Dynamic Optical System Calibration

Abstract

The apparatus includes a flow cell, an imaging assembly, and a processor. The flow cell includes a channel and a plurality of reaction sites. The imaging assembly is operable to receive light emitted from the reaction sites in response to excitation light. The processor is configured to drive relative movement between at least a portion of the imaging assembly and the flow cell along a continuous range of motion, thereby enabling the imaging assembly to capture images along the length of the channel. The processor is also configured to activate the imaging assembly to capture one or more calibration images of one or more calibration regions of the channel during a first portion of the continuous range of motion. The processor is also configured to activate the imaging assembly to capture images of the reaction sites during a second portion of the continuous range of motion.

Description

[Background technology]

[0001] The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, it should not be assumed that the problems mentioned in this section or problems associated with the subject matter provided as background have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which themselves may also correspond to implementations of the claimed technology.

[0002] Aspects of the present disclosure relate generally to biological or chemical analysis, and more particularly to systems and methods that use image sensors for biological or chemical analysis.

[0003] Various protocols in biological or chemical research involve conducting multiple controlled reactions on a localized support surface or within a predetermined reaction chamber. The designated reactions can then be observed or detected, and subsequent analysis can help identify or characterize the chemicals involved in the reactions. For example, in some multiplex assays, an unknown analyte bearing an identifiable label (e.g., a fluorescent label) can be exposed to thousands of known probes under controlled conditions. Each known probe can be deposited in a corresponding well of a flow cell channel. Observing any chemical reactions that occur between the known probes and the unknown analyte in the well can help identify or characterize the analyte. Other examples of such protocols include known DNA sequencing processes, such as sequencing-by-synthesis (SBS) or circular array sequencing.

[0004] Some conventional fluorescence detection protocols use optical systems to direct excitation light onto fluorescently labeled analytes and to detect fluorescent signals that may be emitted from the analytes. Such optical systems may include an arrangement of lenses, filters, and light sources. It may be desirable to provide calibration of such optical systems without substantially affecting overall processing time. [Brief explanation of the drawings]

[0005] [Figure 1] 1 shows a schematic diagram of an example of an imaging assembly that may be implemented in a system for biological or chemical analysis. [Figure 2] 2 shows a perspective view of an example of a flow cell that can be used with the system of FIG. 1. [Figure 3] FIG. 4 shows an enlarged perspective view of the channels of the flow cell of FIG. 3. [Figure 4] 2 shows a plan view of another example of a flow cell that can be used with the system of FIG. 1. [Figure 5] 5 shows an enlarged top view of the channels of the flow cell of FIG. 4. [Figure 6] 10 is a graph showing an example of a capture position in the focus model generation process. [Figure 7] 10 shows a motion profile illustrating an example of an integrated through-focus path for moving an objective lens of an imaging assembly for focus model generation and / or updating. [Figure 8] 10 shows a graph of detecting a focus tracking spot reflected by a first surface and a second surface. [Figure 9] 10 shows a pair of graphs illustrating a substantially linear relationship between the position of the detected focus tracking spot versus the z-position height of the objective lens of the imaging assembly. [Figure 10] 1 shows a graph showing the average spot separation values ​​through the calibration area of ​​the flow cell. [Figure 11] 10 shows a graph illustrating image quality score values ​​through a calibration area of ​​a flow cell. [Figure 12]10 is a graph showing image quality score values ​​versus average spot separation values. [Figure 13] 1 shows a flowchart illustrating an example method for dynamically calibrating optical system components. [Figure 14] 1 shows a flowchart illustrating an example method for dynamically calibrating optical system components. [Figure 15] 1 shows an exemplary set of regions of interest. [Figure 16] Specific approaches that can be taken when performing a calibration operation are presented. DETAILED DESCRIPTION OF THE INVENTION

[0006] I. Overview of Systems for Biological or Chemical Analysis Described herein are devices, systems, and methods for dynamically optically calibrating an imaging assembly for a biological or chemical analytical system. Dynamic optical calibration can improve the performance of a biological or chemical analytical system by improving image quality at one or more points on a substrate of interest during a scanning process. The examples described herein can be used in a variety of biological or chemical processes and systems for academic, commercial, or other analysis. More specifically, the examples described herein can be used in a variety of processes and systems in which it is desirable to detect an event, characteristic, quality, or property indicative of a specified response.

[0007] Bioassay systems as described herein can be configured to perform multiple designated reactions that can be detected individually or collectively. Biosensors and bioassay systems can be configured to perform multiple cycles in which multiple designated reactions occur in parallel. For example, bioassay systems can be used to sequence high-density arrays of nucleic acid features through repeated cycles of enzymatic operation and image acquisition. Cartridges and biosensors used in bioassay systems 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 across a substantially planar surface or can be patterned across the substantially planar surface in a predetermined manner, such as a hexagonal pattern, a rectilinear pattern, or any other repeating pattern. In some variations, the reaction sites are located within reaction chambers that compartmentalize the designated reactions therein.

[0008] Regardless of the form taken by the reaction site, each of the reaction sites can be imaged to detect light from the reaction site. In some examples, one or more image sensors may detect light emitted from the reaction site. The signals indicative of the photons emitted from the reaction site and detected by the individual image sensors can be referred to as illumination values ​​of those sensors. These illumination values ​​can be combined into images indicative of the photons detected from the reaction site. These images can be further analyzed to identify the composition, reaction, conditions, etc. at each reaction site.

[0009] The following detailed description of certain examples will be better understood when read in conjunction with the accompanying 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 piece of hardware (e.g., a general-purpose signal processor or random access memory, a hard disk, etc.). Similarly, a program may be a stand-alone program, may be incorporated as a subroutine within an operating system, may be a function within an installed software package, etc. It should be understood that the various examples are not limited to the arrangements and instrumentality shown in the drawings.

[0010] As used herein, elements or steps described in the singular and followed by the words "a" or "an" should be understood as not excluding a plurality of those elements or steps, unless such exclusion is expressly stated. Furthermore, references to "one example" are not intended to be interpreted as excluding the existence of additional examples that also incorporate the recited features. Furthermore, unless expressly stated to the contrary, examples "comprising" or "having" an element or elements having a particular characteristic can include additional elements, whether or not they have that characteristic.

[0011] As used herein, a "designated reaction" includes a change in at least one of the chemical, electrical, physical, or optical properties (or qualities) of an analyte of interest. In some examples, the designated reaction is a positive binding event (e.g., incorporation of one or more fluorescently labeled biomolecules with the analyte of interest). More generally, the designated reaction can be a chemical conversion, chemical change, or chemical interaction. In some examples, the designated reaction includes incorporation of a fluorescently labeled molecule into the analyte. The analyte can be an oligonucleotide, and the fluorescently labeled molecule can be a nucleotide. The designated reaction can be detected when excitation light is directed at an oligonucleotide bearing a labeled nucleotide and the fluorophore of the labeled nucleotide emits a detectable fluorescent signal. In alternative examples, the detected fluorescence is the result of chemiluminescence or bioluminescence. The specified reaction can also, for example, increase fluorescence (or Förster) resonance energy transfer (FRET) by bringing a donor fluorophore into close proximity with an acceptor fluorophore, decrease FRET by separating the donor and acceptor fluorophores, increase fluorescence by separating a quencher from a fluorophore, or decrease fluorescence by coexisting a quencher and fluorophore.

[0012] 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 may be delivered to a reaction site in solution and / or may be immobilized at the reaction site. Reaction components may interact directly or indirectly with another substance, such as an analyte of interest.

[0013] As used herein, the term "reaction site" refers to a localized region where a specified reaction can occur. A reaction site can include a support surface of a substrate on which a substance can be immobilized or positioned. For example, a reaction site can include a substantially planar surface within a channel of a flow cell having nucleic acid colonies thereon. The nucleic acids in the colonies can have substantially the same sequence, e.g., clonal copies of a single-stranded or double-stranded template. In some examples, the nucleic acids in the colonies can be polyclonal by having two or more populations of clonal copies of different templates. In some implementations, a polyclonal colony can still be detectable if one of the clonal copies is distinguishable from the other clonal copies. However, in some examples, a reaction site may contain only a single nucleic acid molecule, e.g., in single-stranded or double-stranded form. Furthermore, multiple reaction sites can be randomly distributed along the support surface or arranged in a predetermined manner (e.g., side-by-side in a matrix such as a microarray). A reaction site can also include a reaction chamber that at least partially defines a spatial region or volume configured to compartmentalize a specified reaction. As used herein, the term "reaction chamber" includes a spatial region in fluid communication with a flow channel. A reaction chamber may be at least partially isolated from the surrounding environment or another spatial region. For example, multiple reaction chambers may be separated from one another by a shared wall, by differences in the height of their bases, by vertical location along a sidewall, or by other distinguishable separating features. As a more specific example, a reaction chamber may include a cavity defined by the inner surface of a well and have an opening or aperture such that the cavity is in fluid communication with the flow channel. A reaction site need not necessarily be provided within a reaction chamber, but may instead be provided on or within any other suitable type of structure.

[0014] As used herein, the term "adjacent," when used in reference to two reaction sites, means that there are no other reaction sites between the two reaction sites. The term "adjacent" can have a similar meaning when used in reference to adjacent detection paths and adjacent image sensors (e.g., adjacent image sensors have no other image sensors between them). In some cases, a reaction site may not be adjacent to another reaction site but may still be in close proximity to another reaction site. A first reaction site may be in close proximity to a second reaction site if a fluorescent emission signal from the first reaction site is detected by an image sensor associated with the second reaction site. More specifically, a first reaction site may be in close proximity to a second reaction site if an image sensor associated with the second reaction site detects, for example, crosstalk from the first reaction site. Adjacent reaction sites may be contiguous such that they are adjacent to one another, or adjacent sites may be non-contiguous with an intervening space between them, such as an interstitial space.

[0015] As used herein, "substance" includes items or solids such as capture beads, as well as biological or chemical substances. As used herein, "biological or chemical substances" includes biomolecules, samples of interest, analytes of interest, and other chemical compounds. Biological substances or chemical substances may be used to detect, identify, or analyze other chemical compounds, or serve as intermediaries for studying or analyzing other chemical compounds. In certain examples, biological substances or chemical substances include biomolecules. As used herein, "biomolecules" include at least one of biopolymers, nucleotides, nucleic acids, polynucleotides, oligonucleotides, proteins, enzymes, polypeptides, antibodies, antigens, ligands, receptors, polysaccharides, carbohydrates, polyphosphates, cells, tissues, organisms, or fragments thereof, or any other biologically active chemical compounds, such as analogs or mimetics of the foregoing species.

[0016] The biomolecules, samples, and biological or chemical substances may be naturally occurring or synthetic and may be suspended in a solution or mixture within the spatial region. The biomolecules, samples, and biological or chemical substances may also be bound to a solid phase or gel material. The 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.

[0017] As used herein, when the terms “removably” and “coupled” (or “engaged”) are used together to describe a relationship between components, the term is intended to mean that the connection between the components is readily separable without destroying or damaging the components. Components are readily separable if they can be separated from one another without undue effort or significant time expenditure in separating the components. For example, components may be removably coupled or engaged electrically such that mating contacts of the components are not destroyed or damaged. Components may also be removably coupled or engaged in a mechanical manner such that features that retain the components are not destroyed or damaged. Components may also be removably coupled or engaged in a fluidic manner such that ports of the components are not destroyed or damaged. A component is not considered to be destroyed or damaged if, for example, only a simple adjustment (e.g., realignment) or simple replacement (e.g., nozzle replacement) to the component is required.

[0018] As used herein, the terms "fluid communication" or "fluidically coupled" refer to two spatial regions that are connected together such that liquid or gas can flow between the two spatial regions. For example, a microfluidic channel may be in fluid communication with a reaction chamber such that fluid can flow freely from the microfluidic channel into the reaction chamber. The terms "fluid communication" or "fluidically coupled" refer to two spatial regions that are in fluid communication through one or more valves, restrictors, or other fluidic components, allowing for the control or regulation of fluid flow through the system.

[0019] In some instances, nucleic acids can be attached to a surface and amplified. Examples of such amplification are described in U.S. Patent No. 7,741,463, entitled "Method of Preparing Libraries of Template Polynucleotides," issued June 22, 2010, the disclosure of which is incorporated herein by reference in its entirety, and / or U.S. Patent No. 7,270,981, entitled "Recombinase Polymerase Amplification," issued September 18, 2007, the disclosure of which is incorporated herein by reference in its entirety. In some instances, repeated rounds of extension (e.g., amplification) using immobilized and in-solution primers can provide multiple copies of the nucleic acid.

[0020] In certain examples, assay protocols performed by the systems and methods described herein include the use of naturally occurring nucleotides and enzymes configured to interact with naturally occurring nucleotides. Naturally occurring nucleotides include, for example, ribonucleotides or deoxyribonucleotides. Naturally occurring nucleotides may be in monophosphate, diphosphate, or triphosphate form and may have a base selected from adenine (A), thymine (T), uracil (U), guanine (G), or cytosine (C). However, it will be understood that non-naturally occurring nucleotides, modified nucleotides, or analogs of the foregoing nucleotides may be used.

[0021] FIG. 1 illustrates an example of components of a system 100 that can be used to provide biological or chemical analysis. In some examples, the system 100 is a workstation, which can be similar to a benchtop device. For example, most (or all) of the system and components for performing a specified reaction may reside within a common housing. In certain examples, the system 100 is a nucleic acid sequencing system (or sequencer) configured for a variety of applications, including, but not limited to, de novo sequencing, resequencing of whole genomes or targeted genomic regions, and metagenomics. Sequencers may also be used for DNA or RNA analysis. In some variations, the system 100 may also be configured to generate reaction sites within the flow cell 110. For example, the system 100 may be configured to receive a sample and generate surface-attached clusters of clonally or substantially clonally amplified nucleic acids derived from the sample. In some implementations, a cluster may comprise a particular sample that is a distinct portion of the cluster, even if the cluster is polyclonal, as a result of one or more other samples present within the cluster. The system (100) is further configured to utilize an imaging assembly (122) to capture images of the reaction sites on the flow cell (110).

[0022] In a particular example, the system 100 is for performing multiple parallel reactions within a flow cell 110. The flow cell 110 includes one or more reaction sites where designated reactions can occur. The reaction sites may be immobilized or aligned on a solid surface of the flow cell 110, or may be immobilized on beads (or other movable substrates) located within corresponding reaction chambers of the flow cell 110. The reaction sites may include, for example, clusters of clonally amplified nucleic acids. The flow cell 110 may include one or more flow channels that receive solutions from the system 100 and direct the solutions toward the reaction sites. Optionally, the flow cell 110 may be engaged with a thermal element for transferring thermal energy into or out of the flow channels.

[0023] 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 biological or chemical analysis. For example, the system 100 includes a system controller 120 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 120 may include one or more microprocessors, storage devices, and / or any other suitable electrical components configured to cooperate to execute control algorithms, data processing, etc.

[0024] In this example, the imaging assembly (122) includes a light-emitting assembly (150) that emits light that reaches the reaction sites on the flow cell (110). The light-emitting assembly (150) may include an incoherent light emitter (e.g., emitting a light beam output by one or more excitation diodes) or a coherent light emitter, such as an emitter of light output by one or more lasers or laser diodes. In some implementations, the light-emitting assembly (150) may include multiple different light sources (not shown), each emitting light in a different wavelength range. Some variations of the light-emitting assembly (150) may also include one or more collimating lenses (not shown), a light structuring optical assembly (not shown), a projection lens (not shown) operable to adjust the structured beam shape and path, epifluorescence microscope components, and / or other components. While the system (100) is shown as having a single light-emitting assembly (150), in some other implementations, multiple light-emitting assemblies (150) may be included.

[0025] In this example, light from the light emitting assembly (150) is directed by a dichroic mirror assembly (146) through an objective lens assembly (142) onto a sample in a flow cell (110) positioned on a motion stage (170). For fluorescence microscopy of a sample, fluorescent elements associated with the sample of interest fluoresce in response to 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. In some implementations, a tube lens assembly can be positioned between the objective lens assembly (142) and the dichroic mirror assembly (146) or between the dichroic mirror (146) and the image sensor of the camera system (140). The movable lens element can be translatable along the longitudinal axis of the tube lens assembly to account for spherical aberrations introduced by focusing onto the upper or lower inner surface of the flow cell (110) and / or by movement of the objective lens assembly (142).

[0026] In this example, the filter switching assembly (144) is inserted between the dichroic mirror assembly (146) and the camera system (140). The filter switching assembly (144) includes one or more emission filters that can be used to pass specific ranges of emission wavelengths and block (or reflect) other ranges of emission wavelengths. For example, one or more emission filters can be used to direct different wavelength ranges of emitted light to different image sensors of the camera system (140) of the imaging assembly (122). For example, the emission filters may be implemented as dichroic mirrors that direct different wavelengths of emitted light from the flow cell (110) to different image sensors of the camera system (140). In some variations, a projection lens is inserted between the filter switching assembly (144) and the camera system (140). The filter switching assembly (144) may be omitted in some variations.

[0027] In the example system 100, a fluid delivery module or device 190 can direct the flow of reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and through) the flow cell 110 and waste valve 180. The flow cell 110 can include one or more substrates to which a sample is provided. For example, in a system for analyzing a large number of different nucleic acid sequences, the flow cell 110 can include one or more substrates to which nucleic acids to be sequenced are bound, attached, or associated. Substrates can include any inert substrate or substrate to which nucleic acids can be attached, such as glass surfaces, plastic surfaces, latex, dextran, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, and silicon wafers. In some applications, the substrate includes channels formed in a substrate or other area, either in a channel or at multiple locations formed in a matrix or array across the flow cell 110. The system 100 may also include a temperature station actuator 130 and a heater / cooler 132 that may optionally adjust the temperature of the fluid conditions within the flow cell 110. In some implementations, the heater / cooler 132 may be affixed to and / or integrated into the sample stage 170 on which the flow cell 110 is mounted.

[0028] In some variations, the flow cell (110) may be implemented as a patterned flow cell configured to contain a liquid between a transparent cover plate, a substrate, and / or the substrate, and the biological sample may be located on the inner surface of the transparent cover plate and / or the inner surface of the substrate. The flow cell may include a large number (e.g., thousands, millions, or billions) of wells (also referred to as nanowells) or regions within the substrate that are patterned into a defined array (e.g., a hexagonal array, a rectangular array, etc.). Such wells may define reaction chambers that provide reaction sites as described above. Each region may form a cluster (e.g., a monoclonal cluster, a substantially monoclonal cluster, or a polyclonal cluster) or multiple clusters of biological samples, such as DNA, RNA, or other genomic material, that can be sequenced using sequencing-by-synthesis. A substantially monoclonal cluster may be one in which a particular sample forms a distinguishable portion of the cluster, even if the cluster itself is polyclonal as a result of the presence of one or more other samples within the cluster. The flow cell may be further divided into a number of spaced lanes (eg, eight lanes), each of which contains a hexagonal array of clusters or a linear array of clusters.

[0029] The flow cell 110 may be mounted on a sample stage 170, which may provide movement and alignment of the flow cell 110 relative to the objective lens assembly 142. The sample stage 170 may have one or more actuators to enable the sample stage 170 to move in any of three dimensions. For example, with respect to a Cartesian coordinate system, actuators may be provided to enable the sample stage 170 to move in the 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 enable one or more sample locations on the flow cell 110 to be positioned in optical alignment with the objective lens assembly 142. Movement of the sample stage 170 relative to the objective lens assembly 142 may be achieved by moving the sample stage 170 itself, by moving the objective lens assembly 142, by moving some other component of the imaging assembly 122, by moving some other component of the system 100, or by any combination of the foregoing. For example, in some implementations, the sample stage 170 may be actuable in the X and Y directions relative to the objective lens assembly 142, while the focusing component 162 or Z-stage may move the objective lens assembly 142 along the Z direction relative to the sample stage 170. Further implementations may also include moving the imaging assembly 122 over a stationary flow cell 110. Thus, in some variations, the flow cell 110 may be fixed during imaging, while one or more components of the imaging assembly 122 are moved to capture images at different regions of the flow cell 110.

[0030] In some implementations, a focus component (162) may be included to control the positioning of the objective lens relative to the flow cell (110) in a focus direction (e.g., along the z-axis or z-dimension). The focus component (162) may include one or more actuators physically coupled to the objective lens assembly (142), the optical stage, 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 proper focusing for imaging operations. In this example, the focus component (162) utilizes a focus tracking module (160) configured to detect displacement of the objective lens assembly (142) relative to a portion of the flow cell (110) and output data indicative of the focus position to the focus component (162) or a component thereof, or operable to control the focus component (162), such as the controller (120), to move the objective lens assembly (142) and position the corresponding portion of the flow cell (110) at the focus of the objective lens assembly (142). By way of example only, the focus tracking module (160) may be constructed and operable in accordance with at least part of the teachings of U.S. Patent No. 10,416,428, entitled "Systems and Methods for Improved Focus Tracking Using a Light Source Configuration," issued September 17, 2019, the entire disclosure of which is incorporated herein by reference; U.S. Provisional Patent Application No. 63 / 300531, entitled "Dynamic Detilt Focus Tracking," filed January 18, 2022, the entire disclosure of which is incorporated herein by reference; or U.S. Provisional Patent Application No. 63 / 410,961, entitled "Spot Error Handling for Focus Tracking," filed September 28, 2022, the entire disclosure of which is incorporated herein by reference.

[0031] In some implementations, the actuators for the focusing component (162) or sample stage (170) may be physically coupled to the objective lens assembly (142), the optical stage, the sample stage (170), or a combination thereof, such as by direct or indirect mechanical, magnetic, fluidic, or other attachment or contact to or with the stage or its components. The actuators of the focusing component (162) may be configured to move the objective lens assembly (142) in the z-direction while maintaining the sample stage (170) in the same plane (e.g., while maintaining a level or horizontal orientation perpendicular to the optical axis). In some implementations, the sample stage (170) includes an X-direction actuator and a Y-direction actuator to form an XY stage. The sample stage (170) may also be configured to include one or more tilt actuators to tilt the sample stage (170) and / or a portion thereof, such as a flow cell chuck. This may be done, for example, so that the flow cell (110) can be dynamically leveled to account for any tilt of its surface.

[0032] The camera system 140 may include one or more image sensors for monitoring and tracking imaging (e.g., sequencing) of the flow cell 110. The camera system 140 may be implemented, for example, as 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 technologies. Although the camera system 140 and associated optical components are shown in FIG. 1 as being positioned above the flow cell 110, one or more image sensors or other camera components may be incorporated into the system 100 in numerous 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 the flow cell (110), such as in or below the sample stage (170), or may be integrated into the flow cell (110).

[0033] II. Example of flow cell structure 2-3 illustrate an example of a possible configuration for the flow cell 110. Specifically, FIGS. 2-3 illustrate an example of a flow cell 200 including a body 202 defining a plurality of elongated flow channels 210, which may be formed in one or more of the bottom surface 206 or top surface 204 and / or may be formed by one or more of the bottom surface 206, top surface 204, one or more intervening layers, and / or one or more adhesive layers structured in a stacked configuration to form the body 202. The flow channels 210, in this example, are generally parallel to one another and extend along substantially the entire length of the body 302. However, in other implementations, the flow channels 210 may be positioned radially relative to one another, perpendicular to one another, and / or in any other angular relationship within substantially the same plane. Although five flow channels 210 are shown, the flow cell 200 may include any other suitable number of flow channels 210, including more or less than five flow channels 210, such as two flow channels 210, four flow channels 210, eight flow channels 210, etc. The flow cell 200 of this example also includes a set of inlet ports 220 and a set of outlet ports 222, each port 220, 222 associated with a corresponding flow channel 210. Thus, each inlet port 220 may be utilized to communicate fluid (e.g., reagents, etc.) into a corresponding channel 210, while each outlet port 222 may be utilized to communicate fluid from a corresponding flow channel 210. In some implementations, two or more flow channels 210 may be fluidly connected by a connecting channel such that two or more flow channels 210 utilize a single inlet port 220 and outlet port 222. In some implementations, the inlet port 220 and outlet port 222 may be positioned or formed in opposite ends of the flow cell 200, may be positioned or formed in substantially the same end of the flow cell 200, and / or may be positioned or formed in any other fixed location of the flow cell 200.

[0034] The flow channel 210 of the flow cell 200 can receive reagent fluid from a fluid delivery module 190, which can be fluidly coupled to reagents stored in one or more consumable reagent containers (not shown). Additionally or alternatively, the flow channel 210 can be coupled to various other fluid sources, reservoirs, or the like. As another exemplary variation, some consumable cartridge variations can be configured to removably receive or otherwise integrate the flow cell 200 into a consumable cartridge (not shown) that is removably seated within the sample stage 170. In some such variations, the flow channel 210 of the flow cell 200 can receive fluid from a reagent volume (not shown) via an inlet port 220. Alternatively, the flow cell 200 can be incorporated into the system 100 in any other suitable manner.

[0035] FIG. 3 shows the flow channel 210 of the flow cell 200 in more detail. As shown, the flow channel 210 includes a plurality of wells 230 formed within the base surface 212 of the flow channel 210. By way of example only, each well 230 may be configured to contain a nucleic acid strand or other oligonucleotide, thereby providing a reaction site for SBS and / or other types of processes. In some variations, each well 230 has a cylindrical configuration with a generally circular cross-sectional profile. In some other variations, each well 230 has a polygonal (e.g., hexagonal, octagonal, square, rectangular, elliptical, etc.) cross-sectional profile. Alternatively, the wells 230 may have any other suitable configuration. It should also be understood that the wells 230 may be arranged in any suitable pattern, including, but not limited to, a grid pattern.

[0036] III. Example Channel Configurations and Processes for Dynamic Optical System Calibration In various processes (e.g., SBS processes) in which the system 100 may be used, it may be useful to maintain the imaging assembly 122 in a substantially calibrated position to capture suitable images of reaction sites within the flow cell 110, 200. To the extent that a calibration routine is initiated at the beginning of an SBS process (or other process), such as an initial tile or swath of the flow cell 110, 200, such calibration may be less accurate at later positions, such as later tiles or swaths of the SBS process (or other process). For example, thermal and / or other conditions may affect the structural properties of one or more features of the imaging assembly 122 and / or the flow cell 110, 200 throughout the duration of an SBS process, such as radiative heating from laser irradiation, or other processes. The accuracy of the system's calibration can be improved despite thermally induced effects (or other environmental effects) by dynamic recalibration of the imaging assembly (122) to ensure that the images captured by the imaging assembly (122) remain suitable throughout the remainder of the SBS process (or other process).

[0037] Factors other than thermal or other environmental factors can also affect calibration, thus providing situations in which dynamic recalibration may be beneficially applied. For example, errors in the position encoders for the sample stage (170) and / or the encoders for the focusing components (162), such as the z-stage, can accumulate over time or travel distance, resulting in drift that can affect the system's calibration. Similarly, in some cases, implementation of other error correction techniques can result in mechanical changes to the imaging assembly that can affect calibration. For example, in some cases, the imaging assembly may be configured to compensate for differences in coverslip thickness through movement of physical imaging components, such as a zoom lens. In other cases (or when the imaging assembly is configured to make mathematical changes to compensate for coverslip thickness), the imaging assembly may be configured to correct for astigmatism through manipulation of a compensation plate, as described in U.S. Published Patent Application No. 2023 / 0108792, filed September 30, 2022, for "Apparatus and Methods for Transmitting Light," the disclosure of which is incorporated herein by reference in its entirety. In these types of cases, dynamic recalibration as described herein can be used to compensate for the unintended effects of mechanical changes caused by other error correction techniques implemented within the imaging assembly (e.g., dynamic recalibration can be used to change the position of the objective lens to compensate for the effects of zoom lens or compensation plate movement). The improvement provided by dynamic recalibration can increase with the sensitivity of the imaging assembly 122. For example, increasing the sensitivity of the imaging assembly 122 can result in the imaging assembly 122 being more susceptible to thermally induced effects or other accumulated errors. Additionally, or alternatively, increasing the sensitivity of the imaging assembly 122 can simply make thermally induced effects or accumulated errors more apparent than they would be with a less sensitive imaging assembly 122.

[0038] Continuous static calibration methods for the imaging assembly 122 and / or sample stage 170 can increase overall processing or turnaround time, especially if the SBS process (or other process) is interrupted to perform one or more calibration routines after the SBS process (or other process) has begun. For example, in some cases, the SBS process may provide a specific series of movements of the imaging assembly 122 relative to the flow cell 110, 200 (and / or a specific series of movements of the flow cell 110, 200 relative to the imaging assembly 122). An example of such a series of movements may include the imaging assembly 122 capturing images or data indicative of illumination intensity of reaction sites within the channel 210 as the imaging assembly 122 moves in the y-direction along the length of the channel 210. In some such variations, the imaging assembly (122) may move back and forth in the y direction over a particular channel (210) a certain number of times (e.g., making multiple passes along the length of the channel (210) for different swaths) before moving in the x direction to begin imaging reaction sites in the next channel (210).

[0039] If such an SBS process is initiated and a static calibration routine is performed after the SBS process is initiated, the static calibration routine may interrupt the particular series of movements of the imaging assembly 122 relative to the flow cell 110, 200 (and / or the particular series of movements of the flow cell 110, 200 relative to the imaging assembly 122) that are performed during the SBS process. Therefore, it may be desirable to provide an alternative in-service calibration routine that can be performed in a manner that does not increase the overall time required to perform the SBS process. Furthermore, it may be desirable to provide an alternative calibration routine that can be dynamically performed throughout the performance of the SBS process to account for any thermal expansion, deformation, or other structural effects that may occur during the SBS process due to increased heat or other environmental conditions, and / or to correct for potential accumulated errors over the course of the scanning process; such dynamic calibration does not interrupt the imaging process that would normally be performed as part of the SBS process.

[0040] A. Example of a flow cell with a calibration area 4 illustrates an example of a flow cell 300 that can be used to provide dynamic calibration of the imaging assembly 122 during an SBS process (or other process). In other words, the flow cell 300 can be used in a process to allow the controller 120 to acquire focus calibration characteristics (such as the z-position of the focusing element 162 relative to one or more surfaces of the flow channel) while the sample stage 170 and / or imaging assembly 122 are moving, or to account for thermally induced and / or other changes in the imaging assembly 122 and / or flow cell 300 in real time, thereby improving the quality of images captured by the imaging assembly 122 throughout the duration of the SBS process (or other process). The flow cell 300 represents another example of a form that can be taken by the flow cells 110, 200. Except as otherwise described below, the flow cell 300 in this example can be configured and operative similarly to the flow cell 200 described above. The flow cell 300 in this example includes eight flow channels 310. Each flow channel 310 may be formed in one or more of the bottom or top surface 304 and / or may be formed by one or more of the bottom surface, top surface 304, one or more intervening layers, and / or one or more adhesive layers structured in a laminate configuration of the body 302 of the flow cell 300. The flow channels 310 are generally parallel to one another and extend substantially along the entire length of the body 302. The flow cell (300) in this example has eight flow channels (310), but any other suitable number of flow channels (310) may be provided, such as one flow channel (310), two flow channels (310), three flow channels (310), four flow channels (310), five flow channels (310), six flow channels (310), seven flow channels (310), or more than eight flow channels (310).

[0041] Each channel (310) includes a first end (320), a second end (322), and a middle region (324) extending along a length between the ends (320, 322). As shown in FIG. 4, this length extends in the y-direction in this example. Although not shown in FIGS. 4-5, each channel (310) may include multiple wells (230) or other structural features that provide reaction sites. In some variations, such wells or other structural features that provide reaction sites are positioned only along the middle region (324). In some other variations, such wells or other structural features that provide reaction sites extend all the way to the ends (320, 322). In either case, the wells or other structural features that provide reaction sites within the channel (310) may contain nucleic acid strands or other oligonucleotides for SBS and / or other types of processes.

[0042] In this example, a pair of alignment features 330, 332 are provided near each end 320, 322 of alternating channels 310. In some other variations, alignment features 330, 332 are provided in all channels 310, only one end 320 or 322, only one channel 310, or in any other suitable arrangement. As best seen in FIG. 5 , alignment feature 330 is square-shaped, while alignment feature 332 is plus-sign or cross-shaped. Alternatively, alignment features 330, 332 may take any other suitable form. Alignment feature 332 can be used for XY alignment calibration, for example, to align a known pattern of a flow cell having patterned features to a loaded flow cell in the system. The alignment features 330 can be configured to function as optical fiducials to facilitate optical alignment between the imaging assembly 122 and the flow cell 110. For example, the imaging assembly 122 can capture an image of the flow cell 110. The controller 120 can identify the alignment features 330, 332 in the captured image. Based on the identified positions of the alignment features 330, 332 in the captured image and / or the characteristics of the alignment features 330, 332, the positions of one or more features of the imaging assembly 122 and / or the position of the flow cell 110 can be adjusted to provide proper optical alignment between the imaging assembly 122 and the flow cell 110 and / or to match the known positions of a known pattern of patterned-feature flow cells to a loaded flow cell in the system. Additionally, or alternatively, subsequent image processing can be adjusted based on the identified positions of the alignment features 330, 332 in the captured image. Alternatively, optical alignment may be provided in any other suitable manner.

[0043] As shown in FIG. 5, the calibration area (340) is defined by a first boundary (342) within the channel (310) and a second boundary (344) within the channel (310). The calibration area (340) is positioned between the intermediate region (324) and the first end (320). It should be understood that a second calibration area may be positioned between the intermediate region (324) and the second end (322), and the second calibration area may be configured and used like the calibration area (340). Thus, the intermediate region (324) may extend between the two calibration areas (340). Alternatively, only the second calibration area may be utilized instead of the first calibration area (340). As shown in FIG. 5, the alignment features (330, 332) are positioned between the calibration area (340) and the first end (320). The specific configuration of the calibration area (340) may differ from that shown in FIG. 5 . For example, one or more of the alignment features (330, 332) may be incorporated into the calibration area (340). In other examples, the first boundary (342) may be moved closer to or further away from the first end (320). Similarly, the second boundary (344) may be moved closer to or further away from the first end (320). It should also be understood that a second calibration area may be positioned between the intermediate area (324) and the second end (322), with the second calibration area being configured and used like the calibration area (340).

[0044] As described above, the channels 310 include wells or other structural features that provide reaction sites, which may contain nucleic acid strands or other oligonucleotides for SBS and / or other types of processes. In this example, such wells or other structural features that provide reaction sites extend along the length of each calibration region 340. Thus, each calibration region 340 includes wells or other structural features that provide reaction sites that may contain nucleic acid strands or other oligonucleotides for SBS and / or other types of processes. The middle region 324 of each channel 310 also includes wells or other structural features that provide reaction sites that may contain nucleic acid strands or other oligonucleotides for SBS and / or other types of processes. Thus, in some variations, the structural configuration and presence of nucleic acid strands or other oligonucleotides is the same throughout the middle region 324 and both calibration regions 340 of each channel 310.

[0045] B. An example of the calibration process As described above, in some cases, the SBS process may provide a specific sequence of movement of the imaging assembly 122 relative to the flow cell 110, 200, 300 (and / or a specific sequence of movement of the flow cell 110, 200, 300 relative to the imaging assembly 122). Depending on the magnitude of the acceleration initiating this relative movement, the structural configuration of the system 100, and / or other factors, there may be some degree of wobble or vibration of the imaging assembly 122 and / or the flow cell 110, 200, 300 during the initial phase of the relative movement between the imaging assembly 122 and the flow cell 110, 200, 300. In some cases, this wobble or vibration may adversely affect the quality of the images captured by the imaging assembly 122 during the initial phase of the relative movement between the imaging assembly 122 and the flow cell 110, 200, 300. Thus, some conventional SBS systems may tend to avoid capturing images or effectively ignore captured images during the early stages of relative movement between the imaging assembly 122 and the flow cell 110, 200, 300. In such a scenario, regions near the ends 220, 222, 320, 322 of the channels 210, 310 of the flow cells 110, 200, 300 may be ignored regions, such that images of samples within those regions, including those associated with the longitudinal position of the calibration region 340, are not utilized. However, in this example, the system 100 advantageously utilizes focus tracking data captured during movement through the calibration region 340 to determine or update a focus model applied to the system 100 as the imaging assembly 122 scans a swath of the flow channel 310 of the flow cell 300. Such focus tracking data can be used to facilitate calibration of the imaging assembly (122), as described below.

[0046] FIG. 6 illustrates one example of how the focus tracking module 160 and other features of the imaging assembly 122 can be used to provide an initial static calibration by capturing images at different elevations relative to the focusing component 162. Specifically, FIG. 6 illustrates a graph 400 depicting a plot 402 representing depth of image capture (“z-height”) as a function of longitudinal position (“y-travel”) along the channel 310. Each horizontal line 420 in the plot 402 represents a period of image capture by the imaging assembly 122, with images captured between points 430 where the z-height remains constant while the y-position changes. In other examples, the y-position of such points 430 may remain substantially the same. It should be understood that the z and y directions depicted on the axes of the graph 400 correspond to the z and y directions depicted in FIGS. 1, 2, and 4-5. In describing the motion represented by plot 402, the following describes movement of the flow cell 300 relative to the imaging assembly 122 along the y-axis. Such movement of the flow cell 300 relative to the imaging assembly 122 may be driven by one or more actuators of the sample stage 170. Some other variations may provide similar relative movement by moving the imaging assembly 122 relative to the flow cell 300 along the y-axis.

[0047] As shown in FIG. 6 , a first image (or set of images) can be captured at a first z-height. In some cases, the first image is captured while the imaging assembly (122) or sample stage (170) remains stationary. In other examples, the imaging assembly (122) can be moved through a first range of y-motion along the channel (310). Then, after the z-height has changed between points (410), a second image (or set of images) can be captured at a second z-height. The change in z-height between points (410) can be achieved by moving the objective lens assembly (142) relative to the flow cell (300) along the z-axis, such as by using a z-stage motion controller for the focusing component (162). In some implementations, the z-stage can include a voice coil actuator. Alternatively, the change in z-height between points (410) can be achieved by moving the flow cell relative to the objective lens assembly (142) along the z-axis. In either scenario, as shown in FIG. 6, the flow cell 300 can move relative to the imaging assembly 122 along the y-axis during z-height changes between points 410. In other examples, the flow cell 300 can remain stationary relative to the imaging assembly 122 during z-height changes. This process can continue, with the flow cell 300 either moving along the y-axis relative to the imaging assembly 122 or remaining in a substantially stationary y-axis position, until the desired image (or set of images) is captured at the desired z-height. In the example shown in FIG. 6, there are four discrete images (or four discrete image sets) captured at four discrete z-heights.

[0048] After an image (or set of images) is captured as described above with reference to FIG. 6, the images can be processed (e.g., by controller 120) to determine which image (or set of images) provides the best focus for the surface of interest, such as using conventional image processing techniques. The z-height associated with the image (or set of images) providing the best focus may then be utilized for subsequent imaging used as part of an SBS process (or other process involving, for example, nucleotides at reaction sites within channel 310). Thus, the process described above with reference to FIG. 6 can be used to initially calibrate imaging assembly 122.

[0049] In some systems, this calibration may be performed as a separate process that starts and ends before the SBS process (or other processes) begins, or it may interrupt an SBS process being performed. In some such cases, the process described above with reference to FIG. 6 may be performed over the entire length of the channel 310 and / or may be repeated several times before the entire calibration process is complete. Therefore, the SBS process may need to be delayed until the entire calibration process is complete. To the extent that the SBS process ultimately affects the imaging assembly 122, the flow cell 300, and / or other components of the system 100 (e.g., due to thermal expansion, etc.), and that effect warrants recalibration, conventional processes may provide for an interruption of the SBS process to perform the recalibration so that the SBS process does not begin again until the recalibration is complete. This can substantially increase overall processing time, and as a result, system operators may be forced to choose between providing an optimized calibration and providing an optimized processing time. Alternatively, if environmental factors such as thermal effects or accumulated errors such as position encoder errors increase beyond a predetermined threshold, the initial calibration may be inaccurate, the resulting imaging data may be less accurate, require increased post-acquisition processing, and / or a portion of the imaging data may be below a predetermined quality threshold.

[0050] FIG. 7 illustrates a motion profile 500 of how other features of the focus tracking module 160 and imaging assembly 122 can be used to provide calibration during dynamic motion by continuously capturing focus tracking data at different elevation positions relative to the focus component 162. In the illustrated example, the controller of the focus component 162 can be instructed to move the focus component from an initial position to a first position, such as +500 nanometers (nm) as shown, during a first motion 502, and then to a second position, such as −500 nm as shown, during a second motion 504. During this time, the sample stage 170 and / or imaging assembly 122 can move continuously along the y-direction through the calibration region 340. As described in connection with FIGS. 10-12, y-position encoders or other position tracking elements on the sample stage 170 and / or imaging assembly 122 can track the y-position relative to the z-position of the focus component 162. In some implementations, tracking the y position may include outputting the y position data values ​​to a log at predetermined intervals (eg, clock cycles).

[0051] During the movement shown in graph 500 of Figure 7, the focus tracking module sensor can receive focus tracking data such as that shown in graph 600 of Figure 8. In one implementation, features of the focus tracking module 160 or focusing component 162 can utilize a focus tracking illumination source to project a spot through the imaging assembly 122 toward the flow cell 300. In some cases, one or more beam splitters may be implemented to split the projected spot into two or more spots toward the flow cell 300. Because the flow cell 300 includes multiple interfaces between surfaces, the projected spot can be reflected by such interfaces of different surfaces. In an exemplary implementation, the flow cell may include a first surface interface (S1, not shown) at which the outer surface of the upper substrate material reflects the projected spot; a second surface interface (S2) at which the inner surface of the upper substrate and internal fluids or other materials positioned within the flow channel reflect the projected spot; a third surface interface (S3) at which the inner surface of the lower substrate and internal fluids or other materials positioned within the flow channel reflect the projected spot; and a fourth surface interface (S4, not shown) at which the outer surface of the lower substrate material reflects the projected spot. As shown in FIG. 8 , the sensor of the focusing component (162) may be configured to detect the illumination intensity of the spots reflected from the interfaces. In the illustrated implementation, a pair of focus tracking spots is used, and the detected illumination data of the reflected spots (604, 606, 608, 610) relative to the second surface interface (S2) and the third surface interface (S3) may be utilized to determine the x-axis position of each of the reflected spots (604, 606, 608, 610), represented by a pixel number.

[0052] Referring now to Figure 9, as the z-height of the objective lens (142) of the imaging assembly (122) is changed during the first movement (502) or the second movement (504), the x-position of the detected illumination data of the reflected spots varies in a substantially linear relationship with the z-height of the objective lens, as shown by graphs (710, 720). As shown in Figure 9, at a first z-height value of 1090 micrometers, a pair of spots (designated S2L and S2R) reflected from the same surface have a smaller spot separation compared to a pair of the same spots reflected from the same surface at a second z-height value of 1110 micrometers. Although the z-heights are shown in increasing values, the orientation of the z-axis in this graph is relative to the zero-value data, where the objective lens (142) is further away from the flow cell (300) and increasing z-height values ​​move the objective lens (142) closer to the flow cell (300).

[0053] In some implementations, a series of average spot separation values ​​may be obtained as the sample stage (170) and / or imaging assembly (122) move continuously along the Y direction through the calibration area (340) and may be correlated with corresponding y position encoder values ​​or other position tracking elements for the sample stage (170) and / or imaging assembly (122), as shown by graph (800) in Figure 10. The curve (802) shown in graph (800) may substantially correspond to the motion profile (500) shown in Figure 7. As shown, the curve (802) has a first portion (804) corresponding to the first movement (502) and a second portion (806) corresponding to the second movement (504).

[0054] During acquisition of a series of average spot separation values ​​during movement through the calibration area (340) shown in FIG. 10, the system (100) can also acquire a series of data indicative of image quality, such as an image quality score, at each y-position encoder value as the series of average spot separation values ​​is acquired, as shown in FIG. 11. In some implementations, this can be done by synchronizing the acquisition of two different systems. For example, the clock cycle of the acquisition system for the average sports separation values ​​can be used for the acquisition system for the data indicative of image quality. In some implementations, the acquisition system for the average spot separation values ​​can comprise a first printed circuit board assembly (PCBA) and can include a first FPGA, and the acquisition system for the data indicative of image quality can comprise a second printed circuit board assembly (PCBA) and can include a second FPGA. As shown in graph (900) in FIG. 11, multiple data points indicative of image quality at corresponding y-positions within the calibration area (340) can be acquired, and a smoothed curve fit (902) can be applied to the data. Although an image quality score is shown as being used, any other data indicative of image quality, such as a Brenner score, may be used.

[0055] FIG. 12 shows a graph (1000) illustrating the correlation of data points indicating image quality, such as an image quality score, with a series of data points of average spot spacing at the same y-position. In this example, the lower the image quality score, the better the image quality of the image acquired by the imaging assembly (122) of the system (100). A parabolic curve fit (1002) can be applied to the data points, and a minimum image quality score (1004) for this example of the curve fit (1002) can be identified, as well as a corresponding average spot separation value (1006). In some implementations, the average spot separation value (1006) can be used directly for the z-height position of the focal component (162) for subsequent imaging acquisition cycles to position the imaging assembly (122), or if the spot separation value is not used, a specific z-height position value can be identified. In implementations where higher values ​​indicate better image quality, a maximum value can be identified instead.

[0056] While the foregoing depictions in Figures 7-12 show graphical depictions of different values ​​calculated at different points by different components of the system (100) to determine z-height position or other values ​​indicative thereof for improved image quality, it should be understood that the values ​​may be calculated and implemented directly without graphical output.

[0057] FIG. 13 illustrates a process that may be employed using a flow cell, such as flow cell (300), in which calibration may be performed dynamically during the SBS process, such that calibration may remain optimized in real time without significantly increasing the overall time required to obtain SBS results. The process illustrated in FIG. 13 may begin with the imaging assembly (122) positioned above the first end (320). The flow cell (300) may then be moved in a first direction relative to the imaging assembly (122) along the y-axis, as shown in block (1100). As the flow cell (300) moves in this first direction along the y-axis relative to the imaging assembly (122), the field of view of the imaging assembly (122) may effectively move toward the second end (322). In some variations, as represented by the dashed rendering of block (1102) in FIG. 7 , the imaging assembly (122) may capture images and focus tracking data along the calibration region (340) near the first end (320) while simultaneously moving the objective lens of the imaging assembly (122) through a range of z-heights as the flow cell (300) moves along the y-axis. In some variations, this step represented by block (1102) may be omitted. If the step represented by block (1102) is performed, integrated through-focus calibration may be performed according to the teachings provided above in the context of FIGS. 7-12 . The z position and focus model generated by the integrated through-focus calibration may then be used for imaging in block (1104). In other implementations, a static focus model generation process may be performed before block (1100) and used for imaging in block (1104).

[0058] Regardless of whether the step represented by block 1102 is performed when the calibration region 340 near the first end 320 passes through the field of view of the imaging assembly 122, the imaging assembly 122 can capture images of the mid-region 324 of the channel 310, as shown in block 1104. These images of the mid-region 324 of the channel 310 may be the same types of images captured during a conventional SBS process (e.g., to identify nucleotides at reaction sites within the channel 310). As the flow cell 300 continues to move in the first direction along the y-axis relative to the imaging assembly 122, the calibration region 340 near the second end 322 eventually reaches the field of view of the imaging assembly 122. As the calibration region 340 near the second end 322 passes through the field of view of the imaging assembly 122, the imaging assembly 122 can capture an image and perform an integrated through-focus calibration during the calibration region 340 near the second end 322, as shown in block 1106. This integrated through-focus calibration during the calibration region 340 near the second end 322 can be performed according to the teachings provided above in the context of Figures 7-12. The data generated by the integrated through-focus calibration in block 1106 can then be used to update the focus model for subsequent swaths and / or subsequent cycles of imaging in block 1104 and / or imaging in block 1112.

[0059] After the calibration area 340 near the second end 322 passes through the field of view of the imaging assembly 122 (or after the desired integrated through-focus calibration is performed on the calibration area 340 near the second end 322), the flow cell 300 may be incrementally shifted to a new swath, and the movement of the flow cell 300 may be reversed. In other words, the flow cell 300 may be moved in a second direction along the y-axis relative to the imaging assembly 122, as shown in block 1108. As the flow cell 300 moves in this second direction along the y-axis relative to the imaging assembly 122, the field of view of the imaging assembly 122 may effectively move back toward the first end 320. In some variations, as represented by the dashed rendering of block (1110) in Figure 7, the imaging assembly (122) can capture integrated through-focus calibration data along the calibration region (340) near the second end (322) as the flow cell (300) moves along the y-axis. In some other variations, this step represented by block (1110) may be omitted. If the step represented by block (1110) is performed, the integrated through-focus calibration can be performed according to the teachings provided above in the context of Figures 7-12.

[0060] Regardless of whether the step represented by block 1110 is performed when the calibration region 340 near the second end 322 passes through the field of view of the imaging assembly 122, the imaging assembly 122 may capture additional images of the mid-region 324 of the channel 310, as shown in block 1112. These images of the mid-region 324 of the channel 310 may be the same types of images captured during a conventional SBS process (e.g., to identify nucleotides at reaction sites within the channel 310). As the flow cell 300 continues to move in a second direction along the y-axis relative to the imaging assembly 122, the calibration region 340 near the first end 320 eventually reaches the field of view of the imaging assembly 122. Once the calibration region (340) near the first end (320) passes through the field of view of the imaging assembly (122), the imaging assembly (122) may perform an integrated through-focus calibration in the calibration region (340) near the first end (320), as shown in block (1114). This integrated through-focus calibration performed in the calibration region (340) near the first end (320) may be performed in accordance with the teachings provided above in the context of Figures 7-12.

[0061] In this example, the SBS imaging process may include one or more passes of the same channel (310) under the imaging assembly (122). As mentioned above, this may include at least two passes, one in a first direction along the y-axis and one in a second direction along the y-axis. In some variations, the SBS imaging process provides three or more passes of each channel (310) under the imaging assembly (122). In other implementations, a single pass may be performed per channel (310). Thus, the method shown in FIG. 13 further includes a determination step, represented by block (1116), for determining whether imaging of the channel (310) at hand is complete. If imaging of the channel (310) at hand is not yet complete, the above-described process may be repeated for that channel (310). By way of example only, some variations may require each channel (310) to be imaged four times before moving on to the next channel (310).

[0062] In either case, if the decision step represented by block 1116 results in a determination that imaging of the current channel 310 is indeed complete, the process can move to the next channel 310, as represented by block 1118. To move to the next channel 310, the flow cell 300 can be moved relative to the imaging assembly 122 along the x-y plane. As described above, such movement can be provided by one or more actuators of the sample stage 170. Alternatively, the imaging assembly 122 can be moved relative to the flow cell 300 along the x-y plane. In either scenario, once the appropriate ends 320, 322 of the next channel 310 are within the field of view of the imaging assembly 122, the process described above with reference to FIG. 13 can be performed along the next channel 310. This can be repeated until all channels 310 have been imaged.

[0063] It should be appreciated from the foregoing that the calibration imaging step can be seamlessly integrated with the SBS imaging step, such that the SBS imaging need not be delayed or interrupted to provide the calibration imaging. Similarly, it should be appreciated from the above that the calibration data can be captured through the same continuous, uninterrupted movement of the flow cell 300 relative to the imaging assembly 122 along the x-y plane. Thus, the capture of the calibration data need not impose any significant delay on the completion of the SBS process. Furthermore, the calibration method described above with reference to FIG. 13 can effectively provide a feedback loop with real-time calibration data, thereby minimizing any adverse effects of thermal distortion, drift, and / or other phenomena that may occur during the SBS process and that could otherwise adversely affect the SBS images.

[0064] In the example described above with reference to FIG. 13 , one or both of the two calibration regions 340 in each channel 310 of the flow cell 300 are used for calibration purposes. In some other variations, only the calibration region in every other channel 310 is used for calibration purposes. Alternatively, any other suitable number of calibration regions 340 in the flow cell 300 can be used for calibration purposes. In some further implementations, the calibration region 340 can be positioned between two intermediate regions 324, thereby allowing calibration to be performed between one or more intermediate positions as the flow channel 310 is imaged in the same direction. For example, such a calibration region can be implemented in a flow cell having an extended length in the y-direction. In other implementations, the calibration region 340 can be implemented for a radial flow cell with a radial flow channel 310 or for a spiral flow channel 310, such as for wafer-based sequencing methods.

[0065] The controller 120 can provide various types of calibration responses based on the calibration data obtained through the integrated through-focus process, as described above. By way of example only, such calibration responses can include adjusting the position and / or orientation of one or more movable components within the imaging assembly 122. For example, the controller 120 can provide an adjusted z-position of the objective lens assembly 142 during acquisition of an SBS image along the intermediate region 340 (as represented by blocks 504 and 512) based on the calibration data obtained through the integrated through-focus process, as described above. Similarly, the controller 120 can provide an adjusted z-position range of the objective lens assembly 142 during acquisition of a subsequent integrated through-focus process (as represented by blocks 502, 506, 510, and 514) based on the calibration data obtained through calibration, as described above. The controller 120 can also adjust how the SBS image is processed based on the calibration data obtained through the calibration image, as described above. The controller 120 may also adjust the irradiance profile (e.g., adjust the intensity of the excitation light) and / or other characteristics of the light emitting assembly 150 based on the calibration data obtained through the calibration images as described above. Alternatively, the controller 120 may provide any other suitable type of calibration response in addition to or instead of providing the calibration responses outlined above based on the calibration data obtained through the calibration images as described above.

[0066] In variations in which the camera system 140 includes a TDI camera, the calibration routine can determine which spot separation provides the best focus. It should also be understood that the z-motion profile depicted in plot 502 is merely an example. Other forms of the z-motion profile may include a step-shaped profile, a sinusoidal profile, or other types of profile shapes.

[0067] In some variations, the illumination intensity may be enhanced during calibration imaging. In other words, the light emitting assembly 150 may illuminate the channel 310 with a higher intensity during acquisition of the calibration images (as represented by blocks 502, 506, 510, 514) than the illumination intensity that provides for acquisition of the SBS images (as represented by blocks 504, 512). Also, in some variations, the system 100 may intentionally induce physical perturbations in one or more components of the imaging assembly 122 and / or the flow cell 300 during the integrated through-focus process. Data obtained during such induced physical perturbations can further enhance the calibration data.

[0068] In the above example, each calibration region (340) of channel (310) contains nucleotides, as does the mid-region (324) of channel (310). Imaging such nucleotides within calibration region (340) for calibration purposes may be particularly desirable because nucleotides are also positioned along mid-region (324), such that the same type of visual target used for SBS imaging is used for calibration imaging for the integrated through-focus process. However, some other variations may provide other types of visual features within calibration region (340). For example, calibration region (340) may include a two-dimensional calibration pattern, a three-dimensional calibration structure with a known topology, or the like. Another variation of calibration region (340) may extend all the way to each end (320, 322) of channel (310), such that first boundary (342) may be effectively eliminated. In some such variations, alignment features (330, 332) may be effectively formed by the absence of nucleotides within channel (310).

[0069] In addition to accounting for changes that may occur in the imaging assembly 122 and / or the flow cell 300 during the SBS process (e.g., due to thermal deformation or drift, etc.) in real time, the calibration methods described above can also account for local spatial variations within the system 100. For example, by providing calibration regions 340 at various locations along the x-y plane of the flow cell 300, the calibration process can effectively account for tilt, tilt, curvature, or other structural variations of the flow cell 300. Thus, the calibration routine executed by the controller 120 can be tailored to be sensitive to calibration data that varies as a function of x-y positioning, thereby applying corresponding calibration responses as a function of x-y positioning.

[0070] As an example of another type of variation on how the teachings of the present disclosure may be implemented, consider FIG. 14 , which shows another flowchart depicting one example of a method for dynamically calibrating optical system components. As will be understood by those skilled in the art, the logic circuitry of a system for biological or chemical analysis such as that illustrated in FIG. 1 may be configured in a manner such as that shown in FIG. 14 . However, such a method may also be performed, in whole or in part, using other components, such as an external processor or computer, that may process data after it has been generated using a system such as that shown in FIG. 1 . Therefore, the description of the method of FIG. 14 in the context of a system such as that shown in FIG. 1 should be understood as merely exemplary and should not be treated as limiting.

[0071] Referring now to FIG. 14 , in the method shown in this figure, a set of calibration operations is performed in block (1401). These calibration operations may include capturing an image of a region of interest in block (1402). In the method shown in FIG. 14 , the region of interest may be a two-dimensional region on the surface of a channel in a flow cell, which includes multiple reaction sites separated from one another along both the length and width of the channel. Once the image of the region of interest is captured, it may be stored in a first memory in block (1403), and an image quality score for the region of interest may be determined in block (1404). This may be done, for example, using a contrast gradient within the image of the region of interest (e.g., the higher the contrast, the less blurred the image is, and therefore, it is assumed that its quality is higher), or other types of score determination may be used, such as a Brenner score, as described above in the context of FIG. 12 . Simultaneously, one or more image quality proxy values ​​may be determined for the region of interest in block (1405). This may be done by capturing images of the region of interest over time while the image is being captured (e.g., by capturing it row by row as the field of view of the imaging device moves along the length of the flow cell channel), capturing spot separation values ​​as described above in the context of Figure 12, and treating the average of those spot separation values ​​as the image quality proxy value for the region of interest.

[0072] After these calibration operations are performed for each region of interest within the plurality of regions of interest, the image quality scores and image quality proxy values ​​for those regions of interest can be used in block (1406) to generate a calibration curve relating the image quality proxy values ​​to the image quality scores. The parabolic curve shown in FIG. 12 is an example of such a calibration curve, which can be achieved by fitting a second-order polynomial to the coordinates defined by the image quality proxy values ​​and the image quality scores. This calibration curve can then be applied in block (1407) to dynamically update the focus of the imaging device while performing a set of base calling operations. These base calling operations can include acquiring nucleotide data in block (1408), which can be performed by using an imaging assembly to detect light emitted from reactants positioned at reaction sites on the surface of the channel, as described above in the context of camera system (140) of FIG. 1 detecting fluorescence emitted from the sample of interest. Simultaneously, image quality proxy values ​​can be acquired in block (1409), showing how these values ​​change during imaging. These image quality proxy values ​​can then be used continuously during base calling to determine whether a feature of the imaging assembly (e.g., the distance between the objective lens and the surface of the channel) should be adjusted in block (1410). This can be done, for example, by projecting spots slightly in front of the area to be imaged, determining where the separation between those spots lies on a calibration curve, and if the spots indicate that the image captured at the spot location is out of focus, adjusting the imaging assembly to address the problem (e.g., by moving the objective lens closer to or farther from the surface of the channel).

[0073] While the above description of FIG. 14 illustrates how the method represented by the flowchart in that figure may be implemented, it should be understood that the exemplary implementation is intended to be illustrative, and that there are many variations on how the method represented by the flowchart in FIG. 14 may be implemented. For illustrative purposes, consider capturing regions of interest in block (1402) and the relationship between those regions. In some implementations, the regions of interest may be adjacent regions of interest or regions of interest separated from each other by a distance. However, in other implementations, the regions of interest may instead be overlapping regions of interest, such as the first region of interest (ROI), second region of interest, and third region of interest illustrated in FIG. 15. For example, if the separation between the objective lens and the surface of the channel being imaged follows a motion profile such as that shown in FIG. 7 as the field of view of the imaging assembly moves down the length of the channel, the regions of interest may overlap such that the image quality proxy value (e.g., average spot separation value) transitions more smoothly from one region of interest to another, thus providing a smoother calibration curve.

[0074] As another example of the types of variations on how the method reflected in the flowchart of FIG. 14 may be implemented, consider the physical devices used in performing the method. For example, in some cases, a method such as that described in the context of FIG. 14 may be implemented in an analysis system having logic circuitry that includes both a programmed general-purpose processor and a processorless special-purpose logic circuit (e.g., an FPGA). In such cases, the particular capabilities of different aspects of the logic circuitry may be exploited to optimize the performance of the method. An example of this type of optimization is provided in FIG. 16, which illustrates a particular approach that may be taken when performing a calibration operation such as that described above in the context of block (1401).

[0075] In the method shown in Figure 16, an image of the region of interest is stored in a separate memory in block (1601) before the region of interest is stored as described above in block (1403). For example, the image of the region of interest may be stored in a memory accessible via a program stored in firmware before being moved to the memory of the FPGA, where it can be processed using the faster processing speed of the FPGA to generate an image quality score fast enough for a set of calibration operations to be performed continuously during scanning of the flow cell channel without requiring the scanning of the flow cell channel to be slowed or stopped. Thus, in this type of implementation, after the region of interest is stored in block (1601), the region of interest can be stored in a different memory in block (1403) by performing steps including transferring the region of interest from one memory to another.

[0076] This transfer may be optimized in some cases. For illustration, consider the case where regions of interest overlap each other and the memory in which they are stored in block (1403) is configured as a circular buffer. In such a case, for a first region of interest, storing the region of interest in block (1403) may be performed by simply transferring an image of the region of interest from a separate memory in block (1602). Alternatively, if the region of interest is not the first region of interest, the transfer may include transferring a portion of the region of interest in block (1603), which, when combined with another portion already stored in memory (e.g., FPGA memory), combines to provide an image of the region of interest. Additionally, already stored data may be removed so that it can be replaced by the transferred portion of the image of the first region of interest (e.g., if the transferred portion consists of 32 rows of a 512x512 pixel region of interest, the oldest 32 rows stored in memory may be removed as the new 32 rows are transferred).

[0077] As another example, consider the case where the imaging assembly captures more data than will be used to create the calibration curve. This may be the case, for example, when the calibration curve is created using image quality scores derived from a 512 x 512 pixel region of interest, but the imaging assembly captures data having dimensions spanning a range spanning the width of the channel greater than 512 pixels. In such a case, when the image is stored in block (1601), all dimensions of the image may be stored to maximize the data available if the image is later used for sequencing by synthesis, for example. However, only the portion actually corresponding to the region of interest may be stored in the memory of the processorless special purpose logic circuit, reflecting the fact that that logic will be specifically used to generate the quality score; therefore, storing all collected data in that memory may not impose any purpose on its capabilities.

[0078] Implementations of methods such as those represented by the flowchart of FIG. 14 may also differ from one another in the relationship of the sets of calibration and base calling operations. For example, in some cases, calibration operations may be performed in first and second calibration regions (e.g., first and second end regions of a channel) in the same manner as described above in the context of blocks 1102, 1106, 1110, and 1114 of FIG. 13 . However, it is also possible that calibration operations and / or generation of a calibration curve may be performed while acquiring nucleotide data during base calling. For example, as described above, in some implementations (e.g., when an FPGA with a circular memory buffer is used to store and derive image quality scores for regions of interest), calibration operations may be performed quickly enough so as not to interfere with scanning of the channel. In such implementations, image quality proxy values ​​acquired during base calling may be used to continuously generate a calibration curve by continually updating them. To facilitate this, in some cases, the characteristics of the imaging assembly may be continuously varied (e.g., dithered) through a small range around an expected optimum during base calling, thereby providing more diverse data for the ongoing generation of calibration curves.

[0079] Other types of variations are possible. For example, in some cases, adjustments may be made to features of the imaging assembly based on the average separation value of pairs of spots, while in other cases, additional spots (e.g., additional pairs of spots resulting in a square configuration with spots at the vertices) may be used to collect additional data for image optimization. As another example, different implementations may determine whether to make and / or perform adjustments at different frequencies. For example, adjustments may be made per run, per cycle, per swath, or per tile during sequencing. As another example, in some implementations, focus information collected in one pass may be used in other passes. For example, when initially scanning the surface of a channel (e.g., when imaging the first swath), an image profile of the channel may be created. In subsequent scan cycles of that channel, that profile may be used to control the scan speed by slowing down in regions where the profile exhibits a steep slope or by increasing the speed in regions where the profile exhibits a gentle (or no) slope. Further variations are possible and will be readily apparent to those skilled in the art in light of this disclosure. Accordingly, the examples provided herein, as well as variations on those examples, should be understood to be illustrative only and should not be treated as implying a limitation on the protection provided by this document or any related document.

[0080] IV. Combination Examples The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. The following examples are not intended to limit the scope of any claims that may be presented at any time in this application or a subsequent application related to this application. No disclaimer is intended. The following examples are provided for illustrative purposes only. It is contemplated that the various teachings herein may be configured and applied in numerous other ways. It is also contemplated that some variations may omit certain features referenced in the following examples. Accordingly, none of the aspects or features referenced below should be considered critical unless later expressly indicated as such by the inventors or the inventors' successors in interest. If any claim including additional features beyond those referenced below is presented in this application or a subsequent application related to this application, those additional features should not be presumed to have been added for any reasons related to patentability.

[0081] Example 1 1. An apparatus, comprising: a flow cell including: a channel having a first end region, a second end region, and an intermediate region extending between the first end region and the second end region, the channel defining a length including the first end region, the intermediate region, and the second end region, the channel configured to receive a fluid, the channel including one or more calibration regions in the first end region, the second end region, or the intermediate region; and a plurality of reaction sites positioned along the intermediate region, each reaction site configured to contain a biological sample carried by the fluid, each reaction site further configured to receive excitation light. an imaging assembly operable to receive light emitted from reactants positioned at the reaction sites in response to excitation light; and a processor, wherein the processor is configured to drive relative movement between at least a portion of the imaging assembly and the flow cell along a continuous range of motion, thereby enabling the imaging assembly to capture images along the length of the channel, activate the imaging assembly to capture one or more calibration images for one or more calibration regions during a first portion of the continuous range of motion, and activate the imaging assembly to capture images of the reaction sites during a second portion of the continuous range of motion.

[0082] Example 2 2. The apparatus of example 1, wherein the one or more calibration regions are positioned in the first end region.

[0083] Example 3 3. The apparatus of any one of the preceding examples, wherein the one or more calibration regions are positioned in the second end region.

[0084] Example 4 4. The apparatus of any one of Examples 1 to 3, wherein the one or more calibration areas are positioned within the intermediate area.

[0085] Example 5 5. The apparatus of any one of Examples 1 to 4, wherein one or more calibration regions comprise nucleotides.

[0086] Example 6 An apparatus described in any one of Examples 1 to 5, wherein the processor is further configured to adjust characteristics of the imaging assembly based at least in part on data from one or more calibration images.

[0087] Example 7 A method comprising: communicating a fluid through a channel of a flow cell; moving at least a portion of an imaging assembly relative to the flow cell through a range of motion; and, while moving at least a portion of the imaging assembly relative to the flow cell through the range of motion, capturing one or more calibration images of a first calibration area via the imaging assembly, the first calibration area being positioned in a first end region of the channel; and capturing one or more images of a reaction site via the imaging assembly, the reaction site being positioned in an intermediate region of the channel.

[0088] Example 8 The method of Example 7, further comprising capturing one or more calibration images of a second calibration area via the imaging assembly while moving at least a portion of the imaging assembly through a range of motion relative to the flow cell, the second calibration area being positioned at a second end region of the channel.

[0089] Example 9 9. The method of example 7 or 8, further comprising adjusting characteristics of the imaging assembly based at least in part on data from one or more calibration images.

[0090] Example 10 The method of any one of Examples 7-9, further comprising performing a sequencing-by-synthesis analysis based on one or more images of the reaction sites.

[0091] Example 11 11. The method of example 10, wherein the sequencing-by-synthesis analysis is performed while at least a portion of the imaging assembly is moved through a range of motion relative to the flow cell.

[0092] Example 12 A method comprising: communicating fluid through a channel of a flow cell; performing sequencing-by-synthesis through the flow cell; and capturing one or more calibration images of a first calibration area via an imaging assembly while performing sequencing-by-synthesis analysis through the flow cell, wherein a first calibration target is positioned at a first end region of the channel; and capturing one or more images of a reaction site via the imaging assembly, wherein the reaction site is positioned at an intermediate region of the channel.

[0093] Example 13 13. The method of example 12, wherein performing the sequencing-by-synthesis via the flow cell comprises moving at least a portion of the imaging assembly relative to the flow cell through a range of motion.

[0094] Example 14 The method of Example 13, wherein capturing one or more images of the reaction site via the imaging assembly is performed while moving at least a portion of the imaging assembly through a range of motion relative to the flow cell.

[0095] Example 15 The method of Example 14, wherein capturing one or more calibration images of the first calibration area via the imaging assembly is performed while moving at least a portion of the imaging assembly through a range of motion relative to the flow cell.

[0096] Example 16 A processor-readable medium comprising content configured to cause a processor to process data by performing the method of Example 12.

[0097] Example 17 The apparatus includes: a flow cell including one or more channels, each of the one or more channels having a length and a width, the length being greater than the width, and including a surface having a plurality of reaction sites; an imaging assembly that receives light emitted from reactants positioned at the reaction sites in response to excitation light; a focusing component that obtains, for each channel from the one or more channels, an image quality proxy value for the surface of the channel; and a logic circuit, wherein the logic circuit performs the following steps for each of a plurality of target regions of interest from the one or more channels, the target regions of interest being two-dimensional regions on the surface of the target channel having a plurality of reaction sites separated from one another along a length of the target channel and a plurality of reaction sites separated from one another along a width of the channel: capturing an image of the region of interest using the imaging assembly; and storing the image of the region of interest in a first memory; and and performing a set of base calling operations including: determining one or more image quality proxy values ​​for a region of interest using a focusing component; calculating an image quality score for the region of interest; generating a calibration curve relating the image quality score for the region of interest to the image quality proxy value for the region of interest; acquiring nucleotide data based on detecting light emitted from reactants positioned at reaction sites on a surface of the target channel using the imaging assembly while driving relative movement of a field of view of the target channel and the imaging assembly along a length of the target channel; acquiring one or more image quality proxy values ​​using a focusing component while acquiring the nucleotide data; and determining whether to adjust features of the imaging assembly based on the calibration curve and the one or more image quality proxy values ​​acquired while acquiring the nucleotide data.

[0098] Example 18 18. The apparatus of Example 17, wherein for each region of interest from the plurality of regions of interest of the subject, the region of interest overlaps with at least one other region of interest from the plurality of regions of interest of the subject along the length of the subject channel.

[0099] Example 19 The logic circuit includes a programmed general-purpose processor and a processorless special-purpose logic circuit, the first memory is a local memory resident on the processorless special-purpose logic circuit, and the apparatus includes a second memory operatively connected to the programmed general-purpose processor, and for each region of interest from the plurality of regions of interest of the object, the set of calibration operations includes prior to storing an image of that region of interest in the first memory, storing the image of that region of interest in the second memory, and for an initial region of interest from the plurality of regions of interest of the object, storing the image of that region of interest in the first memory includes transferring the image of that region of interest from the second memory to the first memory, and for each region of interest from the plurality of regions of interest of the object other than the initial region of interest, transferring the image of that region of interest to the first memory. 19. The apparatus of example 18, wherein storing the region of interest includes transferring a first portion of an image of the region of interest from the second memory to the first memory when the first memory already includes a second portion of the region of interest as a result of the second portion being included in a different region of interest that was previously stored, wherein the first portion of the image of the region of interest and the second portion of the region of interest are combined to provide the image of the region of interest; and removing data from the first memory, wherein the data removed from the first memory is replaced by the first portion of the image of the region of interest; and wherein the processorless special purpose logic circuit is for calculating, for each region of interest from the plurality of regions of interest, an image quality score for the region of interest.

[0100] Example 20 The apparatus of Example 19, wherein for at least one region of interest from a plurality of regions of interest of the subject, at least a portion of the second portion of the region of interest is made up of a plurality of different previously stored regions of interest.

[0101] Example 21 The apparatus of Example 19, wherein, for each of a plurality of regions of interest in the target, capturing an image of the region of interest using the imaging assembly includes capturing a corresponding image of the target channel, wherein the corresponding image of the target channel has an extent along the width of the target channel that is greater than the extent of the region of interest along the width of the target channel, and the corresponding image of the target channel has an extent along the length of the target channel that is equal to the extent of the region of interest along the length of the target channel, and storing the image of the region of interest in a second memory includes storing the corresponding image of the target channel in the second memory.

[0102] Example 22 18. The apparatus of Example 17, wherein each of the one or more channels comprises a first end region, a second end region, and an intermediate region extending between the first end region and the second end region, and the logic circuit is for: moving a field of view of the imaging assembly along a length of the target channel from the first end region of the target channel, through the intermediate region of the target channel, to the second end region of the target channel during a first period of time; performing a set of calibration operations using the first plurality of regions of interest as the target plurality of regions of interest during the first period of time, the first plurality of regions of interest being regions of interest within the first end region of the target channel; and moving a field of view of the imaging assembly along a length of the target channel from the second end region of the target channel, through the intermediate region of the target channel, to the first end region of the target channel during a second period of time; performing a set of calibration operations using a second plurality of regions as the target plurality of regions of interest during the second period of time, the second plurality of regions of interest being regions of interest within the second end region of the target channel.

[0103] Example 23 23. The apparatus of example 22, wherein the logic circuit is for performing a set of calibration operations using a third plurality of regions of interest as the plurality of regions of interest of the target, the third plurality of regions of interest being regions of interest within an intermediate region of the target channel.

[0104] Example 24 The apparatus of Example 23, wherein the logic circuit is for driving relative movement between the imaging assembly feature and the flow cell along a height, the height being perpendicular to the length and width of the target channel, through a continuous range of motion between a first value and a second value while performing a set of calibration operations using the first plurality of regions of interest as the target plurality of regions of interest, and for driving relative movement between the imaging assembly feature and the flow cell along a height, the height being perpendicular to the length and width of the target channel, through a continuous range of motion between a third value and a fourth value, the third value and the fourth value being between the first value and the second value, respectively, while performing a set of calibration operations using the third plurality of regions of interest as the target plurality of regions of interest.

[0105] Example 25 The imaging assembly features an objective lens, and the logic circuit is for driving relative movement between the objective lens and the surface of the target channel through a continuous range of motion along a height perpendicular to the length and width of the target channel while performing a set of calibration operations for a target channel from one or more channels, driving relative movement of the target channel and the field of view of the imaging assembly along the length of the target channel; the focusing component is for obtaining, for each channel from the one or more channels, an image quality proxy value for the surface of the channel by performing an operation including projecting a set of spots onto the surface of the channel and detecting reflections of the set of spots from the surface of the channel; for each region of interest from a plurality of target regions of interest, the one or more image quality proxy values ​​for the region of interest include an average spot separation value for the region of interest; determining the one or more image quality proxy values ​​for the region of interest using the focusing component includes projecting a set of spots onto the surface of the target channel and detecting reflections of the set of spots from the surface of the target channel while the focusing component captures an image of the region of interest; and determining whether to adjust the imaging assembly features includes determining whether to adjust the relative position of the objective lens and the surface of the target channel along the height.

[0106] Example 26 1. A method comprising: performing a set of calibration operations for each of a plurality of regions of interest in an object, each region of interest being a two-dimensional region on a surface of an object channel, the object channel having a plurality of reaction sites separated from one another along a length of the object channel and a plurality of reaction sites separated from one another along a width of the channel; performing the set of calibration operations includes capturing an image of the region of interest using an imaging assembly; storing the image of the region of interest in a first memory; determining one or more image quality proxy values ​​for the region of interest using a focusing component of a system for analyzing chemical or biological materials; and calculating an image quality score for the region of interest. A method comprising: performing a set of base calling operations including: generating a calibration curve relating scores to image quality proxy values ​​for the region of interest; acquiring nucleotide data based on detecting light emitted from reactants positioned at reaction sites on a surface of the target channel using an imaging assembly while driving relative movement of a field of view of the target channel and the imaging assembly along the length of the target channel; acquiring one or more image quality proxy values ​​using a focusing component while acquiring the nucleotide data; and determining whether to adjust features of the imaging assembly based on the calibration curve and the one or more image quality proxy values ​​acquired while acquiring the nucleotide data.

[0107] Example 27 27. The method of example 26, wherein for each region of interest from the plurality of regions of interest of the subject, the region of interest overlaps with at least one other region of interest from the plurality of regions of interest of the subject along the length of the subject channel.

[0108] Example 28 The first memory is a local memory resident on a processorless special purpose logic circuit, and for each region of interest from the plurality of regions of interest of the object, the set of calibration operations includes storing an image of that region of interest in the first memory before storing the image of that region of interest in the first memory, and storing the image of that region of interest in a second memory, the second memory being operatively connected to the general purpose processor, and for a first region of interest from the plurality of regions of interest of the object, storing the image of that region of interest in the first memory includes transferring the image of that region of interest from the second memory to the first memory, and for each region of interest from the plurality of regions of interest of the object other than the initial region of interest, storing the region of interest in the first memory includes transferring a first portion of the image of that region of interest. 27. The method of example 26, including transferring from the second memory to the first memory when the first memory already contains a second portion of the region of interest as a result of the second portion being included in a different region of interest that was previously stored, wherein the first portion of the image, the region of interest, and the second portion of the region of interest are combined to provide an image of the region of interest; and removing data from the first memory, wherein the data removed from the first memory is replaced by the first portion of the image of the region of interest; and the processorless special purpose logic circuit is for calculating, for each region of interest from the plurality of regions of interest, an image quality score for the region of interest.

[0109] Example 29 29. The method of example 28, wherein for at least one region of interest from the plurality of regions of interest of the subject, at least a portion of the second portion of the region of interest is made up of a plurality of different previously stored regions of interest.

[0110] Example 30 The method of example 28, wherein, for each of a plurality of regions of interest in the target, capturing an image of the region of interest using the imaging assembly includes capturing a corresponding image of the target channel, wherein the corresponding image of the target channel has an extent along the width of the target channel that is greater than the extent of the region of interest along the width of the target channel, and the corresponding image of the target channel has an extent along the length of the target channel that is equal to the extent of the region of interest along the length of the target channel, and storing the image of the region of interest in a second memory includes storing the corresponding image of the target channel in the second memory.

[0111] Example 31 Each of the one or more channels comprises a first end region, a second end region, and an intermediate region extending between the first end region and the second end region, and the method includes: moving, during a first time period, a field of view of an imaging assembly along a length of the target channel from the first end region of the target channel, through the intermediate region of the target channel, to the second end region of the target channel; and capturing, during the first time period, a first plurality of regions of interest as a plurality of regions of interest of the target channel, the first plurality of regions of interest being regions of interest within the first end region of the target channel. 27. The method of example 26, comprising: performing a set of calibration operations using a plurality of regions of interest; during a second period, moving a field of view of the imaging assembly along a length of the target channel from a second end region of the target channel, through an intermediate region of the target channel, to a first end region of the target channel; and during the second period, performing the set of calibration operations using a second plurality of regions as the plurality of regions of interest of the target channel, wherein the second plurality of regions of interest are regions of interest within the second end region of the target channel.

[0112] Example 32 32. The method of claim 31, wherein the method includes performing a set of calibration operations using a third plurality of regions of interest as the plurality of regions of interest of the target, the third plurality of regions of interest being regions of interest within an intermediate region of the target channel.

[0113] Example 33 The method of Example 32, wherein the method includes, while performing a set of calibration operations using a first plurality of regions of interest as the plurality of regions of interest of the target, driving relative movement between a feature of the imaging assembly and a flow cell along a height, the height being perpendicular to the length and width of the target channel, through a continuous range of motion between a first value and a second value, and while performing a set of calibration operations using a third plurality of regions of interest as the plurality of regions of interest of the target, driving relative movement between a feature of the imaging assembly and a flow cell along the height through a continuous range of motion between a third value and a fourth value, the third value and the fourth value being between the first value and the second value, respectively.

[0114] Example 34 33. The method of Example 32, wherein the method includes determining a nucleotide sequence for the sample of biological material by performing sequencing-by-synthesis based on nucleotide data captured from a third plurality of regions of interest.

[0115] Example 35 The feature of the imaging assembly is an objective lens, and the method includes, for a target channel, driving relative movement between the objective lens and a surface of the target channel through a continuous range of motion along a height that is perpendicular to the length and width of the target channel while performing a set of calibration operations, and driving relative movement of the target channel and the field of view of the imaging assembly along the length of the target channel; the focusing component is for obtaining image quality proxy values ​​for the surface of the target channel by performing an operation for the target channel that includes projecting a set of spots onto the surface of the channel and detecting reflections of the set of spots from the surface of the channel; for each region of interest from a plurality of regions of interest of the target, the one or more image quality proxy values ​​for the region of interest include an average spot separation value for the region of interest; determining the one or more image quality proxy values ​​for the region of interest using the focusing component includes projecting a set of spots onto the surface of the target channel and detecting reflections of the set of spots from the surface of the target channel while the focusing component captures an image of the region of interest; and determining whether to adjust the feature of the imaging assembly includes determining whether to adjust the relative position of the objective lens and the surface of the target channel along the height.

[0116] V. Other Although the foregoing examples are provided in the context of a system (100) that may be used in a nucleotide sequencing process, the teachings herein may also be readily applied in other contexts, including systems that perform other processes (i.e., other than nucleotide sequencing procedures). Thus, the teachings herein are not necessarily limited to systems used to perform nucleotide sequencing processes.

[0117] It is to be understood that the subject matter described herein is not limited in its application to the details of construction and the arrangement of components set forth in the description herein or illustrated in the drawings herein. The subject matter described herein is capable of other implementations and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0118] When used in the claims, the term "set" should be understood as one or more things grouped together. Similarly, when used in the claims, "based on" should be understood to indicate that one thing is determined at least in part by what it is specified to be "based on." When something must be determined exclusively by another, it is said to be "exclusively based on" that thing upon which it is determined.

[0119] Unless otherwise specified or limited, the terms "mounted," "connected," "supported," and "coupled," as well as variations thereof, are used broadly and encompass both direct and indirect mounting, connecting, supporting, and coupling. Furthermore, "connected" and "coupled" are not limited to physical or mechanical connections or couplings. It should also be understood that the expressions and terms used herein with respect to the orientation of devices or elements (e.g., terms such as "upper," "lower," "anterior," "posterior," "distal," and "proximal") are used solely to simplify the description of one or more examples described herein and do not solely indicate or imply that the referenced device or element must have a particular orientation. Additionally, terms such as "outer" and "inner" are used herein for descriptive purposes and are not intended to indicate or imply relative importance or significance.

[0120] It should be understood that the above description is intended to be illustrative, and not limiting. For example, the above-described examples (and / or aspects thereof) can be used in combination with each other. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the subject matter described herein without departing from its scope. While the dimensions, material types, and coatings described herein are intended to define the parameters of the disclosed subject matter, they are by no means limiting and are instead exemplary. Many further examples will be apparent to those skilled in the art upon review of the above description. Therefore, the scope of the disclosed subject matter should be determined with reference to the appended claims, along with the full range of equivalents to which such claims are entitled. In the appended claims, the terms "including" and "in which" are used as the plain-English equivalents of the respective terms "comprising" and "wherein." Furthermore, in the following claims, the terms "first," "second," "third," etc. are used merely as labels and are not intended to impose numerical requirements on their objects. Moreover, no limitation in the following claims is written in means-plus-function form, and such claim limitations are not intended to be construed under paragraph 35 U.S.C. 3112(f) unless and until the phrase "means for" is explicitly used followed by a statement of function that does not include further structure.

[0121] The following claims recite certain example aspects of the disclosed subject matter and are considered to be part of the above disclosure. These aspects may be combined with each other.

[0122] [Embodiment] (1) A device comprising: a flow cell including one or more channels, each of the one or more channels having a length and a width, the length being greater than the width, the flow cell including a surface having a plurality of reaction sites; an imaging assembly that receives light emitted from a reactant positioned in the reaction site in response to excitation light; a focus component for obtaining, for each channel from the one or more channels, an image quality proxy value for the surface of that channel; and a logic circuit, wherein the logic circuit performs, for a target channel from the one or more channels: For each of a plurality of regions of interest of the subject, each region of interest being a two-dimensional region on the surface of the subject channel having a plurality of reaction sites separated from one another along the length of the subject channel and a plurality of reaction sites separated from one another along the width of the channel: capturing an image of the region of interest using the imaging assembly; storing the image of the region of interest in a first memory; determining one or more image quality proxy values ​​for the region of interest using the focus component; calculating an image quality score for the region of interest; generating a calibration curve relating image quality scores for the region of interest to image quality proxy values ​​for the region of interest; while driving relative movement of the object channel and the field of view of the imaging assembly along the length of the object channel; acquiring nucleotide data based on detecting light emitted from reactants positioned at reaction sites on the surface of the target channel using the imaging assembly; acquiring one or more image quality proxy values ​​using said focus component while acquiring nucleotide data; and determining whether to adjust characteristics of the imaging assembly based on the calibration curve and the one or more image quality proxy values ​​obtained while acquiring nucleotide data. (2) The apparatus of embodiment 1, wherein for each region of interest from the plurality of regions of interest of the object, the region of interest overlaps with at least one other region of interest from the plurality of regions of interest of the object along the length of the object channel. (3) The logic circuit is a programmed general purpose processor; a processorless special purpose logic circuit; the first memory is a local memory resident in the processor-less special purpose logic circuit; the apparatus comprising a second memory operably connected to the programmed general-purpose processor; For each region of interest from a plurality of regions of interest of the object, the set of calibration operations includes, before storing the image of that region of interest in the first memory, storing the image of that region of interest in the second memory; For a first region of interest from a plurality of regions of interest of the object, storing the image of the region of interest in the first memory includes transferring the image of the region of interest from the second memory to the first memory; For each region of interest from a plurality of regions of interest of the object other than the initial region of interest, storing the region of interest in the first memory includes: transferring a first portion of the image of the region of interest from the second memory to the first memory when the first memory already contains a second portion of the region of interest as a result of the second portion being included in a different region of interest that was previously stored, the first portion of the image of the region of interest and the second portion of the region of interest being combined to provide the image of the region of interest; removing data from the first memory, wherein the data removed from the first memory is replaced by the first portion of the image of the region of interest; the processor-less special purpose logic circuit is for calculating, for each region of interest from the plurality of regions of interest, the image quality score for that region of interest; An apparatus as described in embodiment 1. (4) The apparatus of embodiment 3, wherein for at least one region of interest from the plurality of regions of interest of the object, at least a portion of the second portion of the region of interest is composed of a plurality of different previously stored regions of interest. (5) for each of a plurality of regions of interest of the subject, capturing the image of the region of interest using the imaging assembly includes capturing a corresponding image of the target channel, the corresponding image of the object channel has a degree of extent along the width of the object channel that is greater than a degree of extent of the region of interest along the width of the object channel; capturing the corresponding image of the target channel having an extent of coverage along the length of the target channel equal to an extent of coverage of the region of interest along the length of the target channel; An apparatus as described in embodiment 3, wherein storing the image of the region of interest in the second memory includes storing the corresponding image of the target channel in the second memory.

[0123] (6) each of the one or more channels comprises a first end region, a second end region, and an intermediate region extending between the first end region and the second end region; The logic circuit comprises: moving a field of view of the imaging assembly along the length of the target channel from the first end region of the target channel, through the intermediate region of the target channel, to the second end region of the target channel during a first period of time; performing the set of calibration operations during the first time period using a first plurality of regions of interest as the plurality of regions of interest of the object, the first plurality of regions of interest being regions of interest within the first end region of the channel of interest; During a second time period, moving a field of view of the imaging assembly along the length of the target channel from the second end region of the target channel, through the intermediate region of the target channel, to the first end region of the target channel; performing the set of calibration operations using a second plurality of regions as the plurality of regions of interest of the object during the second time period, the second plurality of regions of interest being regions of interest within the second end region of the object channel; An apparatus as described in embodiment 1. (7) The device of embodiment 6, wherein the logic circuit is for performing the set of calibration operations using a third plurality of regions of interest as the plurality of regions of interest of the object, the third plurality of regions of interest being regions of interest within the intermediate region of the object channel. (8) The logic circuit comprises: driving relative movement between the feature of the imaging assembly and the flow cell along a height that is perpendicular to the length and width of the target channel through a continuous range of motion between a first value and a second value while performing the set of calibration operations using the first plurality of regions of interest as the target plurality of regions of interest; The apparatus of embodiment 7, wherein the apparatus is for driving relative movement between the feature of the imaging assembly and the flow cell along the height through a continuous range of motion between a third value and a fourth value, the third value and the fourth value being between the first value and the second value, while performing the set of calibration operations using the third plurality of regions of interest as the plurality of regions of interest of the object. (9) The feature of the imaging assembly is an objective lens; The logic circuit, while performing the set of calibration operations for the target channel from the one or more channels, driving relative movement between the objective lens and the surface of the object channel through a continuous range of motion along a height that is perpendicular to the length and width of the object channel; for driving relative movement of the object channel and the field of view of the imaging assembly along the length of the object channel; the focusing component is for obtaining, for each channel from the one or more channels, an image quality proxy value for the surface of that channel by performing operations including: projecting a set of spots onto the surface of that channel; and detecting reflections of the set of spots from the surface of that channel; For each region of interest from a plurality of regions of interest of the object: the one or more image quality proxy values ​​for the region of interest include a mean spot separation value for the region of interest; determining the one or more image quality proxy values ​​for the region of interest using the focus component includes projecting the set of spots onto the surface of the target channel and detecting reflections of the set of spots from the surface of the target channel while the focus component is capturing the image of the region of interest; determining whether to adjust the feature of the imaging assembly includes determining whether to adjust a relative position of the objective lens and the surface of the target channel along the height; 2. The device of embodiment 1, comprising: (10) A method comprising: performing a set of calibration operations for each of a plurality of regions of interest in an object, each region of interest being a two-dimensional region on a surface of an object channel, the object channel having a plurality of reaction sites separated from one another along the length of the object channel and a plurality of reaction sites separated from one another along the width of the channel, performing the set of calibration operations includes: capturing an image of the region of interest using an imaging assembly; storing the image of the region of interest in a first memory; determining one or more image quality proxy values ​​for the region of interest using a focusing component of a system for analyzing chemical or biological material; calculating an image quality score for the region of interest; generating a calibration curve relating image quality scores for the region of interest to image quality proxy values ​​for the region of interest; while driving relative movement of the object channel and the field of view of the imaging assembly along the length of the object channel; acquiring nucleotide data based on detecting light emitted from reactants positioned at reaction sites on the surface of the target channel using the imaging assembly; acquiring one or more image quality proxy values ​​using said focus component while acquiring nucleotide data; and performing a set of base calling operations including determining whether to adjust features of the imaging assembly based on the calibration curve and the one or more image quality proxy values ​​obtained while acquiring nucleotide data.

[0124] (11) The method of embodiment 10, wherein for each region of interest from the plurality of regions of interest of the object, the region of interest overlaps with at least one other region of interest from the plurality of regions of interest of the object along the length of the object channel. (12) The first memory is a local memory resident on a processor-less dedicated purpose logic circuit; For each region of interest from a plurality of regions of interest of the object, the set of calibration operations includes, before storing the image of that region of interest in the first memory, storing the image of that region of interest in the second memory, the second memory operably connected to a general purpose processor; For a first region of interest from a plurality of regions of interest of the object, storing the image of the region of interest in the first memory includes transferring the image of the region of interest from the second memory to the first memory; For each region of interest from a plurality of regions of interest of the object other than the initial region of interest, storing the region of interest in the first memory includes: transferring a first portion of the image of the region of interest from the second memory to the first memory when the first memory already contains a second portion of the region of interest as a result of the second portion being included in a different region of interest that was previously stored, the first portion of the image of the region of interest and the second portion of the region of interest being combined to provide the image of the region of interest; removing data from the first memory, wherein the data removed from the first memory is replaced by the first portion of the image of the region of interest; the processor-less special purpose logic circuit is for calculating, for each region of interest from the plurality of regions of interest, the image quality score for that region of interest; The method of embodiment 10. (13) The method of embodiment 12, wherein for at least one region of interest from the plurality of regions of interest of the object, at least a portion of the second portion of the region of interest is made up of a plurality of different previously stored regions of interest. (14) for each of a plurality of regions of interest of the subject, capturing the image of the region of interest using the imaging assembly includes capturing a corresponding image of the target channel, the corresponding image of the object channel has a degree of extent along the width of the object channel that is greater than a degree of extent of the region of interest along the width of the object channel; capturing the corresponding image of the target channel having an extent of coverage along the length of the target channel equal to an extent of coverage of the region of interest along the length of the target channel; 13. The method of claim 12, wherein storing the image of the region of interest in the second memory includes storing the corresponding image of the target channel in the second memory. (15) Each of the one or more channels comprises a first end region, a second end region, and an intermediate region extending between the first end region and the second end region; The method comprises: moving a field of view of the imaging assembly along the length of the target channel from the first end region of the target channel, through the middle region of the target channel, to the second end region of the target channel during a first period of time; performing the set of calibration operations during the first time period using a first plurality of regions of interest as a plurality of regions of interest of the object, the first plurality of regions of interest being regions of interest within the first end region of the object channel; moving a field of view of the imaging assembly along the length of the target channel from the second end region of the target channel, through the intermediate region of the target channel, to the first end region of the target channel during a second time period; performing the set of calibration operations using a second plurality of regions as a plurality of regions of interest of the object during the second time period, the second plurality of regions of interest being regions of interest within the second end region of the object channel; 11. The method of embodiment 10, comprising:

[0125] (16) The method of embodiment 15, wherein the method includes performing the set of calibration operations using a third plurality of regions of interest as the plurality of regions of interest of the target, the third plurality of regions of interest being regions of interest within the intermediate region of the target channel. (17) The method comprises: driving relative movement between the feature of the imaging assembly and the flow cell along a height, the height being perpendicular to the length and width of the target channel, through a continuous range of motion between a first value and a second value while performing the set of calibration operations with the first plurality of regions of interest as a plurality of regions of interest of the target; 17. The method of claim 16, further comprising driving relative movement between the feature of the imaging assembly and the flow cell along the height through a continuous range of motion between a third value and a fourth value, the third value and the fourth value being between the first value and the second value, while performing the set of calibration operations using the third plurality of regions of interest as the plurality of regions of interest of the object. (18) The method of embodiment 16, wherein the method includes determining a nucleotide sequence for the sample of biological material by performing sequencing by synthesis based on nucleotide data captured from the third plurality of regions of interest. (19) The feature of the imaging assembly is an objective lens; The method includes, while performing the set of calibration operations, performing, for the target channel: driving relative movement between the objective lens and the surface of the object channel through a continuous range of motion along a height that is perpendicular to the length and width of the object channel; driving relative movement of the object channel and the field of view of the imaging assembly along the length of the object channel; the focusing component is for obtaining an image quality proxy value for the surface of the channel of interest by performing operations including, for the channel of interest, projecting a set of spots onto the surface of the channel and detecting reflections of the set of spots from the surface of the channel; For each region of interest from a plurality of regions of interest of the object: the one or more image quality proxy values ​​for the region of interest include a mean spot separation value for the region of interest; determining the one or more image quality proxy values ​​for the region of interest using the focus component includes projecting the set of spots onto the surface of the target channel and detecting reflections of the set of spots from the surface of the target channel while the focus component is capturing the image of the region of interest; determining whether to adjust the feature of the imaging assembly includes determining whether to adjust a relative position of the objective lens and the surface of the target channel along the height; 11. The method of embodiment 10, comprising: (20) An apparatus comprising: A flow cell, a channel having a first end region, a second end region, and an intermediate region extending between the first end region and the second end region, the channel defining a length including the first end region, the intermediate region, and the second end region, the channel configured to receive a fluid, the channel including one or more calibration regions in the first end region, the second end region, or the intermediate region; a flow cell including a plurality of reaction sites positioned along the intermediate region, each reaction site configured to contain a biological sample carried by the fluid, and each reaction site further configured to receive excitation light; an imaging assembly operable to receive light emitted from a reactant positioned in the reaction site in response to the excitation light; a processor, the processor comprising: driving relative movement between at least a portion of the imaging assembly and the flow cell along a continuous range of motion, thereby enabling the imaging assembly to capture images along the length of the channel; activating the imaging assembly to capture one or more calibration images related to the one or more calibration areas during a first portion of the continuous range of motion; The apparatus is configured to activate the imaging assembly to capture an image of the reaction site during a second portion of the continuous range of motion.

[0126] (21) The device described in embodiment 20, wherein the one or more calibration areas are positioned in the first end area. (22) The device described in embodiment 20 or 21, wherein the one or more calibration areas are positioned in the second end area. (23) An apparatus according to any one of embodiments 20 to 22, wherein the one or more calibration areas are positioned within the intermediate area. (24) The device described in any one of embodiments 20 to 23, wherein the one or more calibration regions contain nucleotides. (25) An apparatus described in any one of embodiments 20 to 24, wherein the processor is further configured to adjust characteristics of the imaging assembly based at least in part on data from the one or more calibration images.

[0127] (26) A method comprising: communicating fluid through a channel of the flow cell; moving at least a portion of an imaging assembly relative to the flow cell through a range of motion; While moving the at least a portion of the imaging assembly through the range of motion relative to the flow cell, capturing, via the imaging assembly, one or more calibration images of a first calibration area, the first calibration area being positioned at a first end region of the channel; capturing, via the imaging assembly, one or more images of a reaction site, the reaction site being positioned in a mid-region of the channel. (27) The method of embodiment 26, further comprising capturing one or more calibration images of a second calibration area via the imaging assembly while moving at least a portion of the imaging assembly relative to the flow cell through the range of motion, the second calibration area being positioned at a second end area of ​​the channel. (28) The method of any one of embodiments 26 to 27, further comprising adjusting characteristics of the imaging assembly based at least in part on data from the one or more calibration images. (29) The method of any one of embodiments 26 to 28, further comprising performing a sequencing-by-synthesis analysis based on the one or more images of the reaction site. (30) The method of embodiment 29, wherein the sequencing-by-synthesis analysis is performed while the at least a portion of the imaging assembly is moved relative to the flow cell through the range of motion.

[0128] (31) A method comprising: communicating fluid through a channel of the flow cell; performing sequencing by synthesis through said flow cell; While performing a sequencing-by-synthesis analysis through the flow cell, capturing, via an imaging assembly, one or more calibration images of a first calibration area, wherein a first calibration target is positioned at a first end region of the channel; capturing one or more images of a reaction site via the imaging assembly, the reaction site being positioned in a mid-region of the channel. (32) The method of embodiment 31, wherein performing sequencing by synthesis through the flow cell includes moving at least a portion of the imaging assembly relative to the flow cell through a range of motion. (33) The method of embodiment 32, wherein capturing one or more images of the reaction site via the imaging assembly is performed while moving the at least a portion of the imaging assembly relative to the flow cell through the range of motion. (34) The method of embodiment 33, wherein capturing one or more calibration images of the first calibration area via the imaging assembly is performed while moving the at least a portion of the imaging assembly relative to the flow cell through the range of motion. (35) A processor-readable medium including content configured to cause a processor to process data by performing the method of embodiment 31.

Claims

1. 1. An apparatus comprising: a flow cell including one or more channels, each of the one or more channels having a length and a width, the length being greater than the width, the flow cell including a surface having a plurality of reaction sites; an imaging assembly that receives light emitted from a reactant positioned in the reaction site in response to excitation light; a focus component for obtaining, for each channel from the one or more channels, an image quality proxy value for the surface of that channel; and a logic circuit, wherein the logic circuit performs, for a target channel from the one or more channels: For each of a plurality of regions of interest of the subject, each region of interest being a two-dimensional region on the surface of the subject channel having a plurality of reaction sites separated from one another along the length of the subject channel and a plurality of reaction sites separated from one another along the width of the channel: capturing an image of the region of interest using the imaging assembly; storing the image of the region of interest in a first memory; determining one or more image quality proxy values ​​for the region of interest using the focus component; calculating an image quality score for the region of interest; generating a calibration curve relating image quality scores for the region of interest to image quality proxy values ​​for the region of interest; while driving relative movement of the object channel and the field of view of the imaging assembly along the length of the object channel; acquiring nucleotide data based on detecting light emitted from reactants positioned at reaction sites on the surface of the target channel using the imaging assembly; acquiring one or more image quality proxy values ​​using said focus component while acquiring nucleotide data; and determining whether to adjust characteristics of the imaging assembly based on the calibration curve and the one or more image quality proxy values ​​obtained while acquiring nucleotide data.

2. 10. The apparatus of claim 1, wherein for each region of interest from the plurality of regions of interest of the object, that region of interest overlaps with at least one other region of interest from the plurality of regions of interest of the object along the length of the channel of interest.

3. The logic circuit comprises: a programmed general purpose processor; a processorless special purpose logic circuit; the first memory is a local memory resident in the processor-less special purpose logic circuit; the apparatus comprising a second memory operatively connected to the programmed general-purpose processor; For each region of interest from a plurality of regions of interest of the object, the set of calibration operations includes, before storing the image of that region of interest in the first memory, storing the image of that region of interest in the second memory; For a first region of interest from a plurality of regions of interest of the object, storing the image of the region of interest in the first memory includes transferring the image of the region of interest from the second memory to the first memory; For each region of interest from a plurality of regions of interest of the object other than the initial region of interest, storing the region of interest in the first memory includes: transferring a first portion of the image of the region of interest from the second memory to the first memory when the first memory already contains a second portion of the region of interest as a result of the second portion being included in a different region of interest that was previously stored, the first portion of the image, the region of interest, and the second portion of the region of interest being combined to provide the image of the region of interest; removing data from the first memory, wherein the data removed from the first memory is replaced by the first portion of the image of the region of interest; the processor-less special purpose logic circuit is for calculating, for each region of interest from the plurality of regions of interest, the image quality score for that region of interest; 10. The apparatus of claim 1.

4. 4. The apparatus of claim 3, wherein for at least one region of interest from a plurality of regions of interest of the object, at least a portion of the second portion of the region of interest is made up of a plurality of different previously stored regions of interest.

5. for each of a plurality of regions of interest of the object, capturing the image of the region of interest using the imaging assembly includes capturing a corresponding image of the target channel, the corresponding image of the object channel has a degree of extent along the width of the object channel that is greater than a degree of extent of the region of interest along the width of the object channel; capturing the corresponding image of the target channel having an extent of coverage along the length of the target channel equal to an extent of coverage of the region of interest along the length of the target channel; 4. The apparatus of claim 3, wherein storing the image of the region of interest in the second memory comprises storing the corresponding image of the target channel in the second memory.

6. each of the one or more channels comprises a first end region, a second end region, and an intermediate region extending between the first end region and the second end region; The logic circuit comprises: during a first period of time, moving a field of view of the imaging assembly along the length of the target channel from the first end region of the target channel, through the intermediate region of the target channel, to the second end region of the target channel; performing the set of calibration operations during the first time period using a first plurality of regions of interest as a plurality of regions of interest of the object, the first plurality of regions of interest being regions of interest within the first end region of the object channel; During a second time period, moving a field of view of the imaging assembly along the length of the target channel from the second end region of the target channel, through the intermediate region of the target channel, to the first end region of the target channel; performing the set of calibration operations using a second plurality of regions as the plurality of regions of interest of the object during the second time period, the second plurality of regions of interest being regions of interest within the second end region of the object channel; 10. The apparatus of claim 1.

7. 7. The apparatus of claim 6, wherein the logic circuitry is for performing the set of calibration operations using a third plurality of regions of interest as the plurality of regions of interest of the object, the third plurality of regions of interest being regions of interest within the intermediate region of the object channel.

8. The logic circuit comprises: driving relative movement between the feature of the imaging assembly and the flow cell along a height that is perpendicular to the length and width of the target channel through a continuous range of motion between a first value and a second value while performing the set of calibration operations using the first plurality of regions of interest as the target plurality of regions of interest; 8. The apparatus of claim 7, wherein the apparatus is for driving relative movement between the feature of the imaging assembly and the flow cell along the height through a continuous range of motion between a third value and a fourth value, the third value and the fourth value being between the first value and the second value, while performing the set of calibration operations using the third plurality of regions of interest as the plurality of regions of interest of the object.

9. the feature of the imaging assembly is an objective lens; The logic circuit, while performing the set of calibration operations for the target channel from the one or more channels, driving relative movement between the objective lens and the surface of the object channel through a continuous range of motion along a height that is perpendicular to the length and width of the object channel; for driving relative movement of the object channel and the field of view of the imaging assembly along the length of the object channel; the focusing component is for obtaining, for each channel from the one or more channels, an image quality proxy value for the surface of that channel by performing operations including: projecting a set of spots onto the surface of that channel; and detecting reflections of the set of spots from the surface of that channel; For each region of interest from a plurality of regions of interest of the object: the one or more image quality proxy values ​​for the region of interest include a mean spot separation value for the region of interest; determining the one or more image quality proxy values ​​for the region of interest using the focus component includes projecting the set of spots onto the surface of the target channel and detecting reflections of the set of spots from the surface of the target channel while the focus component is capturing the image of the region of interest; determining whether to adjust the feature of the imaging assembly includes determining whether to adjust a relative position of the objective lens and the surface of the target channel along the height; The apparatus of claim 1 , comprising:

10. 1. An apparatus comprising: A flow cell, a channel having a first end region, a second end region, and an intermediate region extending between the first end region and the second end region, the channel defining a length including the first end region, the intermediate region, and the second end region, the channel configured to receive a fluid, the channel including one or more calibration regions in the first end region, the second end region, or the intermediate region; a flow cell including a plurality of reaction sites positioned along the intermediate region, each reaction site configured to contain a biological sample carried by the fluid, and each reaction site further configured to receive excitation light; an imaging assembly operable to receive light emitted from a reactant positioned in the reaction site in response to the excitation light; a processor, the processor comprising: driving relative movement between at least a portion of the imaging assembly and the flow cell along a continuous range of motion, thereby enabling the imaging assembly to capture images along the length of the channel; activating the imaging assembly to capture one or more calibration images related to the one or more calibration areas during a first portion of the continuous range of motion; The apparatus is configured to activate the imaging assembly to capture an image of the reaction site during a second portion of the continuous range of motion.

11. The apparatus of claim 10 , wherein the one or more calibration regions are positioned in the first end region.

12. 12. The apparatus of claim 10 or 11, wherein the one or more calibration regions are positioned in the second end region.

13. The apparatus of claim 10 , wherein the one or more calibration areas are positioned within the intermediate area.

14. The apparatus of claim 10 , wherein the one or more calibration regions comprise nucleotides.

15. The apparatus of claim 10 , wherein the processor is further configured to adjust characteristics of the imaging assembly based at least in part on data from the one or more calibration images.

Images