Apparatus for reducing signal variation in a sequencing system

Abstract

An apparatus includes a sequencing table and an illumination assembly. The sequencing table is configured to receive a flow cell comprising 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 table to illuminate the reaction sites. The lighting assembly includes a light source, a first lenslet array, a second lenslet array, and a diffuser. The first lenslet array is interposed between the light source and the sequencing table. The second lenslet array is also interposed between the light source and the sequencing table. The diffuser is also interposed between the light source and the sequencing table.

Description

[0001] priority

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 357,074, filed on June 30, 2022, entitled “Apparatus for Reduction of Signal Variation in Sequencing System,” the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] The subject matter discussed in this section should not be considered to be prior art simply because it is mentioned in this section. Similarly, problems mentioned in this section or associated with the subject matter provided as background technology should not be considered to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which themselves may also correspond to specific implementations of the technology protected by the claims.

[0004] Aspects of the present disclosure relate generally to biological or chemical analysis, and more particularly to systems and methods for performing biological or chemical analysis using image sensors.

[0005] Various schemes in biological or chemical research involve carrying out a large number of controlled reactions on a local support surface or in a predefined reaction chamber. The specified reaction can then be observed or detected, and subsequent analysis can help identify or reveal the properties of the chemicals involved in the reaction. For example, in some multiple determinations, an unknown analyte with an identifiable marker (e.g., a fluorescent marker) can be exposed to thousands of known probes under controlled conditions. Each known probe can be placed in the corresponding hole of a flow cell channel. Any chemical reaction occurring between the known probe and the unknown analyte in the observation hole can help identify or reveal the characteristics of the analyte. Other examples of such schemes include known DNA sequencing processes, such as sequencing by synthesis (SBS) or cycle array sequencing.

[0006] In some conventional fluorescence detection schemes, an optical system is used to direct excitation light to a fluorescently labeled analyte and also to detect a fluorescent signal that may be emitted from the analyte. Such an optical system may include an arrangement of lenses, filters, and light sources. It may be desirable to provide uniformity in spatial irradiance from the excitation light, thereby obtaining spatial uniformity in the collected fluorescence emission signals. However, there may be challenges in providing such uniformity in spatial irradiance from the excitation light, thereby obtaining spatial uniformity in the collected fluorescence emission signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1Depicted is a schematic diagram of an example of an imaging system that may be implemented in a system for biological or chemical analysis.

[0008] Figure 2 Depicted is a perspective view of an example of an optical fiber having a core with a square cross-sectional profile.

[0009] Figure 3 Depicted as used in two different wavelength ranges Figure 2 Example of a graph of modulation magnitude as a function of frequency obtained for an optical fiber.

[0010] Figure 4 Depicted is a perspective view of an example of an optical fiber having a core with a circular cross-sectional profile.

[0011] Figure 5 Depicted as used in two different wavelength ranges Figure 4 Example of a graph of modulation magnitude as a function of frequency obtained for an optical fiber.

[0012] Figure 6 Depicted is a perspective view of an example of an optical fiber having a core with a rectangular cross-sectional profile.

[0013] Figure 7 Depicted as used in two different wavelength ranges Figure 6 Example of a graph of modulation magnitude as a function of frequency obtained for an optical fiber.

[0014] Figure 8 A schematic diagram depicting an example of a lighting assembly.

[0015] Fig. 9 Depicted Figure 8 A plan view of a lighting footprint of a lighting assembly at a first plane.

[0016] Fig.10 Depicted Figure 8 A plan view of the lighting footprint of the lighting assembly at a second plane.

[0017] Fig.11 A schematic diagram depicting another example of a lighting assembly.

[0018] Fig.12 A schematic diagram depicting another example of a lighting assembly.

[0019] Fig.13 Describes as Fig.12 Used in lighting components Figure 2 Example of a graph of modulation magnitude as a function of frequency obtained for an optical fiber.

[0020] Fig.14 Describes as Fig.12 Used in lighting components Figure 4 Example of a graph of modulation magnitude as a function of frequency obtained for an optical fiber.

[0021] Fig.15 Describes as Fig.12 Used in lighting components Figure 6 Example of a graph of modulation magnitude as a function of frequency obtained for an optical fiber.

[0022] Fig.16 A schematic diagram depicting another example of a lighting assembly.

[0023] Fig.17 A schematic diagram depicting another example of a lighting assembly. DETAILED DESCRIPTION

[0024] I. Overview of Systems for Biological or Chemical Analysis

[0025] This article describes a device, system and method for providing uniformity of spatial irradiance of excitation light emitted from an optical system, thereby obtaining spatial uniformity of fluorescence signals emitted from analytes exposed to the excitation light. The examples described herein can be used in various biological or chemical processes and systems for academic analysis, commercial analysis or other analysis. More specifically, the examples described herein can be used in various processes and systems where it is desired to detect events, attributes, qualities or characteristics indicating a specified reaction.

[0026] Bioassay systems (such as those described herein) can be configured to perform multiple specified reactions that can be detected individually or together. These biosensors and bioassay systems can be configured to perform multiple cycles in which multiple specified reactions occur synchronously. For example, a bioassay system can be used to sequence dense arrays of DNA features through iterative cycles of enzyme manipulation and image acquisition. The cartridges and biosensors used in the bioassay system can include one or more microfluidic channels that deliver reagents or other reaction components to the reaction sites. The reaction sites can be randomly distributed on a substantially planar surface; or they can be patterned across a substantially planar surface in a predetermined manner. In some versions, the reaction sites are located in a reaction chamber that separates the specified reactions therein.

[0027] Regardless of the form the reaction sites take, each of the reaction sites can be imaged to detect light from the reaction sites. In some examples, one or more image sensors can detect light emitted from the reaction sites. Signals indicating photons emitted from the reaction sites and detected by individual image sensors can be referred to as illumination values ​​for those sensors. These illumination values ​​can be combined into images indicating photons detected from the reaction sites. These images can be further analyzed to identify the composition, reaction, conditions, etc. at each reaction site.

[0028] The following detailed description of certain examples will be better understood when read in conjunction with the following drawings. To the extent that the drawings illustrate diagrams of functional blocks of various examples, the functional blocks do not necessarily indicate a division between hardware components. Thus, for example, one or more of the functional blocks (e.g., a processor or memory) may be implemented in a single-chip hardware (e.g., a general-purpose signal processor or random access memory, a hard disk, etc.). Similarly, the programs may be stand-alone programs, may be incorporated into an operating system as subroutines, may be functions in an installed software package, etc. It should be understood that the various examples are not limited to the arrangements and tools shown in the drawings.

[0029] As used herein, an element or step recited in the singular and preceded by the word "a" or "an" should be understood as not excluding a plurality of said elements or steps, unless such exclusion is explicitly stated. Furthermore, reference to "an example" is not intended to be interpreted as excluding the existence of additional examples that also incorporate the recited features. Furthermore, unless explicitly stated to the contrary, an example that "comprises" or "has" one or more elements having a particular property may include additional elements, whether or not they have that property.

[0030] As used herein, a "specified reaction" includes a change in at least one of the chemical, electrical, physical, or optical properties (or mass) of an analyte of interest. In some examples, the specified reaction is a positive binding event (e.g., binding of a fluorescently labeled biomolecule to the analyte of interest). More generally, the specified reaction can be a chemical transformation, a chemical change, or a chemical interaction. In some examples, the specified reaction includes binding a fluorescently labeled molecule to an analyte. The analyte may be an oligonucleotide, and the fluorescently labeled molecule may be a nucleotide. The specified reaction can be detected when excitation light is directed to the oligonucleotide with the labeled nucleotide and the fluorophore emits a detectable fluorescent signal. In alternative examples, the detected fluorescence is the result of chemiluminescence or bioluminescence. The specified reaction can also increase fluorescence (or ) resonance energy transfer (FRET), reducing FRET by separating the donor and acceptor fluorophores, increasing fluorescence by separating the quencher from the fluorophore, or decreasing fluorescence by co-localizing the quencher and fluorophore.

[0031] As used herein, "reaction component" or "reactant" includes any substance that can be used to obtain a specified reaction. For example, reaction components include reagents, enzymes, samples, other biomolecules, and buffers. Reaction components can be delivered to the reaction site in solution and / or fixed at the reaction site. Reaction components can interact directly or indirectly with another substance, such as an analyte of interest.

[0032] As used herein, the term "reaction site" is a local area where a specified reaction may occur. The reaction site may include a support surface of a substrate on which a substance can be fixed. For example, the reaction site may include a substantially planar surface in a channel of a flow cell, on which a nucleic acid population is provided. The nucleic acids in the population may have the same sequence, such as cloned copies of single-stranded or double-stranded templates. However, in some examples, the reaction site may only contain a single nucleic acid molecule, such as a single-stranded or double-stranded form. In addition, a plurality of reaction sites may be randomly distributed along the support surface or arranged in a predetermined manner (e.g., arranged side by side in a matrix, such as in a microarray). The reaction site may also include a reaction chamber, which at least partially defines a spatial region or volume configured to separate a specified reaction. As used herein, the term "reaction chamber" includes a spatial region that is fluidly connected to 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 common wall. As a more specific example, the reaction chamber may include a cavity defined by the inner surface of a hole, and may have an opening or pore so that the cavity may be fluidly connected to the flow channel. The reaction sites do not necessarily need to be provided in a reaction chamber, but may be provided on or in any other suitable kind of structure.

[0033] 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 image detectors (e.g., there are no other image detectors between adjacent image detectors). In some cases, a reaction site may not be adjacent to another reaction site; but may still be in the immediate vicinity of the other reaction site. When a fluorescent emission signal from a first reaction site is detected by an image sensor associated with a second reaction site, the first reaction site may be in the immediate vicinity of a second reaction site. More specifically, when an image sensor associated with the second reaction site detects, for example, crosstalk from the first reaction site, the first reaction site may be in the immediate vicinity of the second reaction site. Adjacent reaction sites may be contiguous, such that they are adjacent to each other; or adjacent sites may be non-contiguous, with a spacing space between them.

[0034] As used herein, "matter" includes articles or solids such as capture beads, as well as biological or chemical substances. As used herein, "biological or chemical substances" include biomolecules, samples of interest, analytes of interest, and other compounds. Biological or chemical substances can be used to detect, identify, or analyze other compounds, or as intermediates for studying or analyzing other compounds. In a specific example, biological or chemical substances include biomolecules. As used herein, "biomolecules" include at least one of biopolymers, nucleosides, 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 compounds such as analogs or mimetics of the foregoing substances.

[0035] Biomolecules, samples, and biological or chemical substances may be naturally occurring or synthetic and may be suspended in a solution or mixture within a spatial region. Biomolecules, samples, and biological or chemical substances may also be bound to a solid phase or gel material. Biomolecules, samples, and biological or chemical substances may also include pharmaceutical compositions. In some cases, the biomolecules, samples, and biological or chemical substances of interest may be referred to as targets, probes, or analytes.

[0036] As used herein, when the term "removably" and "coupled" (or "engaged") are used together to describe the relationship between components, the term is intended to mean that the connection between the components can be easily separated without destroying or damaging the components. Components are easily separable when they can be separated from each other without undue effort or without spending a lot of time to separate the components. For example, the components can be removably coupled or engaged electrically so that the mating contacts of the components are not destroyed or damaged. The components can also be removably coupled or engaged mechanically so that the features that hold the components are not destroyed or damaged. The components can also be removably coupled or engaged fluidically so that the ports of the components are not destroyed or damaged. If, for example, only a simple adjustment (e.g., realignment) or a simple replacement (e.g., replacement of a nozzle) of the component is required, the component is not considered to be destroyed or damaged.

[0037] As used herein, the terms "fluidic communication" and "fluid coupling" refer to two spatial regions connected together so that a liquid or gas can flow between the two spatial regions. For example, a microfluidic channel can be in fluid communication with a reaction chamber so that a fluid can flow freely from the microfluidic channel into the reaction chamber. The terms "fluidic communication" or "fluid coupling" allow two spatial regions to be in fluid communication through one or more valves, restrictors, or other fluid components to control or regulate the flow of fluid through the system.

[0038] As used herein, when used with respect to biomolecules or biological or chemical substances, the term "fixed" includes substantially attaching biomolecules or biological or chemical substances to a surface at a molecular level. For example, adsorption techniques can be used to fix biomolecules or biological or chemical substances to the surface of a substrate material, and these adsorption techniques include non-covalent interactions (e.g., electrostatic forces, van der Waals forces, and dehydration of hydrophobic interfaces) and covalent bonding techniques, wherein functional groups or joints are conducive to attaching biomolecules to a surface. Fixing biomolecules or biological or chemical substances to the surface of a substrate material can be based on the properties of the substrate surface, the liquid medium carrying the biomolecules or biological or chemical substances, and the properties of the biomolecules or biological or chemical substances themselves. In some cases, the substrate surface can be functionalized (e.g., chemically or physically modified) to facilitate fixing biomolecules (or biological or chemical substances) to the substrate surface. The substrate surface can first be modified so that the functional groups are bound to the surface. Then, the functional groups can be bound to biomolecules or biological or chemical substances to be fixed thereon.

[0039] As used herein, the term "magnet" includes permanent magnets and electromagnets.

[0040] In some examples, nucleic acids can be attached to a surface and amplified. Examples of such amplification are described in the following patent application: U.S. Patent No. 7,741,463, entitled "Method ofPreparing Libraries of TemplatePolynucleotides," published on June 22, 2010, the disclosure of which is incorporated herein by reference in its entirety. In some cases, repeated rounds of cycles (e.g., amplification) using fixed primers and primers in solution can provide multiple copies of nucleic acids.

[0041] In a specific example, the assay protocol performed by the systems and methods described herein includes the use of natural nucleotides and enzymes configured to interact with natural nucleotides. Natural nucleotides include, for example, ribonucleotides or deoxyribonucleotides. Natural nucleotides can be in the form of monophosphates, diphosphates, or triphosphates, and can have a base selected from adenine (A), thymine (T), uracil (U), guanine (G), or cytosine (C). However, it should be understood that non-natural nucleotides, modified nucleotides, or analogs of the aforementioned nucleotides can be used.

[0042] Figure 1Examples of components of a system (100) that can be used to provide biological or chemical analysis are depicted. In some examples, the system (100) is a workstation that can be similar to a desktop device. For example, most (or all) of the systems and components for performing a specified reaction can be located in a common housing. In a specific example, the system (100) is a nucleic acid sequencing system (or sequencer) configured for various applications, including but not limited to de novo sequencing, resequencing of whole genomes or target genome regions, and metagenomics. The sequencer can also be used for DNA or RNA analysis. In some versions, the system (100) can also be configured to generate reaction sites in a circulation pool (110). For example, the system (100) can be configured to receive a sample and generate a surface-attached cluster of clonal amplified nucleic acids derived from the sample. In some specific implementations, the cluster can include a specific sample as a distinguishable portion of the cluster, even if the cluster is polyclonal due to the presence of one or more other samples in the cluster. The system (100) is further configured to capture images of reaction sites on the circulation pool (110) using an imaging component (120).

[0043] In a specific example, the system (100) will perform a large number of parallel reactions in a circulation cell (110). The circulation cell (110) includes one or more reaction sites where a specified reaction can occur. The reaction site can be, for example, fixed to a solid surface of the circulation cell (110), or fixed to a bead (or other removable substrate) in a corresponding reaction chamber of the circulation cell (110). The reaction site can include, for example, a cluster of cloned amplified nucleic acids. The circulation cell (110) can include one or more flow channels that receive a solution from the system (100) and direct the solution to the reaction site. Optionally, the circulation cell (110) can be coupled to a thermal element for transferring heat energy into or from the flow channel.

[0044] The system (100) may include various components, assemblies, and systems (or subsystems) that interact with each other to perform a predetermined method or assay protocol for a biological or chemical analysis. For example, the system (100) includes a system controller (195) that can communicate with the various components, assemblies, and subsystems of the system (100). Examples of such components are described in more detail below. The controller (195) may include one or more microprocessors, storage devices, and / or any other suitable electronic components configured to cooperate to execute control algorithms, data processing, etc.

[0045] In this example, the imaging assembly (120) includes a light emitter (150) that emits light that reaches the reaction sites on the circulation pool (110). The light emitter (150) may include an incoherent light emitter (e.g., an emitter that emits a light beam output by one or more excitation diodes) or a coherent light emitter (such as an emitter of light output by one or more lasers or laser diodes). In this example, the light emitter (150) includes an optical fiber (152) for guiding the light beam output via the light emitter. However, other configurations of the light emitter (150) may be used. In some specific implementations, the optical fiber (152) may be optically coupled to a plurality of different light sources (not shown), each light source emitting light of a different wavelength range. Although the system (100) is illustrated as having a single light emitter (150), in some other specific implementations, multiple light emitters (150) may be included.

[0046] In this example, light output from the light emitter (150) is collimated by a collimating lens (154). The collimated light is structured (patterned) by a light structuring optical assembly (156) and reaches a projection lens (158). In some versions, the projection lens (158) includes a lens element that is operable to translate along an axis (i.e., the axis on 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) can be translated along the axis to account for a range of sample thicknesses (e.g., different cover glass thicknesses) of a sample in the flow cell (110). In other embodiments, the projection lens (156) may be fixed and / or omitted, and a movable lens element may be positioned within a tubular lens assembly in the emission optical path to account for focusing on the upper or lower inner surface of the circulation pool (110) and / or spherical aberration introduced by movement of the objective lens assembly (142). The aforementioned illumination components (150, 152, 154, 156, 158) are merely examples. In addition to or in place of any of the aforementioned illumination components (150, 152, 154, 156, 158), the system (100) may alternatively include any other suitable component to provide illumination. For example, some other variations may omit the structured optical assembly (156) to use unstructured illumination and / or any other type of illumination and / or optical device (e.g., a fluorescence microscope, etc.).

[0047] In this example, light directed by the dichroic mirror (160) passes through the objective lens assembly (142) to a sample in a flow cell (110) positioned on a motion stage (170). In the case of fluorescence microscopy of a sample, a fluorescent element associated with the sample of interest fluoresces in response to the excitation light, and the resulting light is collected by the objective lens assembly (142) and directed to an image sensor of a camera system (140) to detect the emitted fluorescence. As mentioned above, in some embodiments, a tubular lens assembly may be positioned between the objective lens assembly (142) and the dichroic mirror (160) or between the dichroic mirror (160) and the image sensor of the camera system (140). The movable lens element may be capable of translating along the longitudinal axis of the tubular lens assembly to account for focusing on the upper or lower inner surface of the flow cell (110) and / or spherical aberration introduced by movement of the objective lens assembly (142). In some embodiments, a filter switching assembly (162) may be included that includes one or more emission filters that can be used to pass specific emission wavelength ranges and block (or reflect) other emission wavelength ranges. For example, one or more emission filters can be used to direct emission light of different wavelength ranges to different image sensors of a camera system (140) of an imaging assembly (120). For example, the emission filter can be implemented as a dichroic mirror that directs emission light of different wavelengths from a flow cell (110) to different image sensors of a camera system (140). In some variations, the projection lens (158) is interposed between the filter switching assembly (162) and the camera system (140), rather than as Figure 1 Positioned as shown. In some versions, filter switching assembly (162) may be omitted.

[0048] In an example of the system (100), a fluid delivery module or device (190) can direct a flow of reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and through) a circulation cell (110) and a waste valve (180). The circulation cell (110) can include one or more substrates on which a sample is provided. For example, in the case of a system for analyzing a large number of different nucleic acid sequences, the circulation cell (110) can include one or more substrates on which nucleic acids to be sequenced are bound, attached, or associated. The substrate can include 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 is within a channel or includes a channel formed at multiple locations in a matrix or array formed in a substrate or other region across the circulation cell (110). The system (100) can also include a temperature station actuator (130) and a heater / cooler (132) that can optionally adjust the temperature of the fluid conditions within the circulation cell (110). In some implementations, the heater / cooler (132) can be secured to and / or integrated into the sample stage (170) on which the flow cell (110) is placed.

[0049] In a specific implementation, the circulation pool (110) can be implemented as a patterned circulation pool, which includes a transparent cover, a substrate, and is configured to contain a liquid therebetween, and the biological sample can be located at the inner surface of the transparent cover and / or the inner surface of the substrate. The circulation pool may include a large number (e.g., thousands, millions, or billions) of holes (also referred to as nanopores) or regions designed to define an array (e.g., a hexagonal array, a rectangular array, etc.) on the substrate. Such holes can define a reaction chamber that provides a reaction site as described above. Each region can form a cluster (e.g., a monoclonal cluster, a substantially monoclonal cluster, or a polyclonal cluster) or more than one cluster) of a biological sample, such as DNA, RNA, or another genomic material that can be sequenced, for example, using sequencing by synthesis. A substantially monoclonal cluster can be a cluster in which a particular sample forms a distinguishable portion of the cluster, even if the cluster itself is polyclonal due to the presence of one or more other samples in the cluster. The flow cell can be further divided into a plurality of spaced-apart lanes (eg, eight lanes), each lane comprising clusters in a hexagonal array or a linear array of clusters.

[0050] The circulation cell (110) may be mounted on a sample stage (170) to provide movement and alignment of the circulation cell (110) relative to the objective lens assembly (142). The sample stage (170) may have one or more actuators to allow the sample stage (170) to move in any of three dimensions. For example, according to a Cartesian coordinate system, an actuator may be provided to allow the sample stage (170) to move in x, y, and z directions relative to the objective lens assembly (142), tilt relative to the objective lens assembly (142), and / or otherwise move relative to the objective lens assembly (142). Movement of the sample stage (170) may allow one or more sample locations on the circulation cell (110) to be positioned to be optically aligned with the objective lens assembly (142). Movement of the sample stage (170) relative to the objective lens assembly (142) can be accomplished by moving the sample stage (170) itself, by moving the objective lens assembly (142), by moving some other component of the imaging assembly (120), by moving some other component of the system (100), or any combination of the foregoing. For example, in some implementations, the sample stage (170) can be actuated in the X-direction and the Y-direction relative to the objective lens assembly (142), while the focusing component (175) or the Z-stage can move the objective lens assembly (142) in the Z-direction relative to the sample stage (170). Additional implementations can also include moving the imaging assembly (120) over a fixed flow cell (110). Thus, in some versions, the flow cell (110) can be fixed during imaging, while one or more components of the imaging assembly (120) are moved to capture images at different areas of the flow cell (110).

[0051] In some implementations, a focusing component (175) may be included to control the positioning of the objective lens relative to the flow cell (110) in a focusing direction (e.g., along the z-axis or z-dimension). The focusing component (175) may include one or more actuators that are physically coupled to the objective lens assembly (142), the optical bench, the sample stage (170), or a combination thereof to move the flow cell (110) on the sample stage (170) relative to the objective lens assembly (142) to provide appropriate focus for the imaging operation. In some implementations, the focusing component (175) may utilize a focus tracking module (not shown) that is configured to detect displacement of the objective lens assembly (142) relative to a portion of the flow cell (110) and output data indicating a focus position to the focusing component (175) to cause the objective lens assembly (142) to move to position a corresponding portion of the flow cell (110) in the focus of the objective lens assembly (142).

[0052] In some embodiments, the focusing component (175) or an actuator for the sample stage (170) may be physically coupled to the objective lens assembly (142), the optical table, the sample stage (170), or a combination thereof, such as by mechanical, magnetic, fluidic, or other direct or indirect attachment or contact to the table or a component thereof. The actuator of the focusing 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., maintaining a horizontal or horizontal attitude perpendicular to the optical axis). In some embodiments, the sample stage (170) includes an X-direction actuator and a Y-direction actuator to form an XY stage. The sample stage (170) may also be configured to include one or more flip or tilt actuators to flip or tilt the sample stage (170) and / or a portion thereof (such as a circulation cell chuck). For example, this can be done so that the circulation cell (110) can be dynamically leveled to account for any slope on its surface.

[0053] The camera system (140) may include one or more image sensors to monitor and track imaging (e.g., sequencing) of the circulation pool (110). The camera system (140) may be implemented as, for example, a CCD or CMOS image sensor camera, although other image sensor technologies (e.g., active pixel sensors) may be used. By way of further example only, the camera system (140) may include a dual sensor time delay integration (TDI) camera, a single sensor camera, a camera with one or more two-dimensional image sensors, and / or other types of camera technology. Although the camera system (140) and associated optical components are generally Figure 1 100, but one or more image sensors or other camera components may be incorporated into system (100) in a variety of other ways, as will be apparent to those skilled in the art in view of the teachings herein. For example, one or more image sensors may be positioned below flow cell (110), such as within or below sample stage (170); or may even be integrated into flow cell (110).

[0054] II. Examples of Features Used to Modify Illumination Footprint at the Flow Cell

[0055] As mentioned above, it may be desirable to provide uniformity in the spatial irradiance of the excitation light emitted from the imaging assembly (120) toward the circulation cell (110) so as to obtain spatial uniformity in the fluorescence emission signal collected by the camera system (140). Otherwise, spatial irradiance non-uniformity in the illumination footprint of the reaction sites in the illumination circulation cell (110) may ultimately result in spatial signal variations in the fluorescence emission signals of the fluorophores at the reaction sites of the circulation cell (110) (as collected via the camera system (140)). Spatial signal variations in the fluorescence emission signals collected via the camera system (140) may in turn result in lower accuracy of the nucleotide sequencing information extracted from the imaging process. For example, the fluorescence emission signals collected via the camera system (140) may typically exhibit a dependence on the position within the imaging field of view. Such dependence may be due to variations in the excitation irradiance above the field of view. That is, if the illumination footprint at the flow cell (110) has higher intensity magnitudes near the center and lower intensity magnitudes at the edges of the footprint, then the response emissions from the fluorophores at the edges may similarly have lower intensities compared to those near the center. Some illumination beam shaping features may tend to impose a periodic modulation on the excitation "lines" that are mapped to the field of view imaged onto the sensor of the camera system (140).

[0056] Uniformity of spatial irradiance from the excitation light emitted from the imaging assembly (120) can be achieved by reducing periodic modulation that may otherwise be found in the excitation light. In other words, it may be desirable to minimize the amount of modulation in the excitation light emitted from the imaging assembly (120) toward the flow cell (110). As will be described in more detail below, providing a rectangular excitation illumination footprint can minimize the amount of modulation in the excitation light, thereby maximizing spatial uniformity in the fluorescence emission signal collected by the camera system (140).

[0057] To the extent that image processing techniques may be applied to images captured by the camera system (140) via software (e.g., executed via a controller (195)) to reduce adverse effects from periodic modulation in the excitation light, it may be desirable to instead provide a hardware-based solution to prevent or at least reduce periodic modulation in the excitation light. Examples of hardware-based solutions for reducing or at least preventing periodic modulation in the excitation light are described in more detail below. Following those examples of hardware-based solutions, examples of software-based solutions will be described. Although examples of hardware-based solutions and examples of software-based solutions are described separately, there may be scenarios in which one or more aspects of the hardware solution may be used in combination with one or more aspects of the software solution, such that examples of hardware-based solutions and examples of software-based solutions should not be viewed as necessarily mutually exclusive.

[0058] A. Examples of different fiber configurations

[0059] Figure 2 An example of an optical fiber (200) that may be included in an imaging assembly (120) is shown as a variation of the optical fiber (152) described above. The optical fiber (200) of this example includes a cladding (202) and a core (204). It should be understood that Figure 2 The depiction of the optical fiber (200) in is simplified, such that the optical fiber (200) may include various other layers and components, including, but not limited to, coatings, strengthening features, jackets, etc. Any suitable material may be used to form the cladding (202), the core (204), and / or other features of the optical fiber (200). In some versions, 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 versions, the laser diode includes a multimode laser diode; and the optical fiber (200) is configured as a multimode optical fiber. By way of further example only, a multimode laser diode used with the optical fiber (200) may include a laser diode that provides multimode light in one axis; and single-mode light in another axis. Alternatively, any other suitable type of light source (e.g., incoherent, etc.) may be optically coupled to the optical fiber (200).

[0060] 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. Where the core (204) has a square cross-sectional profile, light emitted directly from the optical fiber (200) can provide a square illumination footprint that includes a substantially top-hat (uniform) irradiance profile.

[0061] Figure 3 A graph (210) having a first curve (212) and a second curve (214) is shown. The first curve (212) represents an example of modulation magnitude values ​​for a first wavelength range of excitation light (e.g., light in a green wavelength range) from an optical fiber (200) as a function of frequency; while the second curve (214) represents an example of modulation magnitude values ​​for a second wavelength range of excitation light (e.g., light in a blue wavelength range) from an optical fiber (200) as a function of frequency. As can be seen, the curves (212, 214) include a non-negligible spike in modulation magnitude values, which may indicate a lack of increase in uniformity in spatial irradiance from the excitation light emitted from the optical fiber (200). This may in turn indicate that there may be an increased spatial signal variation in the fluorescence emission signal from the fluorophores at the reaction sites of the flow cell (110) illuminated by the excitation light from the optical fiber (200).

[0062] Figure 4Another example of an optical fiber (220) that may be included in the imaging assembly (120) is shown as a variation of the optical fiber (152) described above. The optical fiber (220) of this example includes a cladding layer (222) and a core (224). It should be understood that Figure 2 The depiction of the optical fiber (200) in is simplified, such that the optical fiber (220) may include various other layers and components, including, but not limited to, coatings, strengthening features, jackets, etc. Any suitable material may be used to form the cladding (222), the core (224), and / or other features of the optical fiber (220). In some versions, 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 versions, the laser diode includes a multimode laser diode; and the optical fiber (220) is configured as a multimode optical fiber. By way of further example only, a multimode laser diode used with the optical fiber (220) may include a laser diode that provides multimode light in one axis; and single-mode light in another axis. Alternatively, any other suitable type of light source (e.g., incoherent, etc.) may be optically coupled to the optical fiber (220).

[0063] In this example, the core (224) has a circular cross-sectional profile. Where the core (224) has a circular cross-sectional profile, light emitted directly from the optical fiber (220) can provide a circular illumination footprint that includes some, but not substantially, a top-hat (uniform) irradiance profile.

[0064] Figure 5 A graph (230) having a first curve (232) and a second curve (234) is shown. The first curve (232) represents an example of modulation magnitude values ​​for a first wavelength range of excitation light (e.g., light in a green wavelength range) from an optical fiber (220) as a function of frequency; while the second curve (234) represents an example of modulation magnitude values ​​for a second wavelength range of excitation light (e.g., light in a blue wavelength range) from an optical fiber (220) as a function of frequency. As can be seen, the curves (232, 234) include a non-negligible spike in modulation magnitude values, which may indicate a lack of increase in uniformity in spatial irradiance from the excitation light emitted from the optical fiber (220). This may in turn indicate that there may be an increased spatial signal variation in the fluorescence emission signal of the fluorophores at the reaction sites of the flow cell (110) illuminated by the excitation light from the optical fiber (220). To the extent that the optical fiber (220) provides less modulation magnitude values ​​than the optical fiber (200), it may be desirable to provide a further reduced modulation magnitude value.

[0065] Figure 6Another example of an optical fiber (240) that may be included in the imaging assembly (120) is shown as a variation of the optical fiber (152) described above. The optical fiber (240) of this example includes a cladding (242) and a core (244). It should be understood that Figure 2 The depiction of the optical fiber (240) in is simplified, such that the optical fiber (240) may include various other layers and components, including, but not limited to, coatings, strengthening features, jackets, etc. Any suitable material may be used to form the cladding (242), the core (244), and / or other features of the optical fiber (240). In some versions, 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 versions, the laser diode includes a multimode laser diode; and the optical fiber (240) is configured as a multimode optical fiber. By way of further example only, a multimode laser diode used with the optical fiber (240) may include a laser diode that provides multimode light in one axis; and single-mode light in another axis. Alternatively, any other suitable type of light source (e.g., incoherent, etc.) may be optically coupled to the optical fiber (240).

[0066] In the present example, 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 versions, 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 versions, 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 versions, 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 such that the core (204) has some other aspect ratio based on the rectangular shape of the core (204), including but not limited to an aspect ratio greater than 4:1. By way of further example only, the width (W) of the core (204) may be parallel to the pitch direction of the light structure optical assembly (156). In a version where a lenslet array (380, 390) is used as described below, the width (W) of the core (204) may be parallel to (or perpendicular to) the pitch direction of the lenslet array (380, 390). The cladding (242) of this example also has a rectangular cross-sectional profile. Where the core (244) has a rectangular cross-sectional profile, light emitted directly from the optical fiber (240) may provide a rectangular illumination footprint that includes a substantially top-hat (uniform) irradiance profile.

[0067] Figure 7A graph (250) having a first curve (252) and a second curve (254) is shown. The first curve (252) represents an example of modulation magnitude values ​​for a first wavelength range of excitation light (e.g., light in a green wavelength range) from an optical fiber (240) as a function of frequency; while the second curve (254) represents an example of modulation magnitude values ​​for a second wavelength range of excitation light (e.g., light in a blue wavelength range) from an optical fiber (240) as a function of frequency. As can be seen, the curves (252, 254) include relatively small spikes in modulation magnitude values, which may indicate a substantial uniformity in spatial irradiance from the excitation light emitted from the optical fiber (240). This may in turn indicate that there may be minimal or otherwise acceptable spatial signal variation in the fluorescence emission signal from the fluorophores at the reaction sites of the flow cell (110) illuminated by the excitation light from the optical fiber (240).

[0068] Thus, the optical fiber (240) can ultimately provide better performance in the context of the system (100) compared to the optical fibers (200, 220). This can be at least partially attributed to the rectangular cross-sectional profile of the core (244). Specifically, the rectangular cross-sectional profile of the core (244) can provide a rectangular illumination footprint in a far-field plane illuminated with light from the optical fiber (240) (e.g., on the flow cell (110) or elsewhere).

[0069] B. Examples of beam shaping features used to transform the illumination footprint

[0070] As described above, it may be desirable to provide a rectangular illumination footprint using excitation light emitted from the imaging assembly (120) toward the circulation cell (110). Also as described above, the rectangular illumination footprint can be achieved by using an optical fiber (240) having a rectangular core (244) as the optical fiber (152) in the imaging assembly (120). Alternatively, the rectangular illumination footprint can be achieved using one or more beam shaping features positioned along the optical path between the optical fiber (152) and the circulation cell (110). Several examples of illumination assemblies (300, 330, 360, 500) are described below, which provide beam shaping features configured and arranged to provide a rectangular illumination footprint at the circulation cell (110).

[0071] 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) to replace Figure 1The optical fiber (152), light emitter (150), collimating lens (154), light structure optical assembly (156) and projection lens (158) are shown. Thus, light from the illumination assembly (300, 330, 360, 500) described below can be directly transmitted to the dichroic mirror (160) and pass through the objective lens assembly (142) to reach one or more reaction sites of the flow cell (110). Alternatively, any other suitable optical component (or no additional optical component) can be inserted between the illumination assembly (300, 330, 360, 500) described below and the flow cell (110).

[0072] 1. Example of an illumination assembly having a deformable lens assembly for reshaping the illumination footprint

[0073] Figure 8 An example of an illumination assembly (300) that may be incorporated into an imaging assembly (120) is shown. The illumination assembly (300) of this example includes a light source (302), an optical fiber (310), a collimator (312), and a lens assembly (314). In some versions, the light source (302) includes a laser diode. In some such versions, the laser diode includes a multimode laser diode; and the optical fiber (310) is configured as a multimode optical fiber. By way of further example only, 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 in another axis. Alternatively, the light source (302) may be configured to provide incoherent light and / or any other kind of light to the optical fiber (310).

[0074] In this example, the optical fiber (310) has a circular core, so that the optical fiber (310) is configured similar to the optical fiber (220). Alternatively, the optical fiber (310) may have a square core similar to the optical fiber (200), a rectangular core similar to the optical fiber (240), or any other suitable configuration. Light emitted from the optical fiber (310) arrives at the collimator (312) at the first plane (P1). In this example, the illumination footprint (320) at the first plane (P1) has a circular shape, such as Fig. 9 As shown. It should be understood that even where the optical fiber (310) is configured similar to the optical fiber (200) or similar to the variation of the optical fiber (240), the illumination footprint (320) may still have some circular aspects, but not necessarily a pure circle. The collimator (312) collimates the light from the optical fiber (310). By way of example only, 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 features, and / or may take any other suitable form.

[0075] After passing through the collimator (312), the light reaches the lens assembly (314). By way of example only, 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 features, and / or may take any other suitable form. The lens assembly (314) is optically configured to transform the illumination footprint into a rectangular shape. Specifically, Figure 8 The illumination surface (316) is shown at a second plane (P2). The illumination surface (316) may correspond to the surface of a reaction site in the flow cell (110) (e.g., where the output of the lens assembly (314) is directed through the objective lens assembly (142) to resemble Figure 1 The arrangement shown reaches the flow cell (110)). Fig.10 An illumination footprint (322) at a second plane (P2) is shown having a rectangular shape such that a height (H) and a width (W) of the illumination footprint (322) are not equal to one another. In some versions, the width (W) of the illumination footprint (322) is substantially greater than the height (H) of the illumination footprint (322). In some such versions, the illumination footprint has an aspect ratio greater than or equal to about 10:1. 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 indicated above, the illumination footprint (322) may provide irradiance at the surface of a reaction site in the circulation pool (110) (e.g., where the output of the lens assembly (314) is directed through the objective lens assembly (142) to resemble Figure 1 The arrangement shown reaches the flow cell (110)).

[0076] The illumination assembly (300) can thus produce an illumination output that is effectively the same as the output from an optical fiber (240) having a rectangular core (310), even though the optical fiber (244) has a round, square, or other shaped core. In some variations, the optical fiber (310) can have a rectangular core (e.g., similar to the rectangular core (244) of the optical fiber (240)) such that the lens assembly (314) can further widen the aspect ratio of the illumination footprint. In other words, due to the deformation applied by the lens assembly (314), the rectangular illumination footprint at the second plane (P2) can have an aspect ratio that is wider than the aspect ratio of a square or rectangular optical fiber core (204, 244).

[0077] The aforementioned illumination assembly (300) can be incorporated into the system (100) such that the illumination footprint (322) can be configured to correspond to a corresponding image sensor size and / or a predetermined size for illuminating a portion of the flow pool (110) while utilizing an optical fiber (310) that does not need to be specifically designed for the illumination footprint (322).

[0078] 2. Example of an illumination assembly with a deformable collimator for reshaping the illumination footprint

[0079] Fig.11 Another example of an illumination assembly (330) that may be incorporated into an imaging assembly (120) is shown. The illumination assembly (330) of this example includes a light source (332), an optical fiber (340), and a collimator (342). In some versions, the light source (332) includes a laser diode. In some such versions, the laser diode includes a multimode laser diode; and the optical fiber (340) is configured as a multimode optical fiber. By way of further example only, 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 in another axis. Alternatively, the light source (332) may be configured to provide incoherent light and / or any other kind of light to the optical fiber (340).

[0080] In this example, optical fiber (340) has a circular core, such that optical fiber (340) is configured similar to optical fiber (220). Alternatively, optical fiber (340) may be configured similar to optical fiber (200) or have any other suitable configuration. Light emitted from optical fiber (340) arrives at collimator (342) at a first plane (P1). In this example, the illumination footprint (not shown) at the first plane (P1) has a circular shape, similar to Fig. 9 The lighting footprint (320) is shown.

[0081] The collimator (342) collimates the light from the optical fiber (310). By way of example only, the collimator (342) may include a deformable collimator such that the collimator (342) is optically configured to transform an illumination footprint (not shown) at the second plane (P2) into a rectangular or elliptical shape on the illumination surface (344). The illumination surface (344) may correspond to the surface of the reaction sites in the flow cell (110). The illumination footprint on the illumination surface (344) at the second plane (P2) may be similar to Fig.10 Illumination footprint (322) is shown. Illumination assembly (330) can thus produce an illumination output that is effectively the same as the output from an optical fiber (240) having a rectangular core (340), even if the optical fiber (244) has a round, square, or other shaped core.

[0082] In some variations, the optical fiber (340) may have a rectangular core (e.g., similar to the rectangular core (244) of the optical fiber (240)), such that the collimator (342) may further widen the aspect ratio of the illumination footprint. In other words, due to the deformation imposed by the collimator (342), the rectangular illumination footprint at the second plane (P2) may have an aspect ratio that is wider than the aspect ratio of the rectangular core (244). In some variations, light from the collimator (342) also passes through an anamorphic lens assembly (e.g., similar to the lens assembly (314)), such that the anamorphic lens assembly further emphasizes the deformation imposed by the collimator (342), thereby providing a further wider aspect ratio in the illumination footprint on the illumination surface (344) at the second plane (P2).

[0083] Although in Figure 8 Although not shown, the illumination assembly (330) may also include other optical components interposed between the collimator (342) and the illumination surface (344), including but not limited to one or more microlens arrays and / or other optical features. Examples of other arrangements including microlens arrays are described in more detail below.

[0084] The aforementioned illumination assembly (330) can be incorporated into the system (100) such that the illumination footprint at the illumination surface (344) can be configured to correspond to a corresponding image sensor size and / or a predetermined size for illuminating a portion of the flow cell (110), while utilizing an optical fiber (340) that does not need to be specifically designed for the illumination footprint at the illumination surface (344).

[0085] 3. Example of a lighting assembly with a diffuser and a microlens array for reshaping the lighting footprint

[0086] Fig.12 Another example of an illumination assembly (360) that may be incorporated into an imaging assembly (120) is shown. The illumination assembly (360) of this example includes a light source (362), an optical fiber (370), a collimator (372), a diffuser (374), a first lenslet array (380), and a second lenslet array (390). In some versions, the light source (362) includes a laser diode. In some such versions, the laser diode includes a multimode laser diode; and the optical fiber (370) is configured as a multimode optical fiber. By way of further example only, 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 in another axis. Alternatively, the light source (362) may be configured to provide incoherent light and / or any other kind of light to the optical fiber (370).

[0087] In this example, optical fiber (370) has a circular core, such that optical fiber (370) is configured similar to optical fiber (220). Alternatively, optical fiber (370) may be configured similar to optical fiber (200) or have any other suitable configuration. Light emitted from optical fiber (370) arrives at collimator (372) at a first plane (P1). In this example, the illumination footprint (not shown) at the first plane (P1) has a circular shape, similar to Fig. 9 320 is shown. Collimator (372) collimates light from optical fiber (370). By way of example only, collimator (372) may include an anamorphic collimator, similar to collimator (342) of illumination assembly (330). As another example, collimator (372) may include a rotationally symmetric lens, similar to collimator (312) of illumination assembly (300). In some versions where collimator (372) includes a rotationally symmetric lens, light may pass through an anamorphic lens assembly, similar to lens assembly (314) of illumination assembly (300), before reaching diffuser (374), as described below. Alternatively, collimator (372) may include any other suitable features and / or take any other suitable form.

[0088] After passing through the collimator (372), the light reaches the diffuser (374). The diffuser (374) is configured to increase the field angle of the light entering the lenslet array (380, 390). In this example, the diffuser (374) comprises a one-dimensional diffuser, but some other versions may include a two-dimensional diffuser. The light that leaves the collimator (372) and reaches the diffuser (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 versions where the diffuser (374) comprises a one-dimensional diffuser, the diffuser (374) increases the residual divergence in one direction. In other words, the diffuser (374) increases the field angle of the light.

[0089] After passing through the diffuser (374), the light reaches the first lenslet array (380); and then the second lenslet array (390). Each lenslet array (380, 390) includes a respective plurality of lenslets (382, 392). In some versions, the lenslet arrays (380, 390) are configured substantially identical to one another, but facing in opposite directions. For example, the lenslets (382) of the first lenslet array (380) may be configured and arranged substantially identically to the configuration and arrangement of the lenslets (392) of the second lenslet array (390). By way of further example only, the lenslet arrays (380, 390) may be separated from one another by a distance approximately equal to the focal length of the lenslets (382, 392). The lenslet arrays (380, 390) may also represent an optical component having arrays on two surfaces. In some versions, including but not limited to versions in which the camera system (140) includes a time delay integration (TDI) imaging component, each lenslet array (380, 390) includes a cylindrical microlens array surface (CuLA). Such a CuLA can cause the light beam to fan out in one direction. In this type of version, the one-dimensional version of the diffuser (374) can be positioned and configured to increase the filling of the CU1A lenslets in the pitch direction without increasing the filling of the CU1A lenslets in the non-pitch direction. While some versions of the lenslet array (380, 390) include cylindrical microlens arrays, some other versions of the lenslet array (380, 390) can include two-dimensional lenslet arrays. Alternatively, the lenslet array (380, 390) can have any other suitable configuration.

[0090] As light passes through the first lenslet array (380), each lenslet (382) of the first lenslet array (380) causes an image of the core of the optical fiber (370) to form near the active surface (394) of the second lenslet array (394). The size of the optical fiber (370) core image can be proportional to the apparent size of the optical fiber (370) core. Similarly, the size of the optical fiber (370) core image near the active surface (394) of the second lenslet array (394) can be proportional to the residual divergence of the light. In some cases, as the apparent size of the optical fiber (370) core increases, the filling factor of the second lenslet array (390) can increase accordingly. The filling factor can be understood as the ratio of the active refractive area (i.e., the area of ​​the lenslets (392) of the second lenslet array (390) that directs light to the illumination surface (376)) to the total continuous area occupied by the lenslets (392) of the second lenslet array (390). As the fill factor of the lenslets (392) increases, the modulation depth of the light exiting the second lenslet array (390) may decrease.

[0091] Light exiting the second lenslet array (390) provides a rectangular shaped illumination footprint (not shown) on an illumination surface (376) at a second plane (P2) away from the second lenslet array (390). The illumination surface (376) may correspond to the surface of a reaction site in the flow cell (110) (e.g., where the output of the illumination assembly (360) is directed through the objective lens assembly (142) to resemble Figure 1 The arrangement shown in FIG. 1 reaches the flow cell (110). The illumination footprint on the illumination surface (376) at the second plane (P2) may be similar to Fig.10 The illumination footprint (322) is shown. The combination of the collimator (372), diffuser (374), and lenslet array (380, 390) can effectively generate an illumination footprint of the illumination surface (376) at the second plane (P2) that is substantially larger than the footprint at the first plane (P1). In other words, the collimator (372), diffuser (374), and lenslet array (380, 390) can provide an illumination output similar to the illumination output of a light source that is much larger than the optical fiber (370), which in turn can provide a modulation that is significantly reduced than the modulation that would otherwise be provided in light directly transmitted from the optical fiber (370) in the absence of the collimator (372), diffuser (374), and lenslet array (380, 390). In addition, the illumination assembly (360) can produce an illumination output that is effectively the same as the output from an optical fiber (240) having a rectangular core (370), even if the optical fiber (244) has a round, square, or other shaped core. Some versions of lighting assembly (360) may also utilize optical fibers having rectangular cores, similar to rectangular core (244) of optical fiber (240).

[0092] The aforementioned illumination assembly (360) can be incorporated into the system (100) such that the illumination footprint at the illumination surface (376) can be configured to correspond to a corresponding image sensor size and / or a predetermined size for illuminating a portion of the flow cell (110), while utilizing an optical fiber (370) that does not need to be specifically designed for the illumination footprint at the illumination surface (376).

[0093] Fig.13 A graph (400) is shown having a curve (402) representing an example of the modulation magnitude of the excitation light as a function of frequency, from a version of the illumination assembly (360) in which the optical fiber (200) is used as the optical fiber (370) so that the illumination footprint at the focus of the lenslet (382) of the first lenslet array (380) is square. The curve (402) can be understood as representing the modulation of the illumination footprint at the second plane (P2), which will be associated with the reaction sites in the flow cell (110). As can be seen, the curve (402) has a relatively small spike in the modulation magnitude. However, the spike is substantially smaller than the peak at Figure 3 The peaks seen in the graphs (212, 214) of , may be understood to correspond to modulation in the illumination footprint at the first plane (P1) when the optical fiber (200) is used as the optical fiber (370) in the illumination assembly (360).

[0094] The substantially smaller spike in the modulation magnitude values ​​in graph (402) as compared to the spikes in graphs (212, 214) may indicate how the collimator (372), diffuser (374), and lenslet array (380, 390) together ultimately reduce modulation in the illumination footprint from the optical fiber (200). Thus, the collimator (372), diffuser (374), and lenslet array (380, 390) together may provide better uniformity of spatial irradiance at reaction sites in the flow cell (110), which may provide reduced spatial signal variation in fluorescence emission signals from fluorophores at reaction sites of the flow cell (110) illuminated by excitation light from the illumination assembly (360). Fig.13 Only one curve (402) is shown in FIG. 4 , which represents only one channel of excitation light. However, substantially similar results may be obtained for other channels of excitation light from the optical fiber (370) in the illumination assembly (360).

[0095] Fig.14 A graph (420) is shown having a curve (422) representing an example of the modulation magnitude of the excitation light as a function of frequency, from a version of the illumination assembly (360) in which the optical fiber (220) is used as the optical fiber (370) so that the illumination footprint at the focus of the lenslet (382) of the first lenslet array (380) is circular. The curve (422) can be understood as representing the modulation of the illumination footprint at the second plane (P2), which will be associated with the reaction sites in the flow cell (110). As can be seen, the curve (422) has a relatively small spike in the modulation magnitude. However, the spike is substantially smaller than the peak at Figure 5 The peaks seen in the graphs (232, 234) of , may be understood to correspond to modulation in the illumination footprint at the first plane (P1) when the optical fiber (220) is used as the optical fiber (370) in the illumination assembly (360).

[0096] The substantially smaller spike in the modulation magnitude values ​​in graph (422) as compared to the spikes in graphs (232, 234) may indicate how the collimator (372), diffuser (374), and lenslet array (380, 390) together ultimately reduce modulation in the illumination footprint from the optical fiber (220). Thus, the collimator (372), diffuser (374), and lenslet array (380, 390) together may provide better uniformity of spatial irradiance at reaction sites in the flow cell (110), which may provide reduced spatial signal variation in fluorescence emission signals from fluorophores at reaction sites of the flow cell (110) illuminated by excitation light from the illumination assembly (360). Fig.14 Only one curve (422) is shown in FIG. 4 , which represents only one channel of excitation light. However, substantially similar results can be obtained for other channels of excitation light from the optical fiber (370) in the illumination assembly (360).

[0097] Fig.15 A graph (440) is shown having a curve (442) representing an example of the modulation magnitude of the excitation light as a function of frequency, from a version of the illumination assembly (360) in which the optical fiber (240) is used as the optical fiber (370) so that the illumination footprint at the focus of the lenslet (382) of the first lenslet array (380) is circular. The curve (442) can be understood as representing the modulation of the illumination footprint at the second plane (P2), which will be associated with the reaction sites in the flow cell (110). As can be seen, the curve (442) has a relatively small spike in the modulation magnitude. However, the spike is substantially smaller than the peak at Figure 7 The peaks seen in the graphs (252, 254) of , may be understood to correspond to modulation in the illumination footprint at the first plane (P1) when the optical fiber (240) is used as the optical fiber (370) in the illumination assembly (360).

[0098] The substantially smaller spike in the modulation magnitude values ​​in graph (442) as compared to the spike in graphs (252, 254) may indicate how the collimator (372), diffuser (374), and lenslet array (380, 390) together ultimately reduce modulation in the illumination footprint from the optical fiber (240). Thus, the collimator (372), diffuser (374), and lenslet array (380, 390) together may provide better uniformity of spatial irradiance at reaction sites in the flow cell (110), which may provide reduced spatial signal variation in fluorescence emission signals from fluorophores at reaction sites of the flow cell (110) illuminated by excitation light from the illumination assembly (360). Fig.15Only one curve (442) is shown in FIG. 4 , which represents only one channel of excitation light. However, substantially similar results can be obtained for other channels of excitation light from the optical fiber (370) in the illumination assembly (360).

[0099] Fig.16 Another example of an illumination assembly (500) that can be incorporated into an imaging assembly (120) is shown. The illumination assembly (500) of this example is a variation of the illumination assembly (360) such that the illumination assembly (500) includes the same components of the illumination assembly (360) but has a different arrangement. Specifically, the illumination assembly (500) includes a light source (362), an optical fiber (370), a collimator (372), a diffuser (374), a first lenslet array (380), and a second lenslet array (390). However, unlike the arrangement of the imaging assembly (360) in which the diffuser (374) is positioned between the collimator (372) and the first lenslet array (380), the diffuser (374) is positioned between the first lenslet array (380) and the second lenslet array (390) in the illumination assembly (500). This arrangement of the diffuser (374) between the first lenslet lens (380) and the second lenslet lens (390) in the lighting assembly (500) can produce substantially the same results as a different arrangement in the lighting assembly (360). In other words, the lighting assembly (500) can provide a rectangular shaped lighting footprint on the lighting surface (376) at the second plane (P2) with a substantially reduced modulation magnitude.

[0100] Fig.17 Another example of an illumination assembly (550) that can be incorporated into an imaging assembly (120) is shown. The illumination assembly (550) of this example is a variation of the illumination assembly (500) such that the illumination assembly (500) includes most of the same components of the illumination assembly (500), except that the illumination assembly (500) includes a refractive substrate (560) instead of a diffuser (374). Similar to the illumination assembly (500), the illumination assembly (550) of this example also includes a light source (362), an optical fiber (370), a collimator (372), a first lenslet array (380), and a second lenslet array (390). The refractive substrate (560) of this example includes a first surface (562) and a second surface (564). The first lenslet array (380) is disposed on the first surface (562). The second lenslet array (390) is disposed on the first surface (564). Therefore, in this example, the refractive substrate (560) is directly interposed between the lenslet arrays (380, 390).

[0101] The refractive substrate (560) is configured such that the refractive substrate (560) substantially focuses a light beam incident on the first lenslet array (380) onto the second lenslet array (390). This focusing aspect of the refractive substrate (560) may be due in part to the thickness of the refractive substrate (i.e., the distance between the surfaces (562, 564)) and other parameters. The arrangement of the refractive substrate (560) between the first lenslet lens (380) and the second lenslet lens (390) in the illumination assembly (550) may produce substantially the same results as the different arrangements in the illumination assembly (360, 500). In other words, the illumination assembly (500) may provide a rectangular shaped illumination footprint on the illumination surface (570) at the second plane (P2) with a substantially reduced modulation magnitude.

[0102] C. Examples of Image Processing Techniques

[0103] In some scenarios, image processing techniques may be applied to images captured by the camera system (140) by software (e.g., executed via the controller (195)) to reduce adverse effects from periodic modulation in the excitation light. For example, the elements of each column of an image captured by the camera system (140) may be summed such that the reading of a pixel in the first row and first column is added to the reading of a pixel in the second row and first column. This may be done for all columns of each column. By way of further example only, for an image that is 5120 columns wide and 4096 rows high, the 4096 rows of each column may be summed to generate a one-dimensional array having 5120 elements (i.e., as many elements as there are columns). These numbers are examples only, as an image may have any other suitable number of columns and rows.

[0104] The above one-dimensional array may be referred to as a flat-field array. The flat-field array may be normalized by dividing each element of the array by the sum of the elements of the array. The flat-field array may be further normalized by dividing each element by the average of the flat-field array elements, after which the average of the flat-field elements will be 1.

[0105] The normalized array may be defined as a flat field profile. Each row of the original image captured by the camera system (140) may be divided by the flat field profile. This process may render the modulation from the original image substantially imperceptible. The modulation may also 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 frequencies of the modulation. In other words, where one or more of the images have a spatial pattern of a particular period, one or more frequency domain filters may be applied to reduce the spatial pattern.

[0106] The image processing techniques described above may be used in versions of the system (100) in which at least one of the optical fibers (200, 220, 240) is incorporated into an imaging assembly (120); in versions of the system (100) in which one of the illumination assemblies (300, 330, 360, 500) is incorporated into an imaging assembly (120); and / or in other versions of the system (100). In some versions, the image processing techniques described above are used to computationally reduce periodic modulations in images captured by the camera system (140) when reaction sites in a flow cell (110) are illuminated with modulated excitation light. Thus, the image processing techniques described above may be used as an alternative to hardware-based solutions (e.g., optical fibers (240), illumination assemblies (300, 330, 360, 500), etc.) to address the modulation problem.

[0107] III. Combined Embodiments

[0108] The following examples relate to various non-exhaustive ways in which the teachings of this article can be combined or applied. The following examples are not intended to limit the coverage of any claims that may be provided at any time in the present application or subsequent submissions of this application. It is not intended to make a disclaimer. The following examples are provided only for the purpose of illustration. It is conceivable that the various teachings of this article can be arranged and applied in a variety of other ways. It is also expected that some variations may omit certain features mentioned in the following examples. Therefore, any of the aspects or features mentioned below should not be considered decisive, unless otherwise, for example, by the inventor or the successor of the inventor concerned clearly indicates so at a later date. If any claim proposed in the present application or in the subsequent submissions related to this application includes additional features other than those mentioned below, these additional features should not be assumed to be added for any reason related to patentability.

[0109] Example 1

[0110] An apparatus comprises: a sequencing station, the sequencing station being configured to receive a flow cell comprising a plurality of reaction sites, each reaction site being configured to contain a biological sample; and an illumination assembly, the illumination assembly being configured to project light toward the sequencing station so as to illuminate the reaction sites, the illumination assembly comprising: a light source, a first lenslet array, the first lenslet array being interposed between the light source and the sequencing station, a second lenslet array, the second lenslet array being interposed between the light source and the sequencing station, and a diffuser, the diffuser being interposed between the light source and the sequencing station.

[0111] Example 2

[0112] According to the device of embodiment 1, the diffuser comprises a one-dimensional diffuser.

[0113] Example 3

[0114] According to the device of any one of embodiments 1 to 2, the first lenslet array includes a cylindrical microlens array.

[0115] Example 4

[0116] According to the apparatus of any one of embodiments 1 to 3, the diffuser is interposed between the light source and the first lenslet array.

[0117] Example 5

[0118] According to the apparatus of any one of embodiments 1 to 3, the diffuser is interposed between the first lenslet array and the second lenslet array.

[0119] Example 6

[0120] According to the apparatus of any one of embodiments 1 to 3, the second lenslet array is interposed between the first lenslet array and the diffuser.

[0121] Example 7

[0122] The apparatus of any one of embodiments 1 to 6 further comprises a collimator interposed between the light source and one or both of the first lenslet array or the diffuser.

[0123] Example 8

[0124] According to the device described in embodiment 7, the collimator includes a deformable collimator.

[0125] Example 9

[0126] According to the device described in any one of embodiments 1 to 8, the first lenslet array includes a plurality of lenslets, each lenslet has a focal length, and the first lenslet array and the second lenslet array are spaced apart from each other by a distance approximately equal to the focal length.

[0127] Example 10

[0128] According to the device of any one of embodiments 1 to 9, the light source includes an optical fiber.

[0129] Embodiment 11

[0130] According to the device of embodiment 10, the optical fiber has a core with a square cross-sectional shape.

[0131] Example 12

[0132] According to the device of embodiment 10, the optical fiber has a core with a circular cross-sectional shape.

[0133] Embodiment 13

[0134] In accordance with the apparatus of embodiment 10, the optical fiber has a core with a rectangular cross-sectional shape defined by a length and a width, the width being greater than the length.

[0135] Embodiment 14

[0136] According to the device of embodiment 13, the length and the width provide an aspect ratio of at least 2:1.

[0137] Embodiment 15

[0138] According to the device of embodiment 13, the length and the width provide an aspect ratio of at least 3:1.

[0139] Example 16

[0140] According to the device of embodiment 13, the length and the width provide an aspect ratio of at least 4:1.

[0141] Embodiment 17

[0142] According to the apparatus of any one of Embodiments 10 to 16, the light source comprises a laser diode.

[0143] Embodiment 18

[0144] According to the device of any one of Embodiments 10 to 17, the laser diode comprises a multi-mode diode and the optical fiber is configured to transmit multiple channels of light.

[0145] Embodiment 19

[0146] The apparatus of any one of embodiments 1 to 18 further comprising a camera system operable to capture an image of the reaction site.

[0147] Embodiment 20

[0148] According to the device of embodiment 19, the camera system includes a time delay integration camera.

[0149] Embodiment 21

[0150] According to the device of any of Embodiments 19 to 20, the camera system is operable to capture light emitted by the fluorophore at the reaction site in response to the light projected by the illumination assembly.

[0151] Embodiment 22

[0152] A method includes: transmitting light toward a sequencing station through an illumination assembly, the illumination assembly including: a light source, a first lenslet array, the first lenslet array being interposed between the light source and the sequencing station, a second lenslet array, the second lenslet array being interposed between the light source and the sequencing station, and a diffuser, the diffuser being interposed between the light source and the sequencing station; the transmitted light is further transmitted toward a plurality of reaction sites at the sequencing station, each reaction site comprising a biological sample.

[0153] Embodiment 23

[0154] According to the method of embodiment 22, the diffuser includes a one-dimensional diffuser.

[0155] Embodiment 24

[0156] According to the method described in any one of embodiments 22 to 23, the first small lens array comprises a cylindrical microlens array.

[0157] Embodiment 25

[0158] According to the method of any one of embodiments 22 to 24, the diffuser is interposed between the light source and the first lenslet array.

[0159] Embodiment 26

[0160] According to the method of any one of embodiments 22 to 24, the diffuser is interposed between the first lenslet array and the second lenslet array.

[0161] Embodiment 27

[0162] According to the method of any one of embodiments 22 to 24, the second lenslet array is interposed between the first lenslet array and the diffuser.

[0163] Embodiment 28

[0164] According to the method of any one of embodiments 22 to 27, the lighting assembly also includes a collimator, which is interposed between the light source and one or both of the first lenslet array or the diffuser.

[0165] Embodiment 29

[0166] According to the method of embodiment 26, the collimator includes a deformable collimator.

[0167] Embodiment 30

[0168] According to the method described in any one of embodiments 22 to 29, the first lenslet array includes a plurality of lenslets, each lenslet has a focal length, and the first lenslet array and the second lenslet array are spaced apart from each other by a distance approximately equal to the focal length.

[0169] Embodiment 31

[0170] According to the method of any one of Embodiments 22 to 30, the light source includes an optical fiber.

[0171] Embodiment 32

[0172] According to the method of embodiment 31, the optical fiber has a core with a square cross-sectional shape.

[0173] Embodiment 33

[0174] According to the method of embodiment 31, the optical fiber has a core with a circular cross-sectional shape.

[0175] Embodiment 34

[0176] According to the method of embodiment 31, 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.

[0177] Embodiment 35

[0178] According to the method of embodiment 34, the length and the width provide an aspect ratio of at least 2:1.

[0179] Embodiment 36

[0180] According to the method of embodiment 34, the length and the width provide an aspect ratio of at least 3:1.

[0181] Embodiment 37

[0182] According to the method of Example 34, the length and the width provide an aspect ratio of at least 4:1.

[0183] Embodiment 38

[0184] According to the method of any one of Embodiments 31 to 37, the light source includes a laser diode.

[0185] Embodiment 39

[0186] According to the method of any one of Embodiments 31 to 38, the laser diode includes a multi-mode diode and the optical fiber is configured to transmit multiple channels of light.

[0187] Embodiment 40

[0188] The method of any one of embodiments 22 to 39 further comprises capturing an image of the reaction site with a camera system.

[0189] Embodiment 41

[0190] According to the method described in Example 40, the camera system includes a time delay integration camera.

[0191] Embodiment 42

[0192] According to the method described in any one of Embodiments 40 to 41, the captured image includes an original image having rows and columns, and the method further includes: summing the rows of each column of the original image to form a one-dimensional flat field array having multiple elements; and dividing each column of the original image by the flat field array to produce a demodulated image.

[0193] Embodiment 43

[0194] According to the method described in any one of Examples 40 to 42, the reaction site includes a fluorophore that emits light in response to the transmitted light reaching the reaction site, and capturing an image of the reaction site with the camera system includes capturing the light emitted by the fluorophore at the reaction site.

[0195] Embodiment 44

[0196]

[00136] The method according to Example 43 also includes identifying at least one nucleotide at the reaction site based on captured light emitted by at least one fluorophore at the reaction site among the plurality of reaction sites.

[0197] Embodiment 45

[0198] The method of any one of embodiments 22 to 44, wherein the reaction site is located in a flow cell.

[0199] Embodiment 46

[0200] The method according to Example 45 also includes performing sequencing by synthesis on the flow cell.

[0201] Embodiment 47

[0202] An apparatus comprises: a sequencing station, the sequencing station being configured to receive a flow cell comprising a plurality of reaction sites, each reaction site being configured to contain a biological sample; and an illumination assembly, the illumination assembly being configured to project light toward the sequencing station, thereby illuminating the reaction sites, the illumination assembly comprising: a light source, a collimator, the collimator being interposed between the light source and the sequencing station, and a deformable lens assembly, the deformable lens assembly being interposed between the collimator and the sequencing station.

[0203] Embodiment 48

[0204] According to the device described in embodiment 47, the collimator includes a rotationally symmetric lens.

[0205] Embodiment 49

[0206] According to the device described in any of Embodiments 47 to 48, the deformable lens assembly is configured to convert the illumination footprint from the light source into a rectangular shape.

[0207] Embodiment 50

[0208] According to the device of embodiment 49, the light source includes an optical fiber having a core with a square cross-sectional shape.

[0209] Embodiment 51

[0210] According to the device of embodiment 49, the light source includes an optical fiber having a core with a circular cross-sectional shape.

[0211] Embodiment 52

[0212] According to the apparatus of any one of Embodiments 49 to 51, the rectangular shape has an aspect ratio of at least 10:1.

[0213] Embodiment 53

[0214] According to the device described in any of embodiments 47 to 48, the deformable lens assembly is configured to convert the illumination footprint from the light source into an elliptical shape.

[0215] Embodiment 54

[0216] According to the device of embodiment 53, the elliptical shape has an aspect ratio of at least 10:1.

[0217] Embodiment 55

[0218] According to the device described in any of embodiments 49 to 54, the deformable lens assembly is configured to provide the illumination coverage area at the reaction site.

[0219] Embodiment 56

[0220] An apparatus comprises: a sequencing station, the sequencing station being configured to receive a flow cell comprising a plurality of reaction sites, each reaction site being configured to contain a biological sample; and an illumination assembly, the illumination assembly being configured to project light toward the sequencing station, thereby illuminating the reaction sites, the illumination assembly comprising: a light source, and a deformable collimator, the deformable collimator being interposed between the light source and the sequencing station.

[0221] Embodiment 57

[0222] According to the device of any of Embodiment 56, the deformable lens assembly is configured to convert the illumination footprint from the light source into a rectangular shape.

[0223] Embodiment 58

[0224] According to the device of embodiment 57, the light source includes an optical fiber having a core with a square cross-sectional shape.

[0225] Embodiment 59

[0226] According to the device of embodiment 57, the light source includes an optical fiber having a core with a circular cross-sectional shape.

[0227] Embodiment 60

[0228] According to the apparatus of any one of Embodiments 57 to 59, the rectangular shape has an aspect ratio of at least 10:1.

[0229] Embodiment 61

[0230] According to the device of any of Embodiment 56, the deformable lens assembly is configured to convert the illumination footprint from the light source into an elliptical shape.

[0231] Embodiment 62

[0232] According to the device of embodiment 61, the elliptical shape has an aspect ratio of at least 10:1.

[0233] Embodiment 63

[0234] According to the device described in any one of embodiments 56 to 62, the deformable lens assembly is configured to provide the illumination coverage area at the reaction site.

[0235] Embodiment 64

[0236] A method includes: conveying light through an illumination assembly toward a plurality of reaction sites at a sequencing station, each reaction site containing a biological sample; capturing images of the reaction sites with a camera system, the camera system including a time-delayed integration camera, the captured image including a raw image having rows and columns; summing the rows of the raw images to form a one-dimensional flat-field array having a plurality of elements; and dividing each column of the raw image by the flat-field array to produce a demodulated image.

[0237] Embodiment 65

[0238] According to the method described in Example 64, the demodulated image has a spatial pattern with a specific period, and the method also includes applying one or more frequency domain filters to reduce the spatial pattern.

[0239] Embodiment 66

[0240] A method comprising: conveying light through an illumination assembly toward a plurality of reaction sites at a sequencing station, each reaction site comprising a biological sample; capturing images of the reaction sites with a camera system, the camera system comprising a time-delayed integration camera, the captured images comprising a raw image having a spatial pattern with a specific period; and applying one or more frequency domain filters to reduce the spatial pattern, thereby producing a demodulated image.

[0241] Embodiment 67

[0242] An apparatus comprises: a sequencing station, the sequencing station being configured to receive a flow cell comprising a plurality of reaction sites, each reaction site being configured to contain a biological sample; and an illumination assembly, the illumination assembly being configured to project light toward the sequencing station, thereby illuminating the reaction sites, the illumination assembly comprising: a light source, a first lenslet array, the first lenslet array being interposed between the light source and the sequencing station, and a second lenslet array, the second lenslet array being interposed between the light source and the sequencing station.

[0243] Embodiment 68

[0244] The apparatus according to embodiment 67 also includes a diffuser, which is inserted between the light source and the sequencing station.

[0245] Embodiment 69

[0246] According to the device of embodiment 68, the diffuser is interposed between the light source and the first lenslet array.

[0247] Embodiment 70

[0248] According to the device of embodiment 68, the diffuser is inserted between the first lenslet array and the second lenslet array.

[0249] Embodiment 71

[0250] According to the device of any one of embodiments 68, the second lenslet array is interposed between the first lenslet array and the diffuser.

[0251] Embodiment 72

[0252] The apparatus of any one of Embodiments 68 to 71, further comprising a collimator interposed between the light source and one or both of the first lenslet array or the diffuser.

[0253] Embodiment 73

[0254] According to the device of embodiment 72, the collimator includes a deformable collimator.

[0255] Embodiment 74

[0256] According to the device described in any one of embodiments 67 to 73, the first small lens array includes a cylindrical microlens array.

[0257] Embodiment 75

[0258] According to the device described in any one of embodiments 67 to 74, the first lenslet array includes a plurality of lenslets, each lenslet having a focal length, and the first lenslet array and the second lenslet array are spaced apart from each other by a distance approximately equal to the focal length.

[0259] Embodiment 76

[0260] According to the device of any one of embodiments 1 to 78, the light source includes an optical fiber.

[0261] Embodiment 77

[0262] According to the device of embodiment 76, the optical fiber has a core with a square cross-sectional shape.

[0263] Embodiment 78

[0264] According to the device of embodiment 76, the optical fiber has a core with a circular cross-sectional shape.

[0265] Embodiment 79

[0266] In accordance with the apparatus of embodiment 76, the optical fiber has a core with a rectangular cross-sectional shape defined by a length and a width, the width being greater than the length.

[0267] Embodiment 80

[0268] According to the device of embodiment 79, the length and the width provide an aspect ratio of at least 2:1.

[0269] Embodiment 81

[0270] According to the device of embodiment 79, the length and the width provide an aspect ratio of at least 3:1.

[0271] Embodiment 82

[0272] According to the device of embodiment 79, the length and the width provide an aspect ratio of at least 4:1.

[0273] Embodiment 83

[0274] According to the apparatus of any one of Embodiments 76 to 82, the light source comprises a laser diode.

[0275] Embodiment 84

[0276] According to the device of any one of Embodiments 76 to 83, the laser diode includes a multi-mode diode and the optical fiber is configured to transmit multiple channels of light.

[0277] Embodiment 85

[0278] The apparatus of any one of Embodiments 67 to 84 further comprising a camera system operable to capture images of the reaction site.

[0279] Embodiment 86

[0280] According to the device described in embodiment 85, the camera system includes a time delay integration camera.

[0281] Embodiment 87

[0282] According to the device of any of Embodiments 85 to 86, the camera system is operable to capture light emitted by the fluorophore at the reaction site in response to the light projected by the illumination assembly.

[0283] Embodiment 88

[0284] According to the device described in any one of embodiments 67 to 87, the lighting component also includes a refractive substrate having a first surface and a second surface, the first small lens array is arranged on the first surface of the refractive substrate, and the second small lens array is arranged on the second surface of the refractive substrate.

[0285] Embodiment 89

[0286] According to the device described in embodiment 88, the refractive substrate is configured so that the first surface and the second surface of the refractive substrate are respectively positioned to substantially focus a collimated light beam incident on the first array onto the second array.

[0287] IV. Miscellaneous

[0288] The foregoing teachings may be readily applied in the context of various kinds of camera features in the camera system (140). For example, the foregoing teachings may be applied in the context of a camera system (140) including a dual sensor time delay integration (TDI) camera, a single sensor camera, a camera having one or more two-dimensional image sensors, and / or other kinds of camera features in the camera system (140).

[0289] Although the foregoing examples are provided in the context of a system (100) that can be used for a nucleotide sequencing process, the teachings herein can also be readily applied to other contexts, including systems that perform other processes (i.e., in addition to a nucleotide sequencing process). Therefore, the teachings herein are not necessarily limited to systems for performing a nucleotide sequencing process.

[0290] It should be understood that the subject matter described herein is not limited to the details of the construction and arrangement of the components set forth in the description herein or illustrated in the drawings herein in terms of its application. The subject matter described herein can have other specific implementations and can be practiced or executed in various ways. In addition, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered as restrictive. "Including," "comprising," or "having" and variations thereof used herein are meant to cover the items listed thereafter and their equivalents and other items.

[0291] When used in the claims, the term "group" should be understood as one or more things that are grouped together. Similarly, when used in the claims, "based on" should be understood to indicate that one thing is at least partially determined by the thing designated as "based on." In the case where one thing needs to be exclusively determined by another thing, then that thing will be referred to as being "exclusively based on" the other thing by which it is determined.

[0292] Unless otherwise specified or limited, the terms "mounted," "connected," "supported," and "coupled," and variations thereof, are used broadly and cover both direct and indirect mounting, connection, support, and coupling. Furthermore, "connected" and "coupled" are not limited to physical or mechanical connections or physical or mechanical couplings. Furthermore, it should be understood that the words and terms used herein with reference to device or element orientations (such as, for example, terms like "above," "below," "front," "rear," "distal," "proximal," etc.) are used only to simplify the description of one or more examples described herein, and do not individually indicate or imply that the device or element referred to must have a particular orientation. Additionally, terms such as "external" and "internal" are used herein for descriptive purposes and are not intended to indicate or imply relative importance or significance.

[0293] It should be understood that the above description is intended to be illustrative rather than restrictive. For example, the above examples (and / or aspects thereof) can be used in combination with each other. In addition, without departing from the scope of the invention, many modifications can be made to adapt specific situations or materials to the teachings of the subject matter currently described. Although the sizes and types of materials and coatings described herein are intended to limit the parameters of the disclosed subject matter, they are by no means restrictive but illustrative. When viewing the above description, many other examples will be obvious to those skilled in the art. Therefore, the scope of the subject matter of the present disclosure should be determined with reference to the appended claims and the full range of equivalents granted by these claims. In the appended claims, the terms "including" and "wherein" are used as the plain English equivalents of the corresponding terms "including" and "wherein". In addition, in the following claims, the terms "first", "second" and "third" are used only as labels and are not intended to impose numerical requirements on their objects. In addition, the limitations of the following claims are not written in a means plus function format and are not intended to be interpreted based on the 35 U.S.C. § 112 (f) paragraph unless and until these claim limitations explicitly use the phrase "members for ..." followed by functional statements without other structures.

[0294] The following claims set forth certain exemplary aspects of the disclosed subject matter and are considered a part of the above disclosure. These aspects can be combined with each other.

Claims

1. A device comprising: a sequencing station configured to receive a flow cell comprising a plurality of reaction sites, each reaction site configured to contain a biological sample; and An illumination assembly, the illumination assembly being configured to project light toward the sequencing station, thereby illuminating the reaction site, the illumination assembly comprising: light source, a first lenslet array, wherein the first lenslet array is inserted between the light source and the sequencing station, a second lenslet array, the second lenslet array being interposed between the light source and the sequencing station, and A diffuser is interposed between the light source and the sequencing station.

2. The apparatus of claim 1, the diffuser comprising a one-dimensional diffuser.

3. The apparatus of any one of claims 1 to 2, the first lenslet array comprising an array of cylindrical microlenses.

4. The apparatus of any one of claims 1 to 3, the diffuser being interposed between the light source and the first lenslet array.

5. The apparatus of any one of claims 1 to 3, the diffuser being interposed between the first lenslet array and the second lenslet array.

6. The apparatus of any one of claims 1 to 3, the second lenslet array being interposed between the first lenslet array and the diffuser.

7. The apparatus of any one of claims 1 to 6, further comprising a collimator interposed between the light source and one or both of the first lenslet array or the diffuser.

8. The apparatus of claim 7, the collimator comprising an anamorphic collimator.

9. The apparatus of any one of claims 1 to 8, the first lenslet array comprising a plurality of lenslets, each lenslet having a focal length, the first lenslet array and the second lenslet array being spaced apart from each other by a distance approximately equal to the focal length.

10. Apparatus according to any one of claims 1 to 9, the light source comprising an optical fibre.

11. The apparatus of claim 10, the optical fiber having a core with a square cross-sectional shape.

12. The apparatus of claim 10, the optical fiber having a core with a circular cross-sectional shape.

13. The apparatus of claim 10, the optical fiber having a core with a rectangular cross-sectional shape defined by a length and a width, the width being greater than the length.

14. The apparatus of claim 13, the length and the width providing an aspect ratio of at least 2:

1.

15. The apparatus of claim 13, the length and the width providing an aspect ratio of at least 3:

1.

16. The apparatus of claim 13, the length and the width providing an aspect ratio of at least 4:

1.

17. Apparatus according to any one of claims 10 to 16, the light source comprising a laser diode.

18. The apparatus of any one of claims 10 to 17, the laser diode comprising a multimode diode, the optical fiber being configured to transmit multiple channels of light.

19. The apparatus of any one of claims 1 to 18, further comprising a camera system operable to capture images of the reaction sites.

20. The apparatus of claim 19, the camera system comprising a time delay integration camera.

21. The apparatus of any one of claims 19 to 20, the camera system being 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 comprising: The light is conveyed toward the sequencing station by an illumination assembly, the illumination assembly comprising: light source, a first lenslet array, wherein the first lenslet array is inserted between the light source and the sequencing station, a second lenslet array, the second lenslet array being interposed between the light source and the sequencing station, and a diffuser, the diffuser being interposed between the light source and the sequencing station; The transmitted light is further transmitted toward a plurality of reaction sites at the sequencing station, each reaction site comprising a biological sample.

23. The method of claim 22, the diffuser comprising a one-dimensional diffuser.

24. The method of any one of claims 22 to 23, the first lenslet array comprising a cylindrical microlens array.

25. The method of any one of claims 22 to 24, the diffuser being interposed between the light source and the first lenslet array.

26. The method of any one of claims 22 to 24, the diffuser being interposed between the first lenslet array and the second lenslet array.

27. The method of any one of claims 22 to 24, the second lenslet array being interposed between the first lenslet array and the diffuser.

28. The method of any one of claims 22 to 27, the lighting assembly further comprising a collimator interposed between the light source and one or both of the first lenslet array or the diffuser.

29. The method of claim 26, the collimator comprising an anamorphic collimator.

30. The method of any one of claims 22 to 29, the first lenslet array comprising a plurality of lenslets, each lenslet having a focal length, the first lenslet array and the second lenslet array being spaced apart from each other by a distance approximately equal to the focal length.

31. A method according to any one of claims 22 to 30, wherein the light source comprises an optical fibre.

32. The method of claim 31, wherein the optical fiber has a core with a square cross-sectional shape.

33. The method of claim 31, wherein the optical fiber has a core with a circular cross-sectional shape.

34. The method of claim 31, the optical fiber having a core with a rectangular cross-sectional shape defined by a length and a width, the width being greater than the length.

35. The method of claim 34, the length and the width providing an aspect ratio of at least 2:

1.

36. The method of claim 34, the length and the width providing an aspect ratio of at least 3:

1.

37. The method of claim 34, the length and the width providing an aspect ratio of at least 4:

1.

38. A method according to any one of claims 31 to 37, wherein the light source comprises a laser diode.

39. A method according to any one of claims 31 to 38, the laser diode comprising a multimode diode, the optical fiber being configured to transmit multiple channels of light.

40. The method of any one of claims 22 to 39, further comprising capturing an image of the reaction site with a camera system.

41. The method of claim 40, wherein the camera system comprises a time delay integration camera.

42. The method of any one of claims 40 to 41, wherein the captured image comprises an original image having rows and columns, the method further comprising: summing the rows for each column of the original image to form a one-dimensional flat-field array having a plurality of elements; as well as Each column of the raw image is divided by the flat field array to produce a demodulated image.

43. The method of any one of claims 40 to 42, wherein the reaction site comprises a fluorophore that emits light in response to the transmitted light reaching the reaction site, and capturing an image of the reaction site with the camera system comprises capturing the light emitted by the fluorophore at the reaction site.

44. The method of claim 43, further comprising identifying at least one nucleotide at the reaction site based on captured light emitted by at least one fluorophore at the reaction site in the plurality of reaction sites.

45. The method of any one of claims 22 to 44, wherein the reaction site is located in a flow cell.

46. ​​The method of claim 45, further comprising performing sequencing by synthesis on the flow cell.

47. An apparatus comprising: a sequencing station configured to receive a flow cell comprising a plurality of reaction sites, each reaction site configured to contain a biological sample; and An illumination assembly, the illumination assembly being configured to project light toward the sequencing station, thereby illuminating the reaction site, the illumination assembly comprising: light source, a collimator, the collimator being inserted between the light source and the sequencing station, A deformable lens assembly is interposed between the collimator and the sequencing station.

48. The apparatus of claim 47, the collimator comprising a rotationally symmetric lens.

49. The apparatus of any one of claims 47 to 48, the anamorphic lens assembly being configured to transform an illumination footprint from the light source into a rectangular shape.

50. The apparatus of claim 49, the light source comprising an optical fiber having a core with a square cross-sectional shape.

51. The apparatus of claim 49, the light source comprising an optical fiber having a core with a circular cross-sectional shape.

52. The apparatus of any one of claims 49 to 51, the rectangular shape having an aspect ratio of at least 10:

1.

53. The apparatus of any one of claims 47 to 48, the anamorphic lens assembly being configured to transform an illumination footprint from the light source into an elliptical shape.

54. The apparatus of claim 53, the elliptical shape having an aspect ratio of at least 10:

1.

55. The apparatus of any one of claims 49 to 54, wherein the deformable lens assembly is configured to provide the illumination footprint at the reaction site.

56. An apparatus comprising: a sequencing station configured to receive a flow cell comprising a plurality of reaction sites, each reaction site configured to contain a biological sample; and An illumination assembly, the illumination assembly being configured to project light toward the sequencing station, thereby illuminating the reaction site, the illumination assembly comprising: light source, and A deformable collimator is inserted between the light source and the sequencing station.

57. The apparatus of any one of claims 56, wherein the anamorphic lens assembly is configured to transform an illumination footprint from the light source into a rectangular shape.

58. The apparatus of claim 57, the light source comprising an optical fiber having a core with a square cross-sectional shape.

59. The apparatus of claim 57, the light source comprising an optical fiber having a core with a circular cross-sectional shape.

60. The apparatus of any one of claims 57 to 59, the rectangular shape having an aspect ratio of at least 10:

1.

61. The apparatus of any one of claims 56, wherein the anamorphic lens assembly is configured to transform an illumination footprint from the light source into an elliptical shape.

62. The apparatus of claim 61, the elliptical shape having an aspect ratio of at least 10:

1.

63. The apparatus of any one of claims 56 to 62, wherein the deformable lens assembly is configured to provide the illumination footprint at the reaction site.

64. A method comprising: conveying light through an illumination assembly toward a plurality of reaction sites at a sequencing station, each reaction site comprising a biological sample; capturing an image of the reaction site with a camera system, the camera system comprising a time-delay integration camera, the captured image comprising a raw image having rows and columns; summing the rows of the original image to form a one-dimensional flat-field array having a plurality of elements; as well as Each column of the raw image is divided by the flat field array to produce a demodulated image.

65. The method of claim 64, wherein the demodulated image has a spatial pattern with a specific period, the method further comprising applying one or more frequency domain filters to reduce the spatial pattern.

66. A method comprising: conveying light through an illumination assembly toward a plurality of reaction sites at a sequencing station, each reaction site comprising a biological sample; capturing an image of the reaction site with a camera system, the camera system comprising a time-delay integration camera, the captured image comprising a raw image having a spatial pattern with a specific period; as well as One or more frequency domain filters are applied to reduce the spatial pattern, thereby producing a demodulated image.

67. An apparatus comprising: a sequencing station configured to receive a flow cell comprising a plurality of reaction sites, each reaction site configured to contain a biological sample; and An illumination assembly, the illumination assembly being configured to project light toward the sequencing station, thereby illuminating the reaction site, the illumination assembly comprising: light source, a first lenslet array, the first lenslet array being interposed between the light source and the sequencing station, and A second lenslet array is interposed between the light source and the sequencing station.

68. The apparatus of claim 67, further comprising a diffuser interposed between the light source and the sequencing station.

69. The apparatus of claim 68, the diffuser being interposed between the light source and the first lenslet array.

70. The apparatus of claim 68, the diffuser being interposed between the first lenslet array and the second lenslet array.

71. The apparatus of any one of claims 68, the second lenslet array being interposed between the first lenslet array and the diffuser.

72. The apparatus of any one of claims 68 to 71, further comprising a collimator interposed between the light source and one or both of the first lenslet array or the diffuser.

73. The apparatus of claim 72, wherein the collimator comprises a deformable collimator.

74. An apparatus according to any one of claims 67 to 73, wherein the first lenslet array comprises an array of cylindrical microlenses.

75. The apparatus of any one of claims 67 to 74, the first lenslet array comprising a plurality of lenslets, each lenslet having a focal length, the first lenslet array and the second lenslet array being spaced apart from each other by a distance approximately equal to the focal length.

76. The apparatus of any one of claims 1 to 78, wherein the light source comprises an optical fiber.

77. The apparatus of claim 76, wherein the optical fiber has a core with a square cross-sectional shape.

78. The apparatus of claim 76, wherein the optical fiber has a core with a circular cross-sectional shape.

79. The apparatus of claim 76, the optical fiber having a core with a rectangular cross-sectional shape defined by a length and a width, the width being greater than the length.

80. The apparatus of claim 79, the length and the width providing an aspect ratio of at least 2:

1.

81. The apparatus of claim 79, the length and the width providing an aspect ratio of at least 3:

1.

82. The apparatus of claim 79, the length and the width providing an aspect ratio of at least 4:

1.

83. The apparatus of any one of claims 76 to 82, wherein the light source comprises a laser diode.

84. The apparatus of any one of claims 76 to 83, the laser diode comprising a multimode diode, the optical fiber being configured to transmit multiple channels of light.

85. The apparatus of any one of claims 67 to 84, further comprising a camera system operable to capture images of the reaction sites.

86. The apparatus of claim 85, wherein the camera system comprises a time delay integration camera.

87. The apparatus of any one of claims 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. According to the device described in any one of claims 67 to 87, the lighting component also includes a refractive substrate, the refractive substrate has a first surface and a second surface, the first small lens array is arranged on the first surface of the refractive substrate, and the second small lens array is arranged on the second surface of the refractive substrate.

89. The apparatus of claim 88, the refractive substrate being configured such that the first surface and the second surface of the refractive substrate are respectively positioned to substantially focus a collimated light beam incident on the first array onto the second array.

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