FLOW CELLS WITH LINEAR WAVEGUIDES

Abstract

Flow cells with linear waveguides and a method for them. For example, a flow cell includes: a nanowell layer having a first set of nanowells and a second set of nanowells to receive a sample; a first linear waveguide associated with the first set of nanowells, and a second linear waveguide associated with the second set of nanowells; and a first grid for the first linear waveguide and a second grid for the second linear waveguide, the first and second grids providing differential coupling of first light and second light.

Description

1 / 100 FLOW CELLS WITH LINEAR WAVEGUIDES CROSS-REFERENCE TO RELATED REQUESTS

[001] The present application claims priority to provisional patent application U.S. No. 62 / 868,423, filed June 28, 2019, entitled “FLOWCELLS WITH LINEAR WAVEGUIDES”, the invention of which is incorporated by reference in the present invention in its entirety. BACKGROUND

[002] Samples of different materials can be analyzed using one or more of a variety of analytical processes. For example, sequencing such as high-throughput DNA sequencing can be the basis for genomic analysis and other genetic research. For example, synthesis sequencing (SBS) technology uses modified deoxyribonucleotide thiophosphates (dNTPs) including a terminator and a fluorescent dye having an emission spectrum. In this and other types of sequencing, the characteristics of a sample of genetic material are determined by illuminating the sample, and by detecting emission light (e.g., fluorescent light) that is generated in response to illumination.

[003] It may be desirable to ensure good sample analysis quality as well as to facilitate that the analysis is performed at a relatively high speed. For example, the amount of sample material that is analyzed at each individual stage drives the resulting throughput of the analysis process. It may be attempted to distribute the sample material more densely in the analysis equipment to allow more material to be analyzed at any given time. However, the characteristics of the analysis system, such as the maximum resolution available from imaging optics, may limit the extent to which such an approach can increase throughput. SUMMARY Petition 870200161164, dated 12 / 23 / 2020, page 29 / 263 2 / 100

[004] In a first aspect, a flow cell includes: a layer of nanowells having a first set of nanowells and a second set of nanowells to receive a sample; a first linear waveguide associated with the first set of nanowells and a second linear waveguide associated with the second set of nanowells; and a first grid for the first linear waveguide, and a second grid for the second linear waveguide, the first and second grids providing differential coupling of the first light and the second light.

[005] Implementations may include any or all of the following features. The first and second grids are spatially offset from each other. The first and second linear waveguides are positioned adjacent to each other, the flow cell further comprising: a first linear waveguide positioned adjacent to the second linear waveguide opposite from the first linear waveguide. The third linear waveguide shares the first grid with the first linear waveguide. The flow cell further comprises a third grid for the third linear waveguide. The third grid has the same spatial offset from the second grid as the first grid. The third grid is spatially offset from each of the first and second grids.The first grid is positioned towards one end of the first linear waveguide, wherein the second grid is positioned towards the other end of the second linear waveguide, and wherein the first end is positioned opposite the second end. The first grid is positioned on a triangular substrate. The first and second grids have different grid periods from each other. The first and second linear waveguides are positioned adjacent to each other, the flow cell further comprising: a third linear waveguide positioned adjacent to the second linear waveguide opposite from the first. Petition 870200161164, dated 12 / 23 / 2020, page 30 / 263 3 / 100 first linear waveguide; and a third grid for the third linear waveguide. The third grid has the same grid period as the first grid. The third grid has a grid period different from each of the grid periods of the first and second grids. The nanowells in at least one of the first and second nanowell sets have a spacing from each other that is resolvable according to an emission optics resolution distance for the flow cell. The first and second linear waveguides are positioned closer to each other than the emission optics resolution distance. Differential coupling of the first light comprises coupling the first light in the first linear waveguide and minimizing coupling of the first light in the second linear waveguide. Differential coupling of the second light comprises coupling the second light in the second linear waveguide and minimizing coupling of the second light into the first linear waveguide.Differential coupling is at least partly due to a coupler parameter of one or more of the first or second grids. The coupler parameter comprises at least one selected from the group consisting of: a refractive index, a pitch, a groove width, a groove height, a groove spacing, a grid non-uniformity, a groove orientation, a groove curvature, a coupler shape, and combinations thereof. Differential coupling is at least partly due to a waveguide parameter of one or more of the first linear waveguide or the second linear waveguide. The waveguide parameter comprises at least one selected from the group consisting of: a cross-sectional profile, a refractive index difference, a mode matching, and combinations thereof. The first and second sets of nanowells are arranged in a polygonal arrangement.The polygonal layout comprises either a rectangular layout or a hexagonal layout. The first and the second. Petition 870200161164, dated 12 / 23 / 2020, page 31 / 263 4 / 100 sets of nanowells are arranged in a hexagonal arrangement, forming at least one hexagon, the hexagon including: the first and second nanowells of the first set of nanowells, the first and second nanowells forming part of a first row of nanowells extending along the first linear waveguide; the third, fourth, and fifth nanowells of the second set of nanowells, the third, fourth, and fifth nanowells forming part of a second row of nanowells extending along the second linear waveguide; and the sixth and seventh nanowells of a third set of nanowells, the sixth and seventh nanowells forming part of a third row of nanowells extending along a third linear waveguide. The first set of nanowells comprises a first row of nanowells, and the second set of nanowells comprises a second row of nanowells.Each of the first and second rows of nanowells is aligned with at least one of the first and second linear waveguides. The first row of nanowells extends along the first linear waveguide, wherein the second row of nanowells extends along the second linear waveguide, wherein the first linear waveguide is parallel and adjacent to the second linear waveguide, and wherein the first row of nanowells is in phase with the second row of nanowells, the flow cell further comprising: a third linear waveguide that is parallel and adjacent to the second linear waveguide; and a third row of nanowells extending along the third linear waveguide, wherein the third row of nanowells is out of phase with the first and second rows of nanowells.The flow cell also comprises: a fourth linear waveguide that is parallel and adjacent to the third linear waveguide; and a fourth row of nanowells extending along the fourth linear waveguide, in which the fourth row of nanowells is in phase with the third row of nanowells. The first and second linear waveguides are parallel and... Petition 870200161164, dated 12 / 23 / 2020, p. 32 / 263 5 / 100 adjacent to each other, wherein the first set of nanowells comprises first and second rows of nanowells extending along the first linear waveguide on opposite sides thereof, and wherein the second set of nanowells comprises third and fourth rows of nanowells extending along the second linear waveguide on opposite sides thereof. At least one nanowell of the first and second sets of nanowells has a non-circular aperture. The non-circular aperture comprises an elliptical aperture. The flow cell further comprises a structure between the first and second linear waveguides to reduce cross-coupling. The structure comprises a series of blocks. The structure provides alternating refractive indices along the structure.The first linear waveguide and the first grid are positioned in a first layer of the flow cell, wherein the second linear waveguide and the second grid are positioned in a second layer of the flow cell, wherein the first and second sets of nanowells are positioned in a third layer of the flow cell, and wherein the second layer is positioned further away from the third layer than the first layer.

[006] In a second aspect, a method comprises: applying, in a flow cell, a sample to a first set of nanowells and to a second set of nanowells; differentially coupling, using a first grating, first light to at least one first linear waveguide associated with the first set of nanowells; and differentially coupling, using a second grating, second light to at least one second linear waveguide associated with the second set of nanowells.

[007] Implementations may include any or all of the following features. The first and second grids are spatially offset from each other, the method further comprising controlling a lighting component relating to at least one of the first or second lights. The Petition 870200161164, dated 12 / 23 / 2020, p. 33 / 263 6 / 100 Lighting component control comprises controlling a beam parameter of a light beam generating at least one of the first light or the second light. Controlling the beam parameter comprises at least one selected from the group consisting of: controlling a location of the light beam, controlling an angle of incidence of the light beam, controlling a divergence of the light beam, controlling a mode profile of the light beam, controlling a polarization of the light beam, controlling an aspect ratio of the light beam, controlling a diameter of the light beam, controlling a wavelength of the light beam, and combinations thereof. The first light is differentially coupled during a first scan performed through the flow cell in a first scan direction, and the second light is differentially coupled during a second scan performed through the flow cell in a second scan direction opposite to the first scan direction.The first and second grids have different grid periods from each other, the method further comprising arranging a lighting component so that the first light is differentially coupled, and arranging the lighting component so that the second light is differentially coupled. The first and second waveguides are positioned adjacent to each other, and the flow cell further comprises a third linear waveguide positioned adjacent to the second linear waveguide opposite the first linear waveguide. The flow cell further comprises a third grid for the third linear waveguide. The method further comprises differentially coupling the first light also to the third linear waveguide using the third grid. The method further comprises differentially coupling at least the third light to the third linear waveguide using the third grid. The third linear waveguide shares the first grid with the first linear waveguide.Nanowells, at least. Petition 870200161164, dated 12 / 23 / 2020, page 34 / 263 7 / 100 of the first and second sets of nanowells have a spacing from each other that is resolvable according to an emission optics resolution distance for the flow cell. The first and second linear waveguides are positioned closer to each other than the emission optics resolution distance. Differentially coupling the first light involves coupling the first light to the first linear waveguide and minimizing coupling of the first light to the second linear waveguide. Differentially coupling the second light involves coupling the second light to the second linear waveguide and minimizing coupling of the second light to the first linear waveguide.

[008] It should be recognized that all combinations of the above concepts and additional concepts discussed in greater detail below (provided that such concepts are not mutually inconsistent) are considered to form part of the inventive matter disclosed in the present invention. In particular, all combinations of the claimed matter appearing at the end of this invention are considered to form part of the inventive matter disclosed in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[009] FIG. 1 shows a cross-section of part of an example flow cell with linear waveguides.

[010] FIGS. 2A-2B illustrate examples with a flow cell having stepped grids.

[011] FIGS. 3A-3B illustrate examples with a flow cell having grids with different grid periods.

[012] FIG. 4 shows another example of a flow cell having stepped grids.

[013] FIG. 5 shows a cross-section of part of an example flow cell. Petition 870200161164, dated 12 / 23 / 2020, page 35 / 263 8 / 100

[014] FIG. 6 shows an example of a flow cell where multiple linear waveguides share a common grid.

[015] FIG. 7 is a diagram of an example lighting system.

[016] FIGS. 8-9 are example method flowcharts.

[017] FIG. 10A shows an example of a hexagonal arrangement of noncircular nanowells.

[018] FIG. 10B shows an example of a triangular arrangement of circular nanowells.

[019] FIG. 11 shows another example of a flow cell having stepped grids.

[020] FIG. 12 shows another example of a flow cell having stepped grids.

[021] FIG. 13 shows another example of a flow cell having stepped grids.

[022] FIG. 14 shows schematically a beam of light incident on a surface.

[023] FIGS. 15A-15C show examples of grids.

[024] FIG. 16 shows examples of coupler formats.

[025] FIG. 17 shows examples of cross-sectional profiles for linear waveguides.

[026] FIG. 18 shows a cross-section of part of another example flow cell with linear waveguides.

[027] FIG. 19 is a flowchart of an example method. DETAILED DESCRIPTION

[028] The present invention describes systems, techniques, industrial products and / or material compositions that facilitate improved sample analysis. In some implementations, differential coupling can be Petition 870200161164, dated 12 / 23 / 2020, page 36 / 263 9 / 100 provided in two or more linear waveguides. For example, being able to differentially couple light in linear waveguides can allow a substrate (e.g., a layer of nanowells to contain sample material) to have an increased density of the sample material. In some implementations, one or more parameters relating to the analysis system and / or process can be selected or adjusted in order to achieve differential coupling. For example, such parameter(s) may include one or more beam parameters, one or more coupler parameters, one or more waveguide parameters, or combinations thereof.

[029] In some implementations, analytical imaging can be performed on sample material having an increased density distribution on a substrate, which can increase the throughput of the analytical process. For example, the sample material can be distributed with a density where individual portions of the sample are positioned at closer distances to each other that can be resolved using available imaging technology, such as microscopy equipment. An analytical process can selectively image only first portions of the sample at a time, rather than imaging second portions near the first portions and subsequently imaging the second portions without (again) imaging the first portions. Such an approach can allow a relatively large amount of sample material on a single sample holder (e.g., a substrate) to be imaged and analyzed in a single session.This can increase the throughput of the analysis process compared to an approach where the substrate is changed after analyzing its entire sample material in order to analyze additional sample material on a new substrate, which approach may involve intermediate steps of substrate removal and insertion, sample preparation, and equipment initialization. Petition 870200161164, dated 12 / 23 / 2020, page 37 / 263 10 / 100

[030] In some implementations, differential coupling between, say, first and second linear waveguides may involve coupling light in the first linear waveguide while not coupling any light in the second linear waveguide, or vice versa. Such differential coupling may not always be practical or possible. In some implementations, differential coupling may involve minimizing coupling in, say, the second linear waveguide while coupling light in the first linear waveguide during a portion of the scan. The amount or fraction of minimization may differ depending on the implementation. In some implementations, the minimized coupling (e.g., crosstalk) corresponds to at most about 1%, about 5%, about 15%, about 25%, or about 45% of the coupling in the linear waveguide. Such differential coupling may not always be practical or possible.In some implementations, differential coupling may involve reducing the coupling in, say, the second linear waveguide compared to the first linear waveguide during a portion of the sweep. The amount or fraction of the reduction may differ depending on the implementation. In some implementations, the reduced coupling (e.g., crosstalk) corresponds to at most about 5%, about 15%, about 35%, about 65%, or about 95% of the coupling in the linear waveguide.

[031] The amount of crosstalk (e.g., its magnitude) can be known or calibrated. In some implementations, multiple scans of a sample can be performed, such as a first scan with coupling in the first linear waveguide where coupling in the second linear waveguide is reduced, and a second scan with coupling in the second linear waveguide where coupling in the first linear waveguide is reduced. The scans can cause modulation of the information obtained from the first and second linear waveguides, Petition 870200161164, dated 12 / 23 / 2020, page 38 / 263 11 / 100 respectively. Such modulation can occur in a predictable manner given the magnitude of the crosstalk. For example, linear algebra can be applied to the information obtained from the first and second respective linear waveguides to extract useful analytical information.

[032] The limit imposed by the maximum available resolution of imaging equipment can be referred to as a diffraction limit. It can be said that an imaging system operating at the maximum resolution thus available is diffraction-limited. For microscopic instruments, the spatial resolution that can be obtained given the diffraction limit depends on the wavelength of light and the numerical aperture of the objective or the illumination source. The minimum resolvable distance d can be expressed as d = λ / (2nsin0) where λ is the wavelength of light, n is the refractive index, and Θ is the half-angle (i.e., half the angle between a geometric optical axis of the microscope and the direction of the most oblique light rays captured by the objective). The factor nsin Θ is usually referred to as the numerical aperture (NA), and the minimum resolvable distance can therefore be expressed as d = λ / (2NA).That is, in existing analytical systems, the sample material is generally distributed with a density such that the individual portions of the sample are separated by at least a distance d. Systems and techniques described in the present invention can allow analysis to be performed on sample material that is distributed more densely than the resolution distance d.

[033] Sample analysis may include, but is not limited to, genetic sequencing (e.g., determining the structure of genetic material), genotyping (e.g., determining differences in an individual's genetic makeup), gene expression (e.g., synthesizing a gene product using gene information), proteomics (e.g., Petition 870200161164, dated 12 / 23 / 2020, page 39 / 263 12 / 100 large-scale protein study) or combinations thereof.

[034] Some examples described in the present invention relate to sequencing of genetic material. Sequencing can be performed on a sample to determine which building blocks, called nucleotides, make up the specific genetic material in the sample. Sequencing can be done after the genetic material has first been purified and then replicated several times to prepare a sample of an appropriate size. Imaging can be performed as part of the genetic material sequencing process. This may involve fluorescent imaging, where a sample of genetic material is exposed to light (e.g., a laser beam) to trigger a fluorescent response by one or more markers in the genetic material.Some nucleotides in the genetic material may have fluorescent tags applied to them, which allows for the determination of the nucleotide's presence by shining light on it and looking for a characteristic response from the sample. Fluorescent responses can be detected during the course of the sequencing process and used to build a record of nucleotides in the sample.

[035] The examples described in the present invention refer to flow cells. A flow cell can be considered a substrate that can be used in the preparation and accommodation or transport of one or more samples in at least one stage of an analytical process. The flow cell is made of a material that is compatible with both the sample material (e.g., genetic material), the illumination, and the chemical reactions to which it will be exposed. The substrate may have one or more channels in which the 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 Petition 870200161164, dated 12 / 23 / 2020, page 40 / 263 13 / 100 one or more chemical reactions and / or remove unwanted material. The flow cell can enable imaging by facilitating the exposure of the sample in the flow cell channel to illumination light, allowing any fluorescent responses from the sample to be detected. Some system implementations 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 transport or when delivered to a customer. For example, the flow cell(s) may be installed in an on-site implementation at the customer's premises in order to perform the analysis.

[036] The examples of the present invention relate to the coupling of light (e.g., a laser beam) into and / or out of a waveguide by one or more gratings. A grating can couple incident light by diffraction of at least a portion of the light, thereby causing the portion of light to propagate in one or more other directions. In some implementations, the coupling may involve one or more interactions, including, but not limited to, reflection, refraction, diffraction, interference, and / or transmission of the portion of light. Implementations may be designed to meet one or more requirements, including, but not limited to, those relating to mass production, cost control, and / or high light coupling efficiency. Two or more gratings may be identical or similar to each other, or different types of gratings may be used. The grating(s) may include one or more forms of periodic structure.In some implementations, the grid can be formed by removing or omitting material from a substrate (for example, from a waveguide material that is included in the flow cell) or other material. For example, the flow cell may be provided with a set of grooves and / or slots within it to form the grid. In some implementations, the grid can be formed by adding material to the cell. Petition 870200161164, dated 12 / 23 / 2020, page 41 / 263 14 / 100 flow (for example, a waveguide material that is included in the flow cell) or other material. For example, the flow cell may be provided with a set of ridges, bands, or other longitudinal protruding structures to form the grid. Combinations of these approaches may be used.

[037] Providing a waveguide on a substrate (such as a flow cell) can provide one or more advantages. Excitation using evanescent light based on total internal reflection (TIR) ​​can provide higher illumination efficiency. In some previous approaches, the entirety of a laser beam was used to illuminate the substrate that held the sample, as in a scanning process. Such an approach can cause a larger portion of the light wave to simply propagate through the substrate without effectively illuminating the sample. As a result, only a small portion of the light applied by such systems can actually be used to excite fluorophores in the sample. Evanescent light, in contrast, can penetrate the material (e.g., a coating adjacent to the core layer) only to a certain depth (e.g., about 150-200 nm in one example).For example, the flow cell can be designed with one or more nanowells configured so that the evanescent field is largely confined to the well area. As a result, evanescent light can be a very efficient way to excite fluorophores. For example, a system operating according to a previous illumination approach might involve a laser with a certain power; using evanescent light, conversely, a significantly lower laser power might be sufficient.

[038] The examples of the present invention relate to chemical vapor deposition. Chemical vapor deposition (CVD) may include all techniques where a volatile material (sometimes referred to as a precursor) is induced to undergo reaction and / or decomposition on the surface of a substrate. Petition 870200161164, dated 12 / 23 / 2020, page 42 / 263 15 / 100 forming a deposit on it. CVD can be characterized by one or more aspects. For example, CVD can be characterized by the physical characteristic(s) of the vapor (e.g., whether the CVD is aerosol-assisted or involves direct liquid injection). For example, CVD can be characterized by the type of substrate heating (e.g., whether the substrate is directly heated or indirectly heated, such as a heated chamber). Examples of CVD types that can be used include, but are not limited to, atmospheric pressure CVD, low pressure CVD, very low pressure CVD, ultra-high vacuum CVD, metal-organic CVD, laser-assisted CVD, and plasma-intensified CVD.

[039] The examples of the present invention relate to atomic layer deposition. Atomic layer deposition can be considered a form of CVD and includes all techniques where a film is grown on a substrate by exposure to gases. For example, gaseous precursors can be alternately introduced into a chamber. The molecules of one of the precursors can react with the surface until a layer is formed and the reaction is terminated, and the next gaseous precursor can then be introduced to begin forming a new layer, etc., in one or more cycles.

[040] The examples of the present invention relate to spray coating. Spray coating may include any and all techniques by which a particular material is induced to be deposited onto a substrate. This may include, but is not limited to, thermal spraying, plasma spraying, cold spraying, hot spraying and / or other procedures involving atomized or nebulized material.

[041] The examples of the present invention relate to spin coating. Spin coating may involve applying a quantity of coating material to a substrate, and distributing or spreading the material. Petition 870200161164, dated 12 / 23 / 2020, p. 43 / 263 16 / 100 coating on the substrate by means of centrifugal force due to rotation or spinning of the substrate.

[042] The examples of the present invention relate to nanoprinting. In nanoprinting lithography, a prefabricated nanoscale jig can mechanically displace a fluid resin to mold the desired nanostructures. The resin can then be cured with the nanoscale jig in place. After removal of the nanoscale jig, a molded solid resin fixed to a desired substrate can be produced. In some implementations, a nanoprinting process may begin with full or partial coating of a substrate or wafer with printing resin (e.g., a resin as exemplified below). One or more nanostructures can be formed on the printing resin in a molding process using a nanoscale jig. The printing 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, resin removal can form chamber lanes adjacent to the nanostructures. The substrate or wafer thus formed can have another substrate or a gasket applied to it 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 printing resin can 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 can also be functionalized with a chemical treatment or a biomolecule attachment, depending on the end use. In nanoprinting lithography, a printed photoresist can be a sacrificial material and similarly be used as an intermediate tool to transfer the patterned resist to the substrate. Petition 870200161164, dated 12 / 23 / 2020, page 44 / 263 17 / 100 or a variation of resist can be used so that the printed resist serves as the input for a subsequent coating step. An example of a resist that would remain after patterning is material formed by a process involving the conversion of monomers in a colloidal solution as a precursor to a gel of particles and / or polymers, sometimes referred to as a sol-gel based material.

[043] The examples of the present invention relate to substrates. A substrate may refer to any material that provides at least a substantially rigid structure, or a structure that retains its shape rather than assuming the shape of a vessel in which it is placed. The material may 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, plastic (including acrylic, polystyrene and styrene copolymers and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.Polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, fiber optic bundles, and a variety of other polymers. In general, the substrates allow optical detection and do not fluoresce appreciably themselves.

[044] The examples of the present invention relate to polymers. A polymer layer may include a film of a polymer material. Film-forming polymers of example include, without limitation, acrylamide or C1-C2 copolymers; hydroxyl and aromatic derivatives; acrylate copolymers; vinylpyrrolidine and vinylpyrrolidone copolymers; sugar-based polymers such as starch or polydextrins; other polymers such as polyacrylic acid, Petition 870200161164, dated 12 / 23 / 2020, page 45 / 263 18 / 100 polyethylene glycol, polylactic acid, silicone, siloxanes, polyethylene amines, guar gum, carrageenan, alginate, locust bean gum, methacrylate copolymers, polyimide, a cyclic olefin copolymer, or combinations thereof. In some implementations, a polymer layer comprises at least one photocurable polymer. For example, a photocurable polymer may include urethane, acrylate, silicone, epoxy, polyacrylic acid, polyacrylates, epoxy silicone, epoxy resins, polydimethyl siloxane (PDMS), silsesquioxane, acyloxysilanes, maleate polyesters, vinyl ethers, monomers with vinyl or ethinyl groups, or copolymers or combinations thereof. In some implementations, a layer may include a covalently bonded polymer coating.For example, this may include a polymer coating that forms chemical bonds with a functionalized surface of a substrate, as opposed to surface attachment by other means, such as adhesion or electrostatic interaction. In some implementations, a polymer comprised within a functionalized layer is poly(N-(5-azidoacetamidyl pentyl)acrylamide-coacrylamide), sometimes referred to as PAZAM.

[045] The examples described in the present invention mention that one or more resins can be used. Any suitable resin can be used for nanoprinting in methods described in the present invention. In some implementations, an organic resin can 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, a polyacetal resin, a polyether resin, a polyurethane resin, a polyamide (and / or nylon) resin, a furan resin, a diallyl phthalate resin, or combinations thereof. In some examples, a resin may include an inorganic siloxane polymer including a Si-O-Si bond between compounds (including silicon, oxygen, and hydrogen) and formed using a Petition 870200161164, dated 12 / 23 / 2020, page 46 / 263 19 / 100 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 replaced by an organic group, such as methyl or phenyl, and typified by an alkyl siloxane polymer, an alkyl silsesquioxane polymer, a silsesquioxane hydride polymer, or an alkyl silsesquioxane hydride polymer. Non-limiting examples of siloxane polymers include polyhedral oligomeric silsesquioxane (POSS), polydimethyl siloxane (PDMS), tetraethyl orthosilicate (TEOS), poly(organo)siloxane (silicone), and perfluoropolyether (PEPE). 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 which uses a suitable solvent.Any one of a number of other resins may be employed, as applicable to the application.

[046] Figure 1 shows a cross-section of part of an exemplary flow cell 100 with linear waveguides 102A-102C. The flow cell 100 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. Only a portion of the flow cell 100 is shown, for illustrative purposes. For example, one or more additional layers and / or more or fewer waveguides 102A-102C can be used.

[047] The flow cell 100 includes a substrate 104. The substrate 104 can form a base for the flow cell 100. In some implementations, one or more other layers may be formed on (e.g., in contact with or near) substrate 104 in the fabrication of the flow cell 100. The substrate 104 can serve as a base for forming the linear waveguides 102A-102C. The linear waveguides 102A-102C may initially exist separately. Petition 870200161164, dated 12 / 23 / 2020, page 47 / 263 20 / 100 of substrate 104 and subsequently applied over substrate 104, or linear waveguides 102A-102C can be formed by applying and / or removing one or more materials to or from the substrate. Linear waveguides 102A-102C can be formed directly over substrate 104 or over one or more intermediate layers on substrate 104.

[048] 102A-102C linear waveguides are used to conduct electromagnetic radiation (including, but not limited to, visible light, such as laser light). In some implementations, the electromagnetic radiation performs one or more functions during an imaging process. For example, electromagnetic radiation can be used to excite fluorophores in a sample material for imaging. 102A-102C linear waveguides can be made of any suitable material that facilitates the propagation of one or more types of electromagnetic radiation. In some implementations, the material(s) of the 102A-102C linear waveguides may include a polymer material. In some implementations, the material(s) of the 102A-102C linear waveguides may include Ta2O5 and / or SiNx. For example, 102A102C linear waveguides can be formed by cathodic sublimation, chemical vapor deposition, atomic layer deposition, spin coating, and / or spray coating.

[049] Each of the 102A-102C linear waveguides may have one or more grids (omitted here for clarity) for coupling electromagnetic radiation into and / or out of that 102A-102C linear waveguide. One or more directions of displacement for electromagnetic radiation in 102A-102C linear waveguides may be employed. For example, the direction of displacement may be into and / or out of the plane of the present illustration. Examples of grids are described elsewhere in the present invention.

[050] Each of the 102A-102C linear waveguides can be positioned Petition 870200161164, dated 12 / 23 / 2020, p. 48 / 263 21 / 100 against one or more types of coating. The coating can serve to limit electromagnetic radiation to the respective 102A-102C linear waveguide and prevent, or reduce the extent of, radiation propagation into other 102A-102C linear waveguides or other substrates. Here, 106A-106D coatings are shown as examples. For example, 106A-106B coatings can be positioned against or near the 102A linear waveguide on different (e.g., opposite) sides of it. For example, 106A-106C coatings can be positioned against or near the 102B linear waveguide on different (e.g., opposite) sides of it. For example, 106A-106C coatings can be positioned against or near the 102C linear waveguide on different (e.g., opposite) sides of it. The 106A-106D coatings can be made of one or more suitable materials that serve to separate the 102A-102C linear waveguides from each other.In some implementations, the 106A-106D coatings may be made of a material having a lower refractive index than the refractive index(es) of the 102A-102C linear waveguides. For example, the 102A-102C linear waveguides may have a refractive index of about 1.4–1.6, and the 106A-106D coatings may have a refractive index of about 1.2–1.4. In some implementations, one or more of the 106A-106D coatings include a polymer material. In some implementations, one or more of the 106A-106D coatings include multiple structures, including, but not limited to, structures of a material (e.g., polymer) interspersed with vacuum regions or another material (e.g., air or a liquid).

[051] The flow cell 100 includes at least one nanowell layer 108. In some implementations, the nanowell layer 108 is positioned opposite the linear waveguides 102A-102C from the substrate 104. For example, the nanowell layer may be positioned adjacent (e.g., Petition 870200161164, dated 12 / 23 / 2020, page 49 / 263 22 / 100 touching or near) the 102A-102C linear waveguides and the 106A-106D coatings. The 108 nanowell layer includes one or more nanowells. In some implementations, the 108 nanowell layer includes 108A-108C nanowells. The 108A-108C nanowells can be used to contain one or more sample materials during at least part of the analysis process (e.g., for imaging). For example, one or more genetic materials (e.g., in the form of clusters) can be placed in the 108A-108C nanowells.

[052] One or more of the 108A-108C nanowells may be at least substantially aligned with one or more of the 102A-102C linear waveguides. This may allow interaction between the respective 108A-108C nanowell and the corresponding 102A-102C linear waveguide for imaging purposes (including, but not limited to, by means of evanescent light transmission). For example, nanowell 108A may be at least substantially aligned with linear waveguide 102A; nanowell 108B may be at least substantially aligned with linear waveguide 102B; and / or nanowell 108C may be at least substantially aligned with linear waveguide 102C.

[053] 108A-108C nanowells can be formed by nanoprinting in the 108 nanowell layer, or a lifting process from the 108 nanowell layer. For example, the 108 nanowell layer may include a resin, and the 108A-108C nanowells may be formed by printing using a nanoscale template. In some implementations, the 108A-108C nanowells may have a size such that one or more of their dimensions vary on the order of one or more nanometers. One end (e.g., the bottom) of the 108A-108C nanowells may have a thickness that accommodates evanescent light propagation. For example, the thickness may be about 0-500 nm. A Petition 870200161164, dated 12 / 23 / 2020, page 50 / 263 The 23 / 100 nanowell layer can cover at least substantially the entire coating surface of the layer that includes the 102A-102C linear waveguides and the 106A-106D coatings. In some implementations, the 108 nanowell layer may have an average pitch between the 108A-108C nanowells of at least 10 nm, 0.1 µm, 0.5 µm, 1 µm, 5 µm, 10 µm, 100 µm or more, and / or may have an average pitch of at most 100 µm, 10 µm, 5 µm, 1 µm, 0.5 µm, 0.1 µm or less. In some implementations, the 108 nanowell layer may have a pitch between the 108A-108C wax nanowells of 150 nm or greater. For example, the 108 nanowell layer may have a pitch between 108A-108C nanowells of approximately 160 nm, 220 nm, 250 nm, 300 nm, 450 nm or greater. The depth of each 108A-108C nanowell may be at least 0.1 sq m, 1 sq m, 10 sq m, 100 sq m or more. Alternatively or additionally, the depth may be at most 1x103 sq m, 100 sq m, 10 sq m, 1 sq m, 0.1 sq m or less.

[054] Figures 2A-2B illustrate examples with a flow cell 200 having stepped grids 202. The flow cell 200 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. Only a portion of the flow cell 200 is shown, for illustrative purposes.

[055] The 200 flow cell includes nanowells, including a 204A nanowell, which are illustrated here using circular shapes. Only one of the nanowells will be specifically mentioned, and the other nanowells may be similar or identical to the one(s) discussed. Nanowells can be formed in a nanowell layer (e.g., by nanoprinting or a lifting process). For example, nanowells can be formed in a resin using a nanoscale jig. The nanowell layer is not explicitly shown in this example for clarity. The 204A nanowell is associated here with a 206A linear waveguide. In some Petition 870200161164, dated 12 / 23 / 2020, page 51 / 263 In 24 / 100 implementations, the linear waveguides described with reference to flow cell 200 may be similar or identical to one or more other linear waveguides described in this invention. For example, linear waveguide 206A is positioned adjacent to (e.g., in contact with or near) the nanowell layer that includes nanowell 204A. In some implementations, linear waveguide 206A may include a linear waveguide core 208 and one or more of the grids 202.

[056] Another 204B nanowell is also associated with the 206A linear waveguide. For example, the 204B nanowell is positioned adjacent to the 204A nanowell, and both 204A-204B nanowells can interact with the 206A linear waveguide in an imaging process (e.g., by receiving electromagnetic radiation from the 206A linear waveguide). Another 204C nanowell, conversely, is instead associated with a 206B linear waveguide. In some implementations, the 206B linear waveguide is positioned adjacent to the 206A linear waveguide. For example, the coating (not shown) and / or other material can be positioned between the 206A-206B linear waveguides.

[057] Some examples described in the present invention mention or otherwise refer to nanowell arrays. A nanowell array is a logical or physical group of one or more nanowells having at least one characteristic. One nanowell array may be associated with one linear waveguide, and another nanowell array may be associated with another linear waveguide. In some implementations, a nanowell array may be arranged in a line. Such a line of nanowells may extend along the linear waveguide, either by being coextensive with (e.g., fully overlapping above or below) the linear waveguide, or by being parallel to and positioned adjacent to (e.g., on either or both sides of) the waveguide. Petition 870200161164, dated 12 / 23 / 2020, p. 52 / 263 25 / 100 of a linear waveguide, to cite some examples. Therefore, a nanowell array may include one or more nanowell lines in some implementations. Each of these nanowell lines may be aligned with at least one linear waveguide.

[058] Nanowells can be arranged on a substrate (e.g., in a nanowell layer) in a substantially, and at least one instance entirely, random manner or according to one or more patterns. In some implementations, nanowells are arranged in one or more arrangements, including, but not limited to, a polygonal arrangement. For example, a polygonal arrangement can be a rectangular, triangular, or hexagonal arrangement, or any other arrangement shape where at least some nanowells are arranged in a polygonal shape. Flow cell 200 in this example has a rectangular arrangement of nanowells.

[059] The flow cell 200 can be used in one or more forms of imaging process. For example, sample material in nanowells (including nanowells 204A-204C) can be subjected to electromagnetic radiation from respective linear waveguides (including linear waveguides 206A-206B, respectively). Emissions resulting from such exposure to electromagnetic radiation (an example of emissions being fluorescence from fluorophores) can be captured using equipment (e.g., one or more cameras and / or other imaging devices). Such equipment is sometimes referred to by the expression emission equipment or a similar term. For example, emission equipment may include one or more cameras or other image sensors and at least one lens or other emission optics. In some implementations, the diffraction limit may be at least partially attributable to one or more features of the emission optics.For example, based on emission optics. Petition 870200161164, dated 12 / 23 / 2020, page 53 / 263 26 / 100 used, a resolution distance can be defined, the resolution distance marking the shortest distance that can be resolved using the emission optics. That is, when resolving features that are separated by the resolution distance, it can be said that the imaging system is operating at its highest available resolution level.

[060] Here, a distance 210 is less than the emission optics resolution distance, and a distance 212 is greater than, or approximately equal to, the emission optics resolution distance. The distance 210 here represents the separation between nanowells in one direction. In some implementations, this may be the direction through the linear waveguides. For example, since the linear waveguides are here aligned with nanowell lines in one direction (e.g., the vertical direction as seen in the illustration), the distance 210 may also represent the distance between adjacent linear waveguides (e.g., linear waveguides 206A-206B). For example, nanowells 204A and 204C are separated by a distance of 210. That is, linear waveguides 206A-206B are positioned closer to each other than the emission optics resolution distance.

[061] The distance 212 here represents the separation between nanowells in a direction other than the distance 210. For example, the distances 210 and 212 may be substantially and at least in one instance completely perpendicular to each other. In some implementations, this may be the direction along any individual waveguide of the linear waveguides. For example, since the linear waveguides are here aligned with nanowell lines in one direction (e.g., the vertical direction as seen in the illustration), the distance 212 may represent the distance between adjacent nanowells in any of the linear waveguides (e.g., linear waveguides 206A-206B). For example, nanowells 204A and 204B are Petition 870200161164, dated 12 / 23 / 2020, p. 54 / 263 27 / 100 separated by a distance of 212. That is, the nanowells associated with the linear waveguide 206A have a spacing from each other that is resolvable according to the emission optics resolution distance for the flow cell 200.

[062] The 202 gratings serve to couple electromagnetic radiation into and / or out of the linear waveguides of the 200 flow cell. Here, the linear waveguide 206A has a 202A grating and the linear waveguide 206B has a 202B grating. The 202A-202B gratings may have the same or different periodic structure. In some implementations, either or both of the 202A-202B gratings may include a periodic structure of crests interspersed with another material. For example, crests of the 202A-202B gratings may have a pitch of about 200-300 nm, to cite just one example.

[063] The 202A-202B grids may have one or more features that facilitate the selective coupling of electromagnetic radiation to the corresponding 206A-206B linear waveguide. In some implementations, one or more of the 202 grids is spatially offset from one or more other 202 grids. The offset may be in a direction that is parallel to the 206A-206B linear waveguides. For example, the distance between the 202B grid and the nearest nanowell of the nanowells associated with the 206B linear waveguide is greater here than the distance between the 202A grid and the nearest nanowell of the nanowells associated with the 206A linear waveguide.The feature of the 202A-202B grids being spatially offset from each other facilitates the coupling of electromagnetic radiation (e.g., light) in one of the linear waveguides (e.g., the 206A linear waveguide) without coupling electromagnetic radiation (e.g., light) in another of the linear waveguides (e.g., the 206B linear waveguide).

[064] The 200 flow cell can include multiple linear waveguides, for example, as illustrated. In some implementations, a waveguide Petition 870200161164, dated 12 / 23 / 2020, page 55 / 263 A 28 / 100 linear waveguide 206C is positioned adjacent to a linear waveguide 206B, opposite a linear waveguide 206A. For example, a linear waveguide 206C might have a grid 202C. In some implementations, the grid 202C might be spatially offset from the grid 202B. For example, the grid 202C might have the same spatial offset from the grid 202B, in the direction parallel to the linear waveguide 206C, as the grid 202A has in the direction parallel to the linear waveguide 206A.

[065] The feature of grids 202A and 202C being spatially offset from grid 202B facilitates coupling of electromagnetic radiation (e.g., light) in one of the linear waveguides (e.g., linear waveguide 206A or 206C) without coupling the electromagnetic radiation (e.g., light) in another of the linear waveguides (e.g., linear waveguide 206B). As another example, the feature facilitates coupling of electromagnetic radiation (e.g., light) in one of the linear waveguides (e.g., linear waveguide 206B) without coupling the electromagnetic radiation (e.g., light) in at least one other of the linear waveguides (e.g., linear waveguide 206A or 206C).

[066] A light area 214 is schematically illustrated here as a rectangle with a dashed outline. The light area 214 represents one or more positions where light or other electromagnetic radiation is induced to impinge as part of an imaging process. In some implementations, illumination light generated by a laser can be directed into the light area 214 to eventually be coupled to some of the linear waveguides. For example, the laser light can be selected to match fluorescence properties of one or more fluorophores in the sample material.

[067] An image capture area 216 is schematically illustrated here as a rectangle with a dashed outline. The area of Petition 870200161164, dated 12 / 23 / 2020, page 56 / 263 29 / 100 image capture 216 represents the field of view of the emission optics. For example, a camera or other image sensor can capture one or more types of emissions (e.g., fluorescent light) emanating from the image capture area 216.

[068] The examples described above illustrate that the flow cell 200 includes a nanowell layer having first (e.g., nanowells associated with linear waveguide 206A) and second (e.g., nanowells associated with linear waveguide 206B) sets of nanowells to receive a sample. The flow cell 200 includes a first linear waveguide (e.g., linear waveguide 206A) aligned with the first set of nanowells and a second linear waveguide (e.g., linear waveguide 206B) aligned with the second set of nanowells; and a first grid (e.g., grid 202A) for the first linear waveguide, and a second grid (e.g., grid 202B) for the second linear waveguide. The first large one has a first characteristic (for example, being spatially offset from the 202B grid) to facilitate coupling the first light to the first linear waveguide without coupling the first light to the second linear waveguide.

[069] An image capture process may include one or more scanning operations. In some implementations, the image capture area 216 may be induced to overlap one or more areas of the flow cell 200 to facilitate image capture relative to one or more nanowells in the image capture area 216. Positioning may include movement of the image capture area 216, or the flow cell 200, or both. For example, the emission optics may be relatively stationary in the analysis equipment so that the image capture area 216 does not move during multiple scanning operations. For example, the flow cell 200 Petition 870200161164, dated 12 / 23 / 2020, page 57 / 263 30 / 100 can be moved (for example, by being positioned on a motorized stage that facilitates precise movement in at least one direction) relative to the image capture area 216 in one or more scan positions. Here, an arrow 218 schematically illustrates that the flow cell 200 can be moved so that the image capture area 216 overlaps at least some of the linear waveguides and the nanowells associated with them.

[070] Light area 214 can remain stationary with, or be moved in accordance with, or be moved independently of image capture area 216. In this example, light area 214 is aligned with some of the grids 202 (for example, with grids 202A and 202C) but is not aligned with some of the other grids (for example, with grid 202B). For example, when scanning in the direction of arrow 218 with the current position of light area 214, gratings 202A and 202C (and others having similar spatial offsets) will be illuminated by the light incident on light area 214, whereas some other gratings (e.g., grating 202B) will not be illuminated by the light incident on light area 214. Consequently, illumination light will be coupled to linear waveguides 206A and 206C (and others whose gratings have similar spatial offsets), whereas light will not be coupled to linear waveguide 206B (and others whose gratings have similar spatial offsets).This can facilitate selective illumination of the flow cell nanowells 200. For example, since linear waveguides 206A and 206C have light coupled to them, excitation light can reach nanowells 204A and 204C associated with linear waveguide 206A, and a nanowell 204D associated with linear waveguide 206C. On the other hand, excitation light should not reach nanowell 204C because it is associated with linear waveguide 206B which does not currently have light coupled to it. As such, imaging can proceed successfully even though some portions of the sample material (e.g., nos. Petition 870200161164, dated 12 / 23 / 2020, pp. 58 / 263 31 / 100 nanowells 204A and 204C) are positioned at a distance of 210 from each other; that is, closer to each other than the resolution distance of the emission optics. During the scan corresponding to the movement represented by arrow 218 (which can be characterized as a line scan), only a specific subset of the linear waveguides can have light coupled to them. In some implementations, light is coupled only to every second linear waveguide. For example, light can be coupled only to the first, third, fifth, seventh, etc. linear waveguide, while light is not coupled to the second, fourth, sixth, eighth, etc. linear waveguide.

[071] In some implementations, the 210 distance is shorter than a diffraction limit (e.g., a resolution distance of the emission optics). For example, if the wavelength is about 700 nm with a numerical aperture of 0.75, the diffraction limit is about 466 nm, and the 210 distance may then be shorter than this limit. In some implementations, the 200 flow cell may be designed so that the 204A and 204D nanowells are separated from each other by approximately the diffraction limit (e.g., by approximately 466 nm). For example, the 210 distance may then be about half the diffraction limit (e.g., about 233 nm). As another example, if the wavelength is about 525 nm with a numerical aperture of 0.75, the diffraction limit is about 350 nm, and the distance 210 can then be about 175 nm. The example above involves activating alternating linear waveguides one at a time.In some implementations, a smaller number of alternating linear waveguides can be triggered at a time. For example, if every third linear waveguide is triggered at a time, then the distance 210 might be about one-third of the diffraction limit. As another example, if every fourth linear waveguide is triggered at a time, then the distance 210 might be about one-quarter of the diffraction limit, etc. Petition 870200161164, dated 12 / 23 / 2020, p. 59 / 263 32 / 100

[072] The scan illustrated in Figure 2A can be described as the flow cell 200 being moved to the left in the image, and stopping at one or more selected positions corresponding to the linear waveguides when the image capture area 216 overlaps them, until the flow cell 200 is to the left of the image capture area 216. One or more linear waveguides that do not have light coupled to them during the scan illustrated in Figure 2A, and whose associated nanowells are therefore not subjected to excitation light, can be imaged in another scan operation.

[073] Such a further scanning operation may be performed in the same direction as described above (e.g., along the direction of arrow 218) or in another direction. Figure 2B shows an example where the scanning is done along a direction corresponding to an arrow 220, whose direction is substantially, and in at least one instance entirely, opposite to the direction associated with arrow 218. The scanning being illustrated in Figure 2B may be described as flow cell 200 being moved to the right in the image, and stopping at one or more selected positions corresponding to the linear waveguides when the image capture area 216 overlaps them, until flow cell 200 is to the right of image capture area 216. The positioning may include movement of image capture area 216, or flow cell 200, or both.

[074] In this example, light area 214 is aligned with some of the 202 grids (for example, with grid 202B) but is not aligned with some of the other grids (for example, with grids 202A and 202C). For example, when scanning in the direction of arrow 220 with the current position of light area 214, grid 202B (and others having similar spatial displacement) will be illuminated by the light incident on light area 214, while some of the others Petition 870200161164, dated 12 / 23 / 2020, pp. 60 / 263 33 / 100 grids (e.g., grids 202A and 202C) will not be illuminated by light incident on light area 214. Consequently, illumination light will be coupled to linear waveguide 206B (and others whose grids have similar spatial displacements), whereas light will not be coupled to linear waveguides 206A and 206C (and others whose grids have similar spatial displacements). This can facilitate selective illumination of flow cell nanowells 200. For example, since linear waveguide 206C has light coupled to it, excitation light can reach nanowell 204C and others associated with linear waveguide 206B. On the other hand, excitation light should not reach nanowells 204A-204B, which are associated with linear waveguide 206A, or nanowell 204D, which is associated with linear waveguide 206C, since linear waveguides currently do not have light coupled to them.As such, imaging can proceed successfully even though some portions of the sample material (e.g., in nanowells 204A and 204C) are positioned at a distance of 210 from each other; that is, closer to each other than the resolution distance of the emission optics. During the scan corresponding to the motion represented by arrow 220 (which can be characterized as a line scan), only a specific subset of the linear waveguides can have light coupled to them. In some implementations, light is coupled only to every second linear waveguide. For example, light may be coupled only to the second, fourth, sixth, eighth, etc., linear waveguide, whereas light is not coupled to the first, third, fifth, seventh, etc., linear waveguide.

[075] The examples in Figures 2A-2B refer to differential coupling where the grids 202 are spatially offset from each other. In some implementations, one or more other approaches may instead or also be used for differential coupling. Such approaches may include, but are not limited to, differentiated beam parameters, Petition 870200161164, dated 12 / 23 / 2020, pp. 61 / 263 34 / 100 differentiated coupler parameters and / or differentiated waveguide parameters. Examples are provided below.

[076] Figures 3A-3B illustrate examples with a flow cell 300 having gratings 302. In some implementations, differential coupling for the flow cell 300 is provided based on differentiating one or more parameters of the light beam(s) applied to the gratings 302. In some implementations, differential coupling for the flow cell 300 is provided based on differentiating one or more parameters of the gratings 302. In some implementations, differential coupling for the flow cell 300 is provided based on differentiating one or more parameters of the linear waveguides of the flow cell 300. Combinations of two or more of these approaches may be used. The flow cell 300 may be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. Only a portion of the flow cell 300 is shown, for illustrative purposes.

[077] The flow cell 300 includes nanowells, including a nanowell 304A, which are illustrated here using circular shapes. Only one of the nanowells will be specifically mentioned, and the other nanowells may be similar or identical to the one(s) discussed. The nanowells can be formed in a nanowell layer (e.g., by nanoprinting or a lifting process). For example, the nanowells can be formed in a resin using a nanoscale jig. The nanowell layer is not explicitly shown in this example for clarity. The nanowell 304A is associated here with a linear waveguide 306A. In some implementations, the linear waveguides described with reference to the flow cell 300 may be similar or identical to one or more other linear waveguides described in the present invention. For example, the linear waveguide 306A Petition 870200161164, dated 12 / 23 / 2020, pp. 62 / 263 35 / 100 is positioned adjacent to (e.g., in contact with or near) the nanowell layer that includes nanowell 304A. In some implementations, linear waveguide 306A may include a linear waveguide core 308 and one or more of the grids 302.

[078] Another 304B nanowell is also associated with the 3206A linear waveguide. For example, the 304B nanowell is positioned adjacent to the 304A nanowell, and both 304A-304B nanowells can interact with the 306A linear waveguide in an imaging process (e.g., by receiving electromagnetic radiation from the 306A linear waveguide). Another 304C nanowell, conversely, is instead associated with a 306B linear waveguide. In some implementations, the 306B linear waveguide is positioned adjacent to the 306A linear waveguide. For example, the coating (not shown) and / or other material can be positioned between the 306A-306B linear waveguides.

[079] The 300 flow cell can be used in one or more forms of imaging process. For example, sample material in nanowells (including nanowells 304A-304C) can be subjected to electromagnetic radiation from respective linear waveguides (including linear waveguides 306A-306B, respectively). Emissions resulting from such exposure to electromagnetic radiation (an example of emissions being fluorescence from fluorophores) can be captured using equipment (e.g., one or more cameras and / or other imaging devices). Such equipment is sometimes referred to as emission equipment or a similar term. For example, emission equipment may include one or more cameras or other image sensors and at least one lens or other emission optics. In some implementations, the diffraction limit may be at least partially attributable to one or more Petition 870200161164, dated 12 / 23 / 2020, pp. 63 / 263 36 / 100 characteristics of emission optics. For example, based on the emission optics used, a resolution distance can be defined, the resolution distance marking the shortest distance that can be resolved using the emission optics. That is, when resolving features that are separated by the resolution distance, it can be said that the imaging system is operating at its highest available resolution level.

[080] Here, a distance of 310 is less than the emission optics resolution distance, and a distance of 312 is greater than, or approximately equal to, the emission optics resolution distance. The distance of 310 here represents the separation between nanowells in one direction. In some implementations, this may be the direction through the linear waveguides. For example, since the linear waveguides are here aligned with nanowell lines in one direction (e.g., the vertical direction as seen in the illustration), the distance of 310 may also represent the distance between adjacent linear waveguides (e.g., linear waveguides 306A-306B). For example, nanowells 304A and 304C are separated by a distance of 310. That is, linear waveguides 306A-306B are positioned closer to each other than the emission optics resolution distance.

[081] The distance 312 here represents the separation between nanowells in a direction other than the distance 310. For example, the distances 310 and 312 may be substantially and at least in one instance completely perpendicular to each other. In some implementations, this may be the direction along any individual waveguide of the linear waveguides. For example, since the linear waveguides are here aligned with nanowell lines in one direction (e.g., the vertical direction as seen in the illustration), the distance 312 may represent the distance between adjacent nanowells in any of the linear waveguides (e.g., the linear waveguides (e.g., the waveguides of Petition 870200161164, dated 12 / 23 / 2020, pp. 64 / 263 37 / 100 linear waveguides 306A-306B). For example, nanowells 304A and 304B are separated by a distance of 312. That is, the nanowells associated with linear waveguide 306A have a spacing from each other that is resolvable according to the emission optics resolution distance for the flow cell 300.

[082] The 302 gratings serve to couple electromagnetic radiation into and / or out of the linear waveguides of the 300 flow cell. Here, the linear waveguide 306A has a 302A grating and the linear waveguide 306B has a 302B grating. The 302A-302B gratings here have different periodic structures. In some implementations, either or both of the 302A-302B gratings may include a periodic structure of crests interspersed with another material. For example, crests of the 302A-302B gratings may have a pitch of about 200-300 nm, to cite just one example.

[083] The 302A-302B grids may have one or more features that facilitate the selective coupling of electromagnetic radiation to the corresponding 306A-306B linear waveguide. In some implementations, one or more of the 302 grids has a grid period different from one or more of the other 302 grids. For example, the 302A grid may have a longer grid period than the 302B grid. As another example, the 302B grid may have a longer grid period than the 302A grid. The feature of the 302A-302B grids having different grid periods from each other facilitates the coupling of electromagnetic radiation (e.g., light) to one of the linear waveguides (e.g., the 306A linear waveguide) without coupling the electromagnetic radiation (e.g., light) to another of the linear waveguides (e.g., the 306B linear waveguide).

[084] In some implementations, the coupling in the 302A302C grids may also be differentiated by a coupler parameter rather than the grid period (for example, but not limited to, refractive index, Petition 870200161164, dated 12 / 23 / 2020, pp. 65 / 263 38 / 100 pitch, slot width, slot height, slot spacing, grid non-uniformity, slot orientation, slot curvature, overall coupler shape, and combinations thereof). In some implementations, the coupling in the 302A-302C grids may also be differentiated by a waveguide parameter relative to one or more linear waveguides of the 300 flow cell (for example, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and / or beam, and combinations thereof). In some implementations, the coupling in the 302A-302C grids may also be differentiated by a beam parameter of the light beam(s) applied to the 300 flow cell (for example, but not limited to, light beam location, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof).

[085] The 300 flow cell can include several linear waveguides, for example, as illustrated. In some implementations, a 306C linear waveguide is positioned adjacent to the 306B linear waveguide opposite the 306A linear waveguide. In other words, in this implementation, the 306B linear waveguide is between the 306A linear waveguide and the 306C linear waveguide. For example, the 306C linear waveguide may have a 302C grid. In some implementations, the 302C grid may have a different grid period than the 302B grid. For example, the 302C grid may have the same grid period as the 302A grid. As another example, the 302C grid may have a different grid period than the 302A grid and the 302B grid.The characteristic of at least some of the 302A-302C grids having different grid periods facilitates coupling electromagnetic radiation (e.g., light) to one of the linear waveguides (e.g., the 306A or 306C linear waveguide) without coupling electromagnetic radiation (e.g., light) to another. Petition 870200161164, dated 12 / 23 / 2020, pp. 66 / 263 39 / 100 linear waveguides (e.g., the 306B linear waveguide). As another example, the feature facilitates coupling electromagnetic radiation (e.g., light) in one of the linear waveguides (e.g., the 306B linear waveguide) without coupling electromagnetic radiation (e.g., light) in at least one other linear waveguide (e.g., the 306A or 306C linear waveguide).

[086] A light area 314 is schematically illustrated here as a rectangle with a dashed outline. The light area 314 represents one or more positions where light or other electromagnetic radiation is induced to impinge as part of an imaging process. In some implementations, illumination light generated by a laser can be directed into the light area 314 to eventually be coupled to some of the linear waveguides. For example, the laser light can be selected to match fluorescence properties of one or more fluorophores in the sample material.

[087] An image capture area 216 is here schematically illustrated as a rectangle with a dashed outline. The image capture area 316 represents the field of view of the emitting optics. For example, a camera or other image sensor may capture one or more types of emissions (e.g., fluorescent light) emanating from the image capture area 316.

[088] The examples described above illustrate that the 300 flow cell includes a nanowell layer having first (e.g., nanowells associated with linear waveguide 306A) and second (e.g., nanowells associated with linear waveguide 306B) sets of nanowells to receive a sample. The 300 flow cell includes a first linear waveguide (e.g., linear waveguide 306A) aligned with the first set of nanowells and a second linear waveguide (e.g., linear waveguide 306B) Petition 870200161164, dated 12 / 23 / 2020, pp. 67 / 263 40 / 100 306B) aligned with the second set of nanowells; and a first grid (e.g., grid 302A) for the first linear waveguide, and a second grid (e.g., grid 302B) for the second linear waveguide. The first grid has a first characteristic (e.g., having a different grid period from grid 302B) to facilitate coupling the first light in the first linear waveguide without coupling the first light in the second linear waveguide.

[089] An image capture process may include one or more scanning operations. In some implementations, the image capture area 316 may be induced to overlap one or more areas of the flow cell 300 to facilitate image capture relative to one or more nanowells in the image capture area 316. Positioning may include movement of the image capture area 316, or the flow cell 300, or both. For example, the emission optics may be relatively stationary in the analysis equipment so that the image capture area 316 does not move during multiple scanning operations. For example, the flow cell 300 may be moved (e.g., by being positioned on a motorized stage that facilitates precise movement in at least one direction) relative to the image capture area 316 in one or more scanning positions.Here, an arrow 318 schematically illustrates that the flow cell 300 can be moved so that the image capture area 316 overlaps at least some of the linear waveguides and the nanowells associated with them.

[090] Light area 314 can remain stationary with, or be moved corresponding to, or be moved independently of the image capture area 316. In this example, light area 314 is aligned with all grids 302 of the flow cell 300. Different angles of incidence can be given to the illumination light incident on light area 314 in order to couple Petition 870200161164, dated 12 / 23 / 2020, pp. 68 / 263 41 / 100 selectively couples light onto at least one, but not at least one other, of the linear waveguides of the 300 flow cell. For example, when scanning in the direction of arrow 318, the incident angle can be chosen so that gratings 302A and 302C (and others having similar grating periods) will couple incident light to light area 314, while some other gratings (e.g., grating 302B) will not couple incident light to light area 314. Consequently, illumination light will be coupled to linear waveguides 306A and 306C (and others whose gratings have similar grating periods), while light will not be coupled to linear waveguide 306B (and others whose gratings have similar grating periods). This can facilitate selective illumination of the nanowells of the 300 flow cell.For example, since linear waveguides 306A and 306C have light coupled to them, excitation light can reach nanowells 304A and 304C associated with linear waveguide 306A, and a nanowell 304D associated with linear waveguide 306C. On the other hand, excitation light should not reach nanowell 304C because it is associated with linear waveguide 306B, which does not currently have light coupled to it. As such, imaging can proceed successfully even though some portions of the sample material (e.g., in nanowells 304A and 304C) are positioned at a distance of 310 from each other; that is, closer to each other than the resolution distance of the emission optics. During the scan corresponding to the motion represented by arrow 318 (which can be characterized as a line scan), only a specific subset of the linear waveguides can have light coupled to them. In some implementations, light is coupled only to the entire second linear waveguide.For example, light can only be coupled to the first, third, fifth, seventh, etc. linear waveguide, whereas light is not coupled to the second, fourth, sixth, eighth, etc. linear waveguide. Petition 870200161164, dated 12 / 23 / 2020, pp. 69 / 263 42 / 100

[091] The scan illustrated in Figure 3A can be described as the flow cell 300 being moved to the left in the image, and stopping at one or more selected positions corresponding to the linear waveguides when the image capture area 316 overlaps them, until the flow cell 300 is to the left of the image capture area 316. One or more linear waveguides that do not have light coupled to them during the scan illustrated in Figure 3A, and whose associated nanowells are therefore not subjected to excitation light, can be imaged in another scan operation.

[092] Such a further scanning operation may be performed in the same direction as described above (e.g., along the direction of arrow 318) or in another direction. Figure 3B shows an example where the scanning is done along a direction corresponding to an arrow 320, whose direction is substantially, and in at least one instance entirely, opposite to the direction associated with arrow 318. The scanning being illustrated in Figure 3B can be described as flow cell 300 being moved to the right in the image, and stopping at one or more selected positions corresponding to the linear waveguides when the image capture area 316 overlaps them, until flow cell 300 is to the right of image capture area 316. The positioning may include movement of image capture area 316, or flow cell 300, or both.

[093] In this example, light area 314 is aligned with all grids 302 of flux cell 300. Different angles of incidence can be given to the illumination light incident on light area 314 in order to selectively couple light to at least one, but not at least one other, of the linear waveguides of flux cell 300. For example, when scanning in the direction of arrow 320, the angle of incidence can be chosen so that Petition 870200161164, dated 12 / 23 / 2020, pp. 70 / 263 43 / 100 that the 302B grid (and others having similar grid periods) will couple incident light to light area 314, whereas some other grids (e.g., grids 302A and 302C) will not couple incident light to light area 314. Consequently, illumination light will be coupled to the linear waveguide 306B (and others whose grids have similar grid periods), whereas light will not be coupled to the linear waveguides 306A and 306C (and others whose grids have similar grid periods). This can facilitate selective illumination of the flow cell nanowells 300. For example, since the linear waveguide 306B has light coupled to it, excitation light can reach nanowell 304C and others associated with the linear waveguide 306B.On the other hand, excitation light should not reach nanowells 304A-304B, which are associated with linear waveguide 306A, or nanowell 304D, which is associated with linear waveguide 306C, as these linear waveguides currently do not have light coupled to them. As such, imaging can proceed successfully even though some portions of the sample material (e.g., in nanowells 304A and 304C) are positioned at a distance of 310 from each other; that is, closer to each other than the resolution distance of the emission optics. During the scan corresponding to the movement represented by arrow 320 (which can be characterized as a line scan), only a specific subset of the linear waveguides can have light coupled to them. In some implementations, light is coupled only to every second linear waveguide. For example, light may be coupled only to the second, fourth, sixth, eighth, etc., linear waveguide, whereas light is not coupled in the first, third, fifth, seventh, etc., linear waveguide.

[094] In some implementations, two or more of the 302 grids may instead or also have different refractive indices. For example, this may allow differential coupling with respect to at least some of the guides. Petition 870200161164, dated 12 / 23 / 2020, page 71 / 263 44 / 100 linear waves 306A-306C.

[095] Figure 4 shows another example of a flow cell 400 having stepped grates 402. The flow cell 400 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. Only a portion of the flow cell 400 is shown, for illustrative purposes.

[096] The flow cell 400 includes nanowells, including a nanowell 404A, which are illustrated here using circular shapes. Only one of the nanowells will be specifically mentioned, and the other nanowells may be similar or identical to the one(s) discussed. The nanowells can be formed in a nanowell layer (e.g., by nanoprinting or a lifting process). For example, the nanowells can be formed in a resin using a nanoscale jig. The nanowell layer is not explicitly shown in this example for clarity. The nanowell 404A is associated here with a linear waveguide 406A. In some implementations, the linear waveguides described with reference to the flow cell 4200 may be similar or identical to one or more other linear waveguides described in the present invention. For example, the linear waveguide 406A is positioned adjacent to (e.g., in contact with or near) the nanowell layer that includes nanowell 404A.

[097] Another 404B nanowell is also associated with the 406A linear waveguide. For example, the 404B nanowell is positioned adjacent to the 404A nanowell, and both 404A-404B nanowells can interact with the 406A linear waveguide in an imaging process (e.g., by receiving electromagnetic radiation from the 406A linear waveguide). Another 404C nanowell, conversely, is instead associated with a 406B linear waveguide. In some implementations, the 406B linear waveguide is Petition 870200161164, dated 12 / 23 / 2020, page 72 / 263 45 / 100 positioned adjacent to linear waveguide 406A. For example, the coating (not shown) and / or other material may be positioned between linear waveguides 406A-406B.

[098] The 400 flow cell can be used in one or more forms of imaging process. For example, sample material in nanowells (including nanowells 404A-404C) can be subjected to electromagnetic radiation from respective linear waveguides (including linear waveguides 406A-406B, respectively). Emissions resulting from such exposure to electromagnetic radiation (an example of emissions being fluorescence from fluorophores) can be captured using equipment (e.g., one or more cameras and / or other imaging devices). Such equipment is sometimes referred to by the expression emission equipment or a similar term. For example, emission equipment may include one or more cameras or other image sensors and at least one lens or other emission optics. In some implementations, the diffraction limit may be at least partially attributable to one or more characteristics of the emission optics.For example, based on the emission optics used, a resolution distance can be defined, the resolution distance marking the shortest distance that can be resolved using the emission optics. That is, when resolving features that are separated by the resolution distance, it can be said that the imaging system is operating at its highest available resolution level.

[099] The 402 gratings serve to couple electromagnetic radiation into and / or out of the linear waveguides of the 400 flux cell. Here, the 406A linear waveguide has a 402A grating, the 406B linear waveguide has a 402B grating, and a 406C linear waveguide has a 402C grating. The 402A-402C gratings may have the same or different periodic structure. In some Petition 870200161164, dated 12 / 23 / 2020, p. 73 / 263 In 46 / 100 implementations, any or all 402A-402C lattices may include a periodic structure of ridges interspersed with another material. For example, ridges of 402A-402C lattices may have a pitch of approximately 200-300 nm, to cite just one example.

[100] The 402A-402C grids may have one or more features that facilitate the selective coupling of electromagnetic radiation to the corresponding 406A-406C linear waveguide. In some implementations, one or more of the 402 grids is spatially offset from one or more other 402 grids. The offset may be in a direction that is parallel to the 406A-406C linear waveguides. For example, the distance between the 402B grid and the nearest nanowell of the nanowells associated with the 406B linear waveguide is greater here than the distance between the 402A grid and the nearest nanowell of the nanowells associated with the 406A linear waveguide.As another example, the distance between the 402C grid and the nearest nanowell associated with the 406C linear waveguide is greater here than the distance between the 402A grid and the nearest nanowell associated with the 406A linear waveguide, and also greater than the distance between the 402B grid and the nearest nanowell associated with the 406B linear waveguide. The characteristic of the 402A-402B grids being spatially offset from each other facilitates coupling electromagnetic radiation (e.g., light) to one of the linear waveguides (e.g., the 406A linear waveguide) without coupling electromagnetic radiation (e.g., light) to another of the linear waveguides (e.g., the 406B or 406C linear waveguide). That is, the 402C grid is spatially displaced in a direction parallel to the 406A-406C linear waveguides from each of the 402A-402B grids.

[101] In some implementations, coupling in 402A402C grids may also or instead be differentiated by a beam parameter. Petition 870200161164, dated 12 / 23 / 2020, pp. 74 / 263 47 / 100 different from the location of the light beam (for example, but not limited to, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof). In some implementations, the coupling in the 402A-402C gratings may also or instead be differentiated by a coupler parameter (for example, but not limited to, grating period, refractive index, pitch, slot width, slot height, slot spacing, grating non-uniformity, slot orientation, slot curvature, overall coupler shape, and combinations thereof). In some implementations, the coupling in the 402A-402C gratings may also or instead be differentiated by a waveguide parameter with respect to one or more linear waveguides of the 400 flow cell (for example, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and / or beam, and combinations thereof).

[102] Light areas 408A-408C are schematically illustrated here as rectangles with dashed outlines. Light areas 408A-408C represent positions where light or other electromagnetic radiation is induced to fall as part of an imaging process. In some implementations, illumination light generated by a laser can be directed into one or more of the light areas 408A-408C to eventually be coupled to the corresponding linear waveguide. For example, laser light can be selected to match fluorescence properties of one or more fluorophores in the sample material. Light can be directed to light area 408A to couple light to linear waveguide 406A without coupling the light to linear waveguides 406B-406C. Light can be directed to light area 408B to couple light to linear waveguide 406B without coupling the light to linear waveguides 406A or 408-408C. Light can be directed to light area 408C to couple light to the waveguide. Petition 870200161164, dated 12 / 23 / 2020, pp. 75 / 263 48 / 100 linear wave 406C without coupling the light in linear waveguides 406A-406B.

[103] The 400 flow cell may have other waveguides besides the 406A-406C linear waveguides, with corresponding grids. Individual grids of such other linear waveguides may have spatial offsets similar to the spatial offsets of one of the 402A-402C grids, or they may have different spatial offsets. For example, light may be coupled only to the first, fourth, seventh, tenth, etc. linear waveguide, whereas light is not coupled to the second, third, fifth, sixth, eighth, ninth, eleventh, twelfth, etc., linear waveguide. More generally, in any individual scanning operation (corresponding to the use of a specific light area, such as one of the 408A-408C light areas), the ordinals of the linear waveguides to which light is coupled can form an arithmetic series where the nth ordinal a_n (n=1,2,3,...) can be expressed as a_n=a_1+d(n-1), where a_1 is a positive integer.For example, with a_1=1 ed=3, it is obtained that the linear waveguides to which light is coupled have the ordinals 1, 4, 7, 10, etc., corresponding to the example mentioned above. As another example, with a_1=1 ed=4, it is obtained that the linear waveguides to which light is coupled have the ordinals 1, 5, 9, 13, etc.

[104] The examples described above illustrate that the 400 flow cell includes a nanowell layer having first (e.g., nanowells associated with linear waveguide 406A) and second (e.g., nanowells associated with linear waveguide 406B) sets of nanowells to receive a sample. The 400 flow cell includes a first linear waveguide (e.g., linear waveguide 406A) aligned with the first set of nanowells, and a second linear waveguide (e.g., linear waveguide 406B) aligned with the second set of nanowells; and a first grid (e.g., grid 402A) for the first linear waveguide, and a Petition 870200161164, dated 12 / 23 / 2020, pp. 76 / 263 49 / 100 second grid (e.g., grid 402B-402C) stops the second linear waveguide. The first grid has a first characteristic (e.g., being spatially offset from grids 402B-402C) to facilitate coupling of the first light in the first linear waveguide without coupling of the first light in the second linear waveguide.

[105] Figure 5 shows a cross-section of part of an example flow cell 500. The flow cell 500 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. The flow cell 500 is shown in cross-section and only a portion of the flow cell 500 is shown, for illustrative purposes.

[106] The 500 flow cell includes a 502 nanowell layer which includes 502A-502B nanowells. The 502 nanowell layer can be formed by nanoprinting or a lifting process. For example, 502A-502B nanowells can be formed by applying a nanoscale template to a resin.

[107] The 500 flow cell includes 504A-504B linear waveguides. One or more of the 504A-504B linear waveguides can be aligned with one or more of the 502A-502B nanowells. For example, the 504A linear waveguide is aligned here with the 502A nanowell, and the 504B linear waveguide is aligned here with the 502B nanowell.

[108] Each of the 504A-504B linear waveguides may have one or more grids (omitted here for clarity) for coupling electromagnetic radiation into and / or out of that 504A-504B linear waveguide. One or more directions of displacement for the electromagnetic radiation in the 504A-504B linear waveguides may be employed. For example, the direction of displacement may be into and / or out of the plane of the present illustration. Petition 870200161164, dated 12 / 23 / 2020, page 77 / 263 50 / 100

[109] Each of the 504A-504B linear waveguides can be positioned against one or more types of coating. The coating can serve to limit electromagnetic radiation to the respective 504A-504B linear waveguide and prevent or reduce the extent of radiation propagation into other 504A-504B linear waveguides or other substrates (e.g., to reduce cross-coupling). Here, 506 coatings are shown as an example. In some implementations, the 506 coatings comprise a series of blocks. In some implementations, the 506 coatings provide refractive indices that alternate along the structure of the 506 coatings. For example, a first of the 506 coatings may have a first refractive index, a second of the 506 coatings adjacent to the first may have a second refractive index, a third of the 506 coatings adjacent to the second may have the first refractive index, etc.Coatings 506 can be positioned against or near linear waveguide 102A on different (e.g., opposite) sides thereof. For example, a coating 506A can be positioned against or near linear waveguide 504B. Here, coatings 506 include multiple structures, including coating 506A. Coatings 506 can be made of one or more suitable materials that serve to separate linear waveguides 504A-504B from each other. In some implementations, coatings 506 can be made of a material having a lower refractive index than the refractive index(es) of linear waveguides 504A-504B. In some implementations, one or more of the coatings 506 includes a polymer material. In some implementations, the multiple structures of coatings 506 can be interspersed with vacuum regions or other material (e.g., air or a liquid).

[110] Figure 6 shows an example of a 600 flow cell where Petition 870200161164, dated 12 / 23 / 2020, page 78 / 263 51 / 100 multiple linear waveguides share a common grid. The 600 flow cell can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. The 500 flow cell is shown in a top view, and only a portion of the 600 flow cell is shown, for illustrative purposes.

[111] The 600 flow cell includes a 602 substrate. In some implementations, the 602 substrate serves as a base layer for the 600 flow cell and may support one or more layers and / or other substrates. For example, the 602 substrate may support one or more 604A linear waveguide components and a nanowell layer (not shown).

[112] The linear waveguide component 604A includes a coupling component 606A having a grid 608A. A linear waveguide connector 610 connects the coupling component 606A and a linear waveguide arrangement 612A to each other. The linear waveguide arrangement 612A includes a linear waveguide distributor 614 coupled to the linear waveguide connector 610 and multiple linear waveguides 616A arranged parallel to each other and coupled to the linear waveguide distributor 614. In operation, light incident on the grid 608A can be coupled by the coupling component 606A and the linear waveguide connector 610 in the linear waveguide arrangement 612A. In the linear waveguide arrangement 612A, the linear waveguide distributor 614 can distribute the light into the linear waveguides 616A.In some implementations, the 616A linear waveguides are positioned adjacent to nanowells (not shown) to facilitate imaging as part of sample analysis. For example, rows of nanowells can be positioned along each of the 616A linear waveguides. The 604A linear waveguide component can be made of one. Petition 870200161164, dated 12 / 23 / 2020, pp. 79 / 263 52 / 100 or more suitable materials that facilitate the propagation of electromagnetic radiation. In some implementations, the material(s) of the 604A linear waveguide component may include a polymer material. In some implementations, the material(s) of the 604A linear waveguide component may include Ta205 and / or SiNx.

[113] The linear waveguide arrangement 612A can facilitate the placement of one or more other flow cell components 600. In some implementations, the flow cell 600 includes a linear waveguide component 604B that is positioned on an opposite side of the substrate 602 from the linear waveguide arrangement 612A. The linear waveguide component 604B may include a coupling component 606B coupled to a linear waveguide arrangement 612B. In some implementations, individual linear waveguides 616B of the linear waveguide arrangement 612B may be interleaved between the respective linear waveguides 616A of the linear waveguide arrangement 612A. For example, two of the linear waveguides 616A may be positioned on respective opposite sides of one of the linear waveguides 616B. The two 616A linear waveguides are therefore sharing the same grid, in this example the 608A grid of the 604A linear waveguide component.

[114] In some implementations, the linear waveguide 616A and the linear waveguide 616B may be positioned closer to each other than an emission optics resolution distance. For example, during a first scanning stage, light may be coupled to the linear waveguides 616A of the linear waveguide component 604A and not to the linear waveguides 616B of the linear waveguide component 604B. During a second scanning stage, additionally, light may instead be coupled to the linear waveguides 616B of the linear waveguide component 604B and Petition 870200161164, dated 12 / 23 / 2020, pp. 80 / 263 53 / 100 not in the 616A linear waveguide component of the 604A linear waveguide.

[115] At least one of the 606A-604B coupling components may include a substrate having a substantially, and at least in one instance fully, triangular shape. This may provide advantages in terms of efficient multi-flow cell placement. A 604C linear waveguide component may not be considered as part of the 600 flow cell, but may instead be considered as part of another flow cell (not shown). In some implementations, the triangular substrate of the 606A coupling component, and a corresponding triangular substrate of a 606C coupling component of the 604C linear waveguide component may be positioned adjacent to each other. For example, the 606A and 606C coupling components may be positioned in opposite orientations in order to provide efficient placement of the 604A and 604C linear waveguide components side by side.

[116] A 608A grid can be positioned towards a first end of the 604A linear waveguide component (in this illustration, towards a left end of the same, for example). In addition, a 608B grid can be positioned towards a second end of the 604B linear waveguide component (in this illustration, towards a right end of the same, for example). The first end can be positioned opposite the second end in a direction parallel to nanowell lines (for example, the direction being parallel to the 616A-616B linear waveguides).

[117] More or fewer linear waveguide components than shown may be used. In some implementations, respective 604D-604F linear waveguide components are implemented. For example, Petition 870200161164, dated 12 / 23 / 2020, page 81 / 263 54 / 100 linear waveguide components 604E-604F can be considered part of flow cell 600, whereas linear waveguide component 604D can be considered part of another flow cell (not shown) that is separate from the flow cell of linear waveguide component 604C.

[118] In some implementations, coupling to the 608A and / or other gratings may be differentiated by a beam parameter (for example, but not limited to, light beam location, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof). In some implementations, coupling to the 608A and / or other gratings may also or instead be differentiated by a coupler parameter (for example, but not limited to, grating period, refractive index, pitch, slot width, slot height, slot spacing, grating non-uniformity, slot orientation, slot curvature, overall coupler shape, and combinations thereof).In some implementations, the coupling on the 608A grid and / or others may also be differentiated by a waveguide parameter relative to one or more linear waveguides of the 600 flow cell (for example, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and / or beam, and combinations thereof).

[119] Figure 7 is a diagram of an example lighting system 700. The lighting system 700 can be used with one or more methods described in the present invention, and / or used in combination with one or more systems or apparatus described in the present invention.

[120] The illumination system 700 includes a light source assembly 710, a mirror 728, an objective lens 734, a flow cell 736, a dichroic emission filter 738, a first optical detection subsystem 756, and a Petition 870200161164, dated 12 / 23 / 2020, page 82 / 263 55 / 100 second optical detection subsystem 758. The illumination system 700 allows simultaneous imaging of two color channels. In some implementations, another illumination system may be configured to allow simultaneous imaging of more than two color channels, for example, three color channels, four color channels, or more. Note that there may be other optical configurations that can produce similar simultaneous imaging of multiple color channels.

[121] The light source assembly 710 produces excitation illumination that is incident on the flux cell 736. This excitation illumination will in turn produce emitted illumination, or fluorescent illumination, from one or more fluorescent dyes that will be collected using lenses 742 and 748. The light source assembly 710 includes a first excitation illumination source 712 and corresponding converging lens 714, a second excitation illumination source 716 and corresponding converging lens 718, and a dichroic filter 720.

[122] The first excitation illumination source 712 and the second excitation illumination source 716 illustrate an illumination system that can simultaneously provide respective excitation illumination lights for a sample (e.g., corresponding to respective color channels). In some implementations, each of the first excitation illumination source 712 and second excitation illumination source 716 includes a light-emitting diode (LED). In some implementations, at least one of the first excitation illumination source 712 and second excitation illumination source 716 includes a laser. The converging lenses 714 and 718 are each adjusted at a distance from the respective excitation illumination sources 712 and 716 so that the illumination emerging from each of the converging lenses 714 / 718 is focused on a field aperture 722. The dichroic filter 720 reflects Petition 870200161164, dated 12 / 23 / 2020, page 83 / 263 56 / 100 illumination from the first excitation light source 712 and transmits illumination from the second excitation light source 716.

[123] In some implementations, the mixed excitation illumination output from the dichroic filter 720 may propagate directly towards the objective lens 134. In other implementations, the mixed excitation illumination may be further modified and / or controlled by additional intermediate optical components before emission from the objective lens 734. The mixed excitation illumination may pass through a focus at the field aperture 722 to a filter 724 and then to a color-corrected collimating lens 726. The collimated excitation illumination from the lens 726 is incident on a mirror 728 which reflects and is incident on an emission / excitation dichroic filter 730.The dichroic emission / excitation filter 730 reflects the excitation illumination emitted from the light source assembly 710 while allowing emission illumination, which will be further described below, to pass through the dichroic emission / excitation filter 730 to be received by one or more optical subsystems 756, 758. The optical subsystems 756 and 758 exemplify a light-gathering system that can simultaneously collect multiplexed fluorescent light. The excitation illumination reflected from the dichroic emission / excitation filter 730 is then incident on a mirror 732, from which it is incident on the objective lens 134 towards the flow cell 736.

[124] Objective lens 734 focuses the collimated excitation illumination from mirror 732 onto flow cell 136. In some implementations, objective lens 734 is a microscope objective with a specified magnification factor, for example, 1X, 2X, 4X, 5X, 6X, 8X, 10X, or higher. Objective lens 734 focuses the incident excitation illumination from mirror 732 onto flow cell 736 in a cone of angles, or numerical aperture, Petition 870200161164, dated 12 / 23 / 2020, page 84 / 263 57 / 100 determined by the magnification factor. In some implementations, the objective lens 734 is movable on a geometric axis that is perpendicular to the flow cell (a geometric-z axis). In some implementations, the illumination system 700 adjusts the z-position of the tube lens 748 and tube lens 742 independently.

[125] The flow cell 736 contains a sample, such as a nucleotide sequence or any other material, to be analyzed. The flow cell 736 may include one or more channels 760 (here schematically illustrated by means of a cross-sectional view at magnification) configured to hold sample material and facilitate actions taken with respect to the sample material, including, but not limited to, initiating chemical reactions or adding or removing material. An object plane 762 of the objective lens 734, here schematically illustrated using a dashed line, extends through the flow cell 736. For example, the object plane 762 may be defined so as to be adjacent to the channel(s) 760.

[126] The objective lens 734 can define a field of view. The field of view can define the area on the flow cell 736 from which an image detector captures emitted light using the objective lens 734. One or more image detectors, for example, detectors 746 and 754, can be used. The illumination system 700 can include separate image detectors 746 and 754 for the respective wavelengths (or wavelength ranges) of the emitted light. At least one of the image detectors 746 and 754 can include a charge-coupled device (CCD), such as a time-delay integrating CCD camera or a sensor manufactured based on complementary metal-oxide semiconductor (CMOS) technology, such as chemically sensitive field-effect transistors (chemFETs), ion-sensitive field-effect transistors (ISFETs), and / or field-effect transistors of Petition 870200161164, dated 12 / 23 / 2020, page 85 / 263 58 / 100 metal-oxide-semiconductor (MOSFET).

[127] In some implementations, the illumination system 700 may include a structured illumination microscope (SIM). SIM imaging relies on spatially structured illumination light and reconstruction to result in a higher resolution image than an image produced solely using magnification from the objective lens 734. For example, the structure may consist of or include a pattern or grid that interrupts the illumination excitation light. In some implementations, the structure may include fringe patterns. Light fringes may be generated by incident a light beam onto a diffraction grating so that reflective or transmissive diffraction occurs. The light structure may be projected onto the sample, illuminating the sample according to the respective fringes which may occur according to some periodicity.To reconstruct an image using SIM, two or more patterned images are used where the excitation illumination pattern is at different phase angles from each other. For example, sample images can be acquired at different phases of the fringes in the structured light, sometimes referred to as the respective pattern phases of the images. This can allow multiple locations in the sample to be exposed to a range of illumination intensities. The resulting set of emitted light images can be combined to reconstruct the higher-resolution image.

[128] The sample material in flow cell 736 is brought into contact with fluorescent dyes that couple to corresponding nucleotides. The fluorescent dyes emit fluorescent illumination after being irradiated with corresponding excitation illumination incident on flow cell 736 from objective lens 734. The emitted illumination is identified with wavelength bands, each of which can be categorized with a respective color channel. The fluorescent dyes are chemically Petition 870200161164, dated 12 / 23 / 2020, page 86 / 263 59 / 100 linked with respective nucleotides, for example, containing respective nucleobases. Thus, a dNTP labeled with a fluorescent dye can be identified based on a wavelength of light emitted being in a corresponding wavelength range when detected by an image detector 746, 754.

[129] Objective lens 734 captures fluorescent light emitted by fluorescent dye molecules in flow cell 736. After capturing this emitted light, objective lens 734 collects and transports collimated light. This emitted light then propagates back along the path where the original excitation illumination arrived from the light source assembly 710. It is observed that there is little to no expected interference between the excitation and emitted illumination along this path due to the lack of coherence between the emitted light and excitation illumination. That is, the emitted light is a result of a separate source, namely that of the fluorescent dye in contact with the sample material in flow cell 736.

[130] The emitted light, after reflection by mirror 732, is incident on the emission / excitation dichroic filter 730. The filter 730 transmits the emitted light to a dichroic filter 738.

[131] In some implementations, a dichroic filter 738 transmits illumination associated with the blue color channel and reflects illumination associated with the green color channel. In some implementations, the dichroic filter 738 is selected so that the dichroic filter 738 reflects illumination emitted to an optical subsystem 756 that is within the defined green wavelength range and transmits illumination emitted to an optical subsystem 758 that is within the defined blue wavelength range, as discussed above. The optical subsystem 756 includes a tube lens 742, a filter 744, and the image detector 746. The optical subsystem 758 includes a tube lens 748, Petition 870200161164, dated 12 / 23 / 2020, page 87 / 263 60 / 100, a 750 filter, and the 754 image detector.

[132] In some implementations, the 738 dichroic filter and the 7120 dichroic filter operate similarly to each other (for example, both can reflect light of one color and transmit light of another color). In other implementations, the 738 dichroic filter and the 720 dichroic filter operate differently from each other (for example, the 738 dichroic filter can transmit light of a color that the 720 dichroic filter reflects, and vice versa).

[133] In some implementations, the emitted illumination encounters a mirror 752 before the image detector 754. In the example shown, the optical path in the optical subsystem 758 is inclined so that the illumination system 700 as a whole can meet space or volume requirements. In some implementations, both of these subsystems 756 and 758 have optical paths that are inclined. In some implementations, neither of the optical paths in subsystem 756 nor 758 is inclined. As such, one or more of multiple optical subsystems may have at least one inclined optical path.

[134] Each tube lens 742 and 748 focuses the incident emitted illumination onto respective image detectors 746 and 754. Each detector 746 and 754 includes, in some implementations, a CCD array. In some implementations, each image detector 746 and 754 includes a complementary metal-oxide-semiconductor (CMOS) sensor.

[135] It is not necessary that the lighting system 700 be as shown in figure 7. For example, each of the mirrors 728, 732, 740 can be replaced with a prism or some other optical device that changes the direction of illumination. Each lens can be replaced with a diffraction grating, a diffractive optic, a Fresnel lens, or some other optical device that produces collimated or focused illumination from the incident illumination. Petition 870200161164, dated 12 / 23 / 2020, page 88 / 263 61 / 100

[136] Figures 8-9 are flowcharts of example methods 800 and 900. Method 800 or 900, or both, can be performed using, and / or in combination with, one or more other examples described in the present invention. More or fewer operations can be performed, and / or two or more operations can be performed in a different order, unless otherwise indicated.

[137] In 810, a sample can be applied to the first and second nanowell lines of a flow cell. In some implementations, the sample can be applied to the nanowell lines associated with the 206A-206B linear waveguides in Figure 2A. In some implementations, the sample can be applied to the nanowell lines associated with the 406A-406B linear waveguides in Figure 4. For example, the sample can include genetic material.

[138] In 820, the position of a lighting component can be changed to handle a subset of grids. In some implementations, the position of the lighting component is changed so that lighting will fall on light area 214 in Figure 2A when light area 214 is aligned with grids 202A and 204C and some others, but not with grid 202B and some others. In some implementations, the position of the lighting component is changed so that lighting will fall on light area 408A in Figure 4, which is aligned with grid 402A, but not with grids 402B-402C. For example, mirror 732 in Figure 7 can be adjusted to change the location where light is incident. In some implementations, the flux cell can be moved or adjusted in addition to, or instead of, moving the lighting equipment.

[139] In 830, scanning can start in a first direction. In some implementations, scanning is performed in the direction corresponding to arrow 218 in Figure 2A. Positioning may include movement of an area. Petition 870200161164, dated 12 / 23 / 2020, pp. 89 / 263 62 / 100 image capture (e.g., movement of the image capture device) or the flow cell or both.

[140] In 840, the first light can be directed into a first grid of a first linear waveguide aligned with the first row of nanowells, without coupling the first light into a second linear waveguide aligned with the second row of nanowells. In some implementations, the first light is directed into light area 214 in Figure 2A, where light area 214 at least partially overlaps grids 202A and 202C. Because grid 202B is spatially offset from grids 202A and 202C, the first light is not coupled into linear waveguide 206B. In some implementations, the first light is directed into light area 408A in Figure 4 which at least partially overlaps grid 402A. Because the 402B-402C gratings are spatially offset from the 402A grating, the first light is not coupled to the 406B-406C linear waveguides.

[141] In 850, one or more images can be captured. In some implementations, an image can be captured from image capture area 216 in Figure 2A when image capture area 216 at least partially overlaps some aspect of flow cell 200. In a similar way, one or more images can be captured from flow cell 400 in Figure 4. For example, image capture can include a line scan.

[142] In 860, a lighting component position can be changed to handle another subset of grids. In some implementations, the lighting component position is changed so that lighting will fall on light area 214 in Figure 2B when light area 214 is aligned with grid 202B and some others, but not with grids 202A and 202C and some others. In some implementations, the lighting component position is changed so that lighting will fall on light area 408B in Figure 4, which is aligned Petition 870200161164, dated 12 / 23 / 2020, pp. 90 / 263 63 / 100 with grid 402B, but not with grids 402A or 402C. For example, mirror 732 in Figure 7 can be adjusted to change the location where light is incident. In some implementations, the flux cell can be moved or adjusted in addition to, or instead of, moving the lighting equipment.

[143] In 870, scanning can start in a second direction. The second direction can be the same as, or different from, the first direction. In some implementations, scanning is performed in the direction corresponding to arrow 220 in Figure 2B. Positioning may include movement of an image capture area (e.g., movement of the image capture device) or the flow cell, or both.

[144] In 880, the second light can be directed into a second grid of a second linear waveguide aligned with the second row of nanowells, without coupling the second light to the first linear waveguide. In some implementations, the second light is directed into light area 214 in Figure 2B, when light area 214 at least partially overlaps grid 202B. Because grids 202A and 202C are spatially offset from grid 202B, the second light is not coupled to linear waveguides 2026A or 206C. In some implementations, the second light is directed into light area 408B in Figure 4, when light area 408B at least partially overlaps grid 402B. Since the 402A and 402C gratings are spatially offset from the 402B grating, the second light is not coupled to the 406A and 406C linear waveguides.

[145] In 890, one or more images can be captured. In some implementations, an image can be captured from image capture area 216 in Figure 2B when image capture area 216 at least partially overlaps some aspect of flow cell 200. In a similar way, one or more images can be captured from flow cell 400 in Figure 4. For example, image capture can include a line scan. Petition 870200161164, dated 12 / 23 / 2020, pp. 91 / 263 64 / 100

[146] Returning now to method 900 in figure 9, in 910 a sample can be applied to the first and second nanowell lines of a flow cell. In some implementations, the sample can be applied to the nanowell lines associated with the linear waveguides 306A-306B in figure 3A. For example, the sample can include genetic material.

[147] In 920, the position of a lighting component can be changed to an angle associated with a grid period of a subset of grids. In some implementations, the position of the lighting component is changed so that the illumination light will have or have an angle of incident at which grids 302A and 302C and some others couple light, but at which grid 302B and some others do not couple light. For example, mirror 732 in Figure 7 can be adjusted to change the angle of incident. In some implementations, the flux cell can be moved or adjusted in addition to, or instead of, adjusting the lighting equipment.

[148] In 930, scanning can start in a first direction. In some implementations, scanning is performed in the direction corresponding to arrow 318 in figure 3A. Positioning may include movement of an image capture area (e.g., movement of the image capture device) or the flow cell or both.

[149] In 940, the first light can be directed into a first grid of a first linear waveguide aligned with the first row of nanowells, without coupling the first light into a second linear waveguide aligned with the second row of nanowells. In some implementations, the first light is directed into light area 314 in Figure 3A when light area 314 at least partially overlaps grids 302. Because grid 302B has a period much different from grids 302A and 302C, the first light is not coupled into linear waveguide 306B. Petition 870200161164, dated 12 / 23 / 2020, pp. 92 / 263 65 / 100

[150] In 950, one or more images can be captured. In some implementations, an image can be captured from image capture area 316 in Figure 3A when image capture area 316 at least partially overlaps some aspect of flow cell 300.

[151] In 960, a lighting component position can be changed to an angle associated with a grid period of another subset of grids. In some implementations, the lighting component position is changed so that illumination light will or has an incident angle at which grid 302B and some others couple light, but at which grids 302A and 302C and some others do not couple light. For example, mirror 732 in Figure 7 can be adjusted to change the location where light is incident. In some implementations, the flux cell can be moved or adjusted in addition to, or instead of, moving the lighting equipment.

[152] In 970, the scan can start in a second direction. The second direction can be the same as, or different from, the first direction. In some implementations, the scan is performed in the direction corresponding to arrow 320 in Figure 3B. Positioning may include movement of an image capture area (e.g., movement of the image capture device) or the flow cell, or both.

[153] In 980, the second light can be directed into a second grid of a second linear waveguide aligned with the second row of nanowells, without coupling the second light to the first linear waveguide. In some implementations, the second light is directed into light area 314 in Figure 3B, when light area 314 at least partially overlaps grids 302. Because grids 302A and 302C have different grid periods than grid 302B, the second light is not coupled to linear waveguides 306A or 306C.

[154] In 990, one or more images can be captured. In some Petition 870200161164, dated 12 / 23 / 2020, pp. 93 / 263 In 66 / 100 implementations, an image can be captured from image capture area 316 in Figure 3B when image capture area 316 at least partially overlaps some aspect of flow cell 300. For example, the image capture might include a line scan.

[155] Some examples of the present invention show nanowells having circular openings, for illustrative purposes only. In some implementations, non-circular nanowells may be used. Figure 10A shows an example of a hexagonal arrangement 1000 of non-circular nanowells 1002. The hexagonal arrangement 1000 may be used with one or more methods described in the present invention, and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the hexagonal arrangement 1000 may be used with circular nanowells or non-circular nanowells, or both. One or more of the non-circular nanowells 1002 may be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the nanowells 1002 may be arranged in a hexagonal arrangement or a non-hexagonal arrangement (e.g., otherwise polygonal) or both.

[156] The size and / or shape of the non-circular nanowells 1002 can affect the imaging that is part of the analysis process. In some implementations, a fluorescence signal is collected from some or all of the non-circular nanowells 1002. The fluorescence signal can be affected by the size and / or shape of the non-circular nanowells 1002. For example, changes in the generated fluorescence signal(s) can affect the throughput of an analysis system (e.g., a sequencing system).

[157] In some implementations, one or more of the noncircular nanowells 1002 has an elliptical aperture. An ellipse can be characterized Petition 870200161164, dated 12 / 23 / 2020, pp. 94 / 263 67 / 100 by the respective lengths of the major and minor geometric axes. The length of the minor geometric axis can be expressed as a percentage of the length of the major geometric axis, but not limited to, such as being 5%, 15%, 35%, 65% or 95% of the length of the major geometric axis, to cite just a few examples. Other geometries besides elliptical for non-circular nanowells are also possible.

[158] Figure 10B shows an example of a triangular arrangement 1004 of circular nanowells 1006. The triangular arrangement 1004 can be used with one or more methods described in the present invention, and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the triangular arrangement 1004 can be used with circular nanowells or non-circular nanowells, or both. One or more of the circular nanowells 1006 can be used with one or more methods described in the present invention, and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the circular nanowells 1006 can be arranged in a hexagonal arrangement or a non-hexagonal arrangement (e.g., otherwise polygonal) or both.

[159] Figure 11 shows another example of a flow cell 1100 having stepped grates 1102. The flow cell 1100 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the flow cell 1100 can be used with stepped grates or non-stepped grates, or both. One or more of the stepped grates 1102 can be used with one or more methods described in the present invention, and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the stepped grates 1102 can be used with Petition 870200161164, dated 12 / 23 / 2020, pp. 95 / 263 68 / 100 nanowells arranged in a hexagonal arrangement or a non-hexagonal arrangement (e.g., otherwise polygonal) or both.

[160] The flow cell 1100 includes nanowells, including a nanowell 1104A, which are illustrated here using circular shapes. Only one of the nanowells will be specifically mentioned, and the other nanowells may be similar or identical to the one(s) discussed. The nanowells can be formed in a nanowell layer (e.g., by nanoprinting or a lifting process). For example, the nanowells can be formed in a resin using a nanoscale jig. The nanowell layer is not explicitly shown in this example for clarity. The nanowell 1104A is associated here with a linear waveguide 1106A. In some implementations, the linear waveguides described with reference to the flow cell 1100 may be similar or identical to one or more other linear waveguides described in the present invention.For example, the linear waveguide 1106A is positioned adjacent to (e.g., in contact with or near) the nanowell layer that includes nanowell 1104A.

[161] Another 1104B nanowell is also associated with the 1106A linear waveguide. For example, the 1104B nanowell is positioned adjacent to the 1104A nanowell, and both 1104A-1104B nanowells can interact with the 1106A linear waveguide in an imaging process (e.g., by receiving electromagnetic radiation from the 1106A linear waveguide). Another 1104C nanowell, conversely, is instead associated with a 1106B linear waveguide. In some implementations, the 1106B linear waveguide is positioned adjacent to the 1106A linear waveguide. For example, the coating (not shown) and / or other material can be positioned between the 1106A-1106B linear waveguides.

[162] An 1104D nanowell is here associated with a linear waveguide Petition 870200161164, dated 12 / 23 / 2020, pp. 96 / 263 69 / 100 1106C. In some implementations, the 1106C linear waveguide is positioned adjacent to the 1106B linear waveguide. For example, the coating (not shown) and / or other material may be positioned between the 1106B-1106C linear waveguides.

[163] A nanowell 1104E is here associated with a linear waveguide 1106D. In some implementations, the linear waveguide 1106D is positioned adjacent to the linear waveguide 1106C. For example, the coating (not shown) and / or other material may be positioned between the linear waveguides 1106C-1106D.

[164] Nanowells 1104A-1104B and others in the present invention form a first set of nanowells (e.g., a line of nanowells) extending along the linear waveguide 1106A. Nanowell 1104C and others in the present invention form a second set of nanowells (e.g., a line of nanowells) extending along the linear waveguide 1106B. Nanowell 1104D and others in the present invention form a third set of nanowells (e.g., a line of nanowells) extending along the linear waveguide 1106C. Nanowell 1104E and others in the present invention form a fourth set of nanowells (e.g., a line of nanowells) extending along the linear waveguide 1106D. In some implementations, the first set of nanowells (e.g., nanowells 1104A-1104B and others) is positioned so that it is in phase with the second set of nanowells (e.g., nanowells 1104C and others).The first set of nanowells can be positioned at substantially, and at least in one instance completely, regular intervals along the 1106A linear waveguide. For example, each of the nanowells in the first set of nanowells in the 1106A linear waveguide has a corresponding nanowell in the second set of nanowells in the linear waveguide. Petition 870200161164, dated 12 / 23 / 2020, pp. 97 / 263 70 / 100 1106B. The corresponding nanowell can be positioned directly through the coating or other material between the linear waveguides 1106A-1106B from the nanowell.

[165] Here, nanowell 1104D and the others in the third nanowell set are positioned substantially at, and at least in one instance completely at, regular intervals along the linear waveguide 1106C. The third nanowell set is positioned so as to be out of phase with at least the second nanowell set. In some implementations, none of the nanowells in the second nanowell set has a corresponding nanowell in the third nanowell set directly through the coating or other material. For example, each of the nanowells in the second nanowell set may be equidistantly positioned between two adjacent nanowells of the nanowells in the third nanowell set.

[166] In some implementations, the fourth set of nanowells (e.g., nanowell 1104E and others along linear waveguide 1106D) is positioned so that it is in phase with the third set of nanowells (e.g., nanowells 1104D and others along linear waveguide 1106C). The fourth set of nanowells may be positioned at substantially, and at least in one instance completely, regular intervals along linear waveguide 1106D. For example, each of the nanowells in the fourth set of nanowells in linear waveguide 1106D has a corresponding nanowell in the third set of nanowells in linear waveguide 1106C. The corresponding nanowell may be positioned directly through the cladding or other material between linear waveguides 1106C-1106D from the nanowell.

[167] The 1102 grids serve to couple electromagnetic radiation in Petition 870200161164, dated 12 / 23 / 2020, pp. 98 / 263 71 / 100 and / or outside the linear waveguides of the 1100 flow cell. Here, the 1106A linear waveguide has an 1102A lattice, the 1106B linear waveguide has an 1102B lattice, the 1106C linear waveguide has an 1102C lattice, and the 1106D linear waveguide has an 1102D lattice. Each of the 1102A-1102D lattices may have the same or different periodic structure. In some implementations, some or all of the 1102A-1102D lattices may include a periodic structure of crests interspersed with another material. For example, crests of the 1102A-1102D lattices may have a pitch of about 200-300 nm, to cite just one example.

[168] The 1102A-1102D lattices may have one or more features that at least in part facilitate selective coupling of electromagnetic radiation in the corresponding 1106A-1106D linear waveguide. In some implementations, one or more of the 1102 lattices is spatially offset from one or more other 1102 lattices. The offset may be in a direction that is parallel to the 1106A-1106D linear waveguides. For example, the distance between the 1102B lattice and the nearest nanowell of the nanowells associated with the 1106B linear waveguide is here greater than the distance between the 1102A lattice and the nearest nanowell of the nanowells associated with the 1106A linear waveguide. As another example, the distance between the 1102D grid and the nearest nanowell associated with the 1106D linear waveguide is greater here than the distance between the 1102C grid and the nearest nanowell associated with the 1106C linear waveguide.In some implementations, the 1102A and 1102C grids have equal or similar spatial offsets. In some implementations, the 1102B and 1102D grids have equal or similar spatial offsets. The characteristic of the 1102A and 1102D grids being spatially offset from each other, at least in part, facilitates the coupling of electromagnetic radiation (e.g., light) to one of them. Petition 870200161164, dated 12 / 23 / 2020, pp. 99 / 263 72 / 100 linear waveguides (e.g., linear waveguide 1106A and / or 1106C) without coupling electromagnetic radiation (e.g., light) to another linear waveguide (e.g., linear waveguide 1106B and / or 1106D).

[169] In some implementations, coupling in 1102A-1102D gratings may also or instead be differentiated by a beam parameter other than the location of the light beam (for example, but not limited to, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength and combinations thereof). In some implementations, coupling in 1102A-1102D gratings may also or instead be differentiated by a coupler parameter (for example, but not limited to, grating period, refractive index, pitch, slot width, slot height, slot spacing, grating non-uniformity, slot orientation, slot curvature, overall coupler shape and combinations thereof).In some implementations, the coupling in the 1102A-1102D gratings may also or instead be differentiated by a waveguide parameter in relation to one or more of the 1106A-1106D linear waveguides (for example, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and / or beam, and combinations thereof).

[170] The examples described above illustrate that the 1100 flow cell includes a nanowell layer having first (e.g., nanowells associated with linear waveguide 1106A) and second (e.g., nanowells associated with linear waveguide 1106B) sets of nanowells to receive a sample. The 1100 flow cell includes a first linear waveguide (e.g., linear waveguide 1106A) aligned with the first set of nanowells and a second linear waveguide (e.g., linear waveguide 1106B) aligned with the second set of nanowells; and a first grid Petition 870200161164, dated 12 / 23 / 2020, pp. 100 / 263 73 / 100 (e.g., grid 1102A) for the first linear waveguide, and a second grid (e.g., grid 1102B) for the second linear waveguide. The first grid has a first characteristic (e.g., being spatially offset from grid 1102B) to facilitate coupling the first light in the first linear waveguide without coupling the first light in the second linear waveguide.

[171] Figure 12 shows another example of a flow cell 1200 having stepped gratings 1202. The flow cell 1200 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the flow cell 1200 can be used with stepped gratings or non-stepped gratings, or both. One or more of the stepped gratings 1202 can be used with one or more methods described in the present invention, and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the stepped gratings 1202 can be used with nanowells arranged in a hexagonal arrangement or a non-hexagonal arrangement (e.g., otherwise polygonal) or both.

[172] The 1200 flow cell includes nanowells, including a 1204A nanowell, which are illustrated here using circular shapes. Only one of the nanowells will be specifically mentioned, and the other nanowells may be similar or identical to the one(s) discussed. Nanowells can be formed in a nanowell layer (e.g., by nanoprinting or a lifting process). For example, nanowells can be formed in a resin using a nanoscale jig. The nanowell layer is not explicitly shown in this example for clarity. The 1204A nanowell is associated here with a 1206A linear waveguide. In some implementations, the linear waveguides described with reference to the flow cell Petition 870200161164, dated 12 / 23 / 2020, pp. 101 / 263 74 / 100 flux 1200 may be similar or identical to one or more other linear waveguides described in the present invention. For example, linear waveguide 1206A is positioned adjacent to (e.g., in contact with or near) the nanowell layer that includes nanowell 1204A.

[173] Another 1204B nanowell is also associated with the 1206A linear waveguide. For example, the 1204B nanowell is positioned adjacent to the 1204A nanowell, and both 1204A-1204B nanowells can interact with the 1206A linear waveguide in an imaging process (e.g., by receiving electromagnetic radiation from the 1206A linear waveguide). Another 1204C nanowell, conversely, is instead associated with a 1206B linear waveguide. In some implementations, the 1206B linear waveguide is positioned adjacent to the 1206A linear waveguide. For example, the coating (not shown) and / or other material can be positioned between the 1206A-1206B linear waveguides.

[174] A 1204D nanowell is here associated with a 1206C linear waveguide. In some implementations, the 1206C linear waveguide is positioned adjacent to the 1206B linear waveguide. For example, the coating (not shown) and / or other material may be positioned between the 1206B-1206C linear waveguides.

[175] A 1204E nanowell is here associated with a 1206D linear waveguide. In some implementations, the 1206D linear waveguide is positioned adjacent to the 1206C linear waveguide. For example, the coating (not shown) and / or other material may be positioned between the 1206C-1206D linear waveguides.

[176] Nanowells 1204A-1204B and others in the present invention form a first set of nanowells (e.g., a line of nanowells) that extends along the linear waveguide 1206A. Nanowell 1204C and others in Petition 870200161164, dated 12 / 23 / 2020, pp. 102 / 263 75 / 100 of the present invention form a second set of nanowells (e.g., a line of nanowells) that extends along the linear waveguide 1206B. Nanowell 1204D and others of the present invention form a third set of nanowells (e.g., a line of nanowells) that extends along the linear waveguide 1206C. Nanowell 1204E and others in the present invention form a fourth set of nanowells (e.g., a line of nanowells) that extends along the linear waveguide 1206D. In some implementations, the first set of nanowells (e.g., nanowells 1204A-1204B and others) is positioned so that it is out of phase with the second set of nanowells (e.g., nanowells 1204C and others). In some implementations, none of the nanowells in the first set of nanowells has a corresponding nanowell in the second set of nanowells directly through the coating or other material.For example, one of the nanowells in the first set of nanowells can be positioned equidistant between two adjacent nanowells in the second set of nanowells.

[177] Here, nanowell 1204D and the others in the third nanowell set are positioned substantially at, and at least in one instance completely at, regular intervals along the linear waveguide 1206C. The third nanowell set is positioned so as to be out of phase with at least the second nanowell set. In some implementations, none of the nanowells in the third nanowell set has a corresponding nanowell in the second nanowell set directly through the coating or other material. For example, each of the nanowells in the third nanowell set may be positioned equidistant between two adjacent nanowells in the second nanowell set. The third nanowell set may be Petition 870200161164, dated 12 / 23 / 2020, pp. 103 / 263 76 / 100 positioned to be in phase with at least the first set of nanowells.

[178] In some implementations, the fourth set of nanowells (e.g., nanowell 1204E and others along linear waveguide 1206D) is positioned so that it is out of phase with the third set of nanowells (e.g., nanowells 1204D and others along linear waveguide 1206C). In some implementations, none of the nanowells in the fourth set of nanowells has a corresponding nanowell in the third set of nanowells directly through the coating or other material. For example, each of the nanowells in the fourth set of nanowells may be positioned equidistant between two adjacent nanowells of the nanowells in the third set of nanowells.

[179] The 1202 gratings serve to couple electromagnetic radiation into and / or out of the linear waveguides of the 1200 flow cell. Here, the linear waveguide 1206A has a 1202A grating, the linear waveguide 1206B has a 1202B grating, the linear waveguide 1206C has a 1202C grating, and the linear waveguide 1206D has a 1202D grating. Each of the 1202A-1202D gratings may have the same or different periodic structure. In some implementations, some or all of the 1202A-1202D gratings may include a periodic structure of crests interspersed with another material. For example, crests of the 1202A-1202D gratings may have a pitch of about 200-300 nm, to cite just one example. The 1202A-1202D grids can have one or more of multiple suitable shapes. In some implementations, the 1202A-1202D grids have a truncated triangular shape.

[180] The 1202A-1202D gratings may have one or more features that at least partially facilitate selective coupling of electromagnetic radiation in the corresponding 1206A-1206D linear waveguide. In Petition 870200161164, dated 12 / 23 / 2020, pp. 104 / 263 In some implementations, one or more of the 1202 grids is spatially offset from one or more other 1202 grids. The offset may be in a direction that is parallel to the 1206A-1206D linear waveguides. For example, the distance between the 1202B grid and the other end of the 1206B linear waveguide is shorter here than the distance between the 1202B grid and the other end of the 1206A linear waveguide. As another example, the distance between the 1202D grid and the other end of the 1206D linear waveguide is shorter here than the distance between the 1202C grid and the other end of the 1206C linear waveguide. In some implementations, the 1202A and 1202C grids have equal or similar spatial offsets. In some implementations, grids 1202B and 1202D have equal or similar spatial displacement.The characteristic of the 1202A-1202D gratings being spatially offset from each other, at least in part, facilitates coupling of electromagnetic radiation (e.g., light) to one of the linear waveguides (e.g., the 1206A and / or 1206C linear waveguide) without coupling electromagnetic radiation (e.g., light) to another of the linear waveguides (e.g., the 1206B and / or 1206D linear waveguide).

[181] In some implementations, coupling in 1202A-1202D gratings may also or instead be differentiated by a beam parameter other than the location of the light beam (for example, but not limited to, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength and combinations thereof). In some implementations, coupling in 1202A-1202D gratings may also or instead be differentiated by a coupler parameter (for example, but not limited to, grating period, refractive index, pitch, slot width, slot height, slot spacing, grating non-uniformity, slot orientation, slot curvature, overall coupler shape). Petition 870200161164, dated 12 / 23 / 2020, pp. 105 / 263 78 / 100 and combinations thereof). In some implementations, the coupling in the 1202A-1202D grids may also or instead be differentiated by a waveguide parameter in relation to one or more of the 1206A-1206D linear waveguides (for example, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and / or beam and combinations thereof).

[182] The 1200 flow cell can have the nanowells arranged in any of multiple patterns. In the present example, the nanowells are arranged in a hexagonal arrangement. A hexagonal arrangement forms one or more hexagons. Here, the linear waveguide 1206B further includes nanowells 1204F-1204G, and the linear waveguide 1206C further includes a nanowell 1204H. The nanowells 1204A-1204H are positioned here in the form of a hexagon. The nanowells 1204A-1204B are part of the first set of nanowells here and are associated with the linear waveguide 1206A; the nanowells 1204C and 1204F-1204G are part of the second set of nanowells and are associated with the linear waveguide 1206B; Nanowells 1204D and 1204H are part of the third set of nanowells and are associated with the linear waveguide 1206C.

[183] ​​The examples described above illustrate that the 1200 flow cell includes a nanowell layer having first (e.g., nanowells associated with linear waveguide 1206A) and second (e.g., nanowells associated with linear waveguide 1206B) sets of nanowells to receive a sample. The 1200 flow cell includes a first linear waveguide (e.g., linear waveguide 1206A) aligned with the first set of nanowells and a second linear waveguide (e.g., linear waveguide 1206B) aligned with the second set of nanowells; and a first grid (e.g., grid 1202A) for the first linear waveguide, and a second grid (e.g., grid 1202B) for the second linear waveguide. Petition 870200161164, dated 12 / 23 / 2020, pp. 106 / 263 79 / 100 The first large one has a first characteristic (for example, being spatially offset from the 1202B grid) to facilitate coupling the first light to the first linear waveguide without coupling the first light to the second linear waveguide.

[184] Figure 13 shows another example of a flow cell 1300 having stepped gratings 1302. The flow cell 1300 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the flow cell 1300 can be used with stepped gratings or non-stepped gratings, or both. One or more of the stepped gratings 1302 can be used with one or more methods described in the present invention, and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the stepped gratings 1302 can be used with nanowells arranged in a hexagonal arrangement or a non-hexagonal arrangement (e.g., otherwise polygonal) or both.

[185] The 1300 flow cell includes nanowells, including a 1304A nanowell, which are illustrated here using circular shapes. Only one of the nanowells will be specifically mentioned, and the other nanowells may be similar or identical to the one(s) discussed. The nanowells can be formed in a nanowell layer (e.g., by nanoprinting or a lifting process). For example, the nanowells can be formed in a resin using a nanoscale jig. The nanowell layer is not explicitly shown in this example for clarity. The 1304A nanowell is associated here with a 1306A linear waveguide. In some implementations, the linear waveguides described with reference to the 1300 flow cell may be similar or identical to one or more other linear waveguides described in the present invention. For example, the linear waveguide Petition 870200161164, dated 12 / 23 / 2020, page 107 / 263 80 / 100 1306A is positioned adjacent to (e.g., in contact with or near) the nanowell layer that includes nanowell 1304A. Nanowell 1304A is part of a first set of nanowells (e.g., one or more nanowell rows) for linear waveguide 1306A. Here, the nanowell row of which nanowell 1304A is a part extends along linear waveguide 1306A on one side thereof. For example, the nanowell row does not overlap linear waveguide 1306A in the perspective shown of flow cell 1300.

[186] Another nanowell 1304B is also associated with the linear waveguide 1306A. Like nanowell 1304A, nanowell 1304B is also part of the first set of nanowells (i.e., one or more nanowell lines) for the linear waveguide 1306A. Here, the nanowell line of which nanowell 1304B is a part extends along the linear waveguide 1306A on the opposite side of it. For example, the nanowell line does not overlap the linear waveguide 1306A in the perspective shown of the flow cell 1300 and is positioned on an opposite side of the linear waveguide 1306A from the nanowell line 1304A. Both nanowells 1304A-1304B can interact with the linear waveguide 1306A in an imaging process (e.g., by receiving electromagnetic radiation from the linear waveguide 1306A).

[187] Another nanowell 1304C is associated with a linear waveguide 1306B. In some implementations, the linear waveguide 1306B is parallel to and positioned adjacent to the linear waveguide 1306A. For example, the coating (not shown) and / or other material may be positioned between the linear waveguides 1306A-1306B. The nanowell 1304C is part of a second set of nanowells (e.g., one or more nanowell rows) for the linear waveguide 1306B. Here, the nanowell row of which the nanowell 1304C is a part extends along the linear waveguide. Petition 870200161164, dated 12 / 23 / 2020, pp. 108 / 263 81 / 100 1306B on one side of it. For example, the nanowell row does not overlap the linear waveguide 1306B in the perspective shown of the flow cell 1300. Another nanowell row that is also part of the second nanowell array can be positioned on the opposite side of the linear waveguide 1306B from the nanowell row 1304C.

[188] Another nanowell 1304D is associated with a linear waveguide 1306C. In some implementations, the linear waveguide 1306C is parallel to and positioned adjacent to the linear waveguide 1306B. For example, the coating (not shown) and / or other material may be positioned between the linear waveguides 1306B-1306C. The nanowell 1304D is part of a third set of nanowells (e.g., one or more nanowell rows) for the linear waveguide 1306C. Here, the nanowell row of which the nanowell 1304D is a part extends along the linear waveguide 1306C on one side of it. For example, the nanowell row does not overlap the linear waveguide 1306C in the perspective shown of the flow cell 1300. Another nanowell row, which is also part of the third nanowell array, can be positioned on the opposite side of the linear waveguide 1306C from the nanowell row 1304D.

[189] Having nanowells positioned with offsets from the associated linear waveguide (e.g., as in the 1300 flow cell) can provide one or more advantages. In some implementations, crosstalk between waveguides can be reduced or minimized. For example, this benefit can compensate for a somewhat lower packing density of the nanowells.

[190] In some implementations, the nanowells in the rows of the first set of nanowells (for example, nanowells 1304A-1304B and others) are positioned so that they are in phase with each other. The nanowells in the rows of Petition 870200161164, dated 12 / 23 / 2020, pp. 109 / 263 82 / 100 nanowells on each side of the 1306A linear waveguide can be positioned substantially at, and at least in one instance entirely at, regular intervals along the 1306A linear waveguide. For example, each of the nanowells in one of these rows has a corresponding nanowell in the other row. The corresponding nanowell of the first set of nanowells can be positioned directly across the 1306A linear waveguides from the other nanowell of the first set of nanowells.

[191] In some implementations, the first set of nanowells (e.g., nanowells 1304A-1304B and others) is positioned so as to be in phase with nanowells of the second set of nanowells (e.g., nanowell 1304C and others). The nanowells in the nanowell lines on either side of the linear waveguide 1306B can be positioned substantially at, and at least in one instance entirely at regular intervals along the linear waveguide 1306B. For example, each of the nanowells in at least one of these lines has a corresponding nanowell in at least one of the lines of the first set of nanowells. The corresponding nanowell of the first set of nanowells can be positioned directly through the coating or other material from the nanowell of the second set of nanowells.

[192] The 1302 gratings serve to couple electromagnetic radiation into and / or out of the linear waveguides of the 1300 flow cell. Here, the linear waveguide 1306A has a 1302A grating, the linear waveguide 1306B has a 1302B grating, the linear waveguide 1306C has a 1302C grating, and a linear waveguide 1306D has a 1302D grating. Each of the 1302A-1302D gratings may have the same or different periodic structure. In some implementations, some or all of the 1302A-1302D gratings may include a periodic structure of crests interspersed with another material. For example, crests of the 1302A- Petition 870200161164, dated 12 / 23 / 2020, pp. 110 / 263 83 / 100 1302D electrons can have a step size of around 200-300 nm, to cite just one example.

[193] The 1302A-1302D lattices may have one or more features that at least in part facilitate selective coupling of electromagnetic radiation in the corresponding 1306A-1306D linear waveguide. In some implementations, one or more of the 1302 lattices is spatially offset from one or more other 1302 lattices. The offset may be in a direction that is parallel to the 1306A-1306D linear waveguides. For example, the distance between the 1302B lattice and the nearest nanowell of the nanowells associated with the 1306B linear waveguide is here greater than the distance between the 1302A lattice and the nearest nanowell of the nanowells associated with the 1306A linear waveguide. As another example, the distance between the 1302D grid and the nearest nanowell associated with the 1306D linear waveguide is greater here than the distance between the 1302C grid and the nearest nanowell associated with the 1306C linear waveguide.In some implementations, the 1302A and 1302C gratings have equal or similar spatial offsets. In some implementations, the 1302B and 1302D gratings have equal or similar spatial offsets. The characteristic of the 1302A-1302D gratings being spatially offset from each other at least in part facilitates coupling electromagnetic radiation (e.g., light) to one of the linear waveguides (e.g., the 1306A and / or 1306C linear waveguide) without coupling electromagnetic radiation (e.g., light) to another of the linear waveguides (e.g., the 1306B and / or 1306D linear waveguide).

[194] Here, a distance of 1308 is less than the emission optics resolution distance and a distance of 1310 is greater than, or approximately equal to, the emission optics resolution distance. The distance of 1308 here represents the separation between the nearest nanowells associated with waveguides. Petition 870200161164, dated 12 / 23 / 2020, p. 111 / 263 84 / 100 adjacent linear waveguides. The distance 1310 here represents the distance between nanowells associated with the same linear waveguide.

[195] In some implementations, coupling in 1302A-1302D gratings may also or instead be differentiated by a beam parameter other than the location of the light beam (for example, but not limited to, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength and combinations thereof). In some implementations, coupling in 1302A-1302D gratings may also or instead be differentiated by a coupler parameter (for example, but not limited to, grating period, refractive index, pitch, slot width, slot height, slot spacing, grating non-uniformity, slot orientation, slot curvature, overall coupler shape and combinations thereof).In some implementations, the coupling in the 1302A-1302D gratings may also or instead be differentiated by a waveguide parameter in relation to one or more of the 1306A-1306D linear waveguides (for example, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and / or beam, and combinations thereof).

[196] The examples of the present invention illustrate differential coupling of light in two or more linear waveguides. Differential coupling can be based on one or more parameters that characterize the analysis system, the parameter(s) having an effect on the extent to which light is (or is not) coupled in one or more linear waveguides. In some implementations, one or more of these parameters may refer to the light beam that is the illumination source (e.g., excitation illumination) for the analysis. For example, a coupler (e.g., a grid) may be relatively sensitive to one or more parameters, thus a change Petition 870200161164, dated 12 / 23 / 2020, pp. 112 / 263 85 / 100 being relatively smaller in the parameter(s) may facilitate differential coupling.

[197] The examples described above illustrate that the 1300 flow cell includes a nanowell layer having first (e.g., nanowells associated with linear waveguide 1306A) and second (e.g., nanowells associated with linear waveguide 1306B) sets of nanowells to receive a sample. The 1300 flow cell includes a first linear waveguide (e.g., linear waveguide 1306A) aligned with the first set of nanowells and a second linear waveguide (e.g., linear waveguide 1306B) aligned with the second set of nanowells; and a first grid (e.g., grid 1302A) for the first linear waveguide, and a second grid (e.g., grid 1302B) for the second linear waveguide.The first large one has a first characteristic (for example, being spatially offset from the 1302B grid) to facilitate coupling the first light to the first linear waveguide without coupling the first light to the second linear waveguide.

[198] Figure 14 schematically shows a light beam 1400 incident on a surface 1402. The examples and / or concepts described with reference to the light beam 1400 can be considered and / or employed in relation to one or more methods described in the present invention and / or used in combination with one or more systems or apparatus described in the present invention.

[199] The light beam 1400 is incident here at a location 1404 of the surface 1402. In some implementations, location 1404 is a beam parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the location 1404 where the light beam 1404 is incident can affect the extent to which the light is (or is not) coupled in one or more linear waveguides.

[200] One or more angles can characterize the incidence of the light beam. Petition 870200161164, dated 12 / 23 / 2020, page 113 / 263 86 / 100 1400. Here, the light beam 1400 has an angle of incidence 1406 with respect to a surface perpendicular 1402. In some implementations, the angle of incidence 1406 is a beam parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the angle of incidence 1406 of the light beam 1400 can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[201] One or more features of the 1400 light beam can be considered. Here, the 1400 light beam includes individual 1400A1400B light rays that are not parallel to each other, but instead form a 1408 angle which is a non-zero angle. A 1400 light beam divergence can be defined based on features such as the 1408 angle. In some implementations, the 1400 light beam divergence is a beam parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the divergence can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[202] The 1400 light beam may include coherent light (e.g., a laser beam) propagating in the form of one or more modes. Here, the 1400 light beam has a 1410 mode profile that schematically illustrates (e.g., in terms of intensity and / or spatial distribution) the profile of at least one mode of the 1400 light beam. In some implementations, the 1410 mode profile is a beam parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the 1410 mode profile may affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[203] The 1400 light beam may have one or more polarizations. In some implementations, polarization is a beam parameter that can be selected and / or adjusted to facilitate differential coupling. For example, polarization can affect the extent to which light is (or is not) coupled in a Petition 870200161164, dated 12 / 23 / 2020, page 114 / 263 87 / 100 or more linear waveguides.

[204] The 1400 light beam can have any suitable cross-sectional profile. In some implementations, the 1400 light beam has a rectangular cross-sectional profile 1412A. For example, one or more dimensions of the rectangular cross-sectional profile 1412A (e.g., an aspect ratio thereof) is a beam parameter that can be selected and / or adjusted to facilitate differential coupling. In some implementations, the 1400 light beam has a circular cross-sectional profile 1412B. For example, one or more dimensions of the circular cross-sectional profile 1412B (e.g., a diameter thereof) is a beam parameter that can be selected and / or adjusted to facilitate differential coupling. The dimension(s) of the rectangular cross-sectional profile 1412A and / or the circular cross-sectional profile 1412B can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[205] The 1400 light beam may include electromagnetic radiation of one or more wavelengths. In some implementations, the wavelength(s) of the 1400 light beam is a beam parameter that can be selected and / or adjusted to facilitate differential coupling. The wavelength(s) may affect the extent to which light is (or is not) coupled in one or more linear waveguides. For example, different wavelengths couple in a grid at different angles. A change in the wavelength and angle of the 1400 light beam may enable differential coupling.

[206] In some implementations, one or more parameters affecting differential coupling may refer to the grating that couples light into the linear waveguide for analysis. For example, a coupler (e.g., a grating) may be relatively sensitive to one or more parameters, thus a change Petition 870200161164, dated 12 / 23 / 2020, pp. 115 / 263 88 / 100 relatively lower in the parameter(s) may facilitate differential coupling.

[207] Figures 15A-15B show examples of grids 1500 and 1502. Grid 1500 and / or 1502 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention.

[208] 1500 and 1502 gratings may have the same or different refractive indices. In some implementations, the refractive index is a coupling parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the refractive index can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[209] Grid 1500 includes slots 1504 and grid 1502 includes slots 1506 and 1508. At least one slot pitch 1510 can be defined for each of the grids 1500 and 1502. The slot pitch 1510 can represent the distance from an edge of one of the slots 1504, 1506, or 1508 to the corresponding edge of the adjacent slot of slots 1504, 1506, or 1508. In some implementations, the slot pitch 1510 is a coupler parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the slot pitch 1510 can affect the extent to which light is (or is not) coupled to one or more linear waveguides.

[210] At least one 1512 slot width can be defined for each of the 1504, 1506, or 1508 slots. The 1512 slot width can represent the edge-to-edge width of one of the 1504, 1506, or 1508 slots. In some implementations, the 1512 slot width is a coupler parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the 1512 slot width can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[211] At least one slot height of 1514 can be defined for Petition 870200161164, dated 12 / 23 / 2020, pp. 116 / 263 89 / 100 each of slots 1504, 1506, or 1508. Slot height 1514 may represent the height from the bottom to the opening of one of slots 1504, 1506, or 1508. In some implementations, slot height 1514 is a coupler parameter that can be selected and / or adjusted to facilitate differential coupling. For example, slot height 1514 may affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[212] At least one 1516 slot spacing can be defined for each of the 1504, 1506, or 1508 slots. The 1516 slot spacing can represent the distance from the edge of one of the 1504, 1506, or 1508 slots to the nearest edge of the adjacent slot of the 1504, 1506, or 1508 slots. In some implementations, the 1516 slot spacing is a coupler parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the 1516 slot spacing can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[213] In some implementations, a non-uniform grid may be used. In some implementations, slots 1506 and 1508 of grid 1502 provide a non-uniform grid. For example, slots 1506 and 1508 may have different slot widths 1512. As another example, slots 1506 and 1508 may instead or additionally have different slot pitches 1510, different slot heights 1514, and / or different slot spacing 1516. As such, grid 1502 is an example of grid non-uniformity.

[214] In some implementations, slot orientation is a coupler parameter that can be selected and / or adjusted to facilitate differential coupling. In some implementations, grids generally couple in a transverse electric polarization in which the electric field is parallel. Petition 870200161164, dated 12 / 23 / 2020, page 117 / 263 90 / 100 as the grid slots. The 1500 and / or 1502 grid can be positioned to obtain a specific orientation of the 1504, 1506 and / or 1508 slots. For example, a slot structure can be rotated to another orientation to provide coupling based on a rotated polarization. That is, the slot orientation can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[215] In some implementations, slot curvature is a coupler parameter that can be selected and / or adjusted to facilitate differential coupling. Figure 15C shows a top view of a 1518 grid with 1520 slots having different curvatures. For example, slot curvature can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[216] In some implementations, the coupler shape is a coupler parameter that can be selected and / or adjusted to facilitate differential coupling. Figure 16 shows examples of 1600, 1602, 1604, and 1606 coupler shapes. These examples show illustrative coupler shapes and schematically indicate the grooves of the respective grids. Coupler 1600 may include a rectangular grid (e.g., square). For example, the grid grooves may be oriented along the longer edge, or the shorter edge, of the rectangle. Coupler 1602 may include an elliptical grid (e.g., circular). For example, the grid grooves may be oriented along the longer geometric axis, or along the shorter geometric axis, of the grid. Coupler 1604 may include a truncated triangular grid. For example, the grid grooves may be oriented perpendicular to the base, or perpendicular to the height, of the triangle.As another example, different angles for the lateral edges can be used. The 1606 coupler can include a triangular grid. For example, the grid slots. Petition 870200161164, dated 12 / 23 / 2020, pp. 118 / 263 91 / 100 can be oriented perpendicular to the base, or perpendicular to the height, of the triangle. As another example, different angles of the lateral edges can be used. In some implementations, the shape of the coupler(s) can be selected based on (e.g., optimized) the diameter of the illumination beam, or an aspect ratio of the illumination beam, or combinations thereof, to cite just a few examples. The coupler shape and / or the orientation of the slots can affect the extent to which light is (or is not) coupled in one or more linear waveguides.

[217] The shape of the coupler (including, but not limited to, couplers 1600, 1602, 1604 and 1606) can be selected based on the diameter, aspect ratio, or other characteristic of the light beam. For example, this can allow the resulting structure to be tuned to a specific differential coupling.

[218] The coupler parameter(s) can be selected and / or adjusted based on a mode profile of the illumination beam. This can be done by selecting (e.g., optimizing) the slot structure. In some implementations, a non-uniform grid can be used. For example, a chiseled grid (e.g., a grid with a variation in slot pitch), an apodized grid (e.g., having a refractive index that approaches zero towards one end of the grid), a curved grid, and combinations thereof, can be used. In some implementations, computer-based optimization on one or more coupler parameters (e.g., grid structure) can be performed. For example, this can facilitate differential coupling based on the mode profile of the incident light beam.

[219] In some implementations, one or more parameters affecting differential coupling may refer to the linear waveguide in which light is Petition 870200161164, dated 12 / 23 / 2020, page 119 / 263 92 / 100 coupled for analysis. For example, the coupling can be relatively sensitive to one or more parameters related to the waveguide, so a relatively minor change in the parameter(s) can facilitate differential coupling.

[220] In some implementations, a cross-sectional profile of the linear waveguide is a waveguide parameter that can be selected and / or adjusted to facilitate differential coupling. Figure 17 shows examples of cross-sectional profiles for linear waveguides. A 1700 waveguide may include a rectangular (e.g., square) profile. For example, a nanowell layer may be positioned adjacent to the longer edge, or the shorter edge, of the rectangle. A 1702 waveguide may include an elliptical (e.g., circular) profile. For example, the nanowell layer may be positioned parallel to the major geometric axis, or parallel to the minor geometric axis, of the 1702 waveguide. A 1704 waveguide may include a truncated triangular profile. For example, the nanowell layer may be positioned adjacent to the base, the side edge(s), and / or the truncation face, of the triangle. Different angles of the side edges may be used.A 1706 waveguide may include a triangular profile. For example, the nanowell layer may be positioned adjacent to one or more sides of the triangle. Different angles of the side edges may be used. The cross-sectional profile may affect the extent to which light is (or is not) coupled to one or more linear waveguides.

[221] In some implementations, the refractive index of the linear waveguide is a waveguide parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the difference in refractive index between two or more linear waveguides can affect the extent to which light is (or is not) coupled in the waveguides. Petition 870200161164, dated 12 / 23 / 2020, pp. 120 / 263 93 / 100

[222] In some implementations, the matching of one or more modes between the linear waveguide and the coupler, or between the linear waveguide and the light beam, or both, is a waveguide parameter that can be selected and / or adjusted to facilitate differential coupling. For example, the dimensions and / or aspect ratios of the linear waveguide can be selected to facilitate propagation (or not facilitate propagation) of a specific incoming light mode. That is, mode matching with the coupler and / or light beam can affect the extent to which light is (or is not) coupled in the waveguides.

[223] The examples of the present invention mention that a beam parameter, a coupler parameter, and / or a waveguide parameter can be selected and / or adjusted to facilitate differential coupling. In some implementations, combinations of two or more of these parameters can be selected and / or adjusted. For example, the selection / adjustment may consider at least two beam parameters; or at least one beam parameter and at least one coupler parameter; or at least one beam parameter, at least one coupler parameter, and at least one waveguide parameter. In some implementations, a cross-sectional profile of a waveguide can be used together with a specific grid (e.g., a grid optimized for certain coupling or non-coupling). For example, this may allow the resulting structure to be tuned for different mode profiles, beam diameters, aspect ratios, to cite just a few examples.

[224] Figure 18 shows a cross-section of part of another example flow cell 1800 with linear waveguides 1802, 1804 and 1806. The flow cell 1800 can be used with one or more methods described in the present invention and / or be used in combination with one or more systems or apparatus described in the present invention. For example, the flow cell 1800 Petition 870200161164, dated 12 / 23 / 2020, pp. 121 / 263 94 / 100 can be used with staggered or non-staggered grids, or both. As another example, the 1800 flow cell can be used with nanowells arranged in a hexagonal arrangement or a non-hexagonal arrangement (e.g., otherwise polygonal) or both. Only a portion of the 1800 flow cell is shown, for illustrative purposes. For example, one or more additional layers and / or more or fewer 1802, 1804 and / or 1804 waveguides can be used.

[225] The 1800 flow cell includes a substrate 1808. The substrate 1808 can form a base for the 1800 flow cell. In some implementations, one or more other layers may be formed on (e.g., in contact with or near) substrate 1808 in the fabrication of the 1800 flow cell. The substrate 1808 can serve as a base for forming the linear waveguides 1802, 1804, and / or 1806. The linear waveguides 1802, 1804, and / or 1806 may initially exist separately from the substrate 1808 and subsequently be applied over the substrate 1808, or the linear waveguides 1802, 1804, and / or 1806 may be formed by applying and / or removing one or more materials on or from the substrate. Linear waveguides 1802, 1804 and / or 1806 can be formed directly on substrate 1808 or on one or more intermediate layers on substrate 1808.

[226] 1802, 1804, and / or 1806 linear waveguides are used to conduct electromagnetic radiation (including, but not limited to, visible light such as laser light). In some implementations, the electromagnetic radiation performs one or more functions during an imaging process. For example, electromagnetic radiation can be used to excite fluorophores in a sample material for imaging. 1802, 1804, and / or 1806 linear waveguides can be made of any suitable material that facilitates the propagation of one or more types of electromagnetic radiation. In some Petition 870200161164, dated 12 / 23 / 2020, pp. 122 / 263 In 95 / 100 implementations, the material(s) of the 1802, 1804, and / or 1806 linear waveguides may include a polymer material. In some implementations, the material(s) of the 1802, 1804, and / or 1806 linear waveguides may include Ta205 and / or SiNx. For example, the 1802, 1804, and / or 1806 linear waveguides may be formed by cathodic sublimation, chemical vapor deposition, atomic layer deposition, spin coating, and / or spray coating.

[227] Each of the linear waveguides 1802, 1804 and / or 1806 may have one or more grids (omitted here for clarity) for coupling electromagnetic radiation into and / or out of that linear waveguide 1802, 1804 and / or 1806. The grid(s) may be positioned in the same layer as the corresponding linear waveguide(s). One or more directions of displacement for electromagnetic radiation in the linear waveguides 1802, 1804 and / or 1806 may be employed. For example, the direction of displacement may be into and / or out of the plane of the present illustration. Examples of grids are described elsewhere in the present invention.

[228] Each of the 1802, 1804 and / or 1806 linear waveguides can be positioned against one or more types of coating. The coating can serve to limit electromagnetic radiation to the respective 1802, 1804 and / or 1806 linear waveguide and prevent, or reduce the extent of, radiation propagation to other 1802, 1804 and / or 1806 linear waveguides or other substrates. Here, coatings 1810, 1812, 1814, 1816 and 1818 are shown as examples. In some implementations, coatings 1810, 1812, and 1814, along with linear waveguides 1802 and 1804, can form a first layer in flow cell 1800. For example, coatings 1810 and 1812 can be positioned against or near the linear waveguide. Petition 870200161164, dated 12 / 23 / 2020, pages 123 / 263 96 / 100 1802 on different (e.g., opposite) sides of the same. For example, coatings 1812 and 1814 can be positioned against or near linear waveguide 1804 on different (e.g., opposite) sides of the same. In some implementations, coatings 1816 and 1818, together with linear waveguide 1806, can form a second layer in the flow cell 1800. For example, coatings 1816 and 1818 can be positioned against or near linear waveguide 1806 on different (e.g., opposite) sides of the same. The formation of multiple layers can provide advantages with respect to differential coupling. In some implementations, two or more different materials can be used for the respective waveguides; for example, this can facilitate different refractive indices being given to the respective waveguides and / or couplers. In some implementations, crosstalk between waveguides can be reduced or minimized.

[229] Coatings 1810, 1812, 1814, 1816 and / or 1818 may be made of one or more suitable materials which serve to separate linear waveguides 1802, 1804 and / or 1806 from each other. In some implementations, the 1810, 1812, 1814, 1816, and / or 1818 coatings may be made of a material having a lower refractive index than the refractive index(es) of the 1802, 1804, and / or 1806 linear waveguides. For example, the 1802, 1804, and / or 1806 linear waveguides may have a refractive index of about 1.4–1.6, and the 1810, 1812, 1814, 1816, and / or 1818 coatings may have a refractive index of about 1.2–1.4. In some implementations, one or more of the 1810, 1812, 1814, 1816, and / or 1818 coatings includes a polymer material.In some implementations, one or more of the coatings 1810, 1812, 1814, 1816 and / or 1818 includes multiple structures, including, but not limited to, structures of a material (e.g., polymer) interspersed with regions of vacuum or other material (e.g., air or a liquid). Petition 870200161164, dated 12 / 23 / 2020, pp. 124 / 263 97 / 100

[230] The 1800 flow cell includes at least one 1820 nanowell layer. In some implementations, the 1820 nanowell layer is positioned opposite the first layer from the second layer. For example, the 182 nanowell layer may be positioned adjacent to (e.g., touching or near) the 1802 1804 linear waveguides and the 1810, 1812, and 1814 coatings. The 1820 nanowell layer includes one or more nanowells. In some implementations, the 1820 nanowell layer includes 1822, 1824, and 1826 nanowells. The 1822, 1824, and / or 1826 nanowells may be used to contain one or more sample materials during at least part of the analysis process (e.g., for imaging). For example, one or more genetic materials (e.g., in the form of clusters) can be placed in nanowells 1822, 1824, and / or 1826.

[231] Nanowells 1822, 1824 and / or 1826 may be arranged in any pattern, or without a specific pattern, in the nanowell layer 1820. One or more of the nanowells 1822, 1824 and / or 1826 may be at least substantially aligned with one or more of the linear waveguides 1802, 1804 and / or 1806. This may allow interaction between the respective nanowell 1822, 1824 and / or 1826 and the corresponding linear waveguide 1802, 1804 and / or 1806 for imaging purposes (including, but not limited to, as evanescent light transmission). For example, nanowell 1822 may be at least substantially aligned with linear waveguide 1802; Nanowell 1824 can be at least substantially aligned with linear waveguide 1804; and / or nanowell 1826 can be at least substantially aligned with linear waveguide 1806.In some implementations, the first layer (e.g., coatings 1810, 1812, and 1814, along with linear waveguides 1802 and 1804) can be positioned closer to the nanowell layer 1820 than the second layer (e.g., coatings 1816). Petition 870200161164, dated 12 / 23 / 2020, pp. 125 / 263 98 / 100 and 1818, along with the linear waveguide 1806). As another example, the second layer can be positioned further from the third layer than the first layer.

[232] Figure 19 is a flowchart of an example method 1900. Method 1900 can be performed using, and / or in combination with, one or more other examples described in the present invention. More or fewer operations can be performed, and / or two or more operations can be performed in a different order, unless otherwise indicated.

[233] In 1910, a sample is applied to at least some nanowells of a flow cell. In some implementations, the sample is applied to a first set of nanowells and a second set of nanowells.

[234] In 1920, the first light could be differentially coupled in at least one linear first waveguide associated with the first set of nanowells. In some implementations, the first light could be differentially coupled using a first grid.

[235] In 1930, the second light could be differentially coupled in at least one second linear waveguide associated with the second set of nanowells. In some implementations, the second light could be differentially coupled using a second grid.

[236] The terms substantially and “about” used throughout this Descriptive Report are used to describe and account for small fluctuations, such as due to variations in processing. For example, they may 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 in the present invention, an indefinite article such as “a” or “an” means “at least one.” Petition 870200161164, dated 12 / 23 / 2020, pp. 126 / 263 99 / 100

[237] It should be recognized that all combinations of the above concepts and additional concepts discussed in greater detail below (provided that such concepts are not mutually inconsistent) are considered to form part of the inventive matter disclosed in the present invention. In particular, all combinations of the claimed matter appearing at the end of this invention are considered to form part of the inventive matter disclosed in the present invention.

[238] Several implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the descriptive report.

[239] Furthermore, the logic flows shown in the figures do not require the specific order shown, or sequential order, to obtain 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. Therefore, other implementations are included within the scope of the following claims.

[240] Although certain features of the implementations described have been illustrated as described in the present invention, many modifications, substitutions, alterations, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that claims herein are intended to cover all such modifications and alterations as comprehended within the scope of the implementations. It should be understood that they have been presented only as examples, not limitations, and various alterations in form and detail may be made. Any portion of the apparatus and / or methods described in the present invention may be combined in any combination except mutually exclusive combinations. The implementations Petition 870200161164, dated 12 / 23 / 2020, pp. 127 / 263 The 100 / 100 implementations described in the present invention may include various combinations and / or subcombinations of the functions, components, and / or characteristics of the different implementations described. Petition 870200161164, dated 12 / 23 / 2020, pp. 128 / 263

Claims

1 / 9 CLAIMS 1.Flow cell (200, 600), characterized in that it comprises: a layer of nanowells (204) having a first set of nanowells and a second set of nanowells to receive a sample, wherein the nanowells in at least one of the first and second sets of nanowells have a spacing between them that is resolvable according to an emission optics resolution distance for the flow cell (200, 600); a first linear waveguide (206A) associated with the first set of nanowells, and a second linear waveguide (206B) associated with the second set of nanowells, wherein the first and second linear waveguides (206A, 206B) are positioned closer to each other than the emission optics resolution distance; and a first grid (202A) for the first linear waveguide (206A) and a second grid (202B) for the second linear waveguide (206B), the first and second grids (202A, 202B) providing differential coupling of first light and second light.

2. Flow cell (200, 600), according to claim 1, characterized in that the first and second grids (202A, 202B) are spatially displaced from each other, and in that the direction of displacement is in a direction parallel to the linear waveguides (206A, 206B).

3. Flow cell (200, 600), according to claim 2, characterized in that the first and second linear waveguides (206A, 206B) are positioned adjacent to each other, the flow cell (200) further comprising: a third linear waveguide positioned adjacent to the second linear waveguide (206B) opposite the first linear waveguide (206A). Petition 870250002332, dated 10 / 01 / 2025, page 25 / 33 2 / 9 4. Flow cell (200, 600), according to claim 3, characterized in that the third linear waveguide shares the first grid (202A) with the first linear waveguide (206A).

5. Flow cell (200, 600), according to claim 3, characterized in that it further comprises a third grid for the third linear waveguide.

6. Flow cell (200, 600), according to claim 5, characterized in that the third grid has the same spatial displacement from the second grid (202B) as the first grid (206A) has.

7. Flow cell (200, 600), according to claim 5, characterized in that the third grid is spatially displaced from each of the first and second grids (202A, 202B).

8. Flow cell (200, 600), according to any one of claims 1 to 7, characterized in that the first grid (608A) is positioned towards a first end of the first linear waveguide (616A), wherein the second grid (608B) is positioned towards a second end of the second linear waveguide (616B), and wherein the first end is positioned opposite from the second end.

9. Flow cell (200, 600), according to any one of claims 1 to 8, characterized in that the first grid is positioned on a triangular substrate.

10. Flow cell (200, 600), according to any one of claims 1 to 9, characterized in that the first and second grids have different grid periods from each other.

11. Flow cell (200, 600), according to claim 10, characterized in that the first and second linear waveguides are positioned adjacent to each other, the flow cell further comprising: Petition 870250002332, dated 10 / 01 / 2025, page 26 / 33 3 / 9 a third linear waveguide positioned adjacent to the second linear waveguide opposite from the first linear waveguide; and a third grid for the third linear waveguide.

12. Flow cell (200, 600), according to claim 11, characterized in that the third grid has the same grid period as the first grid.

13. Flow cell (200, 600), according to claim 11, characterized in that the third grid has a grid period different from each of the grid periods of the first and second grids.

14. Flow cell (200, 600), according to any one of claims 1 to 13, characterized in that the differential coupling of the first light comprises coupling the first light in the first linear waveguide and minimizing coupling of the first light in the second linear waveguide.

15. Flow cell (200, 600), according to claim 14, characterized in that the differential coupling of the second light comprises coupling of the second light in the second linear waveguide and minimizing coupling of the second light in the first linear waveguide.

16. Flow cell (200, 600), according to any one of claims 1 to 15, characterized in that the differential coupling is at least partly due to a coupler parameter of one or more of the first grid or the second grid.

17. Flow cell (200, 600), according to claim 16, characterized in that the coupler parameter comprises at least one selected from the group consisting of: a refractive index, a pitch, a groove width, a groove height, a groove spacing, a grid non-uniformity, a groove orientation, a groove curvature, a coupler shape and combinations thereof. Petition 870250002332, dated 10 / 01 / 2025, p. 27 / 33 4 / 9 18. Flow cell (200, 600), according to any one of claims 1 to 17, characterized in that the differential coupling is at least partly due to a waveguide parameter of one or more of the first linear waveguide or second linear waveguide.

19. Flow cell (200, 600), according to claim 18, characterized in that the waveguide parameter comprises at least one selected from the group consisting of: a cross-sectional profile, a refractive index difference, a mode matching and combinations thereof.

20. Flow cell (200, 600), according to any one of claims 1 to 19, characterized in that the first and second sets of nanowells are arranged in a polygonal arrangement.

21. Flow cell (200, 600), according to claim 20, characterized in that the polygonal arrangement comprises a rectangular arrangement or a hexagonal arrangement.

22. Flow cell (200, 600), according to claim 21, characterized in that the first and second sets of nanowells are arranged in a hexagonal arrangement, forming at least one hexagon, the hexagon including: first and second nanowells of the first set of nanowells, the first and second nanowells forming part of a first row of nanowells extending along the first linear waveguide; third, fourth and fifth nanowells of the second set of nanowells, the third, fourth and fifth nanowells forming part of a second row of nanowells extending along the second linear waveguide; and sixth and seventh nanowells of a third set of nanowells, the sixth and seventh nanowells forming part of a third row of nanowells that... Petition 870250002332, dated 10 / 01 / 2025, p. 28 / 33 5 / 9 extends along a third linear waveguide.

23. Flow cell (200, 600), according to any one of claims 1 to 21, characterized in that the first set of nanowells comprises a first row of nanowells, and in that the second set of nanowells comprises a second row of nanowells.

24. Flow cell (200, 600), according to claim 23, characterized in that each of the first and second rows of nanowells is aligned with at least one of the first and second linear waveguides.

25. Flow cell (200, 600), according to claim 24, characterized in that the first row of nanowells extends along the first linear waveguide, wherein the second row of nanowells extends along the second linear waveguide, wherein the first linear waveguide is parallel and adjacent to the second linear waveguide, and wherein the first row of nanowells is in phase with the second row of nanowells, the flow cell (200, 600) further comprising: a third linear waveguide that is parallel and adjacent to the second linear waveguide; and a third row of nanowells extending along the third linear waveguide, wherein the third row of nanowells is out of phase with the first and second rows of nanowells.

26. Flow cell (200, 600), according to claim 25, characterized in that it further comprises: a fourth linear waveguide that is parallel and adjacent to the third linear waveguide; and a fourth row of nanowells extending along the fourth linear waveguide, wherein the fourth row of nanowells is in phase with the third row of nanowells. Petition 870250002332, dated 10 / 01 / 2025, p. 29 / 33 6 / 9 27. Flow cell (200, 600), according to any one of claims 1 to 20, characterized in that the first and second linear waveguides are parallel and adjacent to each other, wherein the first set of nanowells comprises first and second rows of nanowells extending along the first linear waveguide on opposite sides thereof, and wherein the second set of nanowells comprises third and fourth rows of nanowells extending along the second linear waveguide on opposite sides thereof.

28. Flow cell (200, 600), according to any one of claims 1 to 27, characterized in that at least one nanowell of the first and second sets of nanowells has a non-circular aperture.

29. Flow cell (200, 600), according to claim 28, characterized in that the non-circular opening comprises an elliptical opening.

30. Flow cell (200, 600), according to any one of claims 1 to 29, characterized in that it further comprises a structure between the first and second linear waveguides to reduce cross-coupling.

31. Flow cell (200, 600), according to claim 30, characterized in that the structure comprises a series of blocks.

32. Flow cell (200, 600), according to claim 30, characterized in that the structure provides alternating refractive indices along the structure.

33. Flow cell (200, 600), according to any one of claims 1 to 32, characterized in that the first linear waveguide and the first grid are positioned in a first layer of the flow cell (200, 600), wherein the second linear waveguide and the second grid are positioned in a second layer of the flow cell (200, 600), wherein the first and second sets of nanowells are positioned in a third layer of the flow cell (200, 600), and wherein the second layer is positioned further away from the third layer than the first layer.

34. Method, characterized in that it comprises: applying, in a flow cell (200), a sample to a first set of nanowells and to a second set of nanowells, wherein the nanowells in at least one of the first and second sets of nanowells have a spacing between them that is resolvable according to an emission optics resolution distance for the flow cell (200); differentially coupling, using a first grating (202A), first light in at least one first linear waveguide (206A) associated with the first set of nanowells; and differentially coupling, using a second grating (202B), second light in at least one second linear waveguide (206B) associated with the second set of nanowells; wherein the first and second linear waveguides (206A, 206B) are positioned closer to each other than the emission optics resolution distance.

35. Method according to claim 34, characterized in that the first and second grids (202A, 202B) are spatially displaced relative to each other, wherein the direction of displacement is in a direction parallel to the linear waveguides (206A, 206B), the method further comprising controlling a lighting component (820) relative to at least one of the first light or second light.

36. Method according to claim 35, characterized in that the control of the lighting component (820) comprises controlling Petition 870250002332, dated 10 / 01 / 2025, page 31 / 33 8 / 9 a beam parameter (1404) of a light beam (1400) generating at least one of the first light or second light.

37. Method according to claim 36, characterized in that the beam parameter control (1404) comprises at least one selected from the group consisting of: controlling a light beam location (1400), controlling an angle of incidence (1406) of the light beam (1400), controlling a divergence (1408) of the light beam (1400), controlling a mode profile (1410) of the light beam (1400), controlling a polarization of the light beam (1400), controlling an aspect ratio (1412A) of the light beam (1400), controlling a diameter (1412B) of the light beam (1400), controlling a wavelength of the light beam (1400) and combinations thereof.

38. Method, according to any one of claims 34 to 37, characterized in that the first light is differentially coupled during a first scan performed through the flux cell (200) in a first scan direction, and the second light is differentially coupled during a second scan performed through the flux cell (200) in a second scan direction opposite to the first scan direction.

39. Method according to claim 34, characterized in that the first and second grids (302A, 302B) have different grid periods from each other, the method further comprises arranging a lighting component (920) so that the first light is differentially coupled, and arranging the lighting component (920) so that the second light is differentially coupled.

40. Method according to claim 39, characterized in that the first and second linear waveguides (306A, 306B) are positioned adjacent to each other, and wherein the flow cell further comprises Petition 870250002332, dated 10 / 01 / 2025, page 32 / 33 9 / 9 a third linear waveguide positioned adjacent to the second linear waveguide (306B) opposite from the first linear waveguide (306A).

41. Method according to claim 40, characterized in that the flow cell further comprises a third grid for the third linear waveguide.

42. Method according to claim 41, characterized in that it further comprises differentially coupling the first light also in the third linear waveguide using the third grid.

43. Method according to claim 41, characterized in that it further comprises differentially coupling a third light at least in the third linear waveguide using the third grid.

44. Method according to claim 40, characterized in that the third linear waveguide shares the first grid with the first linear waveguide.

45. A method according to any one of claims 34 to 44, characterized in that differentially coupling the first light comprises coupling the first light in the first linear waveguide and minimizing coupling of the first light in the second linear waveguide.

46. ​​Method according to claim 45, characterized in that differentially coupling the second light comprises coupling the second light to the second linear waveguide and minimizing coupling of the second light to the first linear waveguide. Petition 870250002332, dated 10 / 01 / 2025, p. 33 / 33

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