阅读说明：本技术 具有线性波导的流通池 (Flow cell with linear waveguide ) 是由 G·埃文斯 S·S·洪 于 2020-05-19 设计创作，主要内容包括：流通池例如包括：纳米阱层,该纳米阱层具有用以容纳样品的第一组纳米阱和第二组纳米阱；与第一组纳米阱相关联的第一线性波导,和与第二组纳米阱相关联的第二线性波导；以及用于第一线性波导的第一光栅,和用于第二线性波导的第二光栅,第一光栅和第二光栅提供第一光和第二光的差分耦合。(The flow cell includes, for example: a nanowell layer having a first set of nanowells and a second set of nanowells to hold 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 grating for the first linear waveguide and a second grating for the second linear waveguide, the first and second gratings providing differential coupling of the first and second light.)

1. A flow-through cell comprising:

a nanowell layer having a first set of nanowells and a second set of nanowells to hold 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 grating for the first linear waveguide, and a second grating for the second linear waveguide, the first and second gratings providing differential coupling of first and second light.

2. The flow cell of claim 1, wherein the first and second gratings are spatially offset from each other.

3. The flow cell of claim 2, wherein 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 the first linear waveguide.

4. The flow cell of claim 3, wherein the third linear waveguide shares the first grating with the first linear waveguide.

5. The flow cell of claim 3, further comprising a third grating for the third linear waveguide.

6. Flow cell according to claim 5, wherein the third grating has the same spatial offset from the second grating as the first grating does.

7. The flow cell of claim 5, wherein the third grating is spatially offset from each of the first and second gratings.

8. The flow cell of any one of claims 1 to 7, wherein the first grating is positioned towards a first end of the first linear waveguide, wherein the second grating is positioned towards a second end of the second linear waveguide, and wherein the first end is positioned opposite the second end.

9. The flow cell of any one of claims 1 to 8, wherein the first grating is positioned on a triangular substrate.

10. The flow cell according to any of claims 1 to 2 and 8 to 9, wherein the first and second gratings have grating periods different from each other.

11. The flow cell of claim 10, wherein 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 the first linear waveguide; and

a third grating for the third linear waveguide.

12. The flow cell of claim 11, wherein the third grating has the same grating period as the first grating.

13. The flow cell of claim 11, wherein the third grating has a grating period that is different from each of the grating period of the first grating and the grating period of the second grating.

14. The flow cell of any one of claims 1 to 13, wherein the nanowells in at least one of the first and second sets of nanowells have a pitch from each other that is resolvable according to a resolution distance of emission optics for the flow cell.

15. The flow cell of claim 14, wherein the first and second linear waveguides are positioned closer to each other than the resolution distance of the emitting optic.

16. The flow cell of any one of claims 1 to 15, wherein the differential coupling of the first light comprises: coupling the first light into the first linear waveguide; and minimizing coupling of the first light into the second linear waveguide.

17. The flow cell of claim 16, wherein the differential coupling of the second light comprises: coupling the second light into the second linear waveguide; and minimizing coupling of the second light into the first linear waveguide.

18. The flow cell of any one of claims 1 to 17, wherein the differential coupling is due at least in part to coupler parameters of one or more of the first grating or the second grating.

19. The flow cell of claim 18, wherein the coupler parameters comprise at least one selected from the group consisting of: refractive index, pitch, groove width, groove height, groove spacing, grating non-uniformity, groove orientation, groove curvature, coupler shape, and combinations thereof.

20. The flow cell of any one of claims 1 to 19, wherein the differential coupling is due at least in part to a waveguide parameter of one or more of the first linear waveguide or the second linear waveguide.

21. The flow cell of claim 20, wherein the waveguide parameters comprise at least one selected from the group consisting of: cross-sectional profile, refractive index difference, mode matching, and combinations thereof.

22. The flow cell of any one of claims 1 to 21, wherein the first and second sets of nanowells are arranged in a polygonal array.

23. The flow cell of claim 22, wherein the polygonal array comprises a rectangular array or a hexagonal array.

24. The flow cell of claim 23, wherein the first and second sets of nano-wells are arranged in the hexagonal array forming at least one hexagon comprising:

a first and second nanowell of the first set of nanowells that are part of a first row of nanowells extending along the first linear waveguide;

a third, fourth, and fifth nanowell of the second set of nanowells, the third, fourth, and fifth nanowell being part of a second row of nanowells extending along the second linear waveguide; and

a sixth and seventh nanowell of the third set of nanowells that are part of a third row of nanowells extending along a third linear waveguide.

25. The flow cell of any one of claims 1 to 23, wherein the first set of nano-wells comprises a first row of nano-wells, and wherein the second set of nano-wells comprises a second row of nano-wells.

26. The flow cell of claim 25, wherein each of the first and second rows of nano-wells is aligned with at least one of the first and second linear waveguides.

27. The flow cell of claim 26, wherein the first row of nano-wells extends along the first linear waveguide, wherein the second row of nano-wells 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 nano-wells is in phase with the second row of nano-wells, the flow cell further comprising:

a third linear waveguide parallel and adjacent to the second linear waveguide; and

a third row of nano-wells extending along the third linear waveguide, wherein the third row of nano-wells is out of phase with the first and second rows of nano-wells.

28. The flow cell of claim 27, further comprising:

a fourth linear waveguide parallel and adjacent to the third linear waveguide; and

a fourth row of nano-wells extending along the fourth linear waveguide, wherein the fourth row of nano-wells is in phase with the third row of nano-wells.

29. The flow cell of any one of claims 1 to 22, wherein the first and second linear waveguides are parallel and adjacent to each other, wherein the first set of nanowells comprises a first and second row of nanowells extending along the first linear waveguide on opposite sides of the first linear waveguide, and wherein the second set of nanowells comprises a third and fourth row of nanowells extending along the second linear waveguide on opposite sides of the second linear waveguide.

30. The flow cell of any one of claims 1 to 29, wherein at least one of the first and second sets of nanowells has a non-circular opening.

31. The flow-through cell of claim 30, wherein the non-circular opening comprises an elliptical opening.

32. The flow cell of any one of claims 1 to 31, further comprising a structure between the first linear waveguide and the second linear waveguide to reduce cross-coupling.

33. The flow cell of claim 32, wherein the structure comprises a series of blocks.

34. The flow cell of claim 32, wherein the structures provide alternating refractive indices along the structures.

35. The flow cell of any one of claims 1 to 34, wherein the first linear waveguide and the first grating are positioned in a first layer of the flow cell, wherein the second linear waveguide and the second grating are positioned in a second layer of the flow cell, wherein the first set of nanowells and the second set of nanowells are positioned in a third layer of the flow cell, and wherein the second layer is positioned further from the third layer than the first layer.

36. A method, comprising:

applying a sample to the first set of nanowells and the second set of nanowells at the flow cell;

differentially coupling first light into at least a first linear waveguide associated with the first set of nanowells using a first grating; and

differentially coupling second light into at least a second linear waveguide associated with the second set of nanowells using a second grating.

37. The method of claim 36, wherein the first and second gratings are spatially offset from each other, the method further comprising controlling an illumination assembly with respect to at least one of the first or second light.

38. The method of claim 37, wherein controlling the illumination assembly comprises controlling a beam parameter of a light beam that generates at least one of the first light or the second light.

39. The method of claim 38, wherein controlling the beam parameter comprises at least one selected from the group consisting of: controlling a position 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.

40. The method of any one of claims 36 to 39, wherein the first light is differentially coupled during a first scan and the second light is differentially coupled during a second scan, the first scan being performed across the flow cell in a first scan direction and the second scan being performed across the flow cell in a second scan direction opposite the first scan direction.

41. The method of claim 36, wherein the first and second gratings have grating periods that are different from each other, the method further comprising: arranging an illumination assembly such that the first light is differentially coupled; and arranging the illumination assembly such that the second light is differentially coupled.

42. The method of claim 41, wherein the first and second linear waveguides are positioned adjacent to each other, and wherein the flow cell further comprises a third linear waveguide positioned adjacent to the second linear waveguide opposite the first linear waveguide.

43. The method of claim 42, wherein the flow cell further comprises a third grating for the third linear waveguide.

44. The method of claim 43, further comprising also differentially coupling the first light into the third linear waveguide using the third grating.

45. The method of claim 43, further comprising differentially coupling third light into at least the third linear waveguide using the third grating.

46. The method of claim 42, wherein the third linear waveguide shares the first grating with the first linear waveguide.

47. The method of any one of claims 36 to 46, wherein the nanowells in at least one of the first and second sets of nanowells have a pitch from each other that is resolvable according to a resolution distance of emission optics for the flow cell.

48. The method of claim 47, wherein the first and second linear waveguides are positioned closer to each other than the resolution distance of the emitting optic.

49. The method of any one of claims 36 to 48, wherein differentially coupling the first light comprises: coupling the first light into the first linear waveguide and minimizing coupling of the first light into the second linear waveguide.

50. The method of claim 49, wherein differentially coupling the second light comprises: coupling the second light into the second linear waveguide and minimizing coupling of the second light into the first linear waveguide.

Background

Samples of different materials may be analyzed using one or more of a variety of analytical procedures. For example, sequencing, such as high throughput DNA sequencing, can be the basis for genomic analysis and other genetic studies. For example, sequencing-by-synthesis (SBS) techniques use modified deoxyribonucleotide triphosphates (dntps) that include a terminator and a fluorescent dye having an emission spectrum. In this and other types of sequencing, characteristics of a sample of genetic material are determined by illuminating the sample and detecting emitted light (e.g., fluorescence) generated in response to the illumination.

It may be desirable to ensure good quality of sample analysis and to facilitate performing the analysis at relatively high speeds. For example, the amount of sample material analyzed at each individual stage drives the throughput of the resulting analysis process. More dense distribution of sample material in the analytical instrument may be attempted 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 the imaging optics) may limit the extent to which this approach can increase throughput.

Disclosure of Invention

In a first aspect, a flow-through cell comprises: a nanowell layer having a first set of nanowells and a second set of nanowells to hold 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; a first grating for the first linear waveguide, and a second grating for the second linear waveguide, the first and second gratings providing differential coupling of the first and second light.

Implementations may include any or all of the following features. The first and second gratings are spatially offset 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 and opposite the first linear waveguide. The third linear waveguide shares the first grating with the first linear waveguide. The flow cell further comprises a third grating for a third linear waveguide. The third grating has the same spatial offset from the second grating as the first grating does. The third grating is spatially offset from each of the first and second gratings. The first grating is positioned towards a first end of the first linear waveguide, wherein the second grating is positioned towards a second end of the second linear waveguide, and wherein the first end is positioned opposite the second end. The first grating is positioned on the triangular substrate. The first grating and the second grating have grating periods different 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 and opposite the first linear waveguide; a third grating for a third linear waveguide. The third grating has the same grating period as the first grating. The third grating has a grating period different from each of the grating periods of the first and second gratings. The nanowells in at least one of the first and second sets of nanowells are spaced apart from each other with a pitch that is resolvable according to a resolution distance of emission optics for the flow cell. The first and second linear waveguides are positioned closer to each other than a resolution distance of the transmitting optic. The differential coupling of the first light includes coupling the first light into the first linear waveguide and minimizing coupling of the first light into the second linear waveguide. The differential coupling of the second light includes coupling the second light into the second linear waveguide and minimizing the coupling of the second light into the first linear waveguide. The differential coupling is due at least in part to coupler parameters of one or more of the first grating or the second grating. The coupler parameters include at least one selected from the group consisting of: refractive index, pitch, groove width, groove height, groove spacing, grating non-uniformity, groove orientation, groove curvature, coupler shape, and combinations thereof. The differential coupling is due, at least in part, to waveguide parameters of one or more of the first linear waveguide or the second linear waveguide. The waveguide parameters include at least one selected from the group consisting of: cross-sectional profile, refractive index difference, mode matching, and combinations thereof. The first set of nanowells and the second set of nanowells are arranged in a polygonal array. The polygonal array includes a rectangular array or a hexagonal array. The first and second sets of nanowells are arranged in a hexagonal array forming at least one hexagon comprising: first and second nanowells in the first set of nanowells, the first and second nanowells being part of a first row of nanowells extending along the first linear waveguide; a third, fourth, and fifth nanowell of the second set of nanowells, the third, fourth, and fifth nanowell being part of a second row of nanowells extending along the second linear waveguide; and sixth and seventh nano-wells of the third set of nano-wells, the sixth and seventh nano-wells being part of a third row of nano-wells extending along a third linear waveguide. The first set of nanowells comprises a first row of nanowells, and wherein 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. A first row of nanowells extending along the first linear waveguide, wherein a second row of nanowells extends along the second linear waveguide, wherein the first linear waveguide is parallel to and adjacent to the second linear waveguide, and wherein the first row of nanowells are in phase with the second row of nanowells, the flow cell further comprising: a third linear waveguide parallel to 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-through cell further comprises: a fourth linear waveguide parallel to 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. The first and second linear waveguides are parallel and adjacent to each other, wherein the first set of nanowells comprises a first and second row of nanowells extending along opposite sides of the first linear waveguide, and wherein the second set of nanowells comprises a third and fourth row of nanowells extending along opposite sides of the second linear waveguide. At least one of the first set of nanowells and the second set of nanowells has a non-circular opening. The non-circular opening comprises an elliptical opening. The flow cell further comprises a structure between the first linear waveguide and the second linear waveguide to reduce cross-coupling. The structure includes a series of blocks. The structure provides alternating refractive indices along the structure. The first linear waveguide and the first grating are positioned in a first layer of the flow cell, wherein the second linear waveguide and the second grating are positioned in a second layer of the flow cell, wherein the first set of nanowells and the second set of nanowells are positioned in a third layer of the flow cell, wherein the second layer is positioned further from the third layer than the first layer.

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

Implementations may include any or all of the following features. The first and second gratings are spatially offset from each other, the method further comprising controlling the illumination assembly with respect to at least one of the first light or the second light. Controlling the illumination assembly includes controlling a beam parameter of a light beam that generates 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 position 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 and the second light is differentially coupled during a second scan, the first scan being performed across the flow cell in a first scan direction and the second scan being performed across the flow cell in a second scan direction opposite the first scan direction. The first grating and the second grating have grating periods different from each other, the method further comprising: arranging the illumination assembly such that the first light is differentially coupled; and arranging the illumination assembly such that the second light is differentially coupled. The first and second linear waveguides are positioned adjacent to each other, and wherein the flow cell further comprises a third linear waveguide positioned adjacent to the second linear waveguide and opposite the first linear waveguide. The flow cell further comprises a third grating for a third linear waveguide. The method also includes differentially coupling the first light into a third linear waveguide using a third grating. The method also includes differentially coupling third light into at least a third linear waveguide using a third grating. The third linear waveguide shares the first grating with the first linear waveguide. The nanowells in at least one of the first and second sets of nanowells are spaced apart from each other with a pitch that is resolvable according to a resolution distance of emission optics for the flow cell. The first and second linear waveguides are positioned closer to each other than a resolution distance of the transmitting optic. Differentially coupling the first light includes coupling the first light into the first linear waveguide and minimizing coupling of the first light into the second linear waveguide. Differentially coupling the second light includes coupling the second light into the second linear waveguide and minimizing coupling of the second light into the first linear waveguide.

It should be understood that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided that such concepts do not contradict each other) are considered a part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are considered part of the inventive subject matter disclosed herein.

Drawings

Fig. 1 shows a cross-section of a portion of an example flow cell with a linear waveguide.

Fig. 2A-2B illustrate examples of flow cells with staggered gratings.

Fig. 3A to 3B illustrate examples of flow cells with gratings of different grating periods.

Figure 4 shows another example of a flow cell with an interleaved grating.

Fig. 5 shows a cross-section of a portion of an example flow cell.

Figure 6 shows an example of a flow cell in which multiple linear waveguides share a common grating.

FIG. 7 is a diagram of an example illumination system.

Fig. 8-9 are flowcharts of example methods.

Figure 10A shows an example of a hexagonal array of non-circular nanowells.

Fig. 10B shows an example of a triangular array of circular nanowells.

Figure 11 shows another example of a flow cell with an interleaved grating.

Figure 12 shows another example of a flow cell with an interleaved grating.

Figure 13 shows another example of a flow cell with an interleaved grating.

Fig. 14 schematically shows a light beam impinging on a surface.

Fig. 15A to 15C show examples of gratings.

Fig. 16 shows an example of the shape of the coupler.

Fig. 17 shows an example of a cross-sectional profile for a linear waveguide.

Fig. 18 shows a cross-section of a portion of another example flow cell with a linear waveguide.

FIG. 19 is a flow chart of an example method.

Detailed Description

The present disclosure describes systems, techniques, articles of manufacture, and/or compositions of matter that facilitate improved analysis of samples. In some embodiments, differential coupling may be provided into two or more linear waveguides. For example, being able to differentially couple light into a linear waveguide may allow a substrate (e.g., a nanowell layer for holding sample material) to have an increased density of sample material. In some embodiments, one or more parameters relating to the analysis system and/or process may be selected or adjusted in order to obtain 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 a combination thereof.

In some embodiments, analytical imaging may be performed on sample materials having increased distribution densities on a substrate, which may increase throughput of the analytical process. For example, the sample material may be distributed at a density such that: where individual portions of the sample are positioned closer to each other than can be resolved using available imaging techniques, such as microscopy instruments. The analysis process may selectively image only a first portion of the sample at a time without imaging a second portion adjacent to the first portion, and then imaging the second portion without (re-) imaging the first portion. Such a method may allow a relatively large amount of sample material on a single sample holder (e.g., substrate) to be imaged and analyzed in a single pass. This may increase the throughput of the analysis process compared to the following method: a method of replacing a substrate for analysis of additional sample material on a new substrate after analysis of the entire sample material of the substrate (which may involve intermediate steps of substrate removal and insertion, sample preparation and instrument initialization).

In some embodiments, for example, differential coupling between the first and second linear waveguides may include coupling light into the first linear waveguide without coupling any light into the second linear waveguide, and vice versa. Such differential coupling may not always be practical or possible. In some embodiments, the differential coupling may involve: during a portion of the scan, coupling to, for example, the second linear waveguide is minimized while coupling light to the first linear waveguide. The amount or fraction of minimization may vary depending on the implementation. In some embodiments, the minimized coupling (e.g., crosstalk) corresponds to at most about 1%, about 5%, about 15%, about 25%, or about 45% of the coupling into the linear waveguide. Such differential coupling may not always be practical or possible. In some embodiments, the differential coupling may involve: during a portion of the scan, coupling to the second linear waveguide is reduced as compared to the first linear waveguide. The amount or proportion of reduction may vary depending on the implementation. In some embodiments, the reduced coupling (e.g., crosstalk) corresponds to at most about 5%, about 15%, about 35%, about 65%, or about 95% of the coupling into the linear waveguide.

The amount of crosstalk (e.g., its magnitude) may be known or calibrated. In some embodiments, multiple scans of the sample may be performed, such as a first scan coupled into a first linear waveguide, wherein coupling into a second linear waveguide is reduced, and a second scan coupled into the second linear waveguide, wherein coupling into the first linear waveguide is reduced. Scanning may cause modulation of information to be obtained from the first and second linear waveguides, respectively. This modulation may occur in a predictable manner, given the magnitude of the crosstalk. For example, linear algebra may be applied to information obtained from the respective first and second linear waveguides to extract useful analysis information.

The limit imposed by the maximum available resolution of the imaging instrument may be referred to as the diffraction limit. It can therefore be said that imaging systems operating at the maximum resolution available are diffraction limited. For microscopic instruments, the spatial resolution that can be obtained with a given diffraction limit depends on the wavelength of the light and on the numerical aperture of the objective or illumination source. The minimum resolvability distance d may be expressed as d ═ λ/(2nsin θ), where λ is the wavelength of light, n is the refractive index, and θ is the half angle (i.e., half the angle between the microscope optical axis and the direction of the most oblique ray captured by the objective lens). The factor nsin θ is commonly referred to as Numerical Aperture (NA), and thus the minimum resolvable distance may be expressed as d ═ λ/(2 NA). That is, in existing analytical systems, the sample material is typically distributed at a density such that: such that individual portions of the sample are separated by at least a distance d. The systems and techniques described herein may allow analysis of sample materials that are more densely distributed than the resolution distance d.

Sample analysis can include, but is not limited to, genetic sequencing (e.g., determining the structure of genetic material), genotyping (e.g., determining differences in the genetic composition of an individual), gene expression (e.g., synthesizing gene products using genetic information), proteomics (e.g., large scale studies of proteins), or a combination thereof.

Some examples described herein relate to sequencing of genetic material. Sequencing can be performed on a sample to determine which component blocks (called nucleotides) constitute a particular genetic material in the sample. The genetic material may be first purified and then repeated several times to prepare a sample of appropriate size before sequencing. Imaging may be performed as part of the genetic material sequencing process. This may involve fluorescence imaging, in which a sample of the genetic material is subjected to light (e.g. a laser beam) to trigger a fluorescence reaction by one or more markers on the genetic material. Certain nucleotides of the genetic material may have a fluorescent label applied thereto, allowing the presence of the nucleotide to be determined by illuminating light onto the sample and looking for a characteristic response from the sample. The fluorescent response can be detected during the sequencing process and used to establish a record of the nucleotides in the sample.

Examples described herein relate to flow-through cells. A flow cell may be considered a substrate that can be used to prepare and contain or carry one or more samples during at least one stage of an analytical process. The flow cell is made of a material compatible with: sample material (e.g., genetic material), radiation, and chemical reactions to which the flow cell is exposed. The substrate may have one or more channels in which sample material may be deposited. A substance (e.g., a liquid) may flow through the channel where the sample genetic material is present to trigger one or more chemical reactions and/or remove unwanted material. The flow cell may be imaged by: the sample in the flow cell channel is facilitated to be able to be subjected to illumination light and any fluorescent response from the sample can be detected. Some embodiments of the system may be designed for use with at least one flow cell, but may not include flow cell(s) during one or more stages, such as during shipping or when delivered to a customer. For example, the flow cell(s) may be installed into an embodiment at the customer site in order to perform the analysis.

Examples herein relate to coupling light (e.g., a laser beam) into and/or out of a waveguide through one or more gratings. The grating may couple light impinging on the grating by diffracting at least a portion of the light, thereby causing the at least a portion of the light to propagate in one or more other directions. In some embodiments, coupling may involve one or more interactions, including but not limited to reflection, refraction, diffraction, interference, and/or transmission of the at least a portion of light. Embodiments 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. The two or more gratings may be identical or similar to each other, or different types of gratings may be used. The grating(s) may comprise one or more forms of periodic structures. In some embodiments, the grating may be formed by removing or omitting material from the substrate (e.g., from waveguide material included in the flow cell) or other material. For example, the flow cell may be provided with a set of slits and/or grooves therein to form a grating. In some embodiments, the grating may be formed by adding a substance to the flow cell (e.g., to a waveguide material included in the flow cell) or other material. For example, the flow cell may be provided with a longitudinal structure of a set of ridges, bands or other protrusions to form a grating. A combination of these approaches may be used.

Providing the waveguide in a substrate, such as a flow cell, may provide one or more advantages. Excitation using evanescent light based on Total Internal Reflection (TIR) can provide higher illumination efficiency. In some previous methods, the entire laser beam is used to illuminate the substrate holding the sample, such as during scanning. This approach may result in most of the light waves simply propagating through the substrate without effectively illuminating the sample. As a result, only a small portion of the light applied by such a system can actually be used to excite fluorophores in the sample. By contrast, evanescent light may penetrate material (e.g., the cladding of an adjacent core layer) only to a certain depth (e.g., about 150-200nm in one example). For example, the flow cell may be designed with one or more nano-wells configured such that the evanescent field is largely confined to the well region. Therefore, evanescent light may be a very efficient way to excite fluorophores. For example, a system operating according to an earlier irradiation method may involve a laser having a certain power; by contrast, a much lower laser power may be sufficient using evanescent light.

Examples herein relate to chemical vapor deposition. Chemical Vapor Deposition (CVD) may include all such techniques: in which a volatile material (sometimes referred to as a precursor) is caused to react and/or decompose on the surface of a substrate to form a deposit on the substrate. CVD can be characterized by one or more aspects. For example, CVD can be characterized by the physical property(s) of the vapor (e.g., whether CVD is aerosol-assisted or involves direct liquid injection). For example, CVD may be characterized by the type of substrate heating (e.g., whether the substrate is directly heated or indirectly heated such as through a heating chamber). Examples of CVD types that may be used include, but are not limited to: atmospheric pressure CVD, low pressure CVD, ultra high vacuum CVD, metal organic CVD, laser assisted CVD, and plasma enhanced CVD.

Examples herein relate to atomic layer deposition. Atomic layer deposition can be considered a form of CVD and includes all techniques for growing films on substrates by exposure to gases. For example, gaseous precursors may be alternately introduced into the chamber. Molecules of one of the precursors can react with the surface until one layer is formed and the reaction is terminated, then the next gaseous precursor can be introduced to begin forming a new layer, and so on in one or more cycles.

Examples herein relate to spray coating. Spraying may include any or all techniques by which a particular material may be deposited onto a substrate. This may include, but is not limited to: thermal spraying, plasma spraying, cold spraying, warm spraying, and/or other processes that involve atomizing or nebulizing a material.

Examples herein relate to spin coating. Spin coating may include applying a quantity of coating material onto a substrate and distributing or spreading the coating material over the substrate by centrifugal force due to rotation or spinning of the substrate.

Examples herein relate to nanoimprinting. In nanoimprint lithography, a pre-fabricated nanoscale template may mechanically displace a fluid resin to mold the desired nanostructures. The resin may then be cured with the nanoscale template in place. After removal of the nanoscale template, a molded solid resin may be produced that adheres to the desired substrate. In some embodiments, the nanoimprinting process can begin by completely or partially covering the substrate or wafer with an imprint resin (e.g., the resins exemplified below). A nanoscale template may be used to form one or more nanostructures in an imprint resin during a molding process. The imprint resin may be cured to the substrate or wafer, and a resin removal process may be applied to remove residue from the wafer or substrate. For example, resin removal may form a chamber scribe lane for adjacent nanostructures. The substrate or wafer so formed may have another substrate or gasket applied thereto in order to form a flow cell with the described nanostructures and a flow cell chamber formed by closing the chamber scribe lane. In some embodiments, the process of applying the imprint resin may be configured to produce little or no resin residue, and in such embodiments, the resin removal process may be omitted. In some applications, chemical treatment or attachment of biomolecules may also be utilized to functionalize the cured resin, depending on the end use. In nanoimprint lithography, the imprinted photoresist may be a sacrificial material and similarly used as an intermediate tool for transferring the patterned resist into the substrate, or variations of the resist may be used such that the imprinted resist is used as an input for performing subsequent coating steps. One example of a resist that will remain after patterning is a material formed by the following process, sometimes referred to as a sol-gel based material: the monomer is converted to a colloidal solution as a precursor for the particles and/or polymer gel.

Examples herein relate to a substrate. Substrate may refer to any material that provides an at least substantially rigid structure, orRefers to a structure that is to retain its shape rather than assume the shape of a container with which it is placed in contact. The material may have a surface to which another material may be attached, including, for example, smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces), and textured and/or porous materials. Possible substrates include, but are not limited to: glass and modified or functionalized glasses, plastics (including acrylic, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon^TMEtc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glass, plastics, fiber optic strands, and various other polymers. Typically, the substrates allow optical detection and do not themselves emit significant fluorescence.

Examples herein relate to polymers. The polymer layer may comprise a film of a polymer material. Exemplary films forming polymers include, but are not limited to: acrylamide or copolymers having C1-C12; aromatic and hydroxy derivatives; an acrylate copolymer; vinyl pyrrolidine and vinyl pyrrolidone copolymers; sugar-based polymers such as starch or polydextrose; or other polymers such as polyacrylic acid, polyethylene glycol, polylactic acid, silicone, siloxane, polyvinylamine, guar gum, carrageenan, alginate, lotus bean gum, methacrylate copolymers, polyimide, cyclic olefin copolymers, or combinations thereof. In some embodiments, the polymer layer comprises at least one photocurable polymer. For example, the photocurable polymer may include urethane, acrylate, silicone, epoxy, polyacrylic, polyacrylate, epoxysiloxane, epoxy, Polydimethylsiloxane (PDMS), silsesquioxane, acyloxysilane, maleate polyester, vinyl ether, monomer or copolymer having vinyl or ethynyl groups, or a combination thereof. In some embodiments, the layer may comprise a covalently attached polymer coating. For example, it may include a polymer coating that forms a chemical bond with the functionalized surface of the substrate, as opposed to being attached to the surface in other ways, such as adhesion or electrostatic interaction. In some embodiments, the polymer included in the functionalizable layer is poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide), sometimes referred to as PAZAM.

The examples described herein mention that one or more resins may be used. Any suitable resin may be used for nanoimprinting in the methods described herein. In some embodiments, organic resins may be used, including but not limited to: acrylic resins, polyimide resins, melamine resins, polyester resins, polycarbonate resins, phenol resins, epoxy resins, polyoxymethylene resins, polyether resins, polyurethane resins, polyamide resins (and/or nylons), furan resins, terephthalate resins, or combinations thereof. In some examples, the resin may include an inorganic siloxane polymer including Si-O-Si bonds between compounds (including silicon, oxygen, and hydrogen), and is formed by using a siloxane polymer-based material typified by silica glass as a raw material. The resin used may also or alternatively be an organosiloxane polymer in which the hydrogen bonded to the silicon is substituted with an organic group such as a methyl or phenyl group, and is represented by an alkylsiloxane polymer, an alkylsilsesquioxane polymer, a silsesquioxane hydride polymer, or an alkylsilsesquioxane hydride polymer. Non-limiting examples of siloxane polymers include: polyhedral oligomeric silsesquioxanes (POSS), Polydimethylsiloxane (PDMS), Tetraethylorthosilicate (TEOS), poly (organo) siloxanes (silicones), and perfluoropolyethers (PFPEs). The resin may be doped with a metal oxide. In some embodiments, the 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 a suitable solvent is used. Any of a variety of other resins may be suitably employed depending on the application.

FIG. 1 shows a cross-section of a portion of an example flow cell 100 having linear waveguides 102A-102C. The flow cell 100 can be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For illustrative purposes, only a portion of the flow cell 100 is shown. For example, one or more additional layers and/or more or fewer waveguides 102A-102C may be used.

The flow cell 100 comprises a substrate 104. The substrate 104 may form the base of the flow cell 100. In some embodiments, one or more other layers may be formed at (e.g., in contact with or near) substrate 104 in the fabrication of flow cell 100. Substrate 104 may serve as a foundation for forming linear waveguides 102A-102C. The linear waveguides 102A-102C may initially exist separate from the substrate 104 and then be applied to the substrate 104, or the linear waveguides 102A-102C may be formed by applying and/or removing one or more materials from the substrate. The linear waveguides 102A-102C may be formed directly on the substrate 104 or on one or more intermediate layers at the substrate 104.

The linear waveguides 102A-102C are for conducting electromagnetic radiation (including, but not limited to, visible light, such as laser light). In some embodiments, the electromagnetic radiation performs one or more functions during the imaging process. For example, electromagnetic radiation may be used to excite fluorophores in the sample material for imaging. Linear waveguides 102A-102C may be made of any suitable material that facilitates propagation of one or more types of electromagnetic radiation. In some embodiments, the material(s) of linear waveguides 102A-102C may comprise a polymeric material. In some embodiments, the material(s) of linear waveguides 102A-102C may include Ta₂O₅And/or SiN_x. For example, the linear waveguides 102A-102C may be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating, and/or spray coating.

Each linear waveguide 102A-102C may have one or more gratings (omitted here for clarity) to couple electromagnetic radiation into and/or out of the linear waveguides 102A-102C. One or more directions of travel of electromagnetic radiation in linear waveguides 102A-102C may be employed. For example, the direction of travel may be into and/or out of the plane of the present example. Examples of gratings are described elsewhere herein.

Each of the linear waveguides 102A-102C may be positioned against one or more types of cladding layers. The cladding layers may serve to confine electromagnetic radiation to the respective linear waveguides 102A-102C and prevent or reduce the extent to which radiation propagates into other linear waveguides 102A-102C or other substrates. Here, the cladding layers 106A-106D are shown as examples. For example, the cladding layers 106A-106B may be positioned against or near the linear waveguide 102A on different (e.g., opposite) sides of the linear waveguide 102A. For example, the cladding layers 106B-106C may be positioned against or near the linear waveguide 102B on different (e.g., opposite) sides of the linear waveguide 102B. For example, the cladding layers 106C-106D may be positioned against or near the linear waveguide 102C on different (e.g., opposite) sides of the linear waveguide 102C. The cladding layers 106A-106D may be made of one or more suitable materials for separating the linear waveguides 102A-102C from each other. In some embodiments, the cladding layers 106A-106D may be made of a material having a lower index of refraction than one or more of the linear waveguides 102A-102C. For example, linear waveguides 102A-102C may have a refractive index of about 1.4-1.6, and claddings 106A-106D may have a refractive index of about 1.2-1.4. In some embodiments, one or more of the cladding layers 106A-106D comprise a polymeric material. In some embodiments, one or more of the cladding layers 106A-106D include a plurality of structures, including but not limited to structures of one material (e.g., a polymer) interspersed with regions of vacuum or another material (e.g., air or liquid).

The flow cell 100 comprises at least one nanowell layer 108. In some embodiments, the nanowell layer 108 is positioned opposite the linear waveguides 102A-102C from the substrate 104. For example, the nanowall layers may be positioned adjacent (e.g., abutting or close to) the linear waveguides 102A-102C and the cladding layers 106A-106D. The nanowell layer 108 comprises one or more nanowells. In some embodiments, the nanowell layer 108 comprises nanowells 108A-108C. The nanowells 108A-108C may be used to hold one or more sample materials during at least a portion of an analytical process (e.g., for imaging). For example, one or more genetic materials (e.g., in clusters) may be placed in the nanowells 108A-108C.

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

The nanowells 108A-108C may be formed from the nanowell layer 108 by nanoimprinting into the nanowell layer 108 or by a lift-off process. For example, the nanowell layer 108 may comprise a resin, and the nanowells 108A-108C may be formed by imprinting using a nanoscale template. In some embodiments, the nanowells 108A-108C may have dimensions such that: such that one or more of its dimensional ranges is within one or more nanometer orders of magnitude. The ends (e.g., bottoms) of the nanowells 108A-108C may have a thickness that accommodates propagation of evanescent light. For example, the thickness may be about 0-500 nm. The nanowall layer may cover at least substantially the entire facing surface of the layer comprising linear waveguides 102A-102C and cladding layers 106A-106D. In some embodiments, the nanowell layer 108 may have an average pitch between nanowells 108A-108C of at least 10nm, 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 to 0.1 μm or less. In some embodiments, the nanowell layer 108 may have a pitch of about 150nm or greater between the nanowells 108A-108C. For example, the nanowell layer 108 may have a pitch of about 160nm, 220nm, 250nm, 300nm, 450nm, or more, between the nanowells 108A-108C. The depth of each nanowell 108A-108C may be at least 0.1 μm, 1 μm, 10 μm, 100 μm, or greater. Alternatively or additionally, the depth may be at most 1 × 10³μ m, 100 μm, 10 μm, 1 μm, 0.1 μm or less.

Fig. 2A-2B illustrate examples of a flow cell 200 with an interleaved grating 202. Flow cell 200 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For illustrative purposes, only a portion of the flow cell 200 is shown.

The flow cell 200 includes a plurality of nano-wells, including nano-well 204A, which is illustrated herein using a circular shape. Only some of the nanowells will be specifically mentioned, while other nanowells may be similar or identical to the nanowell(s) in question. The nanowells may be formed in a nanowell layer (e.g., by a nanoimprint or lift-off process). For example, a nanoscale template may be used to form a nanowell in a resin. For clarity, the nanowell layer is not explicitly shown in this example. The nano-trap 204A is here associated with a linear waveguide 206A. In some embodiments, the linear waveguide described with reference to flow cell 200 may be similar or identical to one or more other linear waveguides described herein. For example, the linear waveguide 206A is positioned adjacent to (e.g., in contact with or proximate to) a nanowell layer that includes the nanowell 204A. In some implementations, the linear waveguide 206A can include a linear waveguide core 208 and one or more gratings 202.

Another nanowell 204B is also associated with linear waveguide 206A. For example, the nanowell 204B is positioned adjacent to the nanowell 204A, and both nanowells 204A-204B can interact with the linear waveguide 206A during imaging (e.g., by receiving electromagnetic radiation from the linear waveguide 206A). By contrast, another nanowell 204C is alternatively associated with a linear waveguide 206B. In some embodiments, linear waveguide 206B is positioned adjacent to linear waveguide 206A. For example, cladding (not shown) and/or another material may be positioned between linear waveguides 206A-206B.

Some examples described herein refer to or otherwise relate to sets of nanowells. A set of nanowells is a logical or physical group of one or more nanowells having at least one characteristic. One set of nanowells may be associated with one linear waveguide and another set of nanowells may be associated with another linear waveguide. In some implementations, a set of nanowells may be arranged in a row. Such a row of nanowells may extend along the linear waveguide, such as by being coextensive with the linear waveguide (e.g., completely overlapping above or below the linear waveguide) or by being parallel to the linear waveguide and positioned adjacent to the linear waveguide (e.g., on either or both sides thereof), to name a few examples. Thus, in some embodiments, a set of nanowells may comprise one or more rows of nanowells. Each of the rows of nanowells may be aligned with at least one linear waveguide.

The nanowells may be arranged on the substrate (e.g., in a nanowell layer) in a substantially random manner and in at least one instance in a completely random manner or according to one or more patterns. In some embodiments, the nanowells are arranged in one or more arrays (including but not limited to polygonal arrays). For example, the polygonal array may be a rectangular, triangular, or hexagonal array, or any other form of array in which at least some of the nanowells are arranged in a polygonal shape. In this example, the flow cell 200 has a rectangular array of nanowells.

The flow cell 200 may be used in one or more forms of imaging. For example, sample material in a nanowell (including nanowells 204A-204C) may be subjected to electromagnetic radiation from a corresponding linear waveguide (including linear waveguides 206A-206B, respectively). The emissions resulting from such exposure to electromagnetic radiation (an example of an emission is fluorescence from fluorophores) may be captured using an instrument (e.g., one or more cameras and/or other imaging devices). Such instruments are sometimes referred to by the expression "transmitting instruments" or similar terms. For example, the emitting instrument may include one or more cameras or other image sensors and at least one lens or other emitting optics. In some embodiments, the diffraction limit may be due, at least in part, to one or more characteristics of the emission optics. For example, based on the emitting optics used, a resolution distance may be defined that marks the shortest distance that can be resolved using the emitting optics. That is, when resolving features separated by a resolution distance, the imaging system can be said to operate at its highest available resolution level.

Here, distance 210 is less than the resolution distance of the emitting optics, and distance 212 is greater than or about equal to the resolution distance of the emitting optics. Distance 210 here represents the spacing between the nanowells in one direction. In some embodiments, this may be a direction across the linear waveguide. For example, because the linear waveguides are here aligned in one direction (e.g., a vertical direction as seen in the illustration) with the rows of nanowells, distance 210 may also represent the distance between adjacent linear waveguides (e.g., linear waveguides 206A-206B). For example, the nanowells 204A and 204C are separated by a distance 210. That is, linear waveguides 206A-206B are positioned closer to each other than the resolution distance of the emitting optics.

Here, the distance 212 represents a spacing between the nano-wells in another direction different from the distance 210. For example, distances 210 and 212 may be substantially perpendicular to each other, and in at least one instance may be substantially perpendicular to each other. In some embodiments, this may be a direction along any individual one of the linear waveguides. For example, since here the linear waveguide is aligned with the row of nanowells in one direction (e.g., the vertical direction as seen in the illustration), distance 212 may represent the distance between adjacent nanowells on any linear waveguide (e.g., linear waveguides 206A-206B). For example, the nanowells 204A and 204B are separated by a distance 212. That is, the nanowells associated with the linear waveguide 206A have a spacing from each other that is resolvable according to the resolution distance of the emission optics for the flow cell 200.

The grating 202 is used to couple electromagnetic radiation into and/or out of the linear waveguide of the flow cell 200. Here, the linear waveguide 206A has a grating 202A, and the linear waveguide 206B has a grating 202B. The gratings 202A-202B may have the same or different periodic structures. In some implementations, either or both of the gratings 202A-202B may include periodic ridge structures interspersed with another material. For example, the ridges of the gratings 202A-202B may have a pitch of about 200 and 300nm, to name just one example.

The gratings 202A-202B may have one or more characteristics that facilitate selective coupling of electromagnetic radiation into the corresponding linear waveguides 206A-206B. In some implementations, one or more gratings 202 are spatially offset from one or more other gratings 202. The offset may be in a direction parallel to the linear waveguides 206A-206B. For example, here, the distance between grating 202B and the closest of the nanotrap associated with linear waveguide 206B is greater than the distance between grating 202A and the closest of the nanotrap associated with linear waveguide 206A. The spatially offset nature of the gratings 202A-202B facilitates coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguide 206A) without coupling electromagnetic radiation (e.g., light) into another of the linear waveguides (e.g., linear waveguide 206B).

The flow cell 200 may include a plurality of linear waveguides, for example as illustrated. In some implementations, linear waveguide 206C is positioned adjacent to linear waveguide 206B and opposite linear waveguide 206A. For example, the linear waveguide 206C may have a grating 202C. In some implementations, grating 202C may be spatially offset from grating 202B. For example, the grating 202C may have the same spatial offset from the grating 202B in a direction parallel to the linear waveguide 206C as the grating 202A in a direction parallel to the linear waveguide 206A.

The spatially offset nature of the gratings 202A and 202C and the grating 202B facilitates coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguide 206A or 206C) without coupling the electromagnetic radiation (e.g., light) into the other of the linear waveguides (e.g., linear waveguide 206B). As another example, the characteristic facilitates coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguide 206B) without coupling electromagnetic radiation (e.g., light) into at least one other of the linear waveguides (e.g., linear waveguides 206A or 206C).

Here, the light area 214 is schematically illustrated as a rectangle having a dashed outline. Light region 214 represents one or more locations that are impinged upon by light or other electromagnetic radiation as part of the imaging process. In some implementations, illumination light generated by the laser can be guided at the light region 214 to be ultimately coupled into some linear waveguides. For example, the laser may be selected so as to correspond to the fluorescent properties of one or more fluorophores in the sample material.

Here, the image capturing area 216 is schematically illustrated as a rectangle having a dashed outline. Image capture area 216 represents the field of view of the emission optics. For example, a camera or other image sensor may capture one or more types of emissions (e.g., fluorescence) emanating from image capture area 216.

The examples described above illustrate that the flow cell 200 includes a nanowell layer having a first set of nanowells (e.g., the nanowells associated with linear waveguide 206A) and a second set of nanowells (e.g., the nanowells associated with linear waveguide 206B) to accommodate a sample. Flow cell 200 includes a first linear waveguide (e.g., linear waveguide 206A) aligned with a first set of nanowells and a second linear waveguide (e.g., linear waveguide 206B) aligned with a second set of nanowells; and a first grating (e.g., grating 202A) for the first linear waveguide and a second grating (e.g., grating 202B) for the second linear waveguide. The first grating has a first characteristic (e.g., spatially offset from the grating 202B) to facilitate coupling the first light into the first linear waveguide without coupling the first light into the second linear waveguide.

The image capture process may include one or more scanning operations. In some embodiments, the image capture region 216 may be overlaid on one or more regions of the flow cell 200 to facilitate image capture with respect to one or more nanowells in the image capture region 216. Positioning may include moving the image capture area 216, or the flowcell 200, or both. For example, the emission optics may be relatively fixed in the analytical instrument such that the image capture region 216 does not move during various scanning operations. For example, the flow cell 200 can be moved (e.g., by being positioned on a motorized stage that facilitates precise movement in at least one direction) into one or more scanning positions relative to the image capture area 216. Here, arrow 218 schematically illustrates that the flow cell 200 may be moved such that the image capture region 216 covers at least some of the linear waveguide and its associated nanowell.

The light region 214 may remain fixed with the image capture region 216 or move corresponding to the image capture region 216, or may move independently of the image capture region 216. In this example, the optical region 214 is aligned with some of the gratings 202 (e.g., with gratings 202A and 202C), but is misaligned with some other gratings (e.g., with grating 202B). For example, when scanning in the direction of arrow 218 from the current position of light region 214, gratings 202A and 202C (and other gratings having similar spatial offsets) will be illuminated by light impinging on light region 214, while some other gratings (e.g., grating 202B) will not be illuminated by light impinging on light region 214. Thus, illumination light will be coupled into linear waveguides 206A and 206C (and other linear waveguides having gratings like spatial offsets), but light will not be coupled into linear waveguide 206B (and other linear waveguides having gratings like spatial offsets). This may facilitate selective illumination of the nanowells of the flow cell 200. For example, because the linear waveguides 206A and 206C have light coupled into them, the excitation light can reach the nanowells 204A and 204C associated with the linear waveguide 206A, and the nanowell 204D associated with the linear waveguide 206C. On the other hand, the excitation light will not reach the nanowell 204C because it is associated with a linear waveguide 206B into which no light is currently coupled. As such, although some portions of the sample material (e.g., within the nanowells 204A and 204C) are positioned at a distance 210 from each other, imaging can be successfully performed; i.e. closer to each other than the resolution of the emitting optics. During a scan corresponding to the movement represented by arrow 218 (which may be characterized as a line scan), only a particular subset of the linear waveguides may have light coupled into them. In some embodiments, light is coupled into only every other linear waveguide. For example, light may be coupled into only the first, third, fifth, seventh, etc. linear waveguides, while light is not coupled into the second, fourth, sixth, eighth, etc. linear waveguides.

In some implementations, the distance 210 is shorter than the diffraction limit (e.g., the resolution distance of the emitting optics). For example, if the numerical aperture is 0.75, the wavelength is about 700nm, the diffraction limit is about 466nm, and the distance 210 may then be shorter than this limit. In some embodiments, the flow cell 200 can be designed such that the nanowells 204A and 204D are separated from each other by about the diffraction limit (e.g., about 466 nm). For example, the distance 210 may then be about half the diffraction limit (e.g., about 233 nm). As another example, if the numerical aperture is 0.75, the wavelength is about 525nm, the diffraction limit is about 350nm, and the distance 210 may then be about 175 nm. The above example involves activating every other linear waveguide at a time. In some embodiments, less than every other linear waveguide may be actuated at a time. For example, if every third linear waveguide is activated at a time, the distance 210 may be about one third of the diffraction limit. As another example, if every third linear waveguide is activated at a time, the distance 210 may be about one quarter of the diffraction limit, and so on.

The scan illustrated in fig. 2A may be described as the flowcell 200 moving to the left in the image and stopping at one or more selected positions corresponding to the linear waveguide when the image capture region 216 covers the linear waveguide until the flowcell 200 reaches the left side of the image capture region 216. One or more linear waveguides into which no light is coupled and which are associated with a nanowell during the scan illustrated in figure 2A and therefore not subjected to excitation light, can be imaged in another scanning operation.

Such other scanning operations may be performed in the same direction as described above (e.g., in the direction of arrow 218) or in another direction. Fig. 2B illustrates an example in which scanning is performed along a direction corresponding to arrow 220 that is substantially, and in at least one instance, entirely opposite to the direction associated with arrow 218. The scan illustrated in fig. 2B may be described as the flowcell 200 moving to the right in the image and stopping at one or more selected positions corresponding to the linear waveguide when the image capture region 216 overlies the linear waveguide until the flowcell 200 reaches the right side of the image capture region 216. Positioning may include moving the image capture area 216, or the flowcell 200, or both.

In this example, the light region 214 is aligned with some of the gratings 202 (e.g., with grating 202B), but not with some other gratings (e.g., with gratings 202A and 202C). For example, when scanning in the direction of arrow 220 from the current position of optical region 214, grating 202B (and other gratings having similar spatial offsets) will be illuminated by light impinging on optical region 214, while some other gratings (e.g., gratings 202A and 202C) will not be illuminated by light impinging on optical region 214. Thus, illumination light will be coupled into linear waveguide 206B (and other linear waveguides having gratings with similar spatial offsets), but light will not be coupled into linear waveguides 206A and 206C (and other linear waveguides having gratings with similar spatial offsets). This may facilitate selective illumination of the nanowells of the flow cell 200. For example, because linear waveguide 206C has light coupled into it, excitation light may reach the nanowell 204C associated with linear waveguide 206B as well as other nanowells. On the other hand, excitation light will not reach the nanowells 204A-204B associated with linear waveguide 206A or the nanowell 204D associated with linear waveguide 206C, which currently has no light coupled into it. As such, although some portions of the sample material (e.g., within the nanowells 204A and 204C) are positioned at a distance 210 from each other, imaging can be successfully performed; i.e. closer to each other than the resolution of the emitting optics. During a scan corresponding to the movement represented by arrow 220 (which may be characterized as a line scan), only a particular subset of the linear waveguides may have light coupled into them. In some embodiments, light is coupled into only every other linear waveguide. For example, light may be coupled into only the second, fourth, sixth, eighth, etc. linear waveguides, while light is not coupled into the first, third, fifth, seventh, etc. linear waveguides.

The example associated with fig. 2A-2B involves differential coupling, where the gratings 202 are spatially offset from each other. In some embodiments, one or more other methods may alternatively or also be used for differential coupling. Such methods may include, but are not limited to, differential beam parameters, differential coupler parameters, and/or differential waveguide parameters. Examples are provided below.

Fig. 3A-3B illustrate an example of a flow cell 300 having a grating 302. In some embodiments, differential coupling to the flow cell 300 is provided based on differentiating one or more parameters of the light beam(s) applied to the grating 302. In some implementations, differential coupling to the flow cell 300 is provided based on differentiating one or more parameters of the grating 302. In some embodiments, differential coupling to the flow cell 300 is provided based on differentiating one or more parameters of a linear waveguide of the flow cell 300. A combination of two or more of these methods may be used. The flow cell 300 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For illustrative purposes, only a portion of the flow cell 300 is shown.

The flow cell 300 includes a plurality of nano-wells, including nano-well 304A, which is illustrated herein using a circular shape. Only some of the nanowells will be specifically mentioned, while other nanowells may be similar or identical to the nanowell(s) in question. The nanowells may be formed in a nanowell layer (e.g., by a nanoimprint or lift-off process). For example, a nanoscale template may be used to form a nanowell in a resin. For clarity, the nanowell layer is not explicitly shown in this example. Here, the nanowell 304A is associated with a linear waveguide 306A. In some embodiments, the linear waveguide described with reference to flow cell 300 may be similar or identical to one or more other linear waveguides described herein. For example, linear waveguide 306A is positioned adjacent to (e.g., in contact with or proximate to) a nanowell layer that includes nanowell 304A. In some implementations, the linear waveguide 306A can include a linear waveguide core 308 and one or more gratings 302.

Another nanowell 304B is also associated with linear waveguide 3206A. For example, nanowell 304B is positioned adjacent to nanowell 304A, and both nanowells 304A-304B may interact with linear waveguide 306A during imaging (e.g., by receiving electromagnetic radiation from linear waveguide 306A). By contrast, another nanowell 304C is instead associated with a linear waveguide 306B. In some embodiments, linear waveguide 306B is positioned adjacent to linear waveguide 306A. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 306A-306B.

The flow cell 300 may be used in one or more forms of imaging. For example, sample material in a nanowell (including nanowells 304A-304C) may be subjected to electromagnetic radiation from a corresponding linear waveguide (including linear waveguides 306A-306B, respectively). The emissions resulting from such exposure to electromagnetic radiation (an example of an emission is fluorescence from fluorophores) may be captured using an instrument (e.g., one or more cameras and/or other imaging devices). Such instruments are sometimes referred to as transmitting instruments or similar terms. For example, the emitting instrument may include one or more cameras or other image sensors and at least one lens or other emitting optics. In some embodiments, the diffraction limit may be due, at least in part, to one or more characteristics of the emission optics. For example, a resolution distance may be defined based on the emitting optics used, which marks the shortest distance that can be resolved using the emitting optics. That is, when resolving features separated by a resolution distance, the imaging system can be said to operate at its highest available resolution level.

Here, distance 310 is less than the resolution distance of the emitting optics, and distance 312 is greater than or about equal to the resolution distance of the emitting optics. Distance 310 here represents the spacing between the nanowells in one direction. In some embodiments, this may be a direction across the linear waveguide. For example, since here the linear waveguides are aligned with the rows of nanowells in one direction (e.g., the vertical direction as seen in the illustration), distance 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 distance 310. That is, the linear waveguides 306A-306B are positioned closer to each other than the resolution distance of the launch optics.

Here, the distance 312 represents a spacing between the nano-wells in another direction different from the distance 310. For example, the distances 310 and 312 may be substantially perpendicular to each other, and in at least one instance may be substantially perpendicular to each other. In some embodiments, this may be a direction along any individual one of the linear waveguides. For example, since the linear waveguide is aligned here with the row of nanowells in one direction (e.g., the vertical direction as seen in the illustration), distance 312 may represent the distance between adjacent nanowells on any linear waveguide (e.g., linear waveguides 306A-306B). For example, nanowells 304A and 304B are separated by distance 312. That is, the nanowells associated with the linear waveguide 306A have a spacing from each other that is resolvable according to the resolution distance of the emission optics for the flow cell 300.

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

The gratings 302A-302B may have one or more characteristics that facilitate selective coupling of electromagnetic radiation into the corresponding linear waveguides 306A-306B. In some implementations, one or more gratings 302 have a different grating period than one or more other gratings 302. For example, grating 302A may have a higher grating period than grating 302B. As another example, grating 302B may have a higher grating period than grating 302A. The characteristics of the gratings 302A-302B having grating periods that are different from each other facilitate coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguide 306A) without coupling electromagnetic radiation (e.g., light) into another of the linear waveguides (e.g., linear waveguide 306B).

In some embodiments, the coupling into the gratings 302A-302C may also or alternatively be differentiated by coupler parameters other than grating period (such as, but not limited to, refractive index, pitch, groove width, groove height, groove spacing, grating non-uniformity, groove orientation, groove curvature, overall shape of the coupler, and combinations thereof). In some embodiments, the coupling into the gratings 302A-302C may also or alternatively be differentiated by waveguide parameters (such as, but not limited to, cross-sectional profile, refractive index difference, mode matching with couplers and/or optical beams, and combinations thereof) with respect to one or more linear waveguides of the flow cell 300. In some embodiments, the coupling into the gratings 302A-302C may also or alternatively be differentiated by beam parameters of the beam(s) applied to the flow cell 300, such as, but not limited to, the position, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof of the beam(s).

The flow cell 300 may comprise a plurality of linear waveguides, for example as illustrated. In some implementations, the linear waveguide 306C is positioned adjacent to the linear waveguide 306B and opposite the linear waveguide 306A. In other words, in this embodiment, linear waveguide 306B is between linear waveguide 306A and linear waveguide 306C. For example, the linear waveguide 306C may have a grating 302C. In some embodiments, grating 302C may have a different grating period than grating 302B. For example, grating 302C may have the same grating period as grating 302A. As another example, grating 302C may have a different grating period than grating 302A and than grating 302B. At least some of the gratings 302A-302C have different grating periods, which facilitates coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguide 306A or 306C) without coupling electromagnetic radiation (e.g., light) into the other of the linear waveguides (e.g., linear waveguide 306B). As another example, the characteristic facilitates coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguide 306B) without coupling electromagnetic radiation (e.g., light) into at least one other of the linear waveguides (e.g., linear waveguides 306A or 306C).

Here, the light area 314 is schematically illustrated as a rectangle having a dashed outline. Light region 314 represents one or more locations that are impinged upon by light or other electromagnetic radiation as part of an imaging process. In some implementations, illumination light generated by the laser can be guided at the light region 314 to be ultimately coupled into some linear waveguides. For example, the laser light may be selected to correspond to the fluorescent properties of one or more fluorophores in the sample material.

Here, the image capturing area 216 is schematically illustrated as a rectangle having a dashed outline. Image capture area 316 represents the field of view of the emission optics. For example, a camera or other image sensor may capture one or more types of emissions (e.g., fluorescence) emanating from image capture area 316.

The examples described above illustrate that the flow cell 300 includes a nanowell layer having a first set of nanowells (e.g., the nanowells associated with linear waveguide 306A) and a second set of nanowells (e.g., the nanowells associated with linear waveguide 306B) to hold a sample. Flow cell 300 includes a first linear waveguide (e.g., linear waveguide 306A) aligned with a first set of nanowells and a second linear waveguide (e.g., linear waveguide 306B) aligned with a second set of nanowells; and a first grating (e.g., grating 302A) for the first linear waveguide and a second grating (e.g., grating 302B) for the second linear waveguide. The first grating has a first characteristic (e.g., has a different grating period than grating 302B) to facilitate coupling the first light into the first linear waveguide and not the second linear waveguide.

The image capture process may include one or more scanning operations. In some embodiments, the image capture region 316 may be overlaid on one or more regions of the flow cell 300 to facilitate image capture with respect to one or more nanowells in the image capture region 316. Positioning may include moving the image capture area 316, or the flowcell 300, or both. For example, the emission optics may be relatively fixed in the analytical instrument such that the image capture region 316 does not move during various 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) into one or more scanning positions relative to the image capture area 316. Here, arrow 318 schematically illustrates that the flow cell 300 may be moved such that the image capture region 316 covers at least some of the linear waveguide and its associated nanowell.

The light region 314 may remain fixed with the image capture region 316 or move corresponding to the image capture region 316, or may move independently of the image capture region 316. In this example, the light region 314 is aligned with all of the gratings 302 of the flow cell 300. Illumination light impinging on the light region 314 may be given different angles of incidence in order to selectively couple light into at least one but not at least another of the linear waveguides of the flow cell 300. For example, when scanning in the direction of arrow 318, the angle of incidence may be selected such that gratings 302A and 302C (and other gratings having similar grating periods) will couple light impinging on light region 314, while some other gratings (e.g., grating 302B) will not couple light impinging on light region 314. Thus, illumination light will be coupled into linear waveguides 306A and 306C (and other linear waveguides having gratings with similar grating periods), while light will not be coupled into linear waveguide 306B (and other linear waveguides having gratings with similar grating periods). This may facilitate selective illumination of the nanowells of the flow cell 300. For example, because linear waveguides 306A and 306C have light coupled into them, excitation light can reach both the nanowells 304A and 304C associated with linear waveguide 306A, and the nanowell 304D associated with linear waveguide 306C. On the other hand, the excitation light will not reach the nanowell 304C because it is associated with a linear waveguide 306B into which no light is currently coupled. As such, although some portions of the sample material (e.g., within nanowells 304A and 304C) are positioned at a distance 310 from each other, imaging can be successfully performed; i.e. closer to each other than the resolution of the emitting optics. During a scan corresponding to the movement represented by arrow 318 (which may be characterized as a line scan), only a particular subset of the linear waveguides may have light coupled into them. In some embodiments, light is coupled into only every other linear waveguide. For example, light may be coupled into only the first, third, fifth, seventh, etc. linear waveguides, while light is not coupled into the second, fourth, sixth, eighth, etc. linear waveguides.

The scan illustrated in fig. 3A may be described as the flowcell 300 moving to the left in the image and stopping at one or more selected positions corresponding to the linear waveguide when the image capture region 316 covers the linear waveguide until the flowcell 300 reaches the left side of the image capture region 316. One or more linear waveguides into which no light is coupled and which are associated with a nanowell during the scan illustrated in figure 3A and therefore not subjected to excitation light, can be imaged in another scanning operation.

Such other scanning operations may be performed in the same direction as described above (e.g., in the direction of arrow 318) or in another direction. Fig. 3B illustrates an example in which scanning is performed along a direction corresponding to arrow 320, which is substantially, and in at least one instance, entirely opposite to the direction associated with arrow 318. The scan illustrated in fig. 3B may be described as the flowcell 300 moving to the right in the image and stopping at one or more selected positions corresponding to the linear waveguide when the image capture region 316 covers the linear waveguide until the flowcell 300 reaches the right side of the image capture region 316. Positioning may include moving the image capture area 316, or the flowcell 300, or both.

In this example, the light region 314 is aligned with all of the gratings 302 of the flow cell 300. Illumination light impinging on the light region 314 may be given different angles of incidence in order to selectively couple light into at least one but not at least another of the linear waveguides of the flow cell 300. For example, when scanning in the direction of arrow 320, the angle of incidence may be selected such that grating 302B (and other gratings having similar grating periods) will couple light impinging on light region 314, while some other gratings (e.g., gratings 302A and 302C) will not couple light impinging on light region 314. Thus, illumination light will be coupled to linear waveguide 306B (and other linear waveguides having gratings with similar grating periods), while light will not be coupled to linear waveguides 306A and 306C (and other linear waveguides having gratings with similar grating periods). This may facilitate selective illumination of the nanowells of the flow cell 300. For example, because the linear waveguide 306B has light coupled into it, the excitation light may reach the nanowell 304C and other nanowells associated with the linear waveguide 306B. On the other hand, the excitation light will not reach the nanowells 304A-304B associated with linear waveguide 306A or the nanowell 304D associated with linear waveguide 306C, which currently has no light coupled into it. As such, although some portions of the sample material (e.g., within nanowells 304A and 304C) are positioned at a distance 310 from each other, imaging can be successfully performed; i.e. closer to each other than the resolution of the emitting optics. During a scan corresponding to the movement represented by arrow 320 (which may be characterized as a line scan), only a particular subset of the linear waveguides may have light coupled into them. In some embodiments, light is coupled into only every other linear waveguide. For example, light may be coupled into only the second, fourth, sixth, eighth, etc. linear waveguides, while light is not coupled into the first, third, fifth, seventh, etc. linear waveguides.

In some embodiments, two or more gratings 302 may alternatively or also have different refractive indices. This may allow for differential coupling with respect to at least some of the linear waveguides 306A-306C, for example.

Fig. 4 shows another example of a flow cell 400 with an interleaved grating 402. Flow cell 400 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For illustrative purposes, only a portion of the flow cell 400 is shown.

The flow cell 400 includes a plurality of nano-wells, including nano-well 404A, which are illustrated herein using a circular shape. Only some of the nanowells will be specifically mentioned, while other nanowells may be similar or identical to the nanowell(s) in question. The nanowells may be formed in a nanowell layer (e.g., by a nanoimprint or lift-off process). For example, a nanoscale template may be used to form a nanowell in a resin. For clarity, the nanowell layer is not explicitly shown in this example. The nano-trap 404A is here associated with a linear waveguide 406A. In some embodiments, the linear waveguide described with reference to flow cell 4200 may be similar or identical to one or more other linear waveguides described herein. For example, the linear waveguide 406A is positioned adjacent to (e.g., in contact with or proximate to) a nanowell layer that includes the nanowell 404A.

Another nanowell 404B is also associated with linear waveguide 406A. For example, the nanowell 404B is positioned adjacent to the nanowell 404A, and both nanowells 404A-404B may interact with the linear waveguide 406A during imaging (e.g., by receiving electromagnetic radiation from the linear waveguide 406A). By contrast, another nanowell 404C is instead associated with a linear waveguide 406B. In some embodiments, linear waveguide 406B is positioned adjacent to linear waveguide 406A. For example, cladding (not shown) and/or another material may be located between the linear waveguides 406A-406B.

The flow cell 400 may be used in one or more forms of imaging. For example, sample material in a nanowell (including nanowells 404A-404C) may be subjected to electromagnetic radiation from a corresponding linear waveguide (including linear waveguides 406A-406B, respectively). The emissions resulting from such exposure to electromagnetic radiation (an example of an emission is fluorescence from fluorophores) may be captured using an instrument (e.g., one or more cameras and/or other imaging devices). Such instruments are sometimes referred to by the expression "transmitting instruments" or similar terms. For example, the emitting instrument may include one or more cameras or other image sensors, and at least one lens or other emitting optics. In some embodiments, the diffraction limit may be due, at least in part, to one or more characteristics of the emission optics. For example, a resolution distance may be defined based on the emitting optics used, which marks the shortest distance that can be resolved using the emitting optics. That is, when resolving features separated by a resolution distance, the imaging system can be said to operate at its highest available resolution level.

The grating 402 is used to couple electromagnetic radiation into and/or out of the linear waveguide of the flow cell 400. Here, linear waveguide 406A has grating 402A, linear waveguide 406B has grating 402B, and linear waveguide 406C has grating 402C. The gratings 402A-402C may have the same or different periodic structures. In some implementations, any or all of the gratings 402A-402C may include periodic ridge structures interspersed with another material. For example, the ridges of gratings 402A-402C may have a pitch of about 200 and 300nm, to name just one example.

The gratings 402A-402C may have one or more characteristics that facilitate selective coupling of electromagnetic radiation into the corresponding linear waveguides 406A-406C. In some implementations, one or more gratings 402 are spatially offset from one or more other gratings 402. The offset may be in a direction parallel to the linear waveguides 406A-406C. For example, here, the distance between grating 402B and the nearest one of the nanotrap associated with linear waveguide 406B is greater than the distance between grating 402A and the nearest one of the nanotrap associated with linear waveguide 406A. As another example, here, the distance between grating 402C and the closest one of the nanotrap associated with linear waveguide 406C is greater than the distance between grating 402A and the closest one of the nanotrap associated with linear waveguide 406A, and is also greater than the distance between grating 402B and the closest one of the nanotrap associated with linear waveguide 406B. The spatially offset nature of the gratings 402A-402C facilitates coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguide 406A) without coupling electromagnetic radiation (e.g., light) into the other of the linear waveguides (e.g., linear waveguides 406B or 406C). That is, the grating 402C is spatially offset from each of the gratings 402A-402B in a direction parallel to the linear waveguides 406A-406C.

In some embodiments, the coupling into the gratings 402A-402C may also or alternatively be differentiated by beam parameters other than the position of the beam (such as, but not limited to, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof). In some embodiments, the coupling into the gratings 402A-402C may also or alternatively be differentiated by coupler parameters such as, but not limited to, grating period, refractive index, pitch, groove width, groove height, groove spacing, grating non-uniformity, groove orientation, groove curvature, overall shape of the coupler, and combinations thereof. In some embodiments, the coupling into the gratings 402A-402C may also or alternatively be differentiated by waveguide parameters (such as, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and/or the optical beam, and combinations thereof) with respect to one or more linear waveguides of the flow cell 400.

Here, the light areas 408A-408C are schematically illustrated as rectangles having dashed outlines. Light regions 408A-408C represent locations where light or other electromagnetic radiation is impinged upon as part of the imaging process. In some implementations, illumination light generated by the lasers can be directed at one or more light regions 408A-408C to be ultimately coupled into corresponding linear waveguides. For example, the laser may be selected so as to correspond to the fluorescent properties of one or more fluorophores in the sample material. Light may be directed into optical region 408A to couple light into linear waveguide 406A without coupling light into linear waveguides 406B-406C. Light may be directed into light region 408B to couple light into linear waveguide 406B without coupling light into linear waveguides 406A or 408-408C. Light may be directed into light region 408C to couple light into linear waveguide 406C without coupling light into linear waveguides 406A-406B.

In addition to the linear waveguides 406A-406C, the flow cell 400 may also have other linear waveguides with corresponding gratings. The individual gratings of such other linear waveguides may have a similar spatial offset as the spatial offset of one of the gratings 402A-402C, or may have a different spatial offset. For example, light may be coupled into only the first, fourth, seventh, tenth, etc. linear waveguides, while light is not coupled into the second, third, fifth, sixth, eighth, ninth, eleventh, twelfth, etc. linear waveguides. More generally, in any individual scanning operation (corresponding to the use of a particular light region, such as one of light regions 408A-408C), the ordinal number of the linear waveguide into which light is coupled may form an arithmetic progression, wherein the nth ordinal number, a_n(n ═ 1, 2, 3.) can be expressed as a_n＝a₁+ d (n-1) wherein a₁Is the first ordinal number and d is a positive integer. For example, corresponding to the above example, at a₁In the case of 1 and d 3, the linear waveguide into which the obtained light is coupled has the ordinal numbers 1, 4, 7, 10, etc. As another example, in a₁In the case of 1 and d 4, the linear waveguide into which the obtained light is coupled has the ordinal numbers 1, 5, 9, 13, etc.

The examples described above illustrate that the flow cell 400 includes a nanowell layer having a first set of nanowells (e.g., the nanowells associated with the linear waveguide 406A) and a second set of nanowells (e.g., the nanowells associated with the linear waveguide 406B) to accommodate a sample. Flow cell 400 includes a first linear waveguide (e.g., linear waveguide 406A) aligned with a first set of nanowells and a second linear waveguide (e.g., linear waveguide 406B) aligned with a second set of nanowells; and a first grating (e.g., grating 402A) for the first linear waveguide and a second grating (e.g., gratings 402B-402C) for the second linear waveguide. The first grating has a first characteristic (e.g., spatially offset from the gratings 402B-402C) to facilitate coupling the first light into the first linear waveguide without coupling the first light into the second linear waveguide.

Fig. 5 shows a cross-section of a portion of an example flow cell 500. Flow cell 500 can be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For illustrative purposes, the flow cell 500 is shown in cross-section, and only a portion of the flow cell 500 is shown.

Flow cell 500 includes a nanowell layer 502 that includes nanowells 502A-502B. The nanowell layer 502 may be formed by a nanoimprint or lift-off process. For example, the nanowells 502A-502B may be formed by applying a nanoscale template to a resin.

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

Each linear waveguide 504A-504B may have one or more gratings (omitted here for clarity) to couple electromagnetic radiation into and/or out of the linear waveguides 504A-504B. One or more directions of travel of electromagnetic radiation within the linear waveguides 504A-504B may be employed. For example, the direction of travel may be into and/or out of the plane of the present example.

Each linear waveguide 504A-504B may be positioned against one or more types of cladding layers. The cladding layers may serve to confine electromagnetic radiation to the respective linear waveguides 504A-504B and prevent or reduce the extent to which radiation propagates into other linear waveguides 504A-504B or other substrates (e.g., to reduce cross-coupling). Here, cladding 506 is shown as an example. In some embodiments, the cladding 506 comprises a series of blocks. In some embodiments, the cladding 506 provides a refractive index that alternates along the structure of the cladding 506. For example, a first one of claddings 506 may have a first refractive index, a second one of claddings 506 adjacent to the first cladding may have a second refractive index, a third one of claddings 506 adjacent to the second cladding may have the first refractive index, and so on. Cladding 506 may be positioned against or near linear waveguide 102A on different (e.g., opposite) sides of linear waveguide 102A. For example, the cladding 506A may be positioned against or near the linear waveguide 504B. Here, the cladding 506 includes a plurality of structures, including a cladding 506A. Cladding 506 may be made of one or more suitable materials that are used to separate linear waveguides 504A-504B from one another. In some embodiments, the cladding 506 may be made of a material having a lower index of refraction than one or more of the linear waveguides 504A-504B. In some embodiments, one or more of the cladding layers 506 comprise a polymeric material. In some embodiments, the plurality of structures of the cladding 506 may be interspersed with regions of vacuum or another material (e.g., air or liquid).

Fig. 6 shows an example of a flow cell 600 in which multiple linear waveguides share a common grating. The flow cell 600 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For illustrative purposes, the flow cell 500 is shown in a top view and only a portion of the flow cell 600 is shown.

The flow cell 600 comprises a substrate 602. In some embodiments, the substrate 602 serves as a base layer of the flow cell 600 and may support one or more layers and/or other structures. For example, the substrate 602 may support one or more linear waveguide assemblies 604A and a nanowall layer (not shown).

The linear waveguide assembly 604A includes a coupling assembly 606A having a grating 608A. A linear waveguide connector 610 connects coupling assembly 606A and linear waveguide array 612A to each other. The linear waveguide array 612A includes a linear waveguide divider 614 coupled to the linear waveguide connector 610, and a plurality of linear waveguides 616A arranged in parallel to each other and coupled to the linear waveguide divider 614. In operation, light incident on the grating 608A may be coupled into the linear waveguide array 612A by the coupling component 606A and the linear waveguide connector 610. In linear waveguide array 612A, linear waveguide splitter 614 may split light into linear waveguides 616A. In some embodiments, linear waveguide 616A is positioned adjacent to a nanowell (not shown) to facilitate imaging as part of sample analysis. For example, a row of nanowells may be positioned along each linear waveguide 616A. The linear waveguide assembly 604A may be made of one or more suitable materials that facilitate the propagation of electromagnetic radiation. In some implementations, the material(s) of the linear waveguide assembly 604A can include a polymer material. In some embodiments, the material(s) of the linear waveguide assembly 604A may include Ta₂O₅And/or SiN_x。

The linear waveguide array 612A may facilitate placement of one or more other components of the flow cell 600. In some embodiments, the flow cell 600 includes a linear waveguide assembly 604B positioned on an opposite side of the substrate 602 from the linear waveguide array 612A. Linear waveguide assembly 604B may include a coupling assembly 606B coupled to a linear waveguide array 612B. In some implementations, individual linear waveguides 616B of linear waveguide array 612B can be interspersed between corresponding linear waveguides 616A of linear waveguide array 612A. For example, two of linear waveguides 616A may be positioned on respective opposite sides of one of linear waveguides 616B. Two of the linear waveguides 616A then share the same grating, in this example grating 608A of linear waveguide assembly 604A.

In some implementations, linear waveguide 616A and linear waveguide 616B can be positioned closer to each other than the resolution distance of the launch optics. For example, during the first scanning phase, light may be coupled into linear waveguide 616A of linear waveguide assembly 604A and not into linear waveguide 616B of linear waveguide assembly 604B. Further, during the second scanning phase, light may instead be coupled into linear waveguide 616B of linear waveguide assembly 604B and not into linear waveguide 616A of linear waveguide assembly 604A.

At least one of the coupling components 606A-604B may include a substrate having a substantially and in at least one example, entirely triangular shape. This may provide advantages in terms of efficient positioning of the plurality of flow cells. The linear waveguide assembly 604C may not be considered part of the flow cell 600 but may be considered part of another flow cell (not shown). In some embodiments, the triangular substrate of coupling assembly 606A and the corresponding triangular substrate of coupling assembly 606C of linear waveguide assembly 604C may be positioned adjacent to each other. For example, the coupling assemblies 606A and 606C may be positioned in opposite directions in order to provide efficient packaging of the linear waveguide assemblies 604A and 604C adjacent to each other.

The grating 608A may be positioned toward a first end of the linear waveguide assembly 604A (e.g., toward a left end thereof in this illustration). Further, the grating 608B may be positioned toward a second end of the linear waveguide assembly 604B (e.g., toward a right end thereof in this illustration). The first end may be positioned opposite the second end in a direction parallel to the row of nanowells (e.g., a direction parallel to the linear waveguides 616A-616B).

More or fewer linear waveguide assemblies than shown may be used. In some embodiments, respective linear waveguide assemblies 604D-604F are implemented. For example, linear waveguide assemblies 604E-604F may be considered part of flow cell 600, while linear waveguide assembly 604D may be considered part of another flow cell (not shown) separate from the flow cell of linear waveguide assembly 604C.

In some embodiments, coupling into grating 608A and/or other gratings may be differentiated by beam parameters such as, but not limited to, position, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof of the beam. In some embodiments, the coupling into grating 608A and/or other gratings may also or alternatively be differentiated by coupler parameters such as, but not limited to, grating period, refractive index, pitch, groove width, groove height, groove spacing, grating non-uniformity, groove orientation, groove curvature, overall shape of the coupler, and combinations thereof. In some embodiments, coupling into the grating 608A and/or other sources may also or alternatively be differentiated by waveguide parameters (such as, but not limited to, cross-sectional profile, refractive index difference, mode matching with couplers and/or beams, and combinations thereof) with respect to one or more linear waveguides of the flow cell 600.

Fig. 7 is a diagram of an example illumination system 700. The illumination system 700 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein.

The illumination system 700 includes a light source assembly 710, a mirror 728, an objective lens 734, a flow cell 736, a transmissive dichroic filter 738, a first optical detection subsystem 756, and a second optical detection subsystem 758. The illumination system 700 enables simultaneous imaging of two color channels. In some embodiments, another illumination system may be configured to enable simultaneous imaging of more than two color channels (e.g., three color channels, four color channels, or more). Note that similar other optical configurations are possible, enabling simultaneous imaging of multiple color channels.

The light source assembly 710 produces excitation radiation that is incident on the flow cell 736. This excitation illumination will in turn produce emitted or fluorescing illumination from one or more fluorescent dyes, which will be collected using lenses 742 and 748. 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.

The first excitation illumination source 712 and the second excitation illumination source 716 illustrate illumination systems capable of simultaneously providing respective excitation illumination light (e.g., corresponding to respective color channels) to the sample. In some embodiments, each of the first excitation illumination source 712 and the second excitation illumination source 716 includes a Light Emitting Diode (LED). In some embodiments, at least one of the first excitation illumination source 712 and the second excitation illumination source 716 comprises a laser. Converging lenses 714 and 718 are each disposed a distance from the respective excitation illumination sources 712 and 716 such that illumination exiting each converging lens 714/718 is focused on field aperture 722. Dichroic filter 720 reflects illumination from first excitation illumination source 712 and transmits illumination from second excitation illumination source 716.

In some embodiments, the mixed excitation illumination output from dichroic filter 720 may propagate directly toward objective lens 134. In other embodiments, the hybrid excitation illumination may be further modified and/or controlled by additional intermediate optical components prior to emission from objective lens 734. The mixed excitation illumination may pass through a focal point in the field aperture 722 to a filter 724 and then to a color corrected collimating lens 726. The collimated excitation illumination from lens 726 is incident on mirror 728, where it is reflected and incident on excitation/emission dichroic filter 730. Excitation/emission dichroic filter 730 reflects excitation illumination emitted from light source assembly 710 while allowing emission illumination (which will be described further below) to pass through excitation/emission dichroic filter 730 for receipt by one or more optical subsystems 756, 758. Optical subsystems 756 and 758 illustrate light collection systems that can collect multiple fluorescent lights simultaneously. Excitation illumination reflected from excitation/emission dichroic filter 730 is then incident on mirror 732, where it is incident on objective lens 134 toward flow cell 736 from mirror 732.

Objective lens 734 focuses the collimated excitation radiation from mirror 732 onto flow cell 136. In some embodiments, objective 734 is a microscope objective with a specified magnification factor of, for example, 1X, 2X, 4X, 5X, 6X, 8X, 10X, or higher. Objective 734 focuses the excitation illumination incident from mirror 732 onto flow cell 736 with a cone angle or numerical aperture determined by a magnification factor. In some embodiments, objective lens 734 is movable in an axis perpendicular to the flow cell ("z-axis"). In some embodiments, illumination system 700 independently adjusts the z position of tube lens 748 and tube lens 742.

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

The objective lens 734 may define a field of view. The field of view may define an area on the flow cell 736 from which the image detector captures the emitted light using the objective lens 734. One or more image detectors, such as detectors 746 and 754, may be used. The illumination system 700 may include separate image detectors 746 and 754 for respective wavelengths (or wavelength ranges) of the emitted light. At least one of image detectors 746 and 754 may include a Charge Coupled Device (CCD), such as a time delay integration CCD camera, or a sensor fabricated based on Complementary Metal Oxide Semiconductor (CMOS) technology, such as a chemical sensitive field effect transistor (chemFET), an Ion Sensitive Field Effect Transistor (ISFET), and/or a Metal Oxide Semiconductor Field Effect Transistor (MOSFET).

In some embodiments, the illumination system 700 may include a Structured Illumination Microscope (SIM). SIM imaging is based on spatially structured illumination and reconstruction to obtain a higher resolution image than would be produced using only magnification from objective 734. For example, the structure may comprise or consist of a pattern or grating that interrupts the illumination of the excitation light. In some embodiments, the structure may comprise a stripe pattern. Fringes of light may be generated by impinging a light beam on a diffraction grating, whereby reflection or transmission diffraction occurs. Structured light may be projected onto the sample, illuminating the sample according to corresponding fringes, which may occur with a certain periodicity. To reconstruct an image using a SIM, two or more patterned images are used with the patterns of excitation illumination at different phase angles from each other. For example, images of the sample may be acquired with different fringe phases (sometimes referred to as corresponding pattern phases of the image) in the structured light. This may allow various locations on the sample to be exposed to multiple illumination intensities. The resulting set of emitted light images can be combined to reconstruct a higher resolution image.

The sample material in the flow cell 736 is contacted with a fluorescent dye coupled to the corresponding nucleotide. The fluorescent dye emits fluorescent radiation when irradiated with corresponding excitation radiation incident on the flow cell 736 from the objective lens 734. The emitted illumination is identified by wavelength bands, each of which may be classified into a respective color channel. The fluorescent dye is chemically bound to the corresponding nucleotide (e.g., containing the corresponding nucleobase). In this manner, dntps labeled with a fluorescent dye can be identified based on the wavelength of emitted light within the corresponding wavelength band when detected by the image detectors 746, 754.

The objective lens 734 captures fluorescence emitted by fluorescent dye molecules in the flow cell 736. After capturing the emitted light, the objective lens 734 collects and transmits the collimated light. The emitted light then travels back along the path that the original excitation illumination arrived from the light source assembly 710. Note that there is little or no expected interference between the emission and excitation illumination along this path due to the lack of coherence between the emission and excitation illumination. That is, the emitted light is the result of a separate light source, i.e., the result of the fluorescent dye being in contact with the sample material in the flow cell 736.

The emitted light is incident on the excitation/emission dichroic filter 730 after being reflected by the mirror 732. The filter 730 transmits the emitted light to the dichroic filter 738.

In some implementations, the dichroic filter 738 transmits illumination associated with the blue channel and reflects illumination associated with the green channel. In some embodiments, the dichroic filter 738 is selected such that the dichroic filter 738 reflects emitted illumination within the defined green wavelength band to the optical subsystem 756 and transmits emitted illumination within the defined blue wavelength band to the optical subsystem 758, as discussed above. Optical subsystem 756 includes tube lens 742, optical filter 744, and image detector 746. Optical subsystem 758 includes tube lens 748, optical filter 750, and image detector 754.

In some embodiments, dichroic filter 738 and dichroic filter 7120 operate similarly to each other (e.g., both may reflect one color of light and transmit another color of light). In other embodiments, the dichroic filter 738 and the dichroic filter 720 operate differently from each other (e.g., the dichroic filter 738 may transmit light of the color reflected by the dichroic filter 720 and vice versa).

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

Each tube lens 742 and 748 focuses emitted illumination incident thereon onto a respective image detector 746 and 754. In some embodiments, each detector 746 and 754 includes a CCD array. In some embodiments, each image detector 746 and 754 includes a Complementary Metal Oxide Semiconductor (CMOS) sensor.

The illumination system 700 need not be as shown in fig. 7. For example, each mirror 728, 732, 740 may be replaced with a prism or some other optical device that changes the direction of illumination. Each lens may be replaced by a diffraction grating, diffractive optics, a fresnel lens, or some other optical device that produces collimated or focused illumination from incident illumination.

Fig. 8-9 are flowcharts of example methods 800 and 900. Method 800 or 900, or both, may be performed using and/or in conjunction with one or more other examples described herein. More or fewer operations may be performed and/or two or more operations may be performed in a different order, unless otherwise indicated.

At 810, a sample may be applied to a first row of nanowells and a second row of nanowells of a flow cell. In some embodiments, the sample may be applied to a row of nanowells associated with the linear waveguides 206A-206B in fig. 2A. In some embodiments, the sample may be applied to a row of nanowells associated with the linear waveguides 406A-406B in fig. 4. For example, the sample may include genetic material.

At 820, the position of the illumination assembly can be changed to address a subset of the gratings. In some embodiments, the position of the illumination assembly is changed so that illumination light will impinge or does impinge on light region 214 in fig. 2A when light region 214 is aligned with gratings 202A and 202C and some other gratings, but misaligned with grating 202B and some other gratings. In some embodiments, the position of the illumination assembly is changed so that the illumination light will impinge or does impinge on light region 408A, which is aligned with grating 402A but not with gratings 402B-402C in FIG. 4. For example, the mirror 732 in fig. 7 may be adjusted to change the position at which light is incident. In some embodiments, the flow cell may be moved or adjusted in addition to or instead of moving the illumination apparatus.

At 830, the scan may begin in a first direction. In some embodiments, the scanning is performed in a direction corresponding to arrow 218 in fig. 2A. Positioning may include moving an image capture area (e.g., an image capture device) or a flowcell or both.

At 840, the first light may be guided at a first grating 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 at light region 214 in fig. 2A when light region 214 at least partially covers gratings 202A and 202C. Because the grating 202B is spatially offset from the gratings 202A and 202C, the first light is not coupled into the linear waveguide 206B. In some implementations, the first light is directed at light region 408A in fig. 4 that at least partially covers grating 402A. Because the gratings 402B-402C are spatially offset from the grating 402A, the first light is not coupled into the linear waveguides 406B-406C.

At 850, one or more images may be captured. In some embodiments, the image capture area 216 in fig. 2A may capture an image when the image capture area 216 at least partially covers some aspects of the flow cell 200. In a similar manner, the flow cell 400 of fig. 4 may capture one or more images. For example, the image capture may include line scanning.

At 860, the position of the illumination assembly may be changed to address another subset of the gratings. In some embodiments, the position of the illumination assembly is changed so that illumination light will impinge or actually impinge on light region 214 in fig. 2B when light region 214 is aligned with grating 202B and some other gratings, but misaligned with gratings 202A and 202C and some other gratings. In some implementations, the position of the illumination assembly is changed so that the illumination light will impinge or does impinge on light region 408B that is aligned with grating 402B but not aligned with grating 402A or 402C in fig. 4. For example, the mirror 732 in fig. 7 may be adjusted to change the position at which light is incident. In some embodiments, the flow cell may be moved or adjusted in addition to or instead of moving the illumination apparatus.

At 870, the scan may begin in a second direction. The second direction may be the same or different from the first direction. In some embodiments, the scanning is performed in a direction corresponding to arrow 220 in fig. 2B. Positioning may include moving an image capture area (e.g., an image capture device) or a flowcell or both.

At 880, the second light may be guided at the second grating of the second linear waveguide aligned with the second row of nano-wells without coupling the second light into the first linear waveguide. In some implementations, the second light is directed at the light region 214 in fig. 2B when the light region 214 at least partially covers the grating 202B. Because the gratings 202A and 202C are spatially offset from the grating 202B, the second light is not coupled into the linear waveguide 206A or 206C. In some implementations, the second light is directed at light region 408B in fig. 4 when light region 408B at least partially covers grating 402B. Because the gratings 402A and 402C are spatially offset from the grating 402B, the second light is not coupled into the linear waveguides 406A and 406C.

At 890, one or more images may be captured. In some embodiments, the image capture area 216 in fig. 2B may capture an image when the image capture area 216 at least partially covers some aspects of the flow cell 200. In a similar manner, the flow cell 400 of fig. 4 may capture one or more images. For example, the image capture may include line scanning.

Turning now to method 900 in fig. 9. At 910, a sample may be applied to a first row of nanowells and a second row of nanowells of a flow cell. In some embodiments, the sample may be applied to a row of nanowells associated with the linear waveguides 306A-306B in fig. 3A. For example, the sample may include genetic material.

At 920, the position of the illumination assembly can be changed to an angle associated with the grating period of the subset of gratings. In some embodiments, the position of the illumination assembly is changed so that the illumination light will or does have an angle of incidence at which gratings 302A and 302C and some other gratings couple light, but at which grating 302B and some other gratings do not couple light. For example, mirror 732 in fig. 7 may be adjusted to change the angle of incidence. In some embodiments, the flow cell may be moved or adjusted in addition to or in lieu of adjusting the illumination apparatus.

At 930, scanning may begin in a first direction. In some embodiments, the scanning is performed in a direction corresponding to arrow 318 in fig. 3A. Positioning may include moving an image capture area (e.g., an image capture device) or a flowcell or both.

At 940, the first light may be guided at the first grating of the first linear waveguide aligned with the first row of nano-wells without coupling the first light into the second linear waveguide aligned with the second row of nano-wells. In some implementations, the first light is directed at the light region 314 in fig. 3A when the light region 314 at least partially overlaps the grating 302. Because grating 302B has a different grating period than gratings 302A and 302C, the first light is not coupled into linear waveguide 306B.

At 950, one or more images may be captured. In some embodiments, the image capture area 316 in fig. 3A may capture an image when the image capture area 316 at least partially covers some aspects of the flow cell 300.

At 960, the position of the illumination assembly can be changed to an angle associated with a grating period of another subset of the gratings. In some embodiments, the position of the illumination assembly is changed so that the illumination light will or does have an angle of incidence at which grating 302B and some other gratings couple light, but at which gratings 302A and 302C and some other gratings do not couple light. For example, the mirror 732 in fig. 7 may be adjusted to change the position at which light is incident. In some embodiments, the flow cell may be moved or adjusted in addition to or instead of moving the illumination apparatus.

At 970, the scan may begin in a second direction. The second direction may be the same or different from the first direction. In some embodiments, the scanning is performed in a direction corresponding to arrow 320 in fig. 3B. Positioning may include moving an image capture area (e.g., an image capture device) or a flowcell or both.

At 980, the second light may be guided at a second grating of a second linear waveguide aligned with the second row of nano-wells without coupling the second light into the first linear waveguide. In some implementations, the second light is directed at the light region 314 in fig. 3B when the light region 314 at least partially overlaps the grating 302. Because gratings 302A and 302C have a different grating period than grating 302B, the second light is not coupled into linear waveguide 306A or 306C.

At 990, one or more images may be captured. In some embodiments, the image capture area 316 in fig. 3B may capture an image when the image capture area 316 at least partially covers some aspects of the flow cell 300. For example, the image capture may include line scanning.

For illustrative purposes only, some examples herein illustrate nanowells with circular openings. In some embodiments, non-circular nanowells may be used. Fig. 10A shows an example of a hexagonal array 1000 of non-circular nanowells 1002. Hexagonal array 1000 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For example, hexagonal array 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 of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For example, the nanowells 1002 may be arranged in a hexagonal array or a non-hexagonal (e.g., polygonal beyond) array, or both.

The size and/or shape of the non-circular nanowell 1002 may affect imaging as part of the analysis process. In some embodiments, fluorescent signals are collected from some or all of the non-circular nanowells 1002. The fluorescence signal may be affected by the size and/or shape of the non-circular nanowell 1002. For example, variations in the generated fluorescent signal(s) can affect the throughput of an analysis system (e.g., a sequencing system).

In some implementations, one or more non-circular nanowells 1002 have an elliptical opening. The ellipse can be characterized by the respective lengths of the major and minor axes. The minor axis length may be expressed as a percentage of the major axis length, including but not limited to 5%, 15%, 35%, 65%, or 95% of the major axis length, to name a few. Other geometries than elliptical are also possible for non-circular nanowells.

Fig. 10B shows an example of a triangular array 1004 of circular nanowells 1006. Triangular array 1004 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For example, triangular array 1004 may be used with circular nanowells or non-circular nanowells or both. One or more of circular nanowells 1006 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For example, circular nanowells 1006 may be arranged in a hexagonal array or a non-hexagonal (e.g., polygonal beyond) array or both.

Fig. 11 shows another example of a flow cell 1100 with an interleaved grating 1102. Flow cell 1100 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For example, the flow cell 1100 may be used with either an interleaved grating or a non-interleaved grating or both. One or more of the interleaved gratings 1102 may be used with one or more of the methods described herein and/or in conjunction with one or more of the systems or devices described herein. For example, the staggered grating 1102 may be used with nanowells arranged in a hexagonal array or a non-hexagonal (e.g., otherwise polygonal) array, or both.

Flow cell 1100 includes a plurality of nano-wells, including nano-well 1104A, which are illustrated herein using a circular shape. Only some of the nanowells will be specifically mentioned, while other nanowells may be similar or identical to the nanowell(s) in question. The nanowells may be formed in a nanowell layer (e.g., by a nanoimprint or lift-off process). For example, a nanoscale template may be used to form a nanowell in a resin. For clarity, the nanowell layer is not explicitly shown in this example. The nanowell 1104A is here associated with a linear waveguide 1106A. In some embodiments, the linear waveguide described with reference to flow cell 1100 may be similar or identical to one or more other linear waveguides described herein. For example, the linear waveguide 1106A is positioned adjacent to (e.g., in contact with or proximate to) a nanowell layer that includes the nanowell 1104A.

Another nanowell 1104B is also associated with the linear waveguide 1106A. For example, the nanowell 1104B is positioned adjacent to the nanowell 1104A, and both nanowells 1104A-1104B may interact with the linear waveguide 1106A during imaging (e.g., by receiving electromagnetic radiation from the linear waveguide 1106A). By contrast, another nanowell 1104C is instead associated with the linear waveguide 1106B. In some embodiments, linear waveguide 1106B is positioned adjacent to linear waveguide 1106A. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 1106A-1106B.

Here, the nanowell 1104D is associated with a linear waveguide 1106C. In some embodiments, linear waveguide 1106C is positioned adjacent to linear waveguide 1106B. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 1106B-1106C.

Here, the nanowell 1104E is associated with a linear waveguide 1106D. In some embodiments, linear waveguide 1106D is positioned adjacent to linear waveguide 1106C. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 1106C-1106D.

Here, the nanowells 1104A-1104B and other nanowells form a first set of nanowells (e.g., a row of nanowells) that extend along the linear waveguide 1106A. Here, the nanowell 1104C and other nanowells form a second set of nanowells (e.g., a row of nanowells) extending along the linear waveguide 1106B. Here, the nanowell 1104D and other nanowells form a third set of nanowells (e.g., a row of nanowells) extending along the linear waveguide 1106C. Here, the nanowell 1104E and other nanowells form a fourth set of nanowells (e.g., a row of nanowells) extending along the linear waveguide 1106D. In some implementations, the first set of nanowells (e.g., nanowells 1104A-1104B and other nanowells) are positioned so as to be in phase with the second set of nanowells (e.g., nanowell 1104C and other nanowells). The first set of nanowells may be positioned at substantially regular intervals and in at least one instance at substantially regular intervals along the linear waveguide 1106A. For example, each nanowell of the first set of nanowells at the linear waveguide 1106A has a corresponding nanowell of the second set of nanowells at the linear waveguide 1106B. A corresponding nanowell may be positioned from the nanowell directly across the cladding or another material between the linear waveguides 1106A-1106B.

Here, the nanowells 1104D and other nanowells in the third set of nanowells are positioned at substantially regular intervals, and in at least one instance at substantially regular intervals, along the linear waveguide 1106C. The third set of nanowells is positioned out of phase with at least the second set of nanowells. In some embodiments, none of the nanowells of the second set of nanowells have a corresponding nanowell of the third set of nanowells directly across the cladding layer or other material. For example, each nanowell of the second set of nanowells may be positioned equidistant between two adjacent nanowells of the third set of nanowells.

In some embodiments, the fourth set of nanowells (e.g., nanowell 1104E and other nanowells along linear waveguide 1106D) are positioned so as to be in phase with the third set of nanowells (e.g., nanowell 1104D and other nanowells along linear waveguide 1106C). A fourth set of nano-wells may be positioned at substantially regular intervals and in at least one instance at substantially regular intervals along the linear waveguide 1106D. For example, each nanowell of the fourth set of nanowells at linear waveguide 1106D has a corresponding nanowell of the third set of nanowells at linear waveguide 1106C. The corresponding nano-well may be positioned from the nano-well directly across the cladding or another material between the linear waveguides 1106C-1106D.

The grating 1102 is used to couple electromagnetic radiation into and/or out of a linear waveguide of the flow cell 1100. Here, linear waveguide 1106A has grating 1102A, linear waveguide 1106B has grating 1102B, linear waveguide 1106C has grating 1102C, and linear waveguide 1106D has grating 1102D. Each of the gratings 1102A-1102D may have the same or different periodic structures. In some embodiments, some or all of the gratings 1102A-1102D may include periodic ridge structures interspersed with another material. For example, the ridges of gratings 1102A-1102D may have a pitch of about 200 and 300nm, to name just one example.

The gratings 1102A-1102D may have one or more characteristics that facilitate, at least in part, selective coupling of electromagnetic radiation into the corresponding linear waveguides 1106A-1106D. In some implementations, one or more gratings 1102 are spatially offset from one or more other gratings 1102. The offset may be in a direction parallel to the linear waveguides 1106A-1106D. For example, here, the distance between grating 1102B and the closest of the nanotrap associated with linear waveguide 1106B is greater than the distance between grating 1102A and the closest of the nanotrap associated with linear waveguide 1106A. As another example, here, the distance between the grating 1102D and the closest of the nanotrap associated with the linear waveguide 1106D is greater than the distance between the grating 1102C and the closest of the nanotrap associated with the linear waveguide 1106C. In some embodiments, gratings 1102A and 1102C have the same or similar spatial offset. In some embodiments, gratings 1102B and 1102D have the same or similar spatial offset. The characteristic of the gratings 1102A-1102D being at least partially spatially offset from one another facilitates coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguides 1106A and/or 1106C) without coupling electromagnetic radiation (e.g., light) into the other of the linear waveguides (e.g., linear waveguides 1106B and/or 1106D).

In some embodiments, the coupling into the gratings 1102A-1102D may also or alternatively be differentiated by beam parameters other than the position of the beam (such as, but not limited to, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof). In some embodiments, the coupling into the gratings 1102A-1102D may also or alternatively be differentiated by coupler parameters such as, but not limited to, grating period, refractive index, pitch, groove width, groove height, groove spacing, grating non-uniformity, groove orientation, groove curvature, overall shape of the coupler, and combinations thereof. In some embodiments, the coupling into the gratings 1102A-1102D may also or alternatively be differentiated by waveguide parameters (such as, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and/or the optical beam, and combinations thereof) with respect to one or more of the linear waveguides 1106A-1106D.

The examples described above illustrate that the flow cell 1100 includes a nanowell layer having a first set of nanowells (e.g., the nanowells associated with the linear waveguide 1106A) and a second set of nanowells (e.g., the nanowells associated with the linear waveguide 1106B) to hold a sample. Flow cell 1100 includes a first linear waveguide (e.g., linear waveguide 1106A) aligned with a first set of nanowells and a second linear waveguide (e.g., linear waveguide 1106B) aligned with a second set of nanowells; and a first grating (e.g., grating 1102A) for a first linear waveguide and a second grating (e.g., grating 1102B) for a second linear waveguide. The first grating has a first characteristic (e.g., is spatially offset from the grating 1102B) to facilitate coupling the first light into the first linear waveguide without coupling the first light into the second linear waveguide.

Fig. 12 shows another example of a flow cell 1200 with an interleaved grating 1202. Flow cell 1200 can be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For example, the flow cell 1200 may be used with either an interleaved grating or a non-interleaved grating or both. One or more of the interleaved gratings 1202 may be used with one or more of the methods described herein and/or in conjunction with one or more of the systems or devices described herein. For example, the staggered grating 1202 may be used with nanowells arranged in a hexagonal array or a non-hexagonal (e.g., polygonal beyond) array, or both.

Flow cell 1200 includes a plurality of nanowells, including nanowell 1204A, illustrated here using a circular shape. Only some of the nanowells will be specifically mentioned, while other nanowells may be similar or identical to the nanowell(s) in question. The nanowells may be formed in a nanowell layer (e.g., by a nanoimprint or lift-off process). For example, a nanoscale template may be used to form a nanowell in a resin. For clarity, the nanowell layer is not explicitly shown in this example. The nano-well 1204A is here associated with a linear waveguide 1206A. In some embodiments, the linear waveguide described with reference to flow cell 1200 may be similar or identical to one or more other linear waveguides described herein. For example, linear waveguide 1206A is positioned adjacent to (e.g., in contact with or proximate to) a nanowell layer that includes nanowell 1204A.

Another nanowell 1204B is also associated with linear waveguide 1206A. For example, nanowell 1204B is positioned adjacent to nanowell 1204A, and both nanowells 1204A-1204B may interact with linear waveguide 1206A during imaging (e.g., by receiving electromagnetic radiation from linear waveguide 1206A). By contrast, another nanowell 1204C is instead associated with linear waveguide 1206B. In some embodiments, linear waveguide 1206B is positioned adjacent to linear waveguide 1206A. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 1206A-1206B.

Here, the nano-well 1204D is associated with a linear waveguide 1206C. In some embodiments, linear waveguide 1206C is positioned adjacent to linear waveguide 1206B. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 1206B-1206C.

Here, the nano-well 1204E is associated with a linear waveguide 1206D. In some embodiments, linear waveguide 1206D is positioned adjacent to linear waveguide 1206C. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 1206C-1206D.

Here, nanowells 1204A-1204B and other nanowells form a first set of nanowells (e.g., a row of nanowells) that extends along linear waveguide 1206A. Here, the nanowell 1204C and other nanowells form a second set of nanowells (e.g., a row of nanowells) that extends along the linear waveguide 1206B. Here, the nanowell 1204D and other nanowells form a third set of nanowells (e.g., a row of nanowells) that extends along the linear waveguide 1206C. Here, the nanowell 1204E and other nanowells form a fourth set of nanowells (e.g., a row of nanowells) extending along the linear waveguide 1206D. In some implementations, a first set of nanowells (e.g., nanowells 1204A-1204B and other nanowells) are positioned so as to be out of phase with a second set of nanowells (e.g., nanowell 1204C and other nanowells). In some embodiments, none of the nanowells of the first set of nanowells have a corresponding nanowell of the second set of nanowells directly across the cladding layer or other material. For example, each nanowell of the first set of nanowells may be positioned equidistant between two adjacent nanowells of the second set of nanowells.

Here, the nanowells 1204D and other nanowells of the third set of nanowells are positioned at substantially regular intervals, and in at least one instance at substantially regular intervals, along the linear waveguide 1206C. The third set of nanowells is positioned out of phase with at least the second set of nanowells. In some embodiments, none of the nanowells of the third set of nanowells have a corresponding nanowell of the second set of nanowells directly across the cladding layer or other material. For example, each nanowell of the third set of nanowells may be positioned equidistant between two adjacent nanowells of the second set of nanowells. The third set of nanowells may be positioned so as to be in phase with at least the first set of nanowells.

In some embodiments, the fourth set of nanowells (e.g., nanowell 1204E and other nanowells along linear waveguide 1206D) are positioned so as to be out of phase with the third set of nanowells (e.g., nanowell 1204D and other nanowells along linear waveguide 1206C). In some embodiments, none of the nanowells in the fourth set of nanowells have a corresponding nanowell in the third set of nanowells directly across the cladding layer or other material. For example, each nanowell of the fourth set of nanowells may be positioned equidistant between two adjacent nanowells of the third set of nanowells.

The grating 1202 is used to couple electromagnetic radiation into and/or out of a linear waveguide of the flow cell 1200. Here, the linear waveguide 1206A has a grating 1202A, the linear waveguide 1206B has a grating 1202B, the linear waveguide 1206C has a grating 1202C, and the linear waveguide 1206D has a grating 1202D. Each of the gratings 1202A-1202D may have the same or different periodic structure. In some embodiments, some or all of the gratings 1202A-1202D may comprise periodic ridge structures interspersed with another material. For example, the ridges of the gratings 1202A-1202D may have a pitch of about 200 and 300nm, to name just one example. The gratings 1202A-1202D may have one or more of a number of suitable shapes. In some embodiments, the gratings 1202A-1202D have a truncated triangular shape.

The gratings 1202A-1202D may have one or more characteristics that facilitate, at least in part, selective coupling of electromagnetic radiation into corresponding linear waveguides 1206A-1206D. In some implementations, one or more gratings 1202 are spatially offset from one or more other gratings 1202. The offset may be in a direction parallel to the linear waveguides 1206A-1206D. For example, here, the distance between the grating 1202B and the other end of the linear waveguide 1206B is shorter than the distance between the grating 1202A and the other end of the linear waveguide 1206A. As another example, here, the distance between the grating 1202D and the other end of the linear waveguide 1206D is shorter than the distance between the grating 1202C and the other end of the linear waveguide 1206C. In some embodiments, gratings 1202A and 1202C have the same or similar spatial offset. In some embodiments, gratings 1202B and 1202D have the same or similar spatial offset. The features of the gratings 1202A-1202D being at least partially spatially offset from one another facilitate coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguide 1206A and/or 1206C) without coupling electromagnetic radiation (e.g., light) into the other of the linear waveguides (e.g., linear waveguide 1206B and/or 1206D).

In some embodiments, the coupling into the gratings 1202A-1202D may also or alternatively be differentiated by beam parameters other than the position of the beam (such as, but not limited to, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof). In some embodiments, the coupling into the gratings 1202A-1202D may also or alternatively be differentiated by coupler parameters such as, but not limited to, grating period, refractive index, pitch, groove width, groove height, groove spacing, grating non-uniformity, groove orientation, groove curvature, overall shape of the coupler, and combinations thereof. In some implementations, the coupling into the gratings 1202A-1202D can also or alternatively be differentiated by waveguide parameters (such as, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and/or the optical beam, and combinations thereof) with respect to one or more of the linear waveguides 1206A-1206D.

The flow cell 1200 may have nano-wells arranged in any of a variety of patterns. In this example, the nanowells are arranged in a hexagonal array. The hexagonal array forms one or more hexagons. Here, linear waveguide 1206B also includes nanowells 1204F-1204G, and linear waveguide 1206C also includes nanowell 1204H. Here, the nanowells 1204A-1204H are positioned in a hexagonal pattern. Here, nanowells 1204A-1204B are part of a first set of nanowells and are associated with linear waveguide 1206A; nanowells 1204C and 1204F-1204G are part of a second set of nanowells and are associated with linear waveguide 1206B; nanowells 1204D and 1204H are part of a third set of nanowells and are associated with linear waveguide 1206C.

The examples described above illustrate that the flow cell 1200 includes a nanowell layer having a first set of nanowells (e.g., the nanowells associated with linear waveguide 1206A) and a second set of nanowells (e.g., the nanowells associated with linear waveguide 1206B) to accommodate a sample. Flow cell 1200 includes a first linear waveguide (e.g., linear waveguide 1206A) aligned with a first set of nanowells and a second linear waveguide (e.g., linear waveguide 1206B) aligned with a second set of nanowells; and a first grating (e.g., grating 1202A) for the first linear waveguide and a second grating (e.g., grating 1202B) for the second linear waveguide. The first grating has a first characteristic (e.g., spatially offset from the grating 1202B) to facilitate coupling the first light into the first linear waveguide without coupling the first light into the second linear waveguide.

Fig. 13 shows another example of a flow cell 1300 with an interleaved grating 1302. The flow cell 1300 may be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For example, the flow cell 1300 may be used with either an interleaved grating or a non-interleaved grating or both. One or more of the interleaved gratings 1302 may be used with one or more of the methods described herein and/or in conjunction with one or more of the systems or devices described herein. For example, the interleaved grating 1302 may be used with nanowells arranged in a hexagonal array or a non-hexagonal (e.g., polygonal beyond) array, or both.

The flow cell 1300 includes a plurality of nano-wells, including nano-well 1304A, which is illustrated herein using a circular shape. Only some of the nanowells will be specifically mentioned, while other nanowells may be similar or identical to the nanowell(s) in question. The nanowells may be formed in a nanowell layer (e.g., by a nanoimprint or lift-off process). For example, a nanoscale template may be used to form a nanowell in a resin. For clarity, the nanowell layer is not explicitly shown in this example. The nano-well 1304A is here associated with a linear waveguide 1306A. In some embodiments, the linear waveguide described with reference to flow cell 1300 can be similar or identical to one or more other linear waveguides described herein. For example, the linear waveguide 1306A is positioned adjacent to (e.g., in contact with or proximate to) a nanowell layer that includes the nanowell 1304A. The nano-wells 1304A are part of a first set of nano-wells (e.g., one or more rows of nano-wells) for the linear waveguide 1306A. Here, the nano-well 1304A, which is part of the nano-well row, extends along the linear waveguide 1306A on one side thereof. For example, in the perspective view of the flow cell 1300 shown, the rows of nanowells do not overlap the linear waveguide 1306A.

Another nanowell 1304B is also associated with the linear waveguide 1306A. Similar to the nano-wells 1304A, the nano-wells 1304B are also part of a first set of nano-wells (e.g., one or more rows of nano-wells) for the linear waveguide 1306A. Here, the nanowell 1304B, which is a part of the nanowell row, extends on the other side thereof along the linear waveguide 1306A. For example, in the perspective view of the flow cell 1300 shown, the row of nanowells does not overlap the linear waveguide 1306A, but is positioned on the opposite side of the linear waveguide 1306A from the row of nanowells 1304A. The nano-wells 1304A-1304B may all interact with the linear waveguide 1306A during imaging (e.g., by receiving electromagnetic radiation from the linear waveguide 1306A).

Another nano-well 1304C is associated with the linear waveguide 1306B. In some embodiments, linear waveguide 1306B is parallel to linear waveguide 1306A and is positioned adjacent to linear waveguide 1306A. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 1306A-1306B. The nano-wells 1304C are part of a second set of nano-wells (e.g., one or more rows of nano-wells) for the linear waveguide 1306B. Here, the nano-well 1304C, which is a part of the nano-well row, extends on one side thereof along the linear waveguide 1306B. For example, in the perspective view of the flow cell 1300 shown, the rows of nanowells do not overlap the linear waveguide 1306B. Another row of nanowells, which is also part of the second set of nanowells, may be positioned on the opposite side of the linear waveguide 1306B from the row of nanowells 1304C.

Another nano-well 1304D is associated with the linear waveguide 1306C. In some embodiments, linear waveguide 1306C is parallel to linear waveguide 1306B and is positioned adjacent to linear waveguide 1306B. For example, cladding (not shown) and/or another material may be positioned between the linear waveguides 1306B-1306C. The nano-wells 1304D are part of a third set of nano-wells (e.g., one or more rows of nano-wells) for the linear waveguide 1306C. Here, the nanowell 1304D, which is a part of the nanowell row, extends on one side thereof along the linear waveguide 1306C. For example, in the perspective view of the flow cell 1300 shown, the rows of nanowells do not overlap the linear waveguide 1306C. Another row of nanowells, which is also part of the third set of nanowells, may be positioned on the opposite side of the linear waveguide 1306C from the row of nanowells 1304D.

Positioning the nanowells with associated linear waveguides with an offset (e.g., as in flow-through cell 1300) may provide one or more advantages. In some embodiments, cross-talk between waveguides may be reduced or minimized. For example, this benefit may outweigh the lower packing density of the nanowells.

In some implementations, the nanowells (e.g., nanowells 1304A-1304B and other nanowells) in a row of the first set of nanowells are positioned so as to be in phase with each other. The nanowells in the nanowell rows on either side of the linear waveguide 1306A may be positioned at substantially regular intervals, and in at least one instance at substantially regular intervals, along the linear waveguide 1306A. For example, each nanowell in one of the rows has a corresponding nanowell in the other row. A corresponding nanowell of the first set of nanowells may be positioned directly across the linear waveguide 1306A from another nanowell of the first set of nanowells.

In some implementations, the first set of nanowells (e.g., nanowells 1304A-1304B and other nanowells) are positioned so as to be in phase with the second set of nanowells (e.g., nanowell 1304C and other nanowells). The nanowells in the nanowell rows on either side of the linear waveguide 1306B may be positioned at substantially regular intervals, and in at least one instance at substantially regular intervals, along the linear waveguide 1306B. For example, each nanowell in at least one of the rows has a corresponding nanowell in at least one of the rows of the first set of nanowells. A corresponding nanowell of the first set of nanowells may be positioned directly across the cladding layer or other material from a nanowell of the second set of nanowells.

The grating 1302 is used to couple electromagnetic radiation into and/or out of the linear waveguide of the flow cell 1300. Here, linear waveguide 1306A has grating 1302A, linear waveguide 1306B has grating 1302B, linear waveguide 1306C has grating 1302C, and linear waveguide 1306D has grating 1302D. Each of gratings 1302A-1302D may have the same or different periodic structure. In some embodiments, some or all of the gratings 1302A-1302D may comprise periodic ridge structures interspersed with another material. For example, the ridges of gratings 1302A-1302D may have a pitch of about 200 and 300nm, to name just one example.

The gratings 1302A-1302D may have one or more characteristics that facilitate, at least in part, selective coupling of electromagnetic radiation into corresponding linear waveguides 1306A-1306D. In some implementations, one or more gratings 1302 are spatially offset from one or more other gratings 1302. The offset may be in a direction parallel to the linear waveguides 1306A-1306D. For example, here, the distance between grating 1302B and the closest of the nanotrap associated with linear waveguide 1306B is greater than the distance between grating 1302A and the closest of the nanotrap associated with linear waveguide 1306A. As another example, here, the distance between grating 1302D and the closest of the nanotrap associated with linear waveguide 1306D is greater than the distance between grating 1302C and the closest of the nanotrap associated with linear waveguide 1306C. In some embodiments, gratings 1302A and 1302C have the same or similar spatial offset. In some embodiments, gratings 1302B and 1302D have the same or similar spatial offset. The features of the gratings 1302A-1302D that are at least partially spatially offset from one another facilitate coupling electromagnetic radiation (e.g., light) into one of the linear waveguides (e.g., linear waveguides 1306A and/or 1306C) without coupling electromagnetic radiation (e.g., light) into another of the linear waveguides (e.g., linear waveguides 1306B and/or 1306D).

Here, distance 1308 is less than the resolution distance of the emitting optic, and distance 1310 is greater than or about equal to the resolution distance of the emitting optic. Distance 1308 here represents the spacing between the nearest nanowells associated with adjacent linear waveguides. Distance 1310 here represents the distance between the nanowells associated with the same linear waveguide.

In some embodiments, the coupling into the gratings 1302A-1302D may also or alternatively be differentiated by beam parameters other than the position of the beam (such as, but not limited to, angle of incidence, divergence, mode profile, polarization, aspect ratio, diameter, wavelength, and combinations thereof). In some embodiments, the coupling into the gratings 1302A-1302D may also or alternatively be differentiated by coupler parameters such as, but not limited to, grating period, refractive index, pitch, groove width, groove height, groove spacing, grating non-uniformity, groove orientation, groove curvature, overall shape of the coupler, and combinations thereof. In some embodiments, the coupling into the gratings 1302A-1302D may also or alternatively be differentiated by waveguide parameters (such as, but not limited to, cross-sectional profile, refractive index difference, mode matching with the coupler and/or the optical beam, and combinations thereof) with respect to one or more of the linear waveguides 1306A-1306D.

Examples herein illustrate differential coupling of light into two or more linear waveguides. Differential coupling may be based on one or more parameters characterizing the analysis system that have an effect on the degree to which light is (or is not) coupled to the one or more linear waveguides. In some embodiments, one or more such parameters may relate to a light beam as a source of illumination (e.g., excitation illumination) for analysis. For example, the coupler (e.g., grating) may be relatively sensitive to one or more parameters, and thus relatively small variations in the parameter(s) may facilitate differential coupling.

The examples described above illustrate that the flow cell 1300 includes a nanowell layer having a first set of nanowells (e.g., the nanowells associated with linear waveguide 1306A) and a second set of nanowells (e.g., the nanowells associated with linear waveguide 1306B) to hold a sample. The flow cell 1300 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 grating (e.g., grating 1302A) for the first linear waveguide and a second grating (e.g., grating 1302B) for the second linear waveguide. The first grating has a first characteristic (e.g., spatially offset from grating 1302B) to facilitate coupling the first light into the first linear waveguide without coupling the first light into the second linear waveguide.

Fig. 14 schematically shows a light beam 1400 impinging on a surface 1402. The examples and/or concepts described with reference to optical beam 1400 can be employed by and/or in conjunction with one or more of the methods described herein and/or with one or more of the systems or apparatuses described herein.

Here, light beam 1400 is incident at location 1404 on surface 1402. In some embodiments, 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 1400 impinges can affect the degree to which light is (or is not) coupled into one or more linear waveguides.

The one or more angles may be indicative of the incidence of the light beam 1400. Here, the light beam 1400 has an angle of incidence 1406 with respect to a normal to the surface 1402. In some embodiments, the angle of incidence 1406 may be selected and/or adjusted to facilitate differentially coupled beam parameters. For example, the angle of incidence 1406 of the light beam 1400 may affect the degree to which light is (or is not) coupled into one or more linear waveguides.

One or more characteristics of the light beam 1400 may be considered. Here, the light beam 1400 includes individual rays 1400A-1400B that are not parallel to each other but form an angle 1408 (non-zero angle). The divergence of the light beam 1400 may be defined based on characteristics such as angle 1408. In some embodiments, the divergence of the light beam 1400 is a beam parameter that can be selected and/or adjusted to facilitate differential coupling. For example, divergence may affect the degree to which light is (or is not) coupled into one or more linear waveguides.

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

The light beam 1400 may have one or more polarizations. In some embodiments, polarization is a beam parameter that can be selected and/or adjusted to facilitate differential coupling. For example, polarization may affect the degree to which light is (or is not) coupled into one or more linear waveguides.

The light beam 1400 may have any suitable cross-sectional profile. In some embodiments, light beam 1400 has a rectangular cross-sectional profile 1412A. For example, one or more dimensions of the rectangular cross-sectional profile 1412A (e.g., its aspect ratio) are beam parameters that may be selected and/or adjusted to facilitate differential coupling. In some embodiments, light beam 1400 has a circular cross-sectional profile 1412B. For example, one or more dimensions of the circular cross-sectional profile 1412B (e.g., its diameter) are beam parameters that may 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 may affect the degree to which light is (or is not) coupled into the one or more linear waveguides.

Optical beam 1400 may include one or more wavelengths of electromagnetic radiation. In some embodiments, the wavelength of the optical beam 1400 is a beam parameter that can be selected and/or adjusted to facilitate differential coupling. The wavelength may affect the extent to which light is (or is not) coupled into the one or more linear waveguides. For example, different wavelengths are coupled into the grating at different angles. Changes in the wavelength and angle of the light beam 1400 may allow for differential coupling.

In some embodiments, the one or more parameters affecting differential coupling may relate to a grating coupling light into the linear waveguide for analysis. For example, the coupler (e.g., grating) may be relatively sensitive to one or more parameters, and thus, relatively small variations in the parameter(s) may facilitate differential coupling.

Fig. 15A-15B show examples of gratings 1500 and 1502. Grating 1500 and/or 1502 may be used with one or more of the methods described herein and/or in conjunction with one or more of the systems or devices described herein.

The gratings 1500 and 1502 may have the same or different refractive indices from each other. In some embodiments, the refractive index is a coupler parameter that can be selected and/or adjusted to facilitate differential coupling. For example, the refractive index may affect the degree to which light is (or is not) coupled into the one or more linear waveguides.

Here, grating 1500 includes grooves 1504 and grating 1502 includes grooves 1506 and 1508. At least one groove pitch 1510 can be defined for each of the gratings 1500 and 1502. Groove pitch 1510 can represent a distance from an edge of one of grooves 1504, 1506, or 1508 to a corresponding edge of an adjacent one of grooves 1504, 1506, or 1508. In some embodiments, groove pitch 1510 is a coupler parameter that can be selected and/or adjusted to facilitate differential coupling. For example, groove pitch 1510 can affect the degree to which light is (or is not) coupled into one or more linear waveguides.

At least one groove width 1512 may be defined for each groove 1504, 1506, or 1508. Groove width 1512 may represent the width of one of grooves 1504, 1506, or 1508 from edge to edge. In some embodiments, the groove width 1512 is a coupler parameter that can be selected and/or adjusted to facilitate differential coupling. For example, the groove width 1512 can affect the degree to which light is (or is not) coupled into one or more linear waveguides.

At least one groove height 1514 may be defined for each groove 1504, 1506, or 1508. Groove height 1514 may represent a height from a bottom of one of grooves 1504, 1506, or 1508 to the opening. In some embodiments, the groove height 1514 is a coupler parameter that can be selected and/or adjusted to facilitate differential coupling. For example, the groove height 1514 may affect the degree to which light is (or is not) coupled into one or more linear waveguides.

At least one groove spacing 1516 may be defined for each groove 1504, 1506, or 1508. Groove spacing 1516 may represent the distance from an edge of one of grooves 1504, 1506, or 1508 to a nearest edge of an adjacent one of grooves 1504, 1506, or 1508. In some embodiments, the groove spacing 1516 is a coupler parameter that can be selected and/or adjusted to facilitate differential coupling. For example, the groove spacing 1516 can affect the degree to which light is (or is not) coupled into the one or more linear waveguides.

In some embodiments, a non-uniform grating may be used. In some embodiments, grooves 1506 and 1508 of grating 1502 provide a non-uniform grating. For example, the grooves 1506 and 1508 may have different groove widths 1512. As another example, grooves 1506 and 1508 may alternatively or additionally have different groove pitches 1510, different groove heights 1514, and/or different groove pitches 1516. Thus, grating 1502 is an example of grating non-uniformity.

In some embodiments, the groove orientation is a coupler parameter that can be selected and/or adjusted to facilitate differential coupling. In some embodiments, the gratings are typically coupled with transverse electrical polarization, with the electric field parallel to the grating grooves. Gratings 1500 and/or 1502 may be positioned such that a particular orientation of grooves 1504, 1506, and/or 1508 is obtained. For example, the groove structure may be rotated to another orientation to provide coupling based on the rotated polarization. That is, the groove orientation may affect the degree to which light is (or is not) coupled into the one or more linear waveguides.

In some embodiments, the groove curvature is a coupler parameter that can be selected and/or adjusted to facilitate differential coupling. Fig. 15C shows a top view of a grating 1518 with grooves 1520 of different curvatures. For example, the groove curvature may affect the degree to which light is (or is not) coupled into the one or more linear waveguides.

In some embodiments, the coupler shape is a coupler parameter that can be selected and/or adjusted to facilitate differential coupling. Fig. 16 shows an example of the shape of couplers 1600, 1602, 1604, and 1606. These examples show an exemplifying shape of the coupler and schematically indicate the grooves of the respective gratings. Coupler 1600 may include a rectangular (e.g., square) grating. For example, the grooves of the grating may be oriented along the long or short sides of a rectangle. The coupler 1602 may include an elliptical (e.g., circular) grating. For example, the grooves of the grating may be oriented along the long or short axis of the grating. The coupler 1604 may comprise a truncated triangular grating. For example, the grooves of the grating may be oriented perpendicular to the base of the triangle or perpendicular to the height of the triangle. As another example, different angles of the sides may be used. Coupler 1606 may include a triangular grating. For example, the grooves of the grating may be oriented perpendicular to the base of the triangle or perpendicular to the height of the triangle. As another example, different angles of the sides may be used. In some embodiments, the shape of the coupler(s) may be selected based on, for example, an optimized diameter of the illumination beam, or an aspect ratio of the illumination beam, or a combination thereof, to name a few. The coupler shape and/or the orientation of the grooves may affect the degree to which light is (or is not) coupled into the one or more linear waveguides.

The shape of the couplers (including but not limited to couplers 1600, 1602, 1604, and 1606) may be selected in view of the diameter, aspect ratio, or other characteristics of the optical beam. This may allow, for example, tuning the resulting structure for a particular differential coupling.

The coupler parameter(s) may be selected and/or adjusted based on a mode profile of the illuminating beam. This can be done by selecting (e.g. optimizing) the groove structure. In some embodiments, a non-uniform grating may be used. For example, linearly chirped gratings (e.g., gratings with groove pitch variation), apodized gratings (e.g., gratings with a near-zero refractive index towards the ends of the gratings), curved surface gratings, and combinations thereof may be used. In some implementations, computer-based optimization can be performed on one or more coupler parameters (e.g., grating structure). This may facilitate differential coupling based on the mode profile of the incident beam, for example.

In some implementations, the one or more parameters affecting the differential coupling may relate to a linear waveguide into which the light is coupled for analysis. For example, the coupling may be relatively sensitive to one or more parameters associated with the waveguide, and thus, relatively small changes in the parameter(s) may facilitate differential coupling.

In some embodiments, the cross-sectional profile of the linear waveguide is a waveguide parameter that can be selected and/or adjusted to facilitate differential coupling. Fig. 17 shows an example of a cross-sectional profile of a linear waveguide. The waveguide 1700 may include a rectangular (e.g., square) profile. For example, the nanowell layer may be positioned adjacent to a long side or a short side of a rectangle. The waveguide 1702 may include an elliptical (e.g., circular) profile. For example, the nanowell layers may be positioned parallel to the long or short axis of the waveguide 1702. The waveguide 1704 may include a truncated triangular profile. For example, the nanowell layer may be positioned adjacent to the base, side(s), and/or truncation of the triangle. Different angled sides may be used. The waveguide 1706 may include a triangular profile. For example, the nanowell layer may be positioned adjacent to one or more sides of a triangle. Different angles of the sides may be used. The cross-sectional profile may affect the extent to which light is (or is not) coupled into the one or more linear waveguides.

In some embodiments, 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 degree to which light is (or is not) coupled into the waveguides.

In some embodiments, the matching of one or more modes between the linear waveguide and the coupler, or between the linear waveguide and the optical beam, or both, is a waveguide parameter that can be selected and/or adjusted to facilitate differential coupling. For example, the dimensions and/or proportions of the linear waveguide may be selected so as to promote propagation (or not promote propagation) of a particular mode of incoming light. That is, mode matching with the coupler and/or the optical beam may affect the degree to which light is (or is not) coupled into the waveguide.

Examples herein refer to beam parameters, coupler parameters, and/or waveguide parameters that may be selected and/or adjusted to facilitate differential coupling. In some embodiments, a combination of two or more such parameters may be selected and/or adjusted. For example, the selection/adjustment may take into account 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, the cross-sectional profile of the waveguide can be used with a particular grating (e.g., a grating optimized for a certain coupling or non-coupling). For example, this may allow tuning of the resulting structure for different mode profiles, beam diameters, aspect ratios, to name a few.

Fig. 18 shows a cross-section of a portion of another example flow cell 1800 having linear waveguides 1802, 1804 and 1806. Flow cell 1800 can be used with one or more of the methods described herein, and/or in conjunction with one or more of the systems or devices described herein. For example, the flow cell 1800 may be used with either an interleaved grating or a non-interleaved grating or both. As another example, the flow cell 1800 may be used with nano-wells arranged in a hexagonal array or a non-hexagonal (e.g., polygonal among others) array, or both. For illustrative purposes, only a portion of the flow cell 1800 is shown. For example, one or more additional layers and/or more or fewer waveguides 1802, 1804, and/or 1806 may be used.

The flow cell 1800 includes a substrate 1808. The substrate 1808 may form a base for the flow cell 1800. In some embodiments, one or more other layers may be formed at (e.g., in contact with or near) the substrate 1808 during fabrication of the flow cell 1800. The substrate 1808 may serve as a foundation for forming the linear waveguides 1802, 1804, and/or 1806. The linear waveguides 1802, 1804, and/or 1806 may be initially present separate from the substrate 1808 and then applied to the substrate 1808, or the linear waveguides 1802, 1804, and/or 1806 may be formed by applying or removing one or more materials from the substrate. The linear waveguides 1802, 1804, and/or 1806 may be formed directly on the substrate 1808, or on one or more intermediate layers at the substrate 1808.

The linear waveguides 1802, 1804, and/or 1806 are used to conduct electromagnetic radiation (including, but not limited to, visible light, such as laser light). In some embodiments, the electromagnetic radiation performs one or more functions during the imaging process. For example, electromagnetic radiation may be used to excite fluorophores in the sample material for imaging. The linear waveguides 1802, 1804, and/or 1806 may be made of any suitable material that facilitates propagation of one or more types of electromagnetic radiation. In some implementations, the material(s) of the linear waveguides 1802, 1804, and/or 1806 may include a polymer material. In some embodiments, the material of the linear waveguides 1802, 1804, and/or 1806 may include Ta₂O₅And/or SiN_x. For example, the linear waveguides 1802, 1804, and/or 1806 may be formed by sputtering, chemical vapor deposition, atomic layer deposition, spin coating, and/or spray coating.

Each linear waveguide 1802, 1804, and/or 1806 may have one or more gratings (omitted here for clarity) to couple electromagnetic radiation into and/or out of the linear waveguide 1802, 1804, and/or 1806. The grating(s) may be positioned in the same layer as the corresponding linear waveguide(s). Electromagnetic radiation may be employed in one or more directions of travel of the linear waveguides 1802, 1804, and/or 1806. For example, the direction of travel may be into and/or out of the plane of the present example. Examples of gratings are described elsewhere herein.

Each of the linear waveguides 1802, 1804, and/or 1806 may be positioned against one or more types of cladding. The cladding may serve to confine electromagnetic radiation to the respective linear waveguide 1802, 1804, and/or 1806 and prevent or reduce the extent to which radiation propagates to other linear waveguides 1802, 1804, and/or 1806 or other substrates. Here, cladding layers 1810, 1812, 1814, 1816, and 1818 are shown as examples. In some embodiments, the cladding layers 1810, 1812, and 1814, along with the linear waveguides 1802 and 1804, may form a first layer in the flow cell 1800. For example, the cladding layers 1810 and 1812 may be positioned against or near the linear waveguide 1802 on different (e.g., opposite) sides of the linear waveguide 1802. For example, cladding layers 1812 and 1814 may be positioned against or near linear waveguide 1804 on different (e.g., opposite) sides of linear waveguide 1804. In some embodiments, cladding layers 1816 and 1818, together with linear waveguide 1806, may form a second layer in flow cell 1800. For example, cladding layers 1816 and 1818 may be positioned against or near linear waveguide 1806 on different (e.g., opposite) sides of linear waveguide 1806. Forming multiple layers may provide advantages with respect to differential coupling. In some embodiments, two or more different materials may be used for the respective waveguides. This may facilitate imparting different indices of refraction to the respective waveguides and/or couplers, for example. In some embodiments, cross-talk between waveguides may be reduced or minimized.

The cladding layers 1810, 1812, 1814, 1816, and/or 1818 may be made of one or more suitable materials for separating the linear waveguides 1802, 1804, and/or 1806 from one another. In some implementations, the cladding layers 1810, 1812, 1814, 1816, and/or 1818 can be made of a material having a lower index of refraction than one or more of the linear waveguides 1802, 1804, and/or 1806. For example, the linear waveguides 1802, 1804, and/or 1806 may have a refractive index of about 1.4-1.6, and the cladding layers 1810, 1812, 1814, 1816, and/or 1818 may have a refractive index of about 1.2-1.4. In some embodiments, one or more of cladding layers 1810, 1812, 1814, 1816, and/or 1818 comprises a polymeric material. In some embodiments, one or more of cladding layers 1810, 1812, 1814, 1816, and/or 1818 comprise a plurality of structures including, but not limited to: structures of one material (e.g., a polymer) are interspersed with regions of vacuum or another material (e.g., air or liquid).

The flow cell 1800 includes at least one nanowell layer 1820. In some embodiments, the nanowell layer 1820 is positioned opposite the first layer from the second layer. For example, the nanowell layer 1820 may be positioned adjacent (e.g., abutting or proximate) the linear waveguides 1802 and 1804 and the cladding layers 1810, 1812, and 1814. The nanowell layer 1820 comprises one or more nanowells. In some embodiments, the nanowell layer 1820 comprises nanowells 1822, 1824, and 1826. The nano-wells 1822, 1824, and/or 1826 may be used to hold one or more sample materials during at least a portion of an analysis process (e.g., for imaging). For example, one or more genetic materials (e.g., in clusters) can be placed in the nanowells 1822, 1824, and/or 1826.

The nanowells 1822, 1824, and/or 1826 may be arranged at the nanowell layer 1820 in any pattern or without a specific pattern. One or more of the nano-wells 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, transmission through evanescent light). For example, the nanowell 1822 may be at least substantially aligned with the linear waveguide 1802; the nano-trap 1824 may be at least substantially aligned with the linear waveguide 1804; and/or the nano-trap 1826 may be at least substantially aligned with the linear waveguide 1806. In some embodiments, the first layer (e.g., cladding layers 1810, 1812, and 1814, along with linear waveguides 1802 and 1804) may be positioned closer to the nanolayer 1820 than the second layer (e.g., cladding layers 1816 and 1818, along with linear waveguide 1806). As another example, the second layer may be positioned farther away from the third layer than the first layer.

Fig. 19 is a flow chart of an exemplary method 1900. Method 1900 may be performed using and/or in conjunction with one or more other examples described herein. More or fewer operations may be performed, and/or two or more operations may be performed in a different order, unless otherwise indicated.

At 1910, a sample is applied to at least some of the nanowells of the flow cell. In some embodiments, the sample is applied to the first set of nanowells and the second set of nanowells.

At 1920, the first light may be differentially coupled into at least one first linear waveguide associated with the first set of nanowells. In some implementations, the first light can be differentially coupled using the first grating.

At 1930, the second light can be differentially coupled into at least one second linear waveguide associated with a second set of nanowells. In some embodiments, the second grating may be used to differentially couple the second light.

The terms "substantially" and "about" are used throughout the specification to describe and explain small fluctuations such as those 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 herein, the indefinite article "a" or "an" means "at least one".

It should be understood that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided that such concepts do not contradict each other) are considered a part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are considered part of the inventive subject matter disclosed herein.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the description.

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

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