Grating fiducial based machine vision system for placement of a sample in an analysis apparatus

Abstract

The techniques described herein relate to positioning a flow cell on a chuck that is configured for moving the flow cell with respect to an imaging system that captures image data from samples in wells of the flow cell. The techniques include: illuminating, by a light source, a fiducial marker of the flow cell; capturing image data of the illuminated fiducial marker with a camera; determining a position of an illuminated fiducial marker based on the image data captured by the camera of the illuminated fiducial marker based on a determined position of the fiducial marker; positioning a robotic end effector at a first position to pick up the flow cell the end effector; and based on a determined position of the illuminated fiducial marker, moving the flow cell picked up by the end effector to a second position to place the flow cell on the chuck.

Description

Aty Docket No. IP-2803-PCT / 0187-020W01GRATING FIDUCIAL BASED MACHINE VISIONSYSTEM FOR PLACEMENT OF A SAMPLE IN ANANALYSIS APPARATUSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 695,080, filed on September 16, 2024, and entitled “GRATING FIDUCIAL BASED MACHINE VISION SYSTEM FOR PLACEMENT OF A SAMPLE IN AN ANALYSIS APPARATUS,” the disclosure of which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] Aspects of the present disclosure relate generally to biological or chemical analysis and more particularly to grating fiducial based machine vision systems and their use for placement of a sample in an analysis apparatus.BACKGROUND

[0003] Various protocols in biological and chemical research involve performing a large number of controlled reactions on local support surfaces or within predefined reaction chambers. The designated reactions may then be observed or detected, and subsequent analysis may help identify or reveal properties of chemicals involved in the reaction. For example, in some multiplex assays, an unknown analyte having an identifiable label (e.g., fluorescent label) may be exposed to thousands of known probes under controlled conditions. Each known probe may be deposited into a corresponding well of a flow cell channel. Observing any chemical reactions that occur between the known probes and the unknown analyte within the wells may help identify or reveal properties of the analyte. Other examples of such protocols include known DNA sequencing processes, such as sequencing-by- synthesis (SBS) or cyclic-array sequencing.

[0004] In some conventional fluorescent-detection protocols, an optical system is used to direct an excitation light onto fluorescently-labeled analytes and to also detect the fluorescent signals that may be emitted from the analytes. Such optical systems may include an arrangement of lenses, filters, and light sources. Chemical reactions involving the analytes can take place on surfaces, or within predefined reaction chambers, of glass slides referred toAtly Docket No. IP-2803-PCT / 0187-020WQ1 as nanowells patterned onto on a flow cell. Some conventional sequencing systems may use mechanical datums to ensure the sample is in a correct location for imaging the flow cell. However, the use of mechanical datum at times can result in misalignment of the flow cell. Moreover, it is difficult to use different sized flow cells with mechanical datums, because they different sizes of the flow cells may not align well with a single set of mechanical datums. As a result, an optical system may be designed to use only a one size of flow cells, which can result in an inefficient use of space on a wafer from which flow cells are fabricated.SUMMARY

[0005] In a general aspect, the techniques described herein relate to an apparatus that includes a rack configured for holding a plurality of flow cells, where each flow cell includes one or more fiducial markers and a plurality of wells, and each well is configured to receive a sample. The apparatus includes an imaging system configured for capturing images of samples in the wells, a chuck configured for holding a flow cell and for moving a flow cell with respect to the imaging system, and a robotic system. The robotic system includes: an end effector, where the end effector includes a gripper configured for holding one of the plurality of flow cells, a light source configured to illuminate a fiducial marker of the flow cell, and a camera configured to capture image data of an illuminated fiducial marker. The robotic system includes: a processor configured to determine a position of an illuminated fiducial marker based on the image data of the illuminated fiducial marker; and one or more motors configured to move the end effector, based on one or more first determined positions of one or more of the illuminated fiducial markers, to a first position to pick up the flow cell from the rack with the gripper and to move the end effector, based on one or more second determined positions of one or more of the illuminated fiducial markers, to a second position to place the flow cell on the chuck.

[0006] Implementations can include one or more of the following features, alone, or any combination with each other.

[0007] For example, the fiducial markers can include a patterned array of optical elements that form an optical grating.

[0008] In another example, the patterned array of optical elements can be included in a layer of the flow cells that includes the wells.

[0009] In another example, the patterned array of optical elements can have a pitch that is substantially similar to a pitch of an array of the plurality of wells.Atty Docket No. IP-2803-PCT / 0187-020W01

[0010] In another example, the light source can include an LED that emits broadband light.

[0011] In another example, the light source can include a laser that emits narrow band light.

[0012] In another example, the camera can include one or more non-telecentric lenses to image the fiducial markers.

[0013] In another example, lenses of the camera that images the fiducial markers can include only non-telecentric lenses.

[0014] In another example, the camera can be located less than 20 mm from the illuminated fiducial marker from which the camera is configured to capture images.

[0015] In another example, the light source can be located less than 15 mm from the illuminated fiducial marker from which the camera is configured to capture images.

[0016] In another example, the gripper can include vacuum cups.

[0017] In another example, the gripper can include a magnet.

[0018] In another aspect, the techniques described herein relate to a method of positioning a flow cell on a chuck that is configured for moving the flow cell with respect to an imaging system that captures image data from samples in wells of the flow cell. The method includes: illuminating, by a light source, a fiducial marker of the flow cell; generating image data of the illuminated fiducial marker with a camera; determining a position of an illuminated fiducial marker based on the image data generated by the camera of the illuminated fiducial marker based on a determined position of the fiducial marker; positioning a robotic end effector at a first position to pick up the flow cell the end effector; and based on a determined position of the illuminated fiducial marker, moving the flow cell picked up by the end effector to a second position to place the flow cell on the chuck.

[0019] Implementations can include one or more of the following features, alone or any combination with each other.

[0020] For example, the fiducial marker can include a patterned array of optical elements that form an optical grating.

[0021] In another example, the patterned array of optical elements can be included in a layer of the flow cell that includes the wells.

[0022] In another example, the patterned array of optical elements can have a pitch that is substantially similar to a pitch of an array of the wells.

[0023] In another example, the light source can include an LED that emits broadband light.Atty Docket No. IP-2803-PCT / 0187-020W01

[0024] In another example, the camera can include one or more non-telecentric lenses to image the fiducial marker.

[0025] In another example, lenses of the camera can include only non-telecentric lenses.

[0026] In another example, the camera can be located less than 20 mm from the illuminated fiducial marker.

[0027] In another example, the light source can be located less than 15 mm from the illuminated fiducial marker.BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG. 1 depicts a schematic view of an example of a system that may be used to provide biological or chemical analysis.

[0029] FIG. 2 depicts a schematic view of an example of a set of components that may cooperate to provide a fluid path in the system of FIG. 1.

[0030] FIG. 3 depicts a schematic view of another example of a system that may be used to provide biological or chemical analysis.

[0031] FIG. 4 is a schematic diagram of an example analysis system for processing predefined reaction chambers (such as for biological applications), imaging the patterned predefined reaction chambers, and analysis of data derived from the imaging.

[0032] FIG. 5 is a schematic diagram of an analysis system in which one or more flow cells can be provided to an imaging system, where nanowells of the flow cells can be illuminated and imaged.

[0033] FIG. 6 is a schematic diagram of a workflow for picking a flow cell from a rack and placing the flow cell on a chuck for imaging.

[0034] FIG. 7 is schematic diagram of an example flow cell, which can include one or more substates, or substrate layers.

[0035] FIG. 8A is a schematic diagram of a repeating pattern of octagonal wells arranged in a hexagonal pattern.

[0036] FIG. 8B is a schematic diagram of a repeating pattern of circular wells arranged in a hexagonal pattern.

[0037] FIG. 9 is a schematic diagram of a periodic pattern that acts as a diffraction grating to diffract light from light source by an angle 9' along an axis that intersects an axis of a camera.

[0038] FIG. 10 is a schematic perspective view of an example pick and place (PnP)Atty Docket No. IP-2803-PCT / 0187-020W01 robotic drive arm that is configured to move an end effector of a PnP system to automatically pick up a flow cell from a rack and move the flow cell into position on a chuck.

[0039] FIGs. 11 A, 11B, and 11C are schematic end, side, and bottom views, respectively of an end effector.

[0040] FIG. 12 is a schematic perspective view of an example end effector that has picked up, and is mechanically coupled to, a flow cell.

[0041] FIG. 13 A is a perspective view of a portion of an end effector, which includes a light source configured to illuminate a fiducial on a flow cell and a camera configured to image light diffracted from the fiducial.

[0042] FIG. 13B is a bottom view of the portion of the end effector of FIG. 13 A, looking up, from below the flow cell, at the camera.

[0043] FIG. 14 is an example image of a checkerboard pattern that can be used to calibrate a camera for determining XY positions of fiducials.

[0044] FIG. 15Ais a schematic perspective view of an end effector holding a flow cell at a first distance in the Z direction above a chuck.

[0045] FIG. 15B is a schematic top view of ends of the end effector holding the flow cell at a distance in the Z direction above the chuck.

[0046] FIG. 15C is a schematic perspective view of the end effector in position to place the flow cell on the chuck.DETAILED DESCRIPTION

[0047] This disclosure provides methods and systems for locating a flow cell to ensure the features of predefined reaction chambers are in alignment with an imaging system, where the locating is performed though an optical method of datuming. It may be beneficial to automate the process of sample placement and datuming by using a machine vision system. Datuming with a machine vision system can enables a user to load multiple flow cells at once into an analysis apparatus, and then to have the apparatus automatically pick up and place a flow cell for imaging, remove the flow cell when an imaging run is complete, automatically pick up and place a different flow cell for imaging, remove that flow cell when an imaging run is complete, etc., thus improving throughput and decreasing instrument downtime. The system includes the imaging, image data analysis, and placement mechanics that are useful for locating features of a flow cell within a system. The systems and methods may be used to automatically place multiple flow cells in a location relative to an imaging system, such that the image processing system can capture image data from the flow cell forAty Docket No. IP-2803-PCT / 0187-020W01 analysis of the content contained in the captured images. This system can also be used to remove samples after they have been tested in order to make space for the next sample. Relevant to the present techniques are flow cells, the processing of which produces image data (or any other form of detection output of sites within the predefined reaction chambers) of wells, such as those used for the analysis of biological samples. Such flow cells may contain repeating patterns of features or predefined reaction chambers that are to be resolved at sub-micron resolution ranges. These patterns are repeated in other regions and can be leveraged as fiducials for machine vision systems for which the methods and systems of the present disclosure are well suited. Although the systems and methods set forth herein provide advantages when placing regular patterns of features in an array, it will be understood that they can be used for random distributions of features in an array as well. As discussed below, in many applications, the material to be imaged and analyzed will be located on one or more surfaces of one or more supports, such as a glass material. Fiducial markers, or simply “fiducials” are located at known locations of the support of the patterned arrays with respect to the sites to assist in accurately placing the support in the system in relation to an imaging system, so that sites in subsequent image data captured by the imaging system can be accurately located.

[0048] As used in the present disclosure, a “patterned array” may include a microarray, a nanoarray, a sequencing array formed as a patterned flow cell, and so forth. Such devices comprise sites at which analytes may be located for processing and analysis. In practice, the sites may be disposed in a repeating pattern, a non-repeating pattern, or in a random arrangement on one or more surfaces of a support, which itself may comprise a flow cell as discussed below. For simplicity, all such devices are referred to and should be understood as included in the term “patterned array” or sometimes simply as “array.”

[0049] A flow cell is a substrate that can be used in preparing and accommodating or carrying one or more samples in at least one stage of an analysis process. A substrate may refer to any material that provides a substantially rigid structure, or to a structure that retains its shape rather than taking on the shape of a vessel to which it is placed in contact. The material can have a surface to which another material can be attached including, for example, smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces), as well as textured and / or porous materials. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materialsAtly Docket No. IP-2803-PCT / 0187-020W01 including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers. In general, the substrates allow optical detection and do not themselves appreciably fluoresce. A flow cell is made of a material that is compatible with both the genetic material, the illumination and the chemical reactions to which it will be exposed. The substrate can have one or more channels in which sample material can be deposited. A substance (e.g., a liquid) can be flowed through the channel where the sample genetic material is present to trigger one or more chemical reactions and / or to remove unwanted material. A flow cell may enable the imaging by facilitating that the sample in the flow cell channel can be subjected to illuminating light and that any fluorescent responses from the sample can be detected. Some implementations of the system may be designed to be used with at least one flow cell but may not include the flow cell(s) during one or more stages, such as during shipping or when delivered to a customer. For example, flow cell(s) can be installed into an implementation at the customer's premises in order to perform analysis.

[0050] The methods and systems set forth herein are useful for analyzing any of a variety of materials, such as biological samples and molecules, which may be on or in a variety of objects. Useful objects are solid supports or solid-phase surfaces with attached analytes. The methods and systems set forth may provide advantages when used with objects having a repeating pattern of features in an X-Y plane, such as a patterned array having an attached collection of molecules, such as DNA, RNA, biological material from viruses, proteins, antibodies, carbohydrates, small molecules (such as drug candidates), biologically active molecules, or any other analytes of interest.

[0051] An increasing number of applications have been developed for arrays with features having biological molecules, such as nucleic acids and polypeptides. Such patterned arrays may include DNA or RNA probes. These are specific for nucleotide sequences present in plants, animals (e.g., humans), and other organisms. In some applications, for example, individual DNA or RNA probes can be attached at individual features or sites of an array. A test sample, such as from a known or unknown person or organism, can be exposed to the array, such that target nucleic acids (e.g., gene fragments, messenger RNA, or amplicons thereof) hybridize to complementary probes at respective features or sites in the array. The probes can be labeled in a target specific process (e.g., due to labels present on the target nucleic acids or due to enzymatic labeling of the probes or targets that are present in hybridized form at the features). The array can then be examined, such as by scanning specific frequencies of light over the features to identify which target nucleic acids areAty Docket No. IP-2803-PCT / 0187-020W01 present in the sample.

[0052] Biological patterned arrays may be used for genetic sequencing and similar applications. In general, genetic sequencing includes determining the order of nucleotides in a length of target nucleic acid, such as a fragment of DNA or RNA. Relatively short sequences may be sequenced at each feature, and the resulting sequence information may be used in various bioinformatics methods to logically fit the sequence fragments together, so as to reliably determine the sequence of much more extensive lengths of genetic material from which the fragments are available. Automated, computer-based algorithms for characterizing fragments have been developed, and have been used more recently in genome mapping, identification of genes and their function, and so forth. Patterned arrays are useful for characterizing genomic content because a large number of variants are present and this supplants the alternative of performing many experiments on individual probes and targets. The patterned array may be a desirable format for performing such investigations in a practical manner.

[0053] As noted above, any of a variety of analyte arrays (also referred to in the present disclosure as “patterned arrays” or simply as “arrays”) known in the art can be used in methods and systems set forth herein. Such arrays contain features, each having an individual probe or a population of probes. In the latter case, the population of probes at each feature may be homogenous having a single species of probe. For example, in the case of a nucleic acid array, each feature can have multiple nucleic acid molecules each having a common sequence. However, in some other examples, the populations at each feature of an array can be heterogeneous. Similarly, protein arrays can have features with a single protein or a population of proteins, which may or may not have the same amino acid sequence. The probes can be attached to the surface of an array, for example, via covalent linkage of the probes to the surface or via non-covalent interaction of the probes with the surface. In some examples, probes, such as nucleic acid molecules, can be attached to a surface via a gel layer.

[0054] Arrays used for nucleic acid sequencing can include random spatial patterns of nucleic acid features. For example, NovaSeqX™ or NextSeq 1000 / 2000™ sequencing platforms available from Illumina, Inc. utilize flow cells comprising supports or arrays upon which nucleic acid(s) is / are disposed by random seeding followed by bridge amplification. However, patterned arrays also can be used for nucleic acid sequencing or other analytical applications. Example patterned arrays, methods for their manufacture and methods for their use are set forth in U.S. Pat. Nos. 9,512,422; 8,895,249; and 9,012,022; and in U.S. Pat. App. Pub. Nos. 2013 / 0116153 Al; and 2012 / 0316086 Al, each of which is incorporated herein byAtly Docket No. IP-2803-PCT / 0187-020W01 reference in its entirety. The features of such patterned arrays can be used to capture a single nucleic acid template molecule to seed subsequent formation of a homogenous colony, for example, via bridge amplification. Such patterned arrays are useful for nucleic acid sequencing applications.

[0055] The size of features, such as sites on an array (or another object used in a method or system herein), can be selected to suit a desired application. In some examples, a feature of an array can have a size that accommodates only a single nucleic acid molecule. A surface having a plurality of features in this size range is useful for constructing an array of molecules for detection at single molecule resolution. Features in this size range are also useful in arrays having features that each contain a colony of nucleic acid molecules. Thus, the features of an array can each have an area that is no larger than about 1 mm2, no larger than about 500 pm2, no larger than about 100 pm2, no larger than about 10 pm2, no larger than about 1 pm2, no larger than about 500 nm2, or no larger than about 100 nm2, no larger than about 10 nm2, no larger than about 5 nm2, or no larger than about 1 nm2. Alternatively, or additionally, the features of an array will be no smaller than about 1 mm2, no smaller than about 500 pm2, no smaller than about 100 pm2, no smaller than about 10 pm2, no smaller than about 1 pm2, no smaller than about 500 nm2, no smaller than about 100 nm2, no smaller than about 10 nm2, no smaller than about 5 nm2, or no smaller than about 1 nm2. Indeed, a feature can have a size that is in a range between an upper and lower limit selected from those exemplified above. Although several size ranges for features of a surface have been exemplified with respect to nucleic acids and on the scale of nucleic acids, it will be understood that features in these size ranges can be used for applications that do not include nucleic acids. It will be further understood that the size of the features need not necessarily be confined to a scale used for nucleic acid applications.

[0056] For examples that include an object (e.g., an array or support) having a plurality of features or sites, the features can be discrete, being separated with spaces between each other. An array useful in the present techniques can have features that are separated by edge-to-edge distance of at most about 100 pm, about 50 pm, about 10 pm, about 5 pm, about 1 pm, about 0.5 pm, 450 nm, 400 nm, 350 nm, 300 nm or less. Alternatively, or additionally, an array can have features that are separated by an edge to edge distance of at least about 300 nm, 350 nm, 400 nm, 450 nm, 0.5 pm, about 1 pm, about 5 pm, about 10 pm, about 50 pm, about 100 pm, or more. These ranges can apply to the average edge to edge spacing for features, as well as to the minimum or maximum spacing.

[0057] The density of features in an array also can be understood in terms of theAty Docket No. IP-2803-PCT / 0187-020W01 number of features present per unit area. For example, the average density of features for an array can be at least about 1 x 103features / mm2, about 1 x 104features / mm2, about 1 x 105features / mm2, about I x lO6features / mm2, about I x lO7features / mm2, about I x lO8features / mm2, or about I x lO9features / mm2or higher. Alternatively, or additionally, the average density of features for an array can be at most about I x lO9features / mm2, about I x lO8features / mm2, about I x lO7features / mm2, about I x lO6features / mm2, about I x lO5features / mm2, about I x lO4features / mm2, or about I x lO3features / mm2or less.

[0058] The features in a patterned example can have any of a variety of pattern shapes and layouts. For example, when observed in a two-dimensional plane, such as on the surface of an array, the features can appear rounded, circular, oval, rectangular, square, symmetric, asymmetric, triangular, polygonal, or the like. The features can be arranged in a regular repeating pattern including, for example, a hexagonal or rectilinear pattern. A pattern can be selected to achieve a desired level of packing. For example, round features are optimally packed in a hexagonal arrangement. Other packing arrangements also can be used for round features and vice versa. Many patterns can result in an optical gradient. The optical properties (angle of reflection, color, orientation, diffraction order, and diffraction efficiency) of the gradient can be tuned based on the layout of the pattern.

[0059] In general, a pattern can be characterized in terms of the number of features that are present in a subset that forms the smallest geometric unit of the pattern. The subset can include, for example, at least 2, 3, 4, 5, 6, 10 or more features. Depending upon the size and density of the features, the geometric unit can occupy an area of less than about 1 mm2, about 500 pm2, about 100 pm2, about 50 pm2, about 10 pm2, about 1 pm2, about 500 nm2, about 100 nm2, about 50 nm2, or about 10 nm2or less. Alternatively, or additionally, the geometric unit can occupy an area of greater than about 10 nm2, about 50 nm2, about 100 nm2, about 500 nm2, about 1 pm2, about 10 pm2, about 50 pm2, about 100 pm2, about 500 pm2, or about 1 mm2or more. Characteristics of the features in a geometric unit, such as shape, size, pitch and the like, can be selected from those set forth herein more generally with regard to features in an array or pattern.

[0060] An array having a regular pattern of features can be ordered with respect to the relative locations of the features but random with respect to one or more other characteristics of each feature. For example, in the case of a nucleic acid array, the nucleic acid features can be ordered with respect to their relative locations but random with respect to one's knowledge of the sequence for the nucleic acid species present at any feature. As a more specific example, nucleic acid arrays formed by seeding a repeating pattern of features with templateAtly Docket No. IP-2803-PCT / 0187-020W01 nucleic acids and amplifying the template at each feature to form copies of the template at the feature (e.g., via cluster amplification or bridge amplification) will have a regular pattern of nucleic acid features but will be random with regard to the distribution of sequences of the nucleic acids across the array. Thus, detection of the presence of nucleic acid material on the array can yield a repeating pattern of features, whereas sequence specific detection can yield non-repeating distribution of signals across the array.

[0061] As used herein, the term “fiducial” means a distinguishable point of reference in or on an object, such as a support or substrate with sites for molecular materials to be analyzed, as well as in image data of the object. The point of reference can be, for example, a mark, an object, shape, edge, line, area, irregularity, channel, pit, post, or, as in many cases, a pattern or collection of features at known locations that can be used as a reference. The point of reference can be detected in an image of the object or in another data set derived from detecting (e.g., imaging) the object. The point of reference can be specified by an X and / or Y coordinate in a plane of the object (e.g., one or more surfaces of the patterned array). One or more coordinates for a point of reference can be specified relative to one or more other features of an object or of an image or other data set derived from the object.

[0062] Several examples will be described below with respect to fiducials, their form, their configuration, and their use in systems and methods of analysis. It will be understood that systems are also provided for carrying out the methods in an automated or semiautomated way, and such systems can include a processor, a data storage device, and a program for image analysis, the program including instructions for carrying out one or more of the methods discussed below. Accordingly, the methods set forth herein can be carried out on a computer, for example, having components and algorithms needed for that purpose.

[0063] Fiducials are included on or in the arrays contemplated in the present disclosure, such as on one or more surfaces of patterned array supports or substrates (whether in an array or in any random or other layout), as well as in image data of the arrays to facilitate localization of the array with respect to other features of an imaging system, including fiducials on stages, racks, and the like, that are used to support the arrays during imaging of the sites of the arrays. Fiducials are useful for registering the spatial locations of sites or features since the fiducials provide a point of reference for relative locations of such sites or features. Fiducials are especially beneficial for applications in which multiple arrays are used in an imaging system, so that the different arrays can be moved automatically and accurately into position in the imaging system. For example, fiducials can allow an individual array to be picked up from a rack and moved into position on a translation stage that movesAtly Docket No. IP-2803-PCT / 0187-020W01 the array during imaging of the sites of the array.

[0064] Examples described herein refer to gratings. A grating can couple light impinging on the grating by way of diffracting at least a portion of the light, thereby causing the portion of the light to propagate in one or more other directions. In some implementations, the coupling can involve one or more interactions, including, but not limited to, reflection, refraction and / or transmission of the portion of the light. Implementations may be designed to meet one or more requirements, including, but not limited to, those regarding mass production, cost control, and / or high light coupling efficiency.

[0065] Examples described herein mention that one or more gratings can be provided for coupling of light. The gratings can be identical or similar to each other, or can be different types of gratings. The grating(s) can include one or more forms of periodic structure. In some implementations, the grating can be formed by removing or omitting material from a substrate (e.g., from a waveguide material that is included in the flow cell) or other material. For example, the flow cell can be provided with a set of slits and / or grooves therein to form the grating. In some implementations, the grating can be formed by adding matter to the flow cell (e.g., to a waveguide material that is included in the flow cell) or other material. For example, the flow cell can be provided with a set of ridges, bands or other protruding longitudinal structures to form the grating. Combinations of these approaches can be used.

[0066] Examples herein refer to photolithography. Photolithography -based approaches may involve use of a photoresist that is patterned with a stepper or mask aligner, exposed with radiation to transfer the pattern present on a reticle / photomask into the photoresist, and then developed to yield a structured film (photoresist) on top of the substrate. The structured resist may be the final substrate which can be used for subsequent core layer coating. As another example, the pattern in the resist can be transferred into a substrate or other material via additional processing. Follow on process operations may include reactive ion etching (plasma based dry etching) or a wet etch (chemically based) process. If the pattern is transferred into the substrate / material, the patterned photoresist is subsequently removed to yield the patterned substrate (e.g., for subsequent core layer coating). A sacrificial film of a material such as chromium or titanium or another metal under the photoresist, and first transfer the pattern in the photoresist to the metal film and then use that film as a hard mask by which the pattern is transferred into the substrate. After pattern transfer into the substrate, the films may be removed and hence be considered sacrificial to the fabrication process. One or more of a variety of materials may be applied in a photolithographic process.Atty Docket No. IP-2803-PCT / 0187-020W01In some implementations, an oxide material is used. For example, SiCh (silicon dioxide) may be applied. A lift-off process may be similar to a pattern photoresist process: instead of removing material through dry etching or wet etching, one may deposit materials (e.g., Si O2), followed by stripping off, which involves removing the photoresist together with the deposited materials on top of it. Grating structures can also or instead be formed.

[0067] Examples herein refer to nanoimprinting. In nanoimprinting lithography, a pre-fabricated nanoscale template may mechanically displace a fluidic resin to mold the desired nanostructures. The resin may then be cured with the nanoscale template in place. Following the removal of the nanoscale template, a molded solid resin attached to a desired substrate may be produced. In some implementations, a nanoimprinting process may begin with fully or partially covering a substrate or wafer with imprinting resin (e.g., a resin as exemplified below). One or more nanostructures may be formed in the imprinting resin in a molding process using a nanoscale template. The imprinting resin can be cured against the substrate or wafer, and a resin-removal process can be applied to remove residue from the wafer or substrate. For example, the resin removal can form chamber lanes adjacent the nanostructures. The substrate or wafer so formed can have another substrate or a gasket applied thereto so as to form a flow cell having the described nanostructures as well as flow cell chambers formed by enclosing the chamber lanes. In some implementations, the process of applying the imprinting resin may be configured to produce little or no resin residue, and in such implementations a resin-removal process can be omitted. In some applications, the cured resin may also be functionalized with a chemical treatment or an attachment of biomolecules, depending on the end use. In nanoimprinting lithography, an imprinted photoresist can be a sacrificial material and similarly be used as an intermediate tool to transfer the patterned resist into the substrate or a variation of the resist can be used such that the imprinted resist serves as the input to a subsequent coating process. An example of a resist that would remain following patterning is material formed by a process that involves conversion of monomers into a colloidal solution as a precursor to a gel of particles and / or polymers, sometimes referred to as a sol -gel based material.

[0068] Examples described herein mention that one or more resins may be used. Any suitable resin may be used for nanoimprinting in methods described herein. In some implementations, an organic resin may be used, including, but not limited to, an acrylic resin, a polyimide resin, a melamine resin, a polyester resin, a polycarbonate resin, a phenol resin, an epoxy resin, polyacetal resin, polyether resin, polyurethane resin, polyamide resin (and / or nylon), a furan resin, a diallyl phthalate resin, or combinations thereof. In some examples, aAtly Docket No. IP-2803-PCT / 0187-020W01 resin may include an inorganic siloxane polymer including a Si — O — Si bond among compounds (including silicon, oxygen, and hydrogen), and formed by using a siloxane polymer-based material typified by silica glass as a starting material. A resin used may also or instead be an organic siloxane polymer in which hydrogen bonded to silicon is substituted by an organic group, such as methyl or phenyl, and typified by an alkylsiloxane polymer, an alkylsilsesquioxane polymer, a silsesquioxane hydride polymer, or an alkylsilsesquioxane hydride polymer. Non-limiting examples of siloxane polymers include polyhedral oligomeric silsesquioxane (POSS), polydimethylsiloxane (PDMS), tetraethyl ortho silicate (TEOS), poly (organo) siloxane (silicone), and perfluoropolyether (PFPE). An example of POSS can be that described in Kehagias et al. Microelectronic Engineering 86 2009), pp. 776-778, which is hereby incorporated by reference in its entirety. A resin may be doped with a metal oxide. In some implementations, a resin may be a sol-gel material including, but not limited to, titanium oxide, hafnium oxide, zirconium oxide, tin oxide, zinc oxide, or germanium oxide, and that uses a suitable solvent. Any one of a number of other resins may be employed, as appropriate to the application.

[0069] FIG. 1 is a schematic diagram of an system 100 that may be used to perform an analysis on one or more samples of interest. In some implementations, the sample may include one or more clusters of nucleotides (e.g., DNA) that have been linearized to form a single stranded DNA (sstDNA). In the implementation shown, system 100 is configured to receive a flow cell cartridge assembly 102 including a flow cell assembly 103 and a sample cartridge 104. The system 100 includes a flow cell receptacle 122 that receives flow cell cartridge assembly 102, a vacuum chuck 124 that supports flow cell assembly 103, and a flow cell interface 126 that is used to establish a fluidic coupling between system 100 and flow cell assembly 103. The flow cell interface 126 may include one or more manifolds. The system 100 further includes a sipper manifold assembly 106, a sample loading manifold assembly 108, and a pump manifold assembly 110. The system 100 also includes a drive assembly 112, a controller 114, an imaging system 116, and a waste reservoir 118. The controller 114 is electrically and / or communicatively coupled to drive assembly 112 and to imaging system 116 and is configured to cause drive assembly 112 and / or the imaging system 116 to perform various functions as disclosed herein.

[0070] In an example implementation, the flow cell assembly 103 can include a flow cell 128 having a channel 130 and defining a plurality of first openings 132 that are fluidically coupled to the channel 130 and arranged on a first side 134 of the channel 130. The flow cell 128 further includes a plurality of second openings 136 fluidically coupled toAtty Docket No. IP-2803-PCT / 0187-020W01 the channel 130 and arranged on a second side 138 of the channel 130. Fluid may thus flow through the flow cell 128 via channel. While the flow cell 128 is shown as including one channel 130, the flow cell 128 may include two or more channels 130. The flow cell assembly 103 also includes a flow cell manifold assembly 140 coupled to flow cell 128 and having a first manifold fluidic line 142 and a second manifold fluidic line 144. The flow cell manifold assembly 140 may be in the form of a laminate including a plurality of layers as discussed in more detail below.

[0071] In the implementation shown, the first manifold fluidic line 142 has a first fluidic line opening 146 and is fluidically coupled to each of the first openings 132 of flow cell 128, and the second manifold fluidic line 144 has a second fluidic line opening 148 and is fluidically coupled to each of the second openings 136. As shown, the flow cell assembly 103 includes gaskets 150 coupled to flow cell manifold assembly 140 and fluidically coupled to fluidic line openings 146, 148. In some implementations in which the flow cell 128 includes a plurality of channels 130, the flow cell manifold assembly 140 may include additional fluidic lines 152 that couple first fluidic line openings 146 to a single manifold port 154. In such implementations, a single gasket 150 may be coupled to the flow cell manifold assembly 140 that surrounds the manifold port 154 and that is in fluidic communication with a plurality of channels 130. In operation, the flow cell interface 126 engages with corresponding gaskets 150 to establish a fluidic coupling between the system 100 and the flow cell 128. The engagement between the flow cell interface 126 and the gaskets 150 reduces or eliminates fluid leakage between the flow cell interface 126 and the flow cell 128.

[0072] In the implementation shown, the first manifold fluidic line 142 has a portion 156 that is substantially parallel to a longitudinal axis 158 of the channel 130, and the second manifold fluidic line 144 has a portion 160 that is substantially parallel to the longitudinal axis 158 of the channel 130. Additionally, the first manifold fluidic line 142 is shown being at least partially adjacent a first end 162 of the flow cell 128 and spaced from a second end 164 of the flow cell 128, and the second manifold fluidic line 144 is shown being at least partially adjacent the second end 164 of the flow cell 128 and spaced from first end 162. Other arrangements of the manifold fluidic lines 142, 144 may prove suitable.

[0073] In the implementation shown, the system 100 includes a sample cartridge receptacle 166 that receives the sample cartridge 104 that carries one or more samples of interest e.g., an analyte). The system 100 also includes a sample cartridge interface 168 that establishes a fluidic connection with the sample cartridge 104. The sample loading manifold assembly 108 includes one or more sample valves 170. The pump manifold assembly 110Atly Docket No. IP-2803-PCT / 0187-020W01 includes one or more pumps 172, one or more pump valves 174, and a cache 176. Valves 170, 174 and pumps 172 may take any suitable form. The cache 176 may include a serpentine cache and may temporarily store one or more reaction components during, for example, bypass manipulations of the system 100. While cache 176 is shown being included in pump manifold assembly 110, the cache 176 may alternatively be located elsewhere e.g., in sipper manifold assembly 106 or in another manifold downstream of a bypass fluidic line 178, etc..

[0074] The sample loading manifold assembly 108 and the pump manifold assembly 110 flow one or more samples of interest from sample cartridge 104 through a fluidic line 180 toward flow cell cartridge assembly 102. In some implementations, the sample loading manifold assembly 108 may individually load or address each channel 130 of the flow cell 128 with a respective sample of interest. The process of loading a channel 130 with a sample of interest may occur automatically using system 100. As shown in FIG. 1, the sample cartridge 104 and the sample loading manifold assembly 108 are positioned downstream of the flow cell cartridge assembly 102. In the implementation shown, the sample loading manifold assembly 108 is coupled between the flow cell cartridge assembly 102 and the pump manifold assembly 110. To draw a sample of interest from the sample cartridge 104 and toward the pump manifold assembly 110, sample valves 170, pump valves 174, and / or pumps 172 may be selectively actuated to urge the sample of interest toward the pump manifold assembly 110. The sample cartridge 104 may include a plurality of sample reservoirs that are selectively fluidically accessible via the corresponding sample valves 170. To individually flow the sample of interest toward a channel 130 of the flow cell 128 and away from the pump manifold assembly 110, sample valves 170, pump valves 174, and / or pumps 172 may be selectively actuated to urge the sample of interest toward flow cell cartridge assembly 102 and into respective channels 130 of flow cell 128.

[0075] The drive assembly 112 interfaces with the sipper manifold assembly 106 and the pump manifold assembly 110 to flow one or more reagents that interact with the sample within the flow cell 128. In some scenarios, a reversible terminator is attached to the reagent to allow a single nucleotide to be incorporated onto a growing DNA strand. In some such implementations, one or more of the nucleotides has a unique fluorescent label that emits a color when excited. The color (or absence thereof) is used to detect the corresponding nucleotide. In the implementation shown, the imaging system 116 excites one or more of the identifiable labels (e.g., a fluorescent label) and thereafter obtains image data for the identifiable labels. The labels may be excited by incident light and / or a laser and the image data may include one or more colors emitted by the respective labels in response to theAtty Docket No. IP-2803-PCT / 0187-020W01 excitation. The image data (e.g., detection data) may be analyzed by the system 100. Examples of features and functionalities that may be incorporated into imaging system 116 will be described in greater detail below.

[0076] After the image data is obtained, the drive assembly 112 interfaces with the sipper manifold assembly 106 and the pump manifold assembly 110 to flow another reaction component (e.g., a reagent) through the flow cell 128 that is thereafter received by the waste reservoir 118 via a primary waste fluidic line 182 and / or otherwise exhausted by system 100. Some reaction components may perform a flushing operation that chemically cleaves the fluorescent label and the reversible terminator from the sstDNA. The sstDNA may then be ready for another cycle.

[0077] The primary waste fluidic line 182 is coupled between pump manifold assembly 110 and the waste reservoir 118. In some implementations, pumps 172 and / or pump valves 174 of the pump manifold assembly 110 selectively flow the reaction components from the flow cell cartridge assembly 102, through the fluidic line 180 and the sample loading manifold assembly 108 to the primary waste fluidic line 182. The flow cell cartridge assembly 102 is coupled to a central valve 184 via the flow cell interface 126. The central valve 184 is coupled with the flow cell interface 126 via a fluidic line 185. An auxiliary waste fluidic line 186 is coupled to the central valve 184 and to the waste reservoir 118. In some implementations, the auxiliary waste fluidic line 186 receives excess fluid of a sample of interest from the flow cell cartridge assembly 102, via the central valve 184, and flows the excess fluid of the sample of interest to the waste reservoir 118 when back loading the sample of interest into the flow cell 128, as described herein.

[0078] The sipper manifold assembly 106 includes a shared line valve 188 and a bypass valve 190. The shared line valve 188 may be referred to as a reagent selector valve. The central valve 184 and the valves 188, 190 of the sipper manifold assembly 106 may be selectively actuated to control the flow of fluid through the fluidic lines 192, 194, 196. The sipper manifold assembly 106 may be coupled to a corresponding number of reagent reservoirs 198 via reagent sippers 200. The reagent reservoirs 198 may contain fluid (e.g., reagent and / or another reaction component. In some implementations, the sipper manifold assembly 106 includes a plurality of ports. Each port of the sipper manifold assembly 106 may receive one of the reagent sippers 200. The reagent sippers 200 may be referred to as fluidic lines. Some forms of reagent sippers 200 may include an array of sipper tubes extending downwardly along the z-dimension from ports in the body of sipper manifold assembly 106. The reagent reservoirs 198 may be provided in a cartridge, and the tubes of theAtty Docket No. IP-2803-PCT / 0187-020W01 reagent sippers 200 may be configured to be inserted into corresponding reagent reservoirs 198 in the reagent cartridge so that liquid reagent may be drawn from each reagent reservoir 198 into the sipper manifold assembly 106.

[0079] The shared line valve 188 of the sipper manifold assembly 106 is coupled to the central valve 184 via the shared reagent fluidic line 196. Different reagents may flow through the shared reagent fluidic line 196 at different times. In some versions, when performing a flushing operation before changing between one reagent and another, the pump manifold assembly 110 may draw wash buffer through the shared reagent fluidic line 196, the central valve 184, and the flow cell cartridge assembly 102.

[0080] The bypass valve 190 of the sipper manifold assembly 106 is coupled to central valve 184 via dedicated reagent fluidic lines 194, 196. Each of the dedicated reagent fluidic lines 194, 196 may be associated with a single reagent. The fluids that may flow through dedicated reagent fluidic lines 194, 196 may be used during sequencing operations and may include a cleave reagent, an incorporation reagent, a scan reagent, a cleave wash, and / or a wash buffer.

[0081] The bypass valve 190 is also coupled to the cache 176 of the pump manifold assembly 110 via the bypass fluidic line 178. One or more reagent priming operations, hydration operations, mixing operations, and / or transfer operations may be performed using bypass fluidic line 178. The priming operations, the hydration operations, the mixing operations, and / or the transfer operations may be performed independent of flow cell cartridge assembly 102. Thus, the operations using the bypass fluidic line 178 may occur during, for example, incubation of one or more samples of interest within the flow cell cartridge assembly 102. That is, the shared line valve 188 may be utilized independently of the bypass valve 190 such that the bypass valve 190 may utilize the bypass fluidic line 178 and / or the cache 176 to perform one or more operations while the shared line valve 188 and / or the central valve 184 simultaneously, substantially simultaneously, or offset synchronously perform other operations.

[0082] The drive assembly 112 includes a pump drive assembly 202 and a valve drive assembly 204. The pump drive assembly 202 may be adapted to interface with one or more pumps 172 to pump fluid through the flow cell 128 and / or to load one or more samples of interest into the flow cell 128. The valve drive assembly 204 may be adapted to interface with one or more of the valves 170, 174, 184, 188, 190 to control the position of the corresponding valves 170, 174, 184, 188, 190.

[0083] FIG. 2 shows an example of a fluidic arrangement 220 that may beAtty Docket No. IP-2803-PCT / 0187-020W01 incorporated into a variation of system 100. The fluidic arrangement 220 of this example includes a pump manifold assembly 222, which may operate similar to the pump manifold assembly 110 described above, a sample loading manifold assembly 228, which may operate similar to the sample loading manifold assembly 108 described above, a flow cell interface 240, which may operate similar to the flow cell interface 126 described above, a sipper manifold assembly 250, which may operate similar to sipper manifold assembly 106 described above, and a waste reservoir 270, which may operate similar to the waste reservoir 118 described above. The pump manifold assembly 222 is coupled with a port assembly 258 of the sipper manifold assembly 250 via a fluidic line 224, which may be similar to the fluidic line 178, and with the sample loading manifold assembly 228 via a fluidic line 226. The sample loading manifold assembly 228 is coupled with the flow cell interface 240 via fluidic line 230, which may be similar to the fluidic line 180; and with the port assembly 258 via the fluidic lines 232, 234. The flow cell interface 240 is coupled with the sipper manifold assembly 250 via fluidic line 242, which may be similar to the fluidic line 185. The sipper manifold assembly 250 includes a manifold body 252 and a common output port 256, which provides fluid communication via the fluidic line 185. A valve assembly 254 controls fluid flow through the common output port 256 and may operate similar to the central valve 184. The port assembly 258 of the sipper manifold assembly 250 is coupled with the waste reservoir 270 via the fluidic line 272, which may be similar to the fluidic line 186.

[0084] A plurality of reagent sippers 260 extend from manifold body 252 and are fluidically coupled with the valve assembly 254 via respective fluid channels 262 in the manifold body 252. The reagent sippers 260 may operate similar to the reagent sippers 200. The valve assembly 254 is operable to selectively couple the fluid channels 262 with the flow cell interface 240 via the common output port 256 and the fluidic line 230, to thereby selectively provide various reagents to the flow cell interface 240. In other words, when each reagent sipper 260 is disposed in a different respective reagent (e.g., in a respective reagent reservoir 198), a flow cell (e.g., like flow cell 128)) that is coupled with the flow cell interface 240 may selectively receive those different reagents based on control of the valve assembly 254.

[0085] The port assembly 258 may provide a fluidic interface between the pump manifold assembly 222 and the sipper manifold assembly 250, thereby allowing the sipper manifold assembly 250 to receive pressurized fluid from the pump manifold assembly 222. The port assembly 258 may also provide a fluidic interface between the sample loading manifold assembly 228 and the sipper manifold assembly 250, thereby allowing the sipperAtly Docket No. IP-2803-PCT / 0187-020W01 manifold assembly 250 to receive sample fluid from the sample loading manifold assembly 228. In addition, the port assembly 258 may provide a fluidic interface between the waste reservoir 270 and the sipper manifold assembly 250, thereby allowing the sipper manifold assembly 250 to communicate waste fluid to the waste reservoir 270. Communication of fluids via the port assembly 258 may be regulated, at least in part, by the valve assembly 254.

[0086] Referring back to FIG. 1, the controller 114 of the present example includes a user interface 206, a communication interface 208, one or more processors 210, and a memory 212 storing instructions executable by the one or more processors 210 to perform various functions including the disclosed implementations. The user interface 206, the communication interface 133, and the memory 212 are electrically and / or communicatively coupled to the one or more processors 210. The user interface 206 may be adapted to receive input from a user and to provide information to the user associated with the operation of the system 100 and / or an analysis taking place. The user interface 206 may include a touch screen, a display, a keyboard, a speaker(s), a mouse, a track ball, and / or a voice recognition system.

[0087] The communication interface 208 is adapted to enable communication between system 100 and a remote system(s) (e.g., computers) via a network(s) (e.g., the Internet, an intranet, a local-area network (LAN), a wide-area network (WAN), a coaxialcable network, a wireless network, a wired network, a satellite network, a digital subscriber line (DSL) network, a cellular network, a Bluetooth connection, a near field communication (NFC) connection, etc.). Some of the communications provided to the remote system may be associated with analysis results, imaging data, etc. generated or otherwise obtained by the system 100. Some of the communications provided to the system 100 may be associated with a fluidics analysis operation, patient records, and / or a protocol(s) to be executed by the system 100.

[0088] The one or more processors 210 and / or the system 100 may include one or more of a processor-based system(s) or a microprocessor-based system(s). In some implementations, the one or more processors 210 and / or the system 100 includes one or more of a programmable processor, a programmable controller, a microprocessor, a microcontroller, a graphics processing unit (GPU), a digital signal processor (DSP), a reduced-instruction set computer (RISC), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a field programmable logic device (FPLD), a logic circuit, and / or another logic-based device executing various functions including the ones described herein.Atly Docket No. IP-2803-PCT / 0187-020W01

[0089] The memory 212 may include one or more of a semiconductor memory, a magnetically readable memory, an optical memory, a hard disk drive (HDD), an optical storage drive, a solid-state storage device, a solid-state drive (SSD), a flash memory, a readonly memory (ROM), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), a random-access memory (RAM), a non-volatile RAM (NVRAM) memory, a compact disc (CD), a compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a Blu-ray disk, a redundant array of independent disks (RAID) system, a cache and / or any other storage device or storage disk in which information is stored for any duration (e.g., permanently, temporarily, for extended periods of time, for buffering, for caching).

[0090] FIG. 3 illustrates a schematic diagram of another example of a system 300 that may be used to perform an analysis on one or more samples of interest. Except as otherwise described below, the system 300 of this example may be configured and operable like the system 100 described above with reference to FIG. 1. In some instances, the system 100 is used to provide a higher volume throughput, while the system 300 is used to provide a lower volume throughput. Alternatively, the systems 100, 300 may provide any other suitable amount or degree of throughput. The system 300 of the present example receives a reagent cartridge 302 and includes, in part, a gas source 304, a drive assembly 306, a controller 308, an imaging system 310, and a waste reservoir 312. The reagent cartridge 302 may be referred to as a consumable, a reagent reservoir, or a reagent assembly. The controller 308 is electrically and / or communicatively coupled to the drive assembly 306 and to the imaging system 310 and causes the drive assembly 306 and / or the imaging system 310 to perform various functions as disclosed herein.

[0091] The reagent cartridge 302 in the implementation shown includes a well assembly 314 having a body 316. The body 316 has a first wall 318 defining a well 320 having a port 322. The first wall 318 has a distal end 324 that defines an opening 326 having an opening perimeter 328. A second wall 330 surrounds the first wall 318 and has a distal end 332. The distal end 332 may be referred to as an edge or an outer edge. A cover 334 is coupled to the distal end 324 of first wall 318 and covers the opening 326 along opening perimeter 328 at a connected portion 336 and is uncoupled from distal end 324 of first wall 318 at an unconnected portion 338. The connected portion 336 may be referred to as connection sections or connected segments and the unconnected portion 338 may be referred to as unconnected sections or unconnected segments. The first wall 318 has a height, and the second wall 330 has a height that is greater than the height of first wall 318. The first wallAtty Docket No. IP-2803-PCT / 0187-020W01318 and second wall 330 may alternatively be the same or similar heights. An impermeable barrier 340 is coupled to the distal end 332 of the second wall 330 and covers the well 320. The impermeable barrier 340 may be foil, plastic, etc. and may prevent or inhibit moisture from infiltrating the wells 320 of the reagent cartridge 302.

[0092] Unconnected portion 338 of the cover 334 forms a vent 342 that allows air flow out of the well 320. Dried reagent 348 is contained within the well 320, and the vent 342 is sized to substantially retain the dried reagent 348 within the the well 320. The body 316 may include a plurality of wells 320, although one well 320 is shown in FIG. 3. Liquid 346 may flow into well 320 via port 322 in practice to rehydrate the dried reagent 348. The vent 342 may vent gas from the well 320 as liquid 346 flows into well 320, and the cover 334 prevents or inhibits the reagent 348 and / or liquid 346 from escaping from well 320. Put another way, the vents 342 retain reagent 348 and / or liquid 346 within wells 320 and prevent or inhibit reagent 348 and / or liquid 346 from migrating out of wells 320. The vent 342 and the cover 334 prevent or inhibit cross-contamination between reagents when the reagent cartridge 302 includes more than one well 320. Liquid 346 and dried reagent 348 may be flowed into and out of the well 320 to mix liquid 346 from the liquid reservoir 362 and dried reagent 348. The system 300 and / or the reagent cartridge 302 may include a mixing chamber that is used to mix liquid 346 and dried reagent 348 in some implementations. Impermeable barrier 340 may be pierced prior to liquid 346 flowing into well 320.

[0093] The gas source 304 may be used to pressurize the liquid reservoir 362 to flow liquid 346 into the well 320, and / or a pump 350 may draw liquid 346 from the liquid reservoir 362 and flow liquid 346 into the well 320 to rehydrate the reagent 348. The gas source 304 may be provided by the system 300 and / or may be carried by reagent cartridge 302. The gas source 304 may alternatively be omitted. The pump 350 may be implemented by a syringe pump, a peristaltic pump, a diaphragm pump, etc. While the pump 350 may be positioned downstream of the flow cell 368 as shown, the pump 350 may be positioned upstream of the flow cell 368 or omitted entirely.

[0094] The reagent cartridge 302 and / or the system 300 includes valves 352 that may be selectively actuatable to control the flow of fluid through the fluidic lines 356. Such valves 352 may be implemented by a valve manifold, a rotary valve, a selector valve, a pinch valve, a flat valve, a solenoid valve, a check valve, a piezo valve, etc. A regulator 354 may be positioned between the gas source 304 and the valve 352 and may regulate the pressure of the gas provided to valve 352. The regulator 354 may include a valve that controls the flow of the gas from the gas source 304.Atly Docket No. IP-2803-PCT / 0187-020W01

[0095] The body 316 of the well assembly 314 has an edge 364, and the impermeable barrier 340 may be hermetically connected to the body 316 along the edge 364. The impermeable barrier 340 may include foil, plastic, and / or any other suitable material(s). The system 300 may pierce impermeable barrier 340. For example, the impermeable barrier 340 may be pierced by an individual prior to use, or the impermeable barrier 340 may be pierced by some other structure or methodology. The system 300 includes an actuator assembly 360 in the implementation shown that interfaces with the impermeable barrier 340 to pierce the impermeable barrier 340. The system 300 may include a protrusion such as a post having a blunt or sharp end that is movable by the actuator assembly 360 to pierce the impermeable barrier 340. The impermeable barrier 340 may alternatively be pierced by an operator prior to reagent cartridge 302 being positioned in system 300. The system 300 also includes a liquid reservoir 362 containing liquid 346. The liquid 346 may comprise a rehydrating liquid, a wash buffer, and / or any other suitable kind(s) of liquid.

[0096] The system 300 further includes a flow cell receptacle 366 that receives a flow cell 368. The flow cell 368 may be configured and operable like the flow cell 128. In some variations, the flow cell 368 is carried by and / or integrated into the reagent cartridge 302. The flow cell 368 may carry the sample of interest. The gas source 304 and / or the pump 350 may flow liquid 346 to rehydrate dry reagents 348 and to flow one or more liquid reagents through the reagent cartridge 302 that interact with the sample. The imaging system 310 may be configured and operable like the imaging system 116, such that the imaging system 310 may be used to obtain image data from the flow cell 368. After the image data is obtained, the drive assembly 306 may interface with the reagent cartridge 302 to flow another reaction component (e.g., a reagent) through the flow cell 368 that is thereafter received by the waste reservoir 312 and / or otherwise exhausted by the reagent cartridge 302. In the present example, the drive assembly 306 includes a pump drive assembly 370, a valve drive assembly 372, and actuator assembly 360. The pump drive assembly 370 interfaces with the pump 350 to pump fluid through the reagent cartridge 302 and / or the flow cell 368; and the valve drive assembly 372 interfaces with valve 352 to control the position of the valve 352.

[0097] The controller 308 of this example includes a user interface 374, a communication interface 376, a processor 378, and a memory 380. The user interface 374 may be configured and operable like the user interface 206 of the system 100. The communication interface 376 may be configured and operable like the communication interface 208 of the system 100. The processor 378 may be configured and operable like the processor 210 of the system 100. The memory 380 may be configured and operable like theAty Docket No. IP-2803-PCT / 0187-020W01 memory 212 of the system 100.

[0098] Further examples and details of how various features of each system 100, 300 may be configured and operable will be described below.

[0099] FIG. 4 is a schematic diagram of an example analysis system 10 for processing predefined reaction chambers (such as for biological applications), imaging the predefined reaction chambers, and analyzing data derived from the imaging. In the illustrated example, the system 10 is designed to introduce molecules, such as nucleotides, oligonucleotides, and other bioactive reagents, into samples (S) 12 that may be prepared on a flow cell. The system 10 may be designed for synthesizing biopolymers, such as DNA chains, or for sequencing biopolymers. It is noted that the present technique is not limited to sequencing operations, gene expression operations, diagnostic applications, or any one of these, but may be used in any of them for analyzing collected image data for multiple swaths or regions detected in regions of a sample as described below. Other substrates containing arrays of molecules or other detectable features also can be used in the techniques and systems disclosed. Example biopolymers may include nucleic acids, such as DNA, RNA, or analogs of DNA or RNA. Other example biopolymers may include proteins (also referred to as polypeptides), polysaccharides, or analogs thereof. In general, the system of FIG. 4 can act upon samples 12 that may include an array of reaction sites. Here again, the term “array” or “patterned array” refers to a support having a population of different reaction sites on a substrate (e.g., a flow cell), such that different reaction sites can be differentiated from each other according to their relative location. A single species of biopolymer may be attached to each individual reaction site. However, multiple copies of a species of biopolymer can be attached to a reaction site. The array, taken as a whole, may include a plurality of different biopolymers attached at a plurality of different sites. Reaction sites can be located at different addressable locations on the same substrate. Alternatively, an array can include separate substrates, such as beads, each forming a different reaction site. The sites may include fragments of DNA attached at specific locations in an array or may be wells in which a target product is to be synthesized. In some applications, the system may be designed for continuously synthesizing or sequencing molecules, such as polymeric molecules based upon common nucleotides.

[0100] The analysis system 10 may include a processing system 14 designed to process samples 12, such as biological patterned arrays, and to acquire fluorescence data representative of individual sites on the patterned array, as well as spaces between sites, and representations of fiducials provided in or on the patterned array support. A data analysis system 16 can receive the fluorescence data as image data or as digital data derived from theAtty Docket No. IP-2803-PCT / 0187-020W01 fluorescence data and can processes the received data in accordance with the present disclosure, to extract meaningful genomic data values from the fluorescence data as described below. A downstream processing / storage system 18, then, may receive this genomic information and store the information where desired. The downstream processing / storage system 18 may further analyze the genomic data or the data derived from the genomic data, such as to diagnose physiological conditions, compile sequencing lists, analyze gene expression, and so forth.

[0101] The processing system 14 may employ a biomolecule reagent delivery system 20 (shown as a nucleotide delivery system in FIG. 4) for delivering various reagents to a sample 12 as processing progresses. The processing system 14 may perform a plurality of operations on the samples 12 over time. The processing system 14 may be designed for cyclic operation in which reactions are promoted with single nucleotides or with oligonucleotides, followed by flushing, imaging and de-blocking in preparation for a subsequent cycle. In a practical system, the samples 12 are disposed in the system, and an automated or semiautomated sequence of operations is performed for reactions, flushing, imaging, de-blocking, and so forth, in a number of successive cycles for a test sample. Again, the process illustrated in FIG. 4 is not limiting, and the present techniques may operate on fluorescent image data acquired from any suitable system employed for any application. While reference is made in the present disclosure to fluorescence in regards to “imaging” or “image data,” in many practical systems this will entail actual optical imaging and extraction of data from electronic detection circuits (e.g., cameras or imaging electronic circuits or chips), although other detection techniques may also be employed, and the resulting detected data characterizing the molecules of interest should also be considered as “images” or “image data.”

[0102] In the example illustrated in FIG. 4, the nucleotide delivery system 20 provides a process stream 22 to the samples 12. An effluent stream 24 from the array or flow cell may be recaptured and recirculated, for example, in the nucleotide delivery system 20. In the illustrated example, then, the array or flow cell may be flushed by a flush system 26 that, for example, flushes the array or flow cell by actuation of appropriate valving to remove additional reagents and to clarify the sample 12 for imaging. The sample 12 then is exposed to an imaging system 28 (which may be within the same device) that generates image data that can be analyzed, for example, for determination of the sequence of a progressively building nucleotide chain, such as based upon a template. In some implementations, the imaging system 28 may employ confocal line scanning to produce progressive pixilated image data that can be analyzed to locate individual sites in an array and to determine theAtty Docket No. IP-2803-PCT / 0187-020W01 type of nucleotide that was most recently attached or bound to each site. Other imaging techniques may also suitably be employed, such as techniques in which one or more points of radiation are scanned along the sample, or techniques employing “step and shoot” imaging approaches.

[0103] As noted, the imaging components of the imaging system 28 may be more generally considered a “detection apparatus,” and any detection apparatus that is capable of high-resolution imaging of surfaces may be useful. In some examples, the detection apparatus will have sufficient resolution to distinguish features at the densities, pitches and / or feature sizes set forth herein. Examples of the detection apparatus are those that are configured to maintain an object and detector in a static relationship while obtaining an area image. As noted, a scanning apparatus can be used, as well as systems that obtain successive area images (e.g. “step and shoot” detectors). Point scanning detectors mentioned above can be configured to scan a point (i.e., a small detection area) over the surface of an object via a raster motion in the X-Y plane of the surface. Line scanning detectors can be configured to scan a line along the Y dimension of the surface of an object, where the longest dimension of the line occurs along the X dimension. It will be understood that the detection device, object or both can be moved to achieve scanning detection.

[0104] Following imaging (e.g., by the imaging system 28), a deblock system 30 can de-block the samples. For example, a blocking molecule or protecting group can be cleaved from the last added nucleotide, along with a marking dye. If the system 14 is used for sequencing, by way of example, data generated by the imaging system 28 from fluorescence data will be stored and forwarded to a data analysis system 16.

[0105] The analysis system 16 may include a general purpose or application-specific programmed computer, which provides a user interface and automated or semi-automated analysis of the fluorescent image data to determine which of the four common DNA nucleotides may have been last added at each of the sites in an array of each sample, as described below. As will be appreciated by those skilled in the art, such analysis may be performed based upon the fluorescent color of unique tagging dyes for each of the four common DNA nucleotides, by using two different wavelengths for colors of fluorescent tags, by using a single wavelength of color of fluorescent tags, and / or any other combination of fluorescent tags. This fluorescent image data may be further analyzed by the downstream processing / storage system 18, which may store data derived from the fluorescent image data as described below where appropriate. Again, the sequencing application is intended to be one example, and other operations, such as diagnostic applications, clinical applications, geneAtty Docket No. IP-2803-PCT / 0187-020W01 expression experiments, and so forth may be carried out that will generate similar fluorescent imaging data operated on by the present techniques.

[0106] As noted above, in some implementations, the patterned array may remain in a fixed position, and the “stations” referred to may include integrated subsystems that act on the patterned array as described (e.g., for introduction and reaction with desired chemistries, flushing, imaging, image data collection, and so forth). The data analysis may, here again, be performed contemporaneously with the other processing operations, or may be done postprocessing by accessing the fluorescence image data, or data derived from the image data, from an appropriate memory (in the same system, or elsewhere). In many applications, a patterned array “container” will comprise a cartridge in which the patterned array is placed and through which the desired chemistry is circulated. In such applications, fluorescence imaging may be done through and via the flow cell. The flow cell may be appropriately located (e.g., in the X-Y plane) on a translation stage, or a chuck located on the translation stage, and moved by the translation stage (e.g., in X, Y, and Z directions) as needed for imaging. Connections for the desired chemistry may be made directly to the flow cell when it is mounted in the apparatus. Moreover, depending upon the device design and the imaging technique used, the patterned array, positioned in the flow cell, may be initially located in the X-Y plane, and moved in this plane during imaging, or imaging components may be moved parallel to and / or perpendicular to this plane during imaging. In general, here again, the “X-Y plane” is the plane of the patterned array surface that supports the sites, or a plane parallel to this. The flow cell, therefore, may be said to extend in the X-Y plane, with the X direction being the longer direction of the flow cell, and the Y direction being the shorter direction (the flow cells being rectangular). It is to be understood, however, that this orientation could be reversed. The flow cell and patterned array may also be moved in the Z direction, which is the direction orthogonal to both the X and Y directions. Such movements may be useful for securing the flow cell into place, for making fluid connections to the flow cell, and for imaging (e.g., focusing the optic for imaging sites at precise Z depths). In some applications, the optic may be moved in the X direction for precise imaging.

[0107] FIG. 5 is a schematic diagram of an analysis system 500 in which one or more flow cells 502 can be provided to an imaging system 504, where nanowells of the flow cells (e.g., millions or billions of nanowells) can be illuminated and imaged. Subsequent to imaging the nanowells of the flow cells, the images can be processed to analyze the content of the nanowells, and, for example, to determine an oligonucleotide sequence of a sample that has interacted with the nanowells of the flow cell.Aty Docket No. IP-2803-PCT / 0187-020W01

[0108] One or more flow cells 502 can be loaded into the analysis system 500 and stored on one or more racks 506 that hold each flow cell 502 in position until the flow cells are ready to be imaged by the imaging system 504. The rack(s) 506 can include any mechanical structure configured for receiving one or more flow cells 502 and for holding the flow cells 502 in position within a housing of the analysis system 500. For example, the one or more racks 506 can include a platform on which the one or more flow cells 502 can be placed. In another example, the one or more racks 506 can include a plurality of bars on which the one or more flow cells 502 can be placed. In another example, the one or more racks 506 can include one or more slots into which the one or more flow cells 502 can be placed. In some implementations, a plurality of flow cells 502 can be manually loaded into the analysis system 500 and received within the rack 506. In some implementations, the flow cells 502 can be automatically loaded into the analysis system 500 and received within the rack 506. For example, a flow cell can be inserted into a slot within a housing of the analysis system 500 and can be conveyed from the slot to the rack by one or more automatic mechanisms that may include one or more electromechanical assemblies (e.g., including one or more motors) that move the flow cell from the slot to the rack, such as a robotic arm system. The rack 506 can support the one or more flow cells 502 while the flow cells are not being imaged by the processing system.

[0109] A flow cell 502 can be moved by a pick and place (PnP) system 510 from its position on the rack 506 to a position proximate to, or in relation to, the imaging system 504, so that nanowells of the flow cell 502 can be imaged by the imaging system 504. In some implementations, a flow cell 502 can be picked up from the rack 506 by an end effector 512 of the PnP system 510 and then placed on a chuck 530 by the end effector. The chuck 530 can be part of a translation stage to translate the flow cell 502 relative to the imaging system 504, so that the nanowells of the flow cell can be imaged by the imaging system.

[0110] The end effector 512 can be moved (or rotated or translated, etc.) from a location at which it picks up a flow cell 502 from a rack 506 to a location at which it places the flow cell 502 on the chuck 530 by one or more motors 514 that are configured to move the end effector in a plurality of directions (e.g., one or more linear directions and in one or more angular directions). Operation of the motors 514, and other components of the end effector 512 to move the end effector, to pick up the flow cell 502 from the rack, and to place the flow cell 502 on the chuck 530 can be controlled by one or more processors 516 executing computer-readable instructions stored in memory 518.

[0111] The end effector 512 of the PnP system 510 can include a gripper 520 to pickAtty Docket No. IP-2803-PCT / 0187-020W01 up the flow cell 502 from the rack and place it on the chuck 530 and to pick up the flow cell 502 from the chuck 530 and place it back on the rack 506. The end effector 512 can include one or more light sources 522 configured for illuminating a flow cell 502, the rack 506, and / or the chuck 530, and one or more cameras 524 for imaging the flow cell 502, the rack 506, and / or the chuck 530, so that the end effector 512 can accurately pick up the flow cell 502 from the rack and can accurately place the flow cell 502 in a predetermined position on the chuck 530 so that the nanowells of the flow cell 502 can be imaged by the imaging system 504. Likewise, imaging of the fiducials on the flow cell can enable the end effector 512 to accurately pick up the flow cell 502 from the chuck and can accurately place the flow cell 502 in a predetermined position back on the rack.

[0112] FIG. 6 is a schematic diagram of a workflow 600 for picking a flow cell from a rack and placing the flow cell on a chuck for imaging. If the analysis system is currently processing one or more flow cells, then the workflow 600 can begin by interrupting one or more routines of an analysis system, for example, by alerting or interrupting software controlling an operation of the analysis system to cause the system to pause, for example, a process of imaging nanowells (602) and to cause the system to move the chuck to a load position at which the flow cell can be placed on the chuck. Then, an end effector of a PnP system can be moved into a position proximate to the rack that holds the flow cell (604). Once the end effector is close to the flow cell in the rack, a height of the end effector above the flow cell can be determined (606), and the height can be adjusted (608) to bring the end effector closer to, or farther from, the flow cell. As explained in more detail hereinbelow (e.g., in relation to FIGs. 11 A - 13B), the flow cell and the rack can be illuminated by one or more light sources included on the end effector and can be imaged by one or more cameras of the end effector, so that an alignment and relative location between the flow cell and the end effector can be determined. The flow cell and the rack can include one or more fiducials that are imaged to determine the alignment and relative location. Based on the determined alignment and relative location, the end effector can be moved into a pickup location (610) at which the end effector can pick up the flow cell at a proper location. Once the end effector is positioned properly relative to the flow cell, the flow cell is picked up (612).

[0113] After the flow cell is picked up by the end effector, the end effector is moved close to the chuck (614), and then is moved in the Z direction, normal to a surface of the chuck upon which the flow cell is to be placed, until the flow cell reaches a predetermined Z- distance from the surface of the chuck (616). As explained in more detail hereinbelow (e.g., in relation to FIGs. 15A-15C), the flow cell and the chuck can be illuminated by one or moreAtly Docket No. IP-2803-PCT / 0187-020W01 light sources included on the end effector and imaged by one or more cameras of the end effector, so that an alignment and relative location between the flow cell and the chuck can be determined. The flow cell and the chuck can include one or more fiducials that are imaged to determine the alignment and relative location. Based on the determined alignment and relative location, the end effector can be moved into a drop-off location (618) at which the end effector can place the flow cell at a proper location on the chuck. Once the flow cell is positioned properly relative to the chuck, the flow cell is placed on the chuck (620).

[0114] FIG. 7 is schematic diagram of an example flow cell 700, which can include one or more substrates, or substrate layers 702. Axes of a Cartesian coordinate system in which the flow cell is located are shown in FIG. 7. For example, the flow cell 700 can include a first substrate, a second substrate, and an interposer disposed between the first substrate and the second substrate. The first and second substrates can be formed from any suitable material, for example, silicon dioxide, glass, quartz, Pyrex, plastics (e.g., polyethylene terephthalate (PET), high density polyethylene (HDPE), low density polyethylene (LDPE), polyvinyl chloride (PVC), polypropylene (PP), etc.), polymers, TEFLON®, Kapton (i.e., polyimide) or any other suitable material. In some implementations, the first and / or the second substrate may be transparent. In other implementations, the first and / or the second substrate may be opaque. The first and / or and the second substrate can define fluidic inlets or outlets for pumping a fluid to and / or from microfluidic channels defined in the interposer.

[0115] The flow cell 700 can include one or more flow channels 704. Each flow channel 704 may be formed in one or more of a bottom substrate layer or an upper substrate layer, and / or in one or more interposed layers, and / or one or more adhesive layers structured into a stacked configuration the flow cell 700. The flow channels 704 are generally parallel with each other and extend along substantially the entire length the flow cell 700. While the flow cell 700 shown in FIG. 7 has six flow channels 704, any other suitable number of flow channels 704 may be provided, such as one flow channel 704, two flow channels 704, three flow channels 704, four flow channels 704, five flow channels 704, or more than six flow channels 704.

[0116] Each flow channel 704 includes a first end 720, a second end 722, and an intermediate region 724 extending along a length between ends 720, 722. As shown in FIG. 7, this length extends in the x-direction in this example. While not shown in FIG. 7, each flow channel 704 may include a plurality of wells (e.g., nanowells) or other structural features providing reaction sites. In some implementations, such wells or other structural features providing reaction sites are only positioned along intermediate region 724. In someAtty Docket No. IP-2803-PCT / 0187-020W01 other versions, such wells or other structural features providing reaction sites extend all the way to ends 720, 722. In either case, the wells or other structural features providing reaction sites in flow channels 704 may include nucleic acid strands or other oligonucleotides for SBS and / or for other kinds of processes.

[0117] In some implementations, the plurality of wells may be etched in the one or more substrates. For example, the substrate 702 may include glass with an array of wells etched in the substrate using a wet etch (e.g., a buffered hydrofluoric acid etch) process or a dry etch (e.g., using a reactive ion etching (RIE) or deep RIE) process. In some implementations, the plurality of wells may be formed in a resin layer disposed on a surface of the substrate 702. The resin layer may include, for example, polymethyl methacrylate (PMMA), polystyrene, glycerol 1,3 -di glycerolate diacrylate (GDD), Ingacure 907, rhodamine 6G tetrafluoroborate, a UV curable resin (e.g., a novolac epoxy resin, PAK-01, etc.) any other suitable resin or a combination thereof. In particular implementations, the resin layer may include a nanoimprint lithography (NIL) resin (e.g., PMMA), and the plurality of wells can be formed in the resin layer by a NIL process. In some implementations, the plurality of wells may be formed by one or more other patterning processes, for example, photolithography, A biological probe may be disposed in each of the plurality of wells. The biological probes may include, for example, DNA probes, RNA probes, antibodies, antigens, enzymes or cells. In some implementations, chemical or biochemical analytes may be additionally or alternatively disposed in the plurality of wells.

[0118] The plurality of wells may be arranged in a regular repeating pattern. For example, FIG. 8A is a schematic diagram of a repeating pattern of octagonal wells arranged in a hexagonal pattern, and FIG. 8B is a schematic diagram of a repeating pattern of circular wells arranged in a hexagonal pattern. The patterns of FIGs. 8 A and 8B are example layouts, although the geometry and layout are highly modifiable. The shape and size of each well in each of FIG. 8 A and 8B can be substantially similar to other wells of the pattern of FIG. 8 A and FIG. 8B, respectively. For example, the wells may have a diameter or cross-section of about 30 microns or less, about 10 microns or less, about 3 microns of less, about 1 micron or less, or about 300 nm or less. The patterns of the wells can have one or more axes of symmetry 802, 804, 806 in which the pattern of the wells repeats itself. The distance 808 over which the pattern repeats itself over an axis of symmetry can be known as a pitch of the pattern. In some implementations, the pitch of the pattern of wells in FIG. 8A and in FIG. 8B can be about 30 microns or less, about 10 microns or less, about 3 microns of less, about 1 micron or less, or about 300 nm or less.Atly Docket No. IP-2803-PCT / 0187-020W01

[0119] Referring again to FIG. 7, the flow cell 700 can include one or more fiducials 710 that can be used in a machine vision system of a PnP system to precisely locate the flow cell relative to other components of an analysis system. The dashed-line box of FIG. 7 shows an enlarged version of one of the fiducials 710. The fiducials 710 can be located at predetermined locations of the flow cell 700 relative to dimensions (e.g., a length, width, and height of the flow cell), so that the fiducials can be used as proxies to locate the entire flow cell relative to the other components of the analysis system. In some implementations, the fiducials 710 can include one or more symmetric markers, e.g., circles, that can be used to determine locations of the fiducials in space relative to an imaging system and / or relative to one or more other components, which themselves also many include fiducial markers. In some implementations, the fiducials 710 can include one or more symmetry -breaking markers, e.g., crosses, butterfly-patterns, and the like, that can be used to determine both locations and angular orientations of the fiducials in space relative to an imaging system and / or relative to one or more other components, which themselves also many include fiducial markers.

[0120] In some implementations, the fiducials 710 can be defined by a nanowell pattern at a location of the fiducials, where the pattern can be exposed to light that can illuminate the pattern and reflected, transmitted, and / or diffracted from the pattern. The nanowell pattern that defines a fiducial 710 can be included on the same substrate that includes the nanowells that contain the biological probes that are imaged by the analysis system. The shape of a fiducial 710 can be defined by an opening in an opaque layer (e.g., an interposer layer) that is disposed between the substrate that includes the nanowell pattern and a light source that illuminates the fiducial. For example, in the case of a circular fiducial 710, a circular opening formed in an opaque layer can allow light to pass through the opening and to be reflected, diffracted, and / or transmitted by the nanowell pattern in the substrate, and the reflected, diffracted, and / or transmitted light can be used to image the fiducial 710. FIG. 9 illustrates additional examples of a fiducial that may be used. Although four fiducials 710 are shown in FIG. 7, a minimum of two circular fiducials, or a minimum of one symmetrybreaking fiducial can be used to determine both locations and angular orientations of the fiducials in space relative to an imaging system and / or relative to one or more other components on an analysis system.

[0121] While the fiducials 710 of FIG. 7 can include a nanowell pattern, where the pattern can diffract light, other unpatterned fiducials also are contemplated. For example, a fiducial having a reflectivity that differs from material surrounding the fiducial also can beAtly Docket No. IP-2803-PCT / 0187-020W01 used, where the fiducial does not include a predetermined pattern. For example, when the flow cell 700 includes an opaque, low-reflectivity layer, and a high-reflectivity layer below the low-reflectivity layer, an opening in an opaque layer can allow light to be reflected from the high-reflectivity layer, such that a fiducial is defined by the shape of the opening in the opaque layer when the flow cell is illuminated by a light source.

[0122] FIG. 9 is a schematic diagram of a periodic pattern 902 that acts as a diffraction grating to diffract light from light source 904 by an angle 0 along an axis 908 that intersects an axis of a camera 906. By way of example, light source 904 can be a narrowband light source (such as a laser or a narrow band light emitting diode (LED) that emits light in a direction toward the periodic pattern 902. Light waves emitted by the light source 904 interact with the periodic pattern 902 and are diffracted by the pattern. For monochromatic light having a wavelength, X, emitted by the light source 904 and propagating at a normal incidence angle to the periodic pattern 902, the intensity maxima of the diffracted light occur at diffraction angles that satisfy the condition sin 6m=where 6mis the angle betweenthe diffracted ray and a vector normal to the surface of the periodic pattern, where d is the pitch of the periodic pattern along the diffraction direction, and where m is an integer representing the diffraction order. When light from the light source 904 is emitted at an angle 61 to the normal direction of the pattern, then the intensity maxima of the diffracted light occur at diffraction angles that satisfy the condition sin 0(- + sin 0mThus, the pitch ofthe periodic pattern that forms a fiducial marker is a parameter in an equation that defines a diffraction angle of light that is used to illuminate the fiducial, and the direction at which light illuminating the fiducial will be diffracted from the periodic pattern can be calculated, so that a camera 906 can be placed in the path of the diffracted light to image the fiducial.

[0123] FIG. 10 is a schematic perspective view of an example PnP robotic drive arm 1000 that is configured to move an end effector of a PnP system to automatically pick up a flow cell from a rack and move the flow cell into position on a chuck, so that nanowells on the flow cell can be imaged by an imaging system of an analysis apparatus. The end effector (not shown in FIG. 10) can be mechanically coupled, or otherwise attached, to the robotic drive arm 1000, so that the end effector can be moved by the drive arm 1000. The robotic drive arm 1000 can include a plurality of motors that can be actuated to cause the end effector to automatically pick up a flow cell from a rack and move the flow cell into position on a chuck. The motors can include, for example, a Z motor 1002 configured to move the end effector in a Z direction of a Cartesian coordinate system, a Y motor 1004 configured to moveAtly Docket No. IP-2803-PCT / 0187-020W01 the end effector in a Y direction of the coordinate system, and an X motor 1006 configured to move the end effector in an X direction of the coordinate system. The motors also can include one or more motors 1008 configured for rotating the end effector about one or more of the X, Y, and Z directions.

[0124] In addition, the robotic drive arm 1000 can include one or more coupling mechanisms that can be used to assist the end effector in picking up, securely holding, and transporting the flow cell. For example, the robotic drive arm 1000 can include a vacuum hose 1010 that can be coupled to the end effector for providing a low pressure, or vacuum, condition at the end effector for use in picking up and securing the flow cell to the end effector. In other implementations, the coupling mechanism can include electrical contacts, circuits, mechanical connections, and the like, for providing one or more electrical, magnetic, or mechanical connections between the end effector and the flow cell.

[0125] FIGs. 11 A, 11B, and 11C are schematic end, side, and bottom views, respectively of an end effector 1100, with axes of a Cartesian coordinate system shown in the figures. The end effector 1100 includes a housing 1102 that supports, and / or contains, components of the end effector. The housing 1102 is mechanically connected to a shaft clamp 1104 that is configured to couple the end effector 1100 to the robotic drive arm 1000, so that the robotic drive arm can move the end effector into position to pick up a flow cell from a rack, move the flow cell to a position with respect to a chuck, and place the flow cell on the chuck. The end effector 1100 includes one or more vacuum cups 1106 that are configured to draw a vacuum pressure compared to ambient pressure, so that the flow cell can be attached to the end effector by the ambient pressure, as compared to the vacuum pressure, pressing the flow cell against the end effector 1100. In some implementations, the end effector 1100 can include one or more pads 1107 that are configured to mechanically mate with respective portions of a flow cell when a vacuum is drawn by the vacuum cups 1106, so that the respective portions of the flow cell are pressed against the pads 1107, and the force of the vacuum pressure on the flow cell is distributed over the area of the pads 1107 that contacts the flow cell.

[0126] The end effector 1100 can include one or more cameras 1108 that are configured to image one or more portions of a flow cell that is proximate to, and / or currently attached to, the end effector. The portions of the flow cell can include one or more fiducials located at predetermined positions on the flow cell and that can be used to determine a location of the flow cell relative to the end effector. The cameras 1108 also are configured to image one or more portions of the rack that support the flow cell while the flow cell is in theAtly Docket No. IP-2803-PCT / 0187-020W01 analysis system but is not being imaged by the imaging system of the analysis system to image and one or more portions of the chuck that supports the flow cell while the flow cell is being imaged by the imaging system.

[0127] Fiducials on the flow cell, portions of the rack, and portions of the chuck can be illuminated by one or more light sources (e.g., lasers, LEDs) 1112 that are connected to a circuit 1110 (e.g., a printed circuit assembly) that powers and controls the light sources 1112. In some implementations, the light sources can provide narrow band light, for example, having a full-wide at half-max (FWHM) of less than 10 nanometers, which may be advantageous for diffracting off of a periodic pattern of a fiducial marker that is imprinted onto the flow cell. In some implementations, the light sources can provide broadband (e.g., white) light, for example, spanning a wavelength range of over 200 nanometers, which may be advantageous for illuminating one or more fiducial markers that include a periodic pattern (e.g., as imprinted onto a substrate of a flow cell) and fiducial markers that do not include a periodic pattern (e.g., included on a surface of a rack or chuck). In some implementations, each light source 1112 can include both a narrow band light source and a broadband light source.

[0128] FIG. 12 is a schematic perspective view of an example end effector 1200 that has picked up, and is mechanically coupled to, a flow cell 1220, with axes of a Cartesian coordinate system in which the flow cell is located shown in the figure. Circuits 1210 (e.g., a printed circuit assemblies) of the end effector 1200 are configured to power and control the light sources 1212 to illuminate fiducials 1202 imprinted on a substrate of the flow cell, and cameras 1208 are configured to image light diffracted from the fiducials 1202. The cameras 1208 can be mounted on a circuit board 1214 (e.g., a printed circuit board) that communicates image data from the cameras to a processor that processes the image data, and the cameras can include one or more imaging elements (e.g., lenses) and one or more sensor arrays, where the imaging elements focus the light diffracted from the fiducials, and light from other objects in a field of view of the cameras onto the sensor array. In some implementations, the flow cells can be relatively small (e.g., less than 100 mm long and less than 40 mm wide). Furthermore, the dimensions of the end effector also can be relatively small, so that the end effector, and the PnP system to which it is attached, can fit within an assembly apparatus that may be small enough to fit on a typical laboratory bench. Because of this, both the light sources 1212 and the cameras 1208 can be quite close to the fiducials of the flow cell 1220. For example, the light sources 1212 can be less than 15 mm from the fiducials, and the light sources 1212 can be less than 25 mm, or less than 20 mm, from the fiducials. Furthermore, inAtly Docket No. IP-2803-PCT / 0187-020W01 some implementations, the circuit board 1214 can be less than 50 mm away from the flow cell 1220 when the flow cell is picked up by the end effector 1200. To keep the overall length of the cameras short enough to fit within the small space between the circuit board 1214 and the flow cell while still being able to image the fiducials on the flow cell onto a sensor array of the camera, non-telecentric imaging elements can be used in the cameras to image the fiducials onto the sensor array. Telecentric lenses are generally larger, longer, and more complicated than comparable non-telecentric lenses, so that non-telecentric lenses can better fit within the confined geometry of the system. However, if space constraints are not needed, telecentric lenses can be used.

[0129] FIG. 13 A is a perspective view of a portion of the end effector 1200, which includes a light source 1302 configured to illuminate a fiducial 1304 on the flow cell 1220 and a camera 1306 configured to image light diffracted from the fiducial 1304. FIG. 13B is a bottom view of the portion of the end effector 1200, looking up, from below the flow cell 1220, at the camera 1306. Axes of a Cartesian coordinate system in which the flow cell is located are shown in FIGs. 13 A and 13B. The path 1310 of light having a wavelength, , is shown in FIGs. 13A and 13B, as it propagates from the light source 1302 located at a fixed height in the Z direction above the fiducial to the fiducial 1304 and is diffracted by a diffraction angle, 0, from a periodic pattern of the fiducial toward the camera 1306. Wavelengths different from the wavelength, , can be diffracted by angles different from the angle, 9. For example, a broadband light source 1302 that emits, for example, white light, can have light diffracted over a range of angles from the fiducial. Thus, in some implementations, a broadband light source 1302 can be used, to provide a wider range of positions of the light source that will allow the fiducial to be imaged by the camera than if a narrow band light source is used. In some implementations, the light source 1302 can include an LED that emits diffuse white light.

[0130] The camera 1306 can have an angular field of view defined by its imaging elements (e.g., lenses) and the dimensions of its sensor array, and, with the optical axis of the camera being substantially along the Z direction and / or at an angle relative to the Z direction, the angular field of view can correspond to a field of view in the XY plane. For a rectangular sensor array, the camera’s field of view in the XY plane also can be rectangular. As the end effector 1200 moves relative to the flow cell 1220, the field of view of the camera moves relative to the fiducials of the flow cell 1220, but it does not move relative to the end effector. Thus, to guide the location of the end effector 1200 relative to the flow cell 1220, so that the end effector can pick up the flow cell from a rack, images or sensor data indicative of aAty Docket No. IP-2803-PCT / 0187-020W01 fiducial on the flow cell 1220 can be captured by the camera 1306 or image sensor while the end effector is moved relative to the flow cell in the rack, and the sensor data or images can be processed (for example, by a processor 516 to determine a location of the flow cell relative to the field of view of the camera. When the fiducial is located in a predetermined position within the field of view, the end effector can be determined to be located in a correct position to pick up the flow cell from the rack.

[0131] Referring again to FIG. 5, in some implementations, a processor 516 can execute instructions stored in memory 518 to move an end effector 512 into a predetermined position that is known to be close to a flow cell 502 that is located on a rack 506. Once the end effector 512 is in the predetermined position, the height of the camera 524 in the end effector 512 relative to the flow cell 502 can be determined. In some implementations, the height of the camera 524 relative to the flow cell 502 can be determined based on the Z position of the end effector and a known Z position of the flow cell when stored on the rack 506. In some implementations, a fiducial of the flow cell 502 can be imaged by the camera 524, and the image can be processed to determine a height of the camera. For example, with the use of non-telecentric lenses in the camera, an image of the fiducial will be out of focus when it is not located at the focal plane of the camera, so a lateral extent in the XY plane of an image of the fiducial can correspond to a height of the camera in the Z direction relative to the flow cell. The height of the end effector in the Z direction can be adjusted until a desired height is attained, and then the end effector 512 can be moved in the XY plane while images of the fiducial are captured until the fiducial is located at a predetermined position in the field of view of the camera. Then, the flow cell can be picked up.

[0132] While implementations disclosed herein describe a machine vision system including a light source 1302 and a camera 1306 that are integrated with, and that travel with, the end effector 1200, other implementations, in which one or more light sources and cameras can be fixed to other components of the system besides the end effector, such that they would not travel with the end effector but still would be used to optically determine a location of the flow cell relative to the end effector when the end effector is being moved into position to pick up the flow cell. For example, a light source and a camera can be located in a fixed position near the rack, and the end effector may include one or more fiducials, so that when the end effector is moved into position to pick up the flow cell, fiducials on the end effector and on the flow cell can be imaged and the images can be used to determine a relative location of the end effector with respect to the flow cell.

[0133] Once the flow cell 502 is picked up by the end effector 512, the end effectorAtly Docket No. IP-2803-PCT / 0187-020W01 can be moved into position relative to the chuck 530, so that the flow cell can be placed on the chuck at a position that enables the chuck to move the flow cell relative to the imaging system 504, so that nanowells of the flow cell can be imaged to gather information about a sample that interacts with the nanowells.

[0134] FIG. 15Ais a schematic perspective view of an end effector 1500 holding a flow cell 1502 at a first distance in the Z direction above a chuck 1504. FIG. 15B is a schematic top view of ends of opposite ends of the end effector 1500 holding the flow cell 1502 at a distance in the Z direction above the chuck 1504, where the middle portions of the end effector and the flow cell, between their respective ends, are omitted for clarity. FIG. 15C is a schematic perspective view of the end effector 1500 in position to place the flow cell 1502 on the chuck 1504. Axes of a Cartesian coordinate system in which the flow cell is located are shown in FIGs. 15 A, 15B, and 15C.

[0135] In some optional implementations, the end effector 1500 can include one or more mechanical datum features that interact with mechanical features of the chuck 1504 to coarsely align the end effector 1500 to the chuck 1504. For example, the end effector 1500 can include one or more alignment pins 1506 that can couple to corresponding features of the chuck 1504 to provide coarse alignment between the end effector and the chuck 1504. In an optional implementation, an alignment pin 1506 of the end effector 1500 can couple to a slot 1508 of the chuck 1504, and an alignment tube 1506 of the end effector 1500 can couple to a pin 1510 of the chuck 1504. In another optional implementation (not shown) the chuck 1504 can include an alignment pin, and the end effector 1500 can include a slot, and the alignment pin of the chuck can couple to the slot 1508 of the end effector. In addition, the end effector 1500 can have a pin, and the chuck 1504 can include an alignment tube, and the alignment tube of the chuck can couple to the pin 1510 of the end effector 1500.

[0136] The flow cell 1502 can include one or more fiducials 1520, and the chuck 1504 also can include one or more fiducials 1530, and the fiducials 1520 and 1530 can be used to provide precise alignment of the flow cell 1502 with respect to the chuck 1504. In an implementation, a light source (not shown) located on the end effector 1500 can illuminate the fiducials 1520, 1530, and a camera (not shown) located on the end effector 1500 can image the fiducials 1520, 1530 that are located in a field of view of the camera. Then, a processor (not shown) can process the images to determine a position of the flow cell relative to the chuck based on the relative positions of the fiducials in the images. The dotted line rectangles of FIG. 15B show example fields of view 1540 of the camera, which include the fiducials 1520, 1530 of the flow cell 1502 and the chuck 1504, respectively.Atly Docket No. IP-2803-PCT / 0187-020W01

[0137] As shown in FIG. 15 A, the flow cell 1502 that is held by the end effector 1500 can be positioned a short distance above (i.e., along the Z direction) the chuck 1504, and one or more alignment pins 1506 of the end effector 1500 can be coupled to slots on the chuck 1504 to coarsely align the end effector to the chuck.

[0138] A camera on the end effector 1500 can image a fiducial 1530 on the chuck to determine a height of the flow cell above the chuck (e.g., based on a lateral extent in the XY plane of an image of the fiducial, as described above), and the height of the end effector 1500 in the Z direction can be adjusted until a desired height of the flow cell 1502 above the chuck is attained. With the flow cell 1220 at the desired height as shown in FIGs. 15B and 15C, an alignment tube of the end effector 1500 can couple to an alignment pin 1506 of the chuck to provide additional course alignment between the end effector and the chuck. Then, the end effector 1500 can be moved in the XY plane while images of the flow cell fiducial 1520 and the chuck fiducial 1530 are captured until the fiducials 1520, 1530 are located at predetermined positions relative to each other in the field of view of the camera, which can correspond to the flow cell 1502 being located at a desired position relative to the chuck to place the flow cell on the chuck. With such an approach, the flow cell 1502 can be located at a predetermined location relative to the chuck within a tolerance of ± 100 micrometer in the X and Y directions and within an angular tolerance about the Z axis of with ± 1 milliradian. In some implementations, the flow cell 1502 can be aligned, positioned, and located on the chuck within the tolerances described above, based on the processing of images of the flow cell fiducials and the chuck fiducials, without the aid of any mechanical datum features being used to align, position, or locate the flow cell 1502 with respect to the chuck 1504.

[0139] When the flow cell 1502 is above the surface of the chuck 1504 and the end effector 1500 is being moved in the Z direction into position to place the flow cell 502 on the chuck 1504, the flow cell’s fiducials and the chuck’s fiducials may not be located in the same plane (e.g., a focal plane of the camera). Because the magnification of the lens(es) in the camera depends on the distance of an object from the lens(es), images of the flow cell’s fiducials and / or the chuck’s fiducials captured by the camera will appear to be stretched or compressed radially in the XY plane from a principal point in the images and their locations will be shifted radially in the XY plane from the principal point as well. Because the fiducials of the flow cell and of the chuck are located in different planes, in an image of both fiducials, the fiducials of the flow cell will be shifted by a different amount than those of the chuck. Therefore, to facilitate alignment based on the images of the fiducials, a correction can be applied to the XY location of one or more of the imaged fiducials from the different planes,Atly Docket No. IP-2803-PCT / 0187-020W01 so that the corrected locations can be used to align and place the flow cell 1502 on the chuck 1504.

[0140] FIG. 14 is an example image of a checkerboard pattern that can be used to calibrate a camera for determining XY positions of fiducials. The checkerboard pattern includes an array of , for example, 36 squares, each of which is numbered. The solid-line circles represent the locations of the top left comer of each square in an image of the checkerboard pattern captured by a camera, when the pattern is located at a focal plane of the camera. The dotted-line circles represent the locations of the top left comer of each square in an image of the checkerboard pattern captured by a camera, when the pattern is shifted in the Z direction from the focal plane of the camera. The principal point of the image at which the XY position of a point does not change with magnification of the point is located near the top left corner. The “perspective shift” in images of the pattern taken when the pattern is, and is not, located at the focal plane of the camera is indicated by the distance between the solidline circles and the dotted-line circles for each top left corner of a square and increases radially from the principal point. Thus, to compare XY positions of imaged fiducials in different Z planes, the perspective shift for the fiducials in at least one of the planes can be corrected, so that the images of the fiducials in different planes are projected onto a common Z plane.

[0141] To perform such a correction, or calibration, a plurality of images of an object, such as, for example, the checkerboard pattern of FIG. 14, can be captured for different Z positions of the object relative to the camera. For each of the captured images the magnification and the locations of particular features (e.g., the top left comers of the individual squares of the checkerboard pattern) in the images can be determined. One Z position of the object with respect to the camera can be selected as a reference plane, and XY position shifts of the imaged features in each image can be determined as a function of the magnification of the image. For example, the x and y coordinates of a position of a point in an imaged plane that is not the reference plane can be related to the point in the imaged reference plane by:Px(jn) = sx(m - m0+ px,Py(rri) = sy(m - m0) + py, where Px(m) and Py(m) are the x- and y-coordinates of the position of the point in an image taken with magnification equal to m, sxand syare the slope values in the x- and y-directions, mo is the magnification when the point is located in the reference plane, and pxand pyare theAtly Docket No. IP-2803-PCT / 0187-020W01 x- and y-coordinates of the position of the point in the reference plane.

[0142] The slope values, sxand sy, can be determined from the locations of points in images from planes not equal to the reference plane according to:where xo and yo are the x- and y-coordinates of the position of the principal point, x and are the x- and y-coordinates of the position of points at a distance from the principal point, and a and C2 are constants related to optical parameters of the camera, which can be determined empirically.

[0143] From the equations above, the shift of a position of a point in an image plane, for a given magnification, relative to the position of the point at the reference plane is given by: x = X(m) — sx(m — m0), y = Y(m) — sy(m — m0).

[0144] In this matter, the locations of fiducials captured from different Z planes can be shifted, so that they can be compared and used for aligning the position of the flow cell while it is being brought closer to the chuck for placement on the chuck.

[0145] Referring again to FIGs. 15A-C, once the flow cell 1502 is properly aligned relative to the chuck 1504, the flow cell 1502 can be released from the end effector 1500 and secured to the chuck 1504, for example, by vacuum pressure, mechanical coupling, electrical coupling, or magnetic coupling. With the flow cell 1502 secured to the chuck 1504, the chuck can move the flow cell relative to the imaging system of an analysis system. As explained above, in other implementations, one or more light sources and cameras can be fixed to other components of the system besides the end effector, such that they would not travel with the end effector but still would be used to optically determine a location of the flow cell relative to the check when the end effector is being moved into position to place the flow cell on the chuck. For example, a light source and a camera can be located in a fixed position near the chuck, so that when the end effector is moved into position to place the flow cell on the chuck, fiducials on the chuck and on the flow cell can be imaged and the images can be used to determine a relative location of the chuck with respect to the flow cell.

[0146] While techniques are described above for using fiducials on the flow cell and on the chuck to place the flow cell in position on the chuck, these techniques also can be usedAtly Docket No. IP-2803-PCT / 0187-020W01 for placing a flow cell in position back on the rack after the flow cell has been imaged by the imaging system, by aligning and positioning the flow cell relative to the rack based on fiducials on the flow cell and on the rack.

[0147] The terms “substantially” and “about” used throughout this Specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. Also, when used herein, an indefinite article such as “a” or “an” means “at least one.”

[0148] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

[0149] A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the specification.

[0150] In addition, the logic flows depicted in the figures do not require the particular order shown, or sequential order, to achieve desirable results. In addition, other processes may be provided, or processes may be eliminated, from the described flows, and other components may be added to, or removed from, the described systems. Accordingly, other implementations are within the scope of the following claims.

[0151] While certain features of the described implementations have been illustrated as described herein, many modifications, substitutions, changes and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that appended claims are intended to cover all such modifications and changes as fall within the scope of the implementations. It should be understood that they have been presented by way of example only, not limitation, and various changes in form and details may be made. Any portion of the apparatus and / or methods described herein may be combined in any combination, except mutually exclusive combinations. The implementations described herein can include various combinations and / or sub-combinations of the functions, components and / or features of the different implementations described.

Claims

Atty Docket No. IP-2803-PCT / 0187-020W01WHAT IS CLAIMED IS:

1. An apparatus comprising: a rack configured for holding a plurality of flow cells, each flow cell including one or more fiducial markers and a plurality of wells, each well configured to receive a sample; an imaging system configured for capturing image data of samples in the wells; a chuck configured for holding a flow cell and for moving a flow cell with respect to the imaging system; and a robotic system including: an end effector, the end effector including a gripper configured for holding one of the plurality of flow cells, a light source configured to illuminate a fiducial marker of the flow cell, and a camera configured to capture image data of an illuminated fiducial marker, a processor configured to determine a position of an illuminated fiducial marker based on image data captured by the camera of the illuminated fiducial marker; one or more motors configured to move the end effector, based on one or more first determined positions of one or more of the illuminated fiducial markers, to a first position to pick up the flow cell from the rack with the gripper and to move the end effector, based on one or more second determined positions of one or more of the illuminated fiducial markers, to a second position to place the flow cell on the chuck.

2. The apparatus of claim 1, wherein the fiducial markers include a patterned array of optical elements that form an optical grating.

3. The apparatus of claim 2, wherein the patterned array of optical elements are included in a layer of the flow cells that includes the wells.

4. The apparatus of any one of claims 2-3, wherein the patterned array of optical elements has a pitch that is substantially similar to a pitch of an array of the plurality of wells.

5. The apparatus of any one of claims 1-4, wherein the light source includes an LED that emits broadband light.Atly Docket No. IP-2803-PCT / 0187-020W016. The apparatus of any one of claims 1-4, wherein the light source includes a laser that emits narrow band light.

7. The apparatus of any one of claims 1-6, wherein the camera includes one or more non-telecentric lenses to image the fiducial markers.

8. The apparatus of claim 7, wherein lenses of the camera that captures image data of the fiducial markers include only non-telecentric lenses.

9. The apparatus of any one of claims 7-8, wherein the camera is located less than 20 mm from the illuminated fiducial marker from which the camera is configured to capture image data.

10. The apparatus of claim 9, wherein the light source is located less than 15 mm from the illuminated fiducial marker from which the camera is configured to capture image data.

11. The apparatus of any one of claims 1-10, wherein the gripper includes vacuum cups.

12. The apparatus of any one of claims 1-10, wherein the gripper includes a magnet.

13. A method of positioning a flow cell on a chuck configured for moving the flow cell with respect to an imaging system that captures images of samples in wells of the flow cell, the method comprising: illuminating, by a light source, a fiducial marker of the flow cell; capturing images of the illuminated fiducial marker with a camera; determining a position of an illuminated fiducial marker based on image data captured by the camera of the illuminated fiducial marker based on a determined position of the fiducial marker, positioning a robotic end effector at a first position to pick up the flow cell the end effector; and based on a determined position of the illuminated fiducial marker, moving the flow cell picked up by the end effector, to a second position to place the flow cell on the chuck.Atly Docket No. IP-2803-PCT / 0187-020W0114. The method of claim 13, wherein the fiducial marker includes a patterned array of optical elements that form an optical grating.

15. The method of claim 14, wherein the patterned array of optical elements are included in a layer of the flow cell that includes the wells.

16. The method of any one of claims 14-15, wherein the patterned array of optical elements has a pitch that is substantially similar to a pitch of an array of the wells.

17. The method of any one of claims 13-16, wherein the light source includes an LED that emits broadband light.

18. The method of any one of claims 13-17, wherein the camera includes one or more non-telecentric lenses configured to capture image data of the fiducial marker.

19. The method of claim 18, wherein lenses of the camera include only non- telecentric lenses.

20. The method of claim 19, wherein the camera is located less than 20 mm from the illuminated fiducial marker.

21. The method of claim 20, wherein the light source is located less than 15 mm from the illuminated fiducial marker.

Images