arrays (380, 390) in this embodiment.

[0077] The refractive substrate (560) is configured such that the beam incident on the first small lens array (380) by the refractive substrate (560) is substantially focused onto the second small lens array (390). This focusing mode of the refractive substrate (560) may be partially due to, among other parameters, the thickness of the refractive substrate (i.e., the distance between the surfaces (562, 564)). The arrangement of the refractive substrate (560) between the first small lens array (380) and the second small lens array (390) in the illumination assembly (550) may result in substantially the same result as the different arrangements in the illumination assemblies (360, 500). In other words, the illumination assembly (500) may provide a rectangular-shaped illumination footprint on the illuminated surface (570) in the second plane (P2) with a substantially reduced modulation amplitude.

[0078] C. Examples of Image Processing Techniques In some scenarios, to reduce the adverse effects from the periodic modulation of the excitation light, image processing techniques may be applied to the images captured by the camera system (140) through software (e.g., executed via the controller (195)). For example, the elements of each column of the image captured by the camera system (140) may be summed such that the readings of the pixels in the first row and the first column are added to the readings of the pixels in the second row and the first column. This may be done for all rows for each column. As a mere further example, for an image that is 5120 columns wide and 4096 rows high, the 4096 rows of each column may be summed to produce a one-dimensional array having 5120 elements (i.e., the same number of elements as columns). Since the image may have any other suitable number of columns and rows, these numbers are merely examples.

[0079] The one-dimensional array described above may be referred to as a flat-fielding array. This flat-fielding array may be normalized by dividing each element of the array by the sum of the elements of the array. The flat-fielding array may further be normalized by dividing all elements by the average of the flat-fielding array elements, and thereafter, the average of the flat-fielding elements becomes 1.

[0080] This normalized array may be defined as a flat-fielding profile. Each row of the original image captured by the camera system (140) may be divided by the flat-fielding profile. This process may make the modulation from the original image substantially imperceptible. The modulation may further be removed via frequency domain filtering. For example, such filtering may utilize a high-pass filter or a notch filter that does not pass the spatial frequency of the modulation. In other words, if one or more of the images have a spatial pattern having a specific period, one or more frequency domain filters may be applied to reduce the spatial pattern.

[0081] The above-described image processing technique may be used in a variant of the system (100) that incorporates at least one of the optical fibers (200, 220, 240) within the imaging assembly (120), in a variant of the system (100) that incorporates one of the illumination assemblies (300, 330, 360, 500) into the imaging assembly (120), and / or in other variants of the system (100). In some variants, the above-described image processing technique is used to computationally reduce the periodic modulation in the images captured by the camera system (140) when the reaction site within the flow cell (110) is illuminated with modulated excitation light. Thus, the above-described image processing technique may function as an alternative to hardware-based solutions (e.g., optical fiber (240), illumination assemblies (300, 330, 360, 500), etc.) for dealing with modulation.

[0082] III. Examples of Combinations The following examples relate to various non-exhaustive ways in which the teachings of this specification may be combined or applied. The following examples are not intended to limit the scope of any claims that may be presented at any time in this application or in a later application of this application. No waiver of rights is intended. The following examples are provided for illustrative purposes only. It is contemplated that the various teachings of this specification may be configured and applied in numerous other ways. Furthermore, it is contemplated that some variations may omit certain features specified in the following examples. Accordingly, none of the aspects or features mentioned below should be considered important unless so explicitly indicated by the inventors or by successors in interest to the inventors at a later date. If any claims containing additional features beyond those mentioned below are presented in this application or in a subsequent application related to this application, those additional features should not be assumed to have been added for any reason related to patentability.

[0083] Example 1 An apparatus comprising: a sequencing stage configured to receive a flow cell including a plurality of reaction sites, each reaction site being configured to contain a biological sample; and an illumination assembly configured to project light toward the sequencing stage to thereby illuminate the reaction sites, the illumination assembly including a light source, a first small lens array interposed between the light source and the sequencing stage, a second small lens array interposed between the light source and the sequencing stage, and a diffuser plate interposed between the light source and the sequencing stage.

[0084] Example 2 The apparatus according to Example 1, wherein the diffuser plate includes a one-dimensional diffuser plate.

[0085] Example 3 The apparatus according to any one of Examples 1 to 2, wherein the first small lens array includes a cylindrical microlens array.

[0086] Example 4 The apparatus according to any one of Examples 1 to 3, wherein the diffuser plate is interposed between the light source and the first small lens array.

[0087] Example 5 The apparatus according to any one of Examples 1 to 3, wherein the diffuser plate is interposed between the first small lens array and the second small lens array.

[0088] Example 6 The apparatus according to any one of Examples 1 to 3, wherein the second small lens array is interposed between the first small lens array and the diffuser plate.

[0089] Example 7 The apparatus according to any one of Examples 1 to 6, further comprising a collimator interposed between the light source and one or both of the first small lens array or the diffuser plate.

[0090] Example 8 The collimator is the device described in Example 7, including an anamorphic collimator.

[0091] Example 9 The first small lens array includes a plurality of small lenses, each small lens having a focal length, and the first small lens array and the second small lens array are separated from each other by a distance approximately equal to the focal length, the device described in any one of Examples 1 to 8.

[0092] Example 10 The light source includes an optical fiber, the device described in any one of Examples 1 to 9.

[0093] Example 11 The optical fiber has a core with a square cross-sectional shape, the device described in Example 10.

[0094] Example 12 The optical fiber has a core with a circular cross-sectional shape, the device described in Example 10.

[0095] Example 13 The optical fiber has a core with a rectangular cross-sectional shape defined by a length and a width, and the width is greater than the length, the device described in Example 10.

[0096] Example 14 The length and the width provide an aspect ratio of at least 2:1, the device described in Example 13.

[0097] Example 15 The length and the width provide an aspect ratio of at least 3:1, the device described in Example 13.

[0098] Example 16 The length and the width provide an aspect ratio of at least 4:1, the device described in Example 13.

[0099] Example 17 The apparatus according to any one of Examples 10 to 16, wherein the light source includes a laser diode.

[0100] Example 18 The apparatus according to any one of Examples 10 to 17, wherein the laser diode includes a multimode diode, and the optical fiber is configured to transmit a plurality of optical channels.

[0101] Example 19 The apparatus according to any one of Examples 1 to 18, further comprising a camera system, the camera system being operable to capture an image of the reaction site.

[0102] Example 20 The apparatus according to Example 19, wherein the camera system includes a time delay integration camera.

[0103] Example 21 The apparatus according to any one of Examples 19 to 20, wherein the camera system is operable to capture light emitted by a fluorophore at the reaction site in response to the light projected by the illumination assembly.

[0104] Example 22 A method, the method including transmitting light through an illumination assembly towards a sequencing stage, the illumination assembly including a light source, a first small lens array interposed between the light source and the sequencing stage, a second small lens array interposed between the light source and the sequencing stage, and a diffuser interposed between the light source and the sequencing stage, the transmitted light being further transmitted towards a plurality of reaction sites at the sequencing stage, each reaction site containing a biological sample.

[0105] Example 23 The method according to Example 22, wherein the diffuser includes a one-dimensional diffuser.

[0106] Example 24 The method according to any one of Examples 22 to 23, wherein the first small lens array includes a cylindrical microlens array.

[0107] Example 25 The method according to any one of Examples 22 to 24, wherein the diffusion plate is interposed between the light source and the first small lens array.

[0108] Example 26 The method according to any one of Examples 22 to 24, wherein the diffusion plate is interposed between the first small lens array and the second small lens array.

[0109] Example 27 The method according to any one of Examples 22 to 24, wherein the second small lens array is interposed between the first small lens array and the diffusion plate.

[0110] Example 28 The method according to any one of Examples 22 to 27, wherein the lighting assembly further includes a collimator interposed between the light source and one or both of the first small lens array or the diffusion plate.

[0111] Example 29 The method according to Example 26, wherein the collimator includes an anamorphic collimator.

[0112] Example 30 The method according to any one of Examples 22 to 29, wherein the first small lens array includes a plurality of small lenses, each small lens has a focal length, and the first small lens array and the second small lens array are separated from each other by a distance substantially equal to the focal length.

[0113] Example 31 The method according to any one of Examples 22 to 30, wherein the light source includes an optical fiber.

[0114] Example 32 The method according to Example 31, wherein the optical fiber has a core having a square cross-sectional shape.

[0115] Example 33 The optical fiber has a core with a circular cross-sectional shape, and the method according to Example 31.

[0116] Example 34 The optical fiber has a core with a rectangular cross-sectional shape defined by a length and a width, and the width is greater than the length, and the method according to Example 31.

[0117] Example 35 The length and the width provide an aspect ratio of at least 2:1, and the method according to Example 34.

[0118] Example 36 The length and the width provide an aspect ratio of at least 3:1, and the method according to Example 34.

[0119] Example 37 The length and the width provide an aspect ratio of at least 4:1, and the method according to Example 34.

[0120] Example 38 The light source includes a laser diode, and the method according to any one of Examples 31 to 37.

[0121] Example 39 The laser diode includes a multimode diode, and the optical fiber is configured to transmit a plurality of optical channels, and the method according to any one of Examples 31 to 38.

[0122] Example 40 Further including capturing an image of the reaction site with a camera system, and the method according to any one of Examples 22 to 39.

[0123] Example 41 The camera system includes a time delay integration camera, and the method according to Example 40.

[0124] Example 42 When the captured image includes an original image having rows and columns, the method further includes summing the rows of each column of the original image to form a one-dimensional flat-fielding array having a plurality of elements, and dividing each column of the original image by the flat-fielding array to generate a demodulated image. The method according to any one of Examples 40 to 41.

[0125] Example 43 The reaction site includes a fluorophore, and the fluorophore emits light in response to the transmitted light reaching the reaction site. Capturing an image of the reaction site using the camera system includes capturing the light emitted by the fluorophore at the reaction site. The method according to any one of Examples 40 to 42.

[0126] Example 44 The method according to Example 43 further includes identifying at least one nucleotide at one of the plurality of reaction sites based on the captured light emitted by at least one fluorophore at the reaction site.

[0127] Example 45 The reaction site is located within a flow cell. The method according to any one of Examples 22 to 44.

[0128] Example 46 The method according to Example 45 further includes performing sequencing by synthesis on the flow cell.

[0129] Example 47 An apparatus comprising: a sequencing stage configured to receive a flow cell including a plurality of reaction sites, each reaction site being configured to contain a biological sample; and an illumination assembly configured to project light toward the sequencing stage so as to illuminate the reaction sites, the illumination assembly including a light source, a collimator interposed between the light source and the sequencing stage, and an anamorphic lens assembly interposed between the collimator and the sequencing stage.

[0130] Example 48 The apparatus according to Example 47, wherein the collimator comprises a rotationally symmetric lens.

[0131] Example 49 The apparatus according to any one of Examples 47 to 48, wherein the anamorphic lens assembly is configured to convert an illumination footprint from the light source into a rectangular shape.

[0132] Example 50 The apparatus according to Example 49, wherein the light source includes an optical fiber having a core with a square cross-sectional shape.

[0133] Example 51 The apparatus according to Example 49, wherein the light source includes an optical fiber having a core with a circular cross-sectional shape.

[0134] Example 52 The apparatus according to any one of Examples 49 to 51, wherein the rectangular shape has an aspect ratio of at least 10:1.

[0135] Example 53 The apparatus according to any one of Examples 47 to 48, wherein the anamorphic lens assembly is configured to convert an illumination footprint from the light source into an elliptical shape.

[0136] Example 54 The device according to Example 53, wherein the elliptical shape has an aspect ratio of at least 10:1.

[0137] Example 55 The device according to any one of Examples 49 to 54, wherein the anamorphic lens assembly is configured to provide the illumination footprint at the reaction site.

[0138] Example 56 A device comprising: a sequencing stage configured to receive a flow cell including a plurality of reaction sites, each reaction site being configured to contain a biological sample; and an illumination assembly configured to project light toward the sequencing stage so as to illuminate the reaction sites, the illumination assembly including a light source and an anamorphic collimator interposed between the light source and the sequencing stage.

[0139] Example 57 The device according to Example 56, wherein the anamorphic lens assembly is configured to convert the illumination footprint from the light source into a rectangular shape.

[0140] Example 58 The device according to Example 57, wherein the light source includes an optical fiber having a core with a square cross-sectional shape.

[0141] Example 59 The device according to Example 57, wherein the light source includes an optical fiber having a core with a circular cross-sectional shape.

[0142] Example 60 The device according to any one of Examples 57 to 59, wherein the rectangular shape has an aspect ratio of at least 10:1.

[0143] Example 61 The apparatus according to Example 56, wherein the anamorphic lens assembly is configured to convert the illumination footprint from the light source into an elliptical shape.

[0144] Example 62 The apparatus according to Example 61, wherein the elliptical shape has an aspect ratio of at least 10:1.

[0145] Example 63 The apparatus according to any one of Examples 56 to 62, wherein the anamorphic lens assembly is configured to provide the illumination footprint at the reaction site.

[0146] Example 64 A method comprising: transmitting light through an illumination assembly towards a plurality of reaction sites in a sequencing stage, each reaction site containing a biological sample, and capturing an image of the reaction site using a camera system, the camera system including a time delay integration camera, the captured image including an original image having rows and columns, summing the rows of the original image to form a one-dimensional flat fielding array having a plurality of elements, and dividing each column of the original image by the flat fielding array to generate a demodulated image.

[0147] Example 65 The method according to Example 64, wherein the demodulated image has a spatial pattern having a specific period, and the method further includes applying one or more frequency domain filters to reduce the spatial pattern.

[0148] Example 66 A method, the method comprising: transmitting light through an illumination assembly towards a plurality of reaction sites in a sequencing stage, each reaction site containing a biological sample, capturing an image of the reaction site using a camera system, the camera system including a time delay integration camera, the captured image including an original image having a spatial pattern with a specific period, applying one or more frequency domain filters to reduce the spatial pattern, thereby generating a demodulated image.

[0149] Example 67 An apparatus, the apparatus comprising: a sequencing stage configured to receive a flow cell including a plurality of reaction sites, each reaction site being configured to contain a biological sample; an illumination assembly configured to project light towards the sequencing stage, thereby illuminating the reaction site, the illumination assembly including a light source, a first small lens array interposed between the light source and the sequencing stage, and a second small lens array interposed between the light source and the sequencing stage.

[0150] Example 68 The apparatus according to Example 67, further comprising a diffuser plate interposed between the light source and the sequencing stage.

[0151] Example 69 The apparatus according to Example 68, wherein the diffuser plate is interposed between the light source and the first small lens array.

[0152] Example 70 The apparatus according to Example 68, wherein the diffuser plate is interposed between the first small lens array and the second small lens array.

[0153] Example 71 The apparatus according to Example 68, wherein the second small lens array is interposed between the first small lens array and the diffuser plate.

[0154] Example 72 The device according to any one of Examples 68 to 71, further comprising a collimator interposed between the light source and one or both of the first small lens array and the diffusion plate.

[0155] Example 73 The device according to Example 72, wherein the collimator includes an anamorphic collimator.

[0156] Example 74 The device according to any one of Examples 67 to 73, wherein the first small lens array includes a cylindrical microlens array.

[0157] Example 75 The device according to any one of Examples 67 to 74, wherein the first small lens array includes a plurality of small lenses, each small lens having a focal length, and the first small lens array and the second small lens array are separated from each other by a distance substantially equal to the focal length.

[0158] Example 76 The device according to any one of Examples 1 to 78, wherein the light source includes an optical fiber.

[0159] Example 77 The device according to Example 76, wherein the optical fiber has a core having a square cross-sectional shape.

[0160] Example 78 The device according to Example 76, wherein the optical fiber has a core having a circular cross-sectional shape.

[0161] Example 79 The device according to Example 76, wherein the optical fiber has a core having a rectangular cross-sectional shape defined by a length and a width, and the width is greater than the length.

[0162] Example 80 The device according to Example 79, wherein the length and the width provide an aspect ratio of at least 2:1.

[0163] Example 81 The device according to Example 79, wherein the length and the width provide an aspect ratio of at least 3:1.

[0164] Example 82 The device according to Example 79, wherein the length and the width provide an aspect ratio of at least 4:1.

[0165] Example 83 The device according to any one of Examples 76 to 82, wherein the light source includes a laser diode.

[0166] Example 84 The device according to any one of Examples 76 to 83, wherein the laser diode includes a multimode diode, and the optical fiber is configured to transmit a plurality of optical channels.

[0167] Example 85 The device according to any one of Examples 67 to 84, further comprising a camera system, the camera system being operable to capture an image of the reaction site.

[0168] Example 86 The device according to Example 85, wherein the camera system includes a time delay integration camera.

[0169] Example 87 The device according to any one of Examples 85 to 86, wherein the camera system is operable to capture light emitted by a fluorophore at the reaction site in response to the light projected by the illumination assembly.

[0170] Example 88 When the illumination assembly further includes a refractive substrate having a first surface and a second surface, and the first small lens array is disposed on the first surface of the refractive substrate, and the second small lens array is disposed on the second surface of the refractive substrate, the device according to any one of Examples 67 to 87.

[0171] Example 89 The refractive substrate, wherein the first surface and the second surface of the refractive substrate are configured to be positioned so as to substantially focus a parallel beam incident on the first array onto the second array, the device according to Example 88.

[0172] IV. Others The foregoing teachings may be readily applied in the context of various types of camera mechanisms in a camera system (140). For example, the foregoing teachings may be applied in the context of a dual sensor time delay integration (TDI) camera, a single sensor camera, a camera having one or more two-dimensional imaging sensors, and / or other types of camera functions within a camera system (140).

[0173] The foregoing examples are provided in the context of a system (100) that can be used in a nucleotide sequencing process, but the teachings herein can be further readily applied in other contexts, including systems that perform other processes (i.e., other than nucleotide sequencing procedures). Accordingly, the teachings herein are not necessarily limited to systems used to perform nucleotide sequencing processes.

[0174] It should be understood that the subject matter described in this specification is not limited, in its application, to the details of construction and the arrangement of components set forth in the description of this specification or shown in the drawings of this specification. The subject matter described in this specification can have other implementations and can be practiced or carried out in various ways. Further, it should be understood that the expressions and terms used in this specification are for the purpose of description and should not be regarded as limiting. The use of "including", "comprising", or "having" and their variants in this specification means including the items listed thereafter and their equivalents as well as additional items.

[0175] When used in the claims, the term "set" should be understood as one or more things grouped together. Similarly, when used in the claims, "based on" should be understood as indicating that one thing is determined at least in part by what it is specified to be "based on". If something needs to be determined exclusively by another thing, then that thing is said to be "exclusively based on" what it is determined by.

[0176] Unless otherwise specified or limited, the terms "mounted," "connected," "supported," and "coupled," and variations thereof, are used broadly and encompass both direct and indirect attachment, connection, support, and coupling. Further, "connected" and "coupled" are not limited to physical or mechanical connection or coupling. Additionally, the expressions and terms used herein with respect to the orientation of a device or element (e.g., terms such as "above," "below," "front," "rear," "distal," "proximal," etc.) are used only to simplify the description of one or more examples described herein and are not intended to singly indicate or imply that the device or element being referred to must have a particular orientation. It should be understood that terms such as "outer" and "inner" are used herein for purposes of explanation and are not intended to indicate or imply relative importance or significance.

[0177] It should be understood that the above description is intended to be illustrative and not restrictive. For example, the embodiments (and / or aspects thereof) described above may be used in combination with each other. Many modifications may be made without departing from the scope thereof to adapt the specific situations or materials to the teachings of the subject matter described herein. The dimensions, types of materials, and coatings described herein are intended to define the parameters of the disclosed subject matter, but they are in no way limiting and are instead illustrative. Many further examples will be apparent to those skilled in the art upon consideration of the above description. Accordingly, the scope of the disclosed subject matter should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. In the appended claims, the terms "including" and "in which" are used as the plain English equivalents of the respective terms "comprising" and "wherein". Further, in the following claims, the terms "first", "second", "third", etc. are used merely as labels and are not intended to impose numerical requirements on their objects. Further, the limitations in the following claims are not set forth in means-plus-function format and are not intended to be construed under paragraph (f) of 35 U.S.C. Section 112, unless such claim limitations expressly use the phrase "means for" followed by a further structure for performing a function and do not recite a corresponding structure for performing that function.

[0178] The following claims enumerate specific example aspects of the disclosed subject matter and are considered to be a part of the above disclosure. These aspects may be combined with each other.

[0179] 〔Embodiment〕 (1) An apparatus, wherein the apparatus A sequencing stage configured to receive a flow cell including a plurality of reaction sites, each reaction site being configured to contain a biological sample, and the sequencing stage; An illumination assembly configured to project light toward the sequencing stage, thereby illuminating the reaction sites, the illumination assembly including: A light source; A first small lens array interposed between the light source and the sequencing stage; A second small lens array interposed between the light source and the sequencing stage; A diffuser plate interposed between the light source and the sequencing stage, and the illumination assembly; and an apparatus comprising the illumination assembly. (2) The apparatus according to Embodiment 1, wherein the diffuser plate includes a one-dimensional diffuser plate. (3) The apparatus according to Embodiment 1 or 2, wherein the first small lens array includes a cylindrical microlens array. (4) The apparatus according to any one of Embodiments 1 to 3, wherein the diffuser plate is interposed between the light source and the first small lens array. (5) The apparatus according to any one of Embodiments 1 to 3, wherein the diffuser plate is interposed between the first small lens array and the second small lens array.

[0180] (6) The apparatus according to any one of Embodiments 1 to 3, wherein the second small lens array is interposed between the first small lens array and the diffuser plate. (7) The apparatus according to any one of Embodiments 1 to 6, further comprising a collimator interposed between the light source and one or both of the first small lens array or the diffuser plate. (8) The apparatus according to Embodiment 7, wherein the collimator includes an anamorphic collimator. (9) The apparatus according to any one of Embodiments 1 to 8, wherein the first small lens array includes a plurality of small lenses, each small lens having a focal length, and the first small lens array and the second small lens array are spaced apart from each other by a distance substantially equal to the focal length. (10) The apparatus according to any one of embodiments 1 to 9, wherein the light source includes an optical fiber.

[0181] (11) The apparatus according to embodiment 10, wherein the optical fiber has a core having a square cross-sectional shape. (12) The apparatus according to embodiment 10, wherein the optical fiber has a core having a circular cross-sectional shape. (13) The apparatus according to embodiment 10, wherein the optical fiber has a core having a rectangular cross-sectional shape defined by a length and a width, and the width is greater than the length. (14) The apparatus according to embodiment 13, wherein the length and the width provide an aspect ratio of at least 2:1. (15) The apparatus according to embodiment 13, wherein the length and the width provide an aspect ratio of at least 3:1.

[0182] (16) The apparatus according to embodiment 13, wherein the length and the width provide an aspect ratio of at least 4:1. (17) The apparatus according to any one of embodiments 10 to 16, wherein the light source includes a laser diode. (18) The apparatus according to any one of embodiments 10 to 17, wherein the laser diode includes a multimode diode, and the optical fiber is configured to transmit a plurality of optical channels. (19) The apparatus according to any one of embodiments 1 to 18, further comprising a camera system, the camera system being operable to capture an image of the reaction site. (20) The apparatus according to embodiment 19, wherein the camera system includes a time delay integration camera.

[0183] (21) The apparatus according to embodiment 19 or 20, wherein the camera system is operable to capture light emitted by a fluorophore at the reaction site in response to the light projected by the illumination assembly. (22) A method, the method comprising Transmitting light through an illumination assembly towards a sequencing stage, wherein the illumination assembly comprises: a light source; a first small lens array interposed between the light source and the sequencing stage; a second small lens array interposed between the light source and the sequencing stage; a diffuser plate interposed between the light source and the sequencing stage, and transmitting light through the illumination assembly towards the sequencing stage, and wherein the transmitted light is further transmitted towards a plurality of reaction sites on the sequencing stage, and each reaction site contains a biological sample. (23) The method according to embodiment 22, wherein the diffuser plate comprises a one-dimensional diffuser plate. (24) The method according to embodiment 22 or 23, wherein the first small lens array comprises a cylindrical microlens array. (25) The method according to any one of embodiments 22 to 24, wherein the diffuser plate is interposed between the light source and the first small lens array.

[0184] (26) The method according to any one of embodiments 22 to 24, wherein the diffuser plate is interposed between the first small lens array and the second small lens array. (27) The method according to any one of embodiments 22 to 24, wherein the second small lens array is interposed between the first small lens array and the diffuser plate. (28) The method according to any one of embodiments 22 to 27, wherein the illumination assembly further comprises a collimator interposed between the light source and one or both of the first small lens array or the diffuser plate. (29) The method according to embodiment 26, wherein the collimator comprises an anamorphic collimator. (30) The method according to any one of embodiments 22 to 29, wherein the first small lens array includes a plurality of small lenses, each small lens has a focal length, and the first small lens array and the second small lens array are separated from each other by a distance substantially equal to the focal length.

[0185] (31) The method according to any one of embodiments 22 to 30, wherein the light source includes an optical fiber. (32) The method according to embodiment 31, wherein the optical fiber has a core with a square cross-sectional shape. (33) The method according to embodiment 31, wherein the optical fiber has a core with a circular cross-sectional shape. (34) The method according to embodiment 31, wherein the optical fiber has a core with a rectangular cross-sectional shape defined by a length and a width, and the width is greater than the length. (35) The method according to embodiment 34, wherein the length and the width provide an aspect ratio of at least 2:1.

[0186] (36) The method according to embodiment 34, wherein the length and the width provide an aspect ratio of at least 3:1. (37) The method according to embodiment 34, wherein the length and the width provide an aspect ratio of at least 4:1. (38) The method according to any one of embodiments 31 to 37, wherein the light source includes a laser diode. (39) The method according to any one of embodiments 31 to 38, wherein the laser diode includes a multimode diode, and the optical fiber is configured to transmit a plurality of optical channels. (40) The method according to any one of embodiments 22 to 39, further comprising capturing an image of the reaction site with a camera system.

[0187] (41) The method according to embodiment 40, wherein the camera system includes a time delay integration camera. (42) The captured image includes an original image having rows and columns, and the method Summing up the rows of each column of the original image to form a one-dimensional flat fielding array having a plurality of elements; Dividing each column of the original image by the flat fielding array to generate a demodulated image, the method according to embodiment 40 or 41, further comprising. (43) The reaction site includes a fluorophore, and the fluorophore emits light in response to the transmitted light reaching the reaction site, and capturing an image of the reaction site using the camera system includes capturing the light emitted by the fluorophore at the reaction site, the method according to any one of embodiments 40 to 42. (44) further comprising identifying at least one nucleotide at one reaction site among the plurality of reaction sites based on the captured light emitted by at least one fluorophore at the reaction site, the method according to embodiment 43. (45) The reaction site is located within a flow cell, the method according to any one of embodiments 22 to 44.

[0188] (46) further comprising performing sequencing by synthesis on the flow cell, the method according to embodiment 45. (47) An apparatus, the apparatus comprising: A sequencing stage configured to receive a flow cell including a plurality of reaction sites, each reaction site being configured to contain a biological sample; An illumination assembly configured to project light toward the sequencing stage to thereby illuminate the reaction sites, the illumination assembly including: A light source; A collimator interposed between the light source and the sequencing stage; An anamorphic lens assembly interposed between the collimator and the sequencing stage, the apparatus comprising the illumination assembly. (48) The apparatus according to embodiment 47, wherein the collimator includes a rotationally symmetric lens. (49) The apparatus according to embodiment 47 or 48, wherein the anamorphic lens assembly is configured to convert the illumination footprint from the light source into a rectangular shape. (50) The apparatus according to embodiment 49, wherein the light source includes an optical fiber having a core with a square cross-sectional shape.

[0189] (51) The apparatus according to embodiment 49, wherein the light source includes an optical fiber having a core with a circular cross-sectional shape. (52) The apparatus according to any one of embodiments 49 to 51, wherein the rectangular shape has an aspect ratio of at least 10:1. (53) The apparatus according to embodiment 47 or 48, wherein the anamorphic lens assembly is configured to convert the illumination footprint from the light source into an elliptical shape. (54) The apparatus according to embodiment 53, wherein the elliptical shape has an aspect ratio of at least 10:1. (55) The apparatus according to any one of embodiments 49 to 54, wherein the anamorphic lens assembly is configured to provide the illumination footprint at the reaction site.

[0190] (56) An apparatus, wherein the apparatus a sequencing stage configured to receive a flow cell including a plurality of reaction sites, each reaction site being configured to contain a biological sample, an illumination assembly configured to project light toward the sequencing stage so as to illuminate the reaction sites, the illumination assembly including a light source, an anamorphic collimator interposed between the light source and the sequencing stage, (57) The apparatus according to embodiment 56, wherein the anamorphic lens assembly is configured to convert the illumination footprint from the light source into a rectangular shape. (58) The apparatus according to embodiment 57, wherein the light source includes an optical fiber having a core with a square cross-sectional shape. (59) The apparatus according to embodiment 57, wherein the light source includes an optical fiber having a core with a circular cross-sectional shape. (60) The apparatus according to any one of embodiments 57 to 59, wherein the rectangular shape has an aspect ratio of at least 10:1.

[0191] (61) The apparatus according to embodiment 56, wherein the anamorphic lens assembly is configured to convert the illumination footprint from the light source into an elliptical shape. (62) The apparatus according to embodiment 61, wherein the elliptical shape has an aspect ratio of at least 10:1. (63) The apparatus according to any one of embodiments 56 to 62, wherein the anamorphic lens assembly is configured to provide the illumination footprint at the reaction site. (64) A method, the method comprising: transmitting light through an illumination assembly towards a plurality of reaction sites in a sequencing stage, each reaction site containing a biological sample, capturing an image of the reaction site using a camera system, the camera system including a time delay integration camera, the captured image including an original image having rows and columns, summing the rows of the original image to form a one-dimensional flat fielding array having a plurality of elements, dividing each column of the original image by the flat fielding array to generate a demodulated image. (65) The method according to embodiment 64, wherein the demodulated image has a spatial pattern having a specific period, and the method further includes applying one or more frequency domain filters to reduce the spatial pattern.

[0192] (66) A method, wherein the method comprises: transmitting light through an illumination assembly towards a plurality of reaction sites in a sequencing stage, each reaction site containing a biological sample, capturing an image of the reaction sites using a camera system, the camera system including a time delay integration camera, the captured image including an original image having a spatial pattern with a specific period, applying one or more frequency domain filters to reduce the spatial pattern, thereby generating a demodulated image. (67) An apparatus, wherein the apparatus comprises: a sequencing stage configured to receive a flow cell including a plurality of reaction sites, each reaction site being configured to contain a biological sample, an illumination assembly configured to project light towards the sequencing stage, thereby illuminating the reaction sites, the illumination assembly including: a light source, a first small lens array interposed between the light source and the sequencing stage, a second small lens array interposed between the light source and the sequencing stage. (68) The apparatus according to embodiment 67, further comprising a diffuser plate interposed between the light source and the sequencing stage. (69) The apparatus according to embodiment 68, wherein the diffuser plate is interposed between the light source and the first small lens array. (70) The apparatus according to embodiment 68, wherein the diffuser plate is interposed between the first small lens array and the second small lens array.

[0193] (71) The apparatus according to embodiment 68, wherein the second small lens array is interposed between the first small lens array and the diffuser plate. (72) The apparatus according to any one of embodiments 68 to 71, further comprising a collimator interposed between the light source and one or both of the first small lens array and the diffuser plate. (73) The apparatus according to embodiment 72, wherein the collimator includes an anamorphic collimator. (74) The apparatus according to any one of embodiments 67 to 73, wherein the first small lens array includes a cylindrical microlens array. (75) The apparatus according to any one of embodiments 67 to 74, wherein the first small lens array includes a plurality of small lenses, each small lens having a focal length, and the first small lens array and the second small lens array are separated from each other by a distance substantially equal to the focal length.

[0194] (76) The apparatus according to any one of embodiments 1 to 78, wherein the light source includes an optical fiber. (77) The apparatus according to embodiment 76, wherein the optical fiber has a core having a square cross-sectional shape. (78) The apparatus according to embodiment 76, wherein the optical fiber has a core having a circular cross-sectional shape. (79) The apparatus according to embodiment 76, wherein the optical fiber has a core having a rectangular cross-sectional shape defined by a length and a width, and the width is greater than the length. (80) The apparatus according to embodiment 79, wherein the length and the width provide an aspect ratio of at least 2:1.

[0195] (81) The apparatus according to embodiment 79, wherein the length and the width provide an aspect ratio of at least 3:1. (82) The apparatus according to embodiment 79, wherein the length and the width provide an aspect ratio of at least 4:1. (83) The apparatus according to any one of embodiments 76 to 82, wherein the light source includes a laser diode. (84) The apparatus according to any one of embodiments 76 to 83, wherein the laser diode includes a multimode diode, and the optical fiber is configured to transmit a plurality of optical channels. (85) The apparatus according to any one of embodiments 67 to 84, further comprising a camera system, wherein the camera system is operable to capture an image of the reaction site.

[0196] (86) The apparatus according to embodiment 85, wherein the camera system includes a time delay integration camera. (87) The apparatus according to any one of embodiments 85 to 86, wherein the camera system is operable to capture light emitted by a fluorophore at the reaction site in response to the light projected by the illumination assembly. (88) The apparatus according to any one of embodiments 67 to 87, wherein the illumination assembly further includes a refractive substrate having a first surface and a second surface, the first small lens array being disposed on the first surface of the refractive substrate, and the second small lens array being disposed on the second surface of the refractive substrate. (89) The apparatus according to embodiment 88, wherein the refractive substrate is configured such that the first surface and the second surface of the refractive substrate are positioned to substantially focus a parallel beam incident on the first array onto the second array.

Claims

1. A device, wherein the device is A sequencing stage configured to receive a flow cell containing multiple reaction sites, wherein each reaction site is configured to contain a biological sample; A lighting assembly configured to illuminate the reaction site by projecting light toward the sequencing stage, wherein the lighting assembly is Light source and A first miniature lens array interposed between the light source and the sequencing stage, A second miniature lens array is interposed between the light source and the sequencing stage, An apparatus comprising a lighting assembly, which includes a diffuser plate interposed between the light source and the sequencing stage.

2. The apparatus according to claim 1, wherein the diffusion plate includes a one-dimensional diffusion plate.

3. The apparatus according to claim 1 or 2, wherein the first miniature lens array includes a cylindrical microlens array.

4. The apparatus according to claim 1, wherein the diffuser plate is interposed between the light source and the first miniature lens array.

5. The apparatus according to claim 1, wherein the diffuser plate is interposed between the first miniature lens array and the second miniature lens array.

6. The apparatus according to claim 1, wherein the second miniature lens array is interposed between the first miniature lens array and the diffuser plate.

7. The apparatus according to claim 1, further comprising a collimator interposed between the light source and one or both of the first miniature lens array or the diffuser plate.

8. The apparatus according to claim 7, wherein the collimator includes an anamorphic collimator.

9. The apparatus according to claim 1, wherein the first miniature lens array includes a plurality of miniature lenses, each miniature lens having a focal length, and the first miniature lens array and the second miniature lens array are spaced apart from each other by a distance substantially equal to the focal length.

10. The apparatus according to claim 1, wherein the light source includes an optical fiber.

11. The apparatus according to claim 10, wherein the optical fiber has a core having a square cross-sectional shape.

12. The apparatus according to claim 10, wherein the optical fiber has a core having a circular cross-sectional shape.

13. The apparatus according to claim 10, wherein the optical fiber has a core having a rectangular cross-sectional shape defined by its length and width, and the width is greater than the length.

14. The apparatus according to claim 13, wherein the length and width provide an aspect ratio of at least 2:

1.

15. The apparatus according to claim 10, wherein the light source includes a laser diode.

16. The apparatus according to claim 15, wherein the laser diode includes a multimode diode, and the optical fiber is configured to transmit a plurality of optical channels.

17. The apparatus according to claim 1, further comprising a camera system, wherein the camera system is operable to capture an image of the reaction site.

18. The apparatus according to claim 17, wherein the camera system includes a time-delay integrating camera.

19. A method, wherein the method is Transmitting light to a sequencing stage through a lighting assembly, wherein the lighting assembly is Light source and A first miniature lens array interposed between the light source and the sequencing stage, A second miniature lens array is interposed between the light source and the sequencing stage, This includes transmitting light toward the sequencing stage through a lighting assembly that includes a diffuser plate interposed between the light source and the sequencing stage, A method wherein the transmitted light is further transmitted to a plurality of reaction sites in the sequencing stage, and each reaction site contains a biological sample.

20. A device, wherein the device is A sequencing stage configured to receive a flow cell containing multiple reaction sites, wherein each reaction site is configured to contain a biological sample; A lighting assembly configured to illuminate the reaction site by projecting light toward the sequencing stage, wherein the lighting assembly is Light source and A first miniature lens array interposed between the light source and the sequencing stage, An apparatus comprising a lighting assembly including a second miniature lens array interposed between the light source and the sequencing stage.

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