Nanostructured substrates for enhancing fluorescence emission

Abstract

A nanostructured substrate is provided for use in connection with analyzing a biological material in an optical detection system. Nanostructures imprinted in an optic support substrate are configured as a nanoantennae array for supporting fluorescence-based assays involving the detection and characterization of target analytes of a biological material, where each nanoantenna includes a conically-formed cavity and a conformal shell of high refractive index medium supporting an interior volume of the cavity that behaves as a single mode resonator to promote strong resonant field coupling between optical modes of the nanoantenna and a resident fluorescent species under excitation.

Description

Nanostructured Substrates for Enhancing Fluorescence EmissionCROSS- REFERENCE TO RELATED APPLICATION(0001] This application claims the benefit of Provisional U.S. Patent Application No.63 / 564,580, filed March 13, 2024, the entire disclosure of which is hereby incorporated by reference herein in its entirety.BACKGROUND OO02| The technology disclosed relates to nanostructured substrates for supporting opticsbased biological and chemical assay protocols in various genomic, exogenomic, transcriptomic, and proteomic domains, including, e.g., high throughput screening (HTP), nucleic acid sequencing implemented using Next Generation Sequencing (NGS), variant detection and gene expression analyses, and the like. By way of example, sequencing methodologies for nucleic acid materials on NGS platforms commonly deploy deoxyribonucleic acid (DNA) libraries in which a DNA target (e.g., genomic DNA (gDNA), or complimentary DNA (cDNA)) is processed into fragments and ligated with technology-specific adaptors. NGS workflow using, e.g., a sequence- by-synthesis (SBS) technique, involves loading a DNA library onto a flow cell and hybridizing individual DNA fragments to adapter-specific complimentary oligonucleotides (oligos) covalently bound to a solid support surface of the flow cell; clustering the individual fragments into thousands of identical DNA template strands (amplicons) through bridge or exclusion amplification; and, finally, sequencing, in which copy strands are simultaneously synthesized and sequenced on the DNA templates using a reversible terminator-based process that detects signals emitted from fluorophore-labeled single bases as they are added round by round to the copy strands. Because the multiple template strands of each cluster have the same sequence, base pairs incorporated into the corresponding copy strands in each round will be the same, and thus the signal generated from each round will be enhanced proportional to the number of copies of the template strand in the cluster.(0003] NGS operates on massively parallel multiplex platforms that can process sequencing volumes of nucleotides in the billions within very short runtimes and at low cost. For example, Illumina’s NovaSeq 6000 sequencing system can generate output in the range of 1.6-40 billion paired end reads at a run time ranging between 13 and 44 hours. By comparison, the HumanGenome Project, which sequenced the first human genome using capillary sequencing, took around 10 years.|0004j Despite vast improvements in output volume, run times, and cost effectiveness, certain operating constraints persist that lock out additional gains in processing efficiency. As just one example, a typical step and shoot sequencing operation using a flow cell format and 300 cycle SBS chemistry may obtain two images per sequencing cycle per tile (using two-channel chemistry), where the total number of cycles is equal to the length in bp of the DNA template strand, viz., 300 bp, and the total number of tiles image may be 130 or greater, resulting in a total number of imaging steps that can exceed 78,000 (130 x 300 x 2). Accordingly, any reduction in the power draw required to generate a signal with sufficient intensity to pass filter can lead to outsized efficiencies in overall power consumption and related costs. jOOOSj For these and other reasons that will become apparent from the present disclosure, there remains a need for enhancement of cost and operational efficiency of HTS, NGS, and other optics-based assay protocols while also maintaining, or even improving, system integrity and reliability.SUMMARY| 06] In one aspect, a nanostructured substrate is provided for use in connection with analyzing a biological material in an optical detection system. In certain implementations, nanostructures imprinted in an optic support substrate can be configured as a nanoantennae array for supporting fluorescence-based assays involving the detection and characterization of target analytes of a biological material, including, e.g., sequencing nucleic acid materials on NGS platforms. Each nanoantenna can include a conically-formed cavity and a conformal shell of high refractive index medium supporting an interior volume of the cavity that behaves as a single mode resonator to promote strong resonant field coupling between optical modes of the nanoantenna and a resident fluorescent species under excitation. Resonant enhancement associated with nanoantennae technology herein results in increased power density and confinement of excitation radiation within the nanoantenna cavity, leading, in turn, to an increased intensity in output signals with SNR values in excess of operating requirements associated with conventional optical detection systems.(0007 Nanostructured substrates can be implemented in single-layer or multi-layer constructs and can be implemented in any format appropriate for use in supporting a given assay protocol. For example, in certain flow cell implementations, a nanostructured substrate is configured as an optic support layer constructed of a cured resin material, such as high-index UV resin, and supported on a base layer a low background material, such as glass. The optic support layer can include a nanoantennae array separated by interstitial regions of featureless resin surface and arranged into one or more lanes along a common axis of the optic support layer. Each nanoantenna of the array can include a conically-formed cavity impressed in the optic support layer. The cavity can define an interior volume and can include an opening coplanar with the featureless resin surface, a base portion providing a solid support for optic detection of a target analyte, and an inwardly conical wall portion joining the opening and the base portion. A reaction site can be collocated with at least a portion of the solid support, which can be functionalized for interaction with a target analyte. Each nanoantenna can further include a conformal shell of high refractive index medium supporting the interior volume of the cavity, which, through resonant interaction with a resident target analyte under fluorescence, can localize the optic field response of the analyte to the vicinity of the nanoantenna and within a volume (hot spot) roughly equivalent in diameter to the wavelength of incident light. f OOOSj In some embodiments, the opening of each nanoantennae can define a radius R) and perpendicular axis, and the base portion can be coaxially located on the axis and defines a second radius (r) such that the cavity forms a frustrum. Dimensions of the first second radii can be characterized in terms of a ratio (R:r), which can be anywhere from —10:1 to -1000:1, depending, e.g., on operational requirements of a particular assay protocol, the desired resonance effect, as well as exigent considerations such as the stochastics of efficient analyte and / or reagent distribution across nanoantennae arrays.|0009j A cured resin material herein can be characterized according to its refraction index (n) within a range of wavelength (X) spectra. By way of example, a cured resin consistent with the disclosure can have a standard refraction index in the range of 1.45 < n i 55 at 403nm < < 848nm. In one such example, the cured resin material may have a refraction index in the range of 1.5 < n < 1.51 at 403nm < X < 848nm. In other examples, the cured resin can have a high refractive index of 1.70 < n > 1.80 at 403nm < X >848nm. In one such example, the cured resinmaterial is UV Resin #18247 (NTT Advanced Technology Corp.), which has a refraction index of n > -1.75 at 403nm < >848nm.|001G| For certain applications herein, the conformal shell can be constructed of a dielectric material having a high refractive index, e.g., n > -2. Dielectric materials consistent with the disclosure may include metal oxides, in either amorphous or crystalline form, such as, e g., tantalum pentoxide (Ta2O5), aluminum oxide (A12O3), and titanium oxide (TiO2), hafnium oxide (HfO2), niobium oxide (NbO), cerium oxide (CeO2), and gallium oxide (GaO2), tungsten oxide (WO), zirconium oxide (ZrO2), and tin oxide (SnO2). Dielectric materials can also include non-metal oxides such as, e.g., silicon nitride (Si3N4), indium phosphide (InP), gallium phosphide (GaP) arsenic phosphide (AsP), and germanium (Ge). According to examples, the conformal shell may have an average thickness of between 10 nm and 60 nm, 20 nm and 50 nm, 30 nm and 40 nm. The dielectric material forming the conformal shell can be applied as a uniform fdm or as densely packed nanoparticles. 00111 In another aspect, a biological assay implemented on a nanostructured substrate can be utilized in a method for optical detection of a biological material or one or more constituent analytes thereof. The method can include providing an optical detection system comprising an excitation source, one or more optical sensors, and a signal processor. In example embodiments, the method can further include irradiating, via the light source, at least a portion of the optic support layer of the biological assay with an incident light; detecting, via the one or more optical sensors, an output signal emitted by the biological material or constituent analyte(s) as an optic field response to the incident light, and obtaining from the output signal, via the signal processor, data indicative of a characteristic of the sample or constituent analyte(s).(0012 The biological material can be a nucleic acid material, e.g., as target analytes, primers, templates, or probes. Target analytes can be gDNA, including DNA variants (e.g., alleles, polymorphs, missense), mtDNA, mRNA, cDNA transcribed from mRNA, non-coding RNA, small RNA, or constituents thereof. Nucleic acid materials herein can also include polynucleotide analogues, amplicons, conjugates, and substitutions, crosslinked polynucleotides, polynucleotide complexes, and non-natural polynucleotides, including, but not limited to, dideoxynucleotides, or biotinylated, aminated, deaminated, alkylated, benzylated, flourophor- labeled polynucleotides. The biological material can also be a protein or peptide-based material, e g., as target analytes, substrates, or reagent enzymes. Protein materials herein can includefunctional proteins or protein domains acting, e.g., as effectors, inhibitors, modulators, mediators, transporters, or stimulators in connection with a specific activity affected by a target molecule. Reagent enzymes can include functioning proteins or peptide-based materials involved in nucleic acid synthesis, extension, fragmentation, amplification, or ligation. 0013| In one example, a method for optical detection of a nucleic acid sample in a sequencing protocol is provided. Flow cells provide a convenient format in the example sequencing protocol, which can involve multiple cycles of repeated chemical delivery and image capture. According to the example method, a DNA sample (e.g., gDNA) or an RNA sample (e.g., as cDNA) in the form of a DNA library can be obtained in which the DNA sample is processed into constituent fragment strands and ligated with technology-specific adaptors. According to the example method, the DNA library can be loaded onto an optic support layer of a flow cell device, where constituent fragment strands are flowed across an array of nanoantennae impressed in the optic support layer and individual strands are absorbed by nanoantennae of the array at a substantially 1 : 1 basis, where each absorbed strand is immobilized at a reaction site of respective nanoantennae through interaction between the strand adaptor and a capture primer covalently bound to the surface of the reaction site. Each immobilized strand can then be amplified (e.g., using bridge or exclusion amplification) to yield a substantially monoclonal cluster of template strands within each respective nanoantennae.|0 I4j Each of the resulting clusters is then sequenced using, e.g., a sequencing-by-synthesis (SBS) technique. SBS in general involves the enzymatic extension of a nascent copy strand through iterative addition and simultaneous detection of nucleoside monomers against a template strands. For each iteration (or cycle), labeled nucleoside monomers can be detected through induced fluorescence as each monomer is added to the copy strand then replaced in a reverseterminator process with non-labeled analogues before the start of a subsequent cycle. According to the example method, in a first cycle, (1) a buffer solution containing a mixture of four different labeled nucleoside derivatives (one for each of the four DNA nucleobases (A, G, C, T)) can be provided to a cluster of template strands in the presence of polymerase; (2) a complementary nucleoside derivative can be added to a nascent copy strand hybridized to each template strand via polymerase primer extension; (3) the added nucleoside derivatives can be irradiated with incident light via an excitation source to induce fluorescence and emission of an output signal from the cluster; (4) the output signal can be detected by one or more opticalsensors through imaging of the signal as a point source at an addressed location on the optic substrate; and (5) information imparted in the imaged output signal can be processed by a signal processor to record the nucleobase of the added nucleoside derivative and an address of the cluster on the optic substrate. Steps ( 1 )-(5) can be performed simultaneously for a plurality of clusters with a given frame (or tile) of the one or more optic sensors. Steps ( 1 )-(5) can then be repeated in subsequent cycles to n number of total cycles, where n is equal to the size of the template strands in base pairs (bp).

[0015] Here, the increased power density and localization of emission radiation within the individual nanoantenna cavity can result in an increased intensity in the output signal and improved detection of intensity clouds associated with adjacent clusters as distinct point sources. Given that, the increased signal intensity engendered by nanoantennae herein can improve detection of intensity clouds associated with adjacent analytes as distinct point sources, which can improve quality measures of the information imparted in the signal.BRIEF DESCRIPTION OF THE DRAWINGS]0016| FIG. 1A provides a schematic illustration of a non-format specific nanostructure architecture supporting a nanoantennae array of the disclosure.| 017] FIG. IB illustrates dimensions for height (H), slant height (1), pitch (P), opening diameter (D), and base diameter (d) of the illustrated nanoantennae of Fig. 1A.

[0018] FIG. 1C illustrates surface area and volume measurements of the illustrated nanoantennae of Fig. 1A.|00 9] FIG. ID demonstrates an implementation of the illustrated nanoantennae of Fig. 1A in an imaging operation.

[0020] FIG. 2A presents FDTD-simulated optical field enhancement distribution and intensity profiles of 264 nm pitch conical nanowells with n = 1.8 resin.{0021 ] FIG. 2B presents FDTD-simulated optical field enhancement distribution and intensity profiles of standard cylindrical nanowells with n = 1.51 resin.(0022] FIG. 3A is a top view of an example flow cell.

[0023] FIG. 3B is an enlarged and partially cutaway view of an example nanostructured substrate supported on the example flow cell of FIG. 3A.(0 24| FIGS. 4A-H are schematic perspective views which together depict an example method of fabricating a nanostructured substrate of the disclosure in a flow cell format.|0025| FIG. 5 illustrates one, two and four channel chemistries appropriate for use with NGSDETAILED DESCRIPTIONImage quality in the context of the present disclosure is, in part, a function of the quality of optic information imparted in a signal emitted by a target under fluorescence and captured by a sensor into an image. In turn, the quality of optic information in a target image is reflected in the Signal -to-Noise Ratio (SNR) of the image, which compares the power of a desired signal to the level of attending background noise captured in the image. As referred to herein, both signal and noise may be measured according to relative intensity (I) such that SNR is given by the formula:J0 27| In one aspect, optical detection systems implementing nanostructured solid support substrates are provided that are capable of generating high quality data from signals of interest with exceedingly high SNR using conventional imaging components and at lower power consumption. Accordingly, optic detection systems herein may provide enhancement of cost and operational efficiency of NGS and other biological target analyses while also maintaining, or even improving, system integrity and reliability.|0028| Optical nanoantennae structures of the present disclosure are capable of amplifying light emission intensity from spatially localized optic fields of fluorescing species to generate information-enhanced point sources for signal detection in fluorescence- and other optic-based assays. The nanoantennae may be imprinted in a high index resin substrate as discrete conically formed cavities organized as a patterned nanoantennae array and the substrate may be supported on any number of formats depending on the assay including flow cells, microarrays, bead chips, microwell plates, and the like. Each nanoantenna may define an opening in the resin surface and its cavity may include a base portion serving as a solid support, an inwardly conical wall portion joining the opening and the base portion, and, in use, a reaction site collocated with at least a portion of the solid support for locating a subject analyte constituting, conjugated with, or in theproximity of a fluorescent species within the nanoantenna cavity. A conformal shell of high refractive index medium (e.g., n > ~2) may be applied to the interior surfaces of the cavity as support for the interior volume of the cavity (ambient void, n=l; fluid (e.g., buffer solution), n ~ 1.38) to increase refractive index contrast (An) at interfaces and thus enhance optical functionality of the cavity. 0O29| Signal enhancements may be achieved by tuning both the cavity geometry and refractive index contrast to a resonant frequency coincident with the emission or excitation spectra of a resident fluorescent species so as to generate enhanced emission. For example, when a fluorescent species is placed within a nanoantenna cavity and fluoresced the cavity behaves as a single mode resonator to promote strong resonant field coupling between optical modes of the nanoantenna and the fluorescent species as a quantum emitter (QE). The strong resonant field coupling dramatically enhances the local density of states (LDOS) at the emitter position which proportionally increases the spontaneous emission rate of photons from the QE field (the Purcell effect) resulting in proportional amplification of a signal emitted from the nanoantenna and enhanced signal intensity.(0030 LDOS, as a measure of local field response at a given point in space, may be affected by a resonant system according to the Purcell factor Q / V, where Q is the mode quality factor and V is the mode volume of the resonant system. Generally, many commonly used design schemes rely on maximizing Q or minimizing V depending on the system size and optics application. For example, systems at nanoscale, i.e., smaller than incident wavelengths, enhance LDOS through intrinsically small mode volumes, whereas the structured medium may have low Q, single-mode resonance. By contrast, systems at microscale, i.e., larger than incident light wavelengths, enhance LDOS through scaling in Q using metal-based plasmonic or crystal-based photonic waveguide resonator structures. For instance, in whispering gallery modes of ring resonators and defect modes of photonic crystals exhibit exponential scaling in Q with increasing system size, while their mode volumes either increase polynomially or remain constant, respectively, leading to exponential growth in the Purcell factor. f 00311 Here, LDOS enhancement is provided both by scaling in Q through increased refractive index contrast (An) at interfaces and reducing V through confinement of the emitter site to a narrowed volume at the base of the conical cavity. In operation, nanoantennae herein induce resonance enhancement of the emission field limited to the LDOS and a reduction intransmission efficiency at the resonant frequency, engendering steep gains in quantum efficiency of the system, which, in turn, provides maximalization of signal strength and minimalization of parasitic autofluorescence in the interstitial substrate. As a result, signals emitted at each nanoantenna are amplified well above the noise floor. 0032| The refractive index of an optical medium, as referenced herein, is a dimensionless number that gives the indication of the light bending ability of that medium. The refractive index may determine how much the path of light is bent, or refracted, when entering a material, as described by Snell's law of refraction, nl sin 01 = n2 sin 02, where 01 and 02 are the angle of incidence and angle of refraction, respectively, of a ray crossing the interface between two media with refractive indices nl and n2. The refractive indices also determine the amount of light that is reflected when reaching the interface, as well as the critical angle for total internal reflection, their intensity (Fresnel's equations) and Brewster's angle. The refractive index may also reflect the factor by which the speed and the wavelength of the radiation are reduced with respect to their vacuum values: the speed of light in a medium is v = c / n, and similarly the wavelength in that medium is X = XO / n, where XO is the wavelength of that light in vacuum. This implies that vacuum has a refractive index of 1 and assumes that the frequency (f = v / X) of the wave is not affected by the refractive index. Optical media herein to which a refractive index may be relevant the substrate layer, nanowell surfaces, interstitial substate layer surfaces, solid supports, and reaction sites, herein.|O033 As used herein, “optic loss” (or simply “loss”) refers to the portion of light absorbed by a material at a given wavelength. Optic loss may be measured using the wavelength dependent extermination coefficient or imaginary part of the refractive index, which may be expressed as n* = n’ + ik, where n* is the complex index of refraction, n is the real part of the refractive index and k is the extinction coefficient or imaginary part of the refractive index. For dielectric materials, these values are generally 2-4 orders of magnitude smaller than for metallic substrates. For example, the value of k for dielectric Ta2Os is 0.00081579 at 532 nm, whereas the value of k for gold (Au) is 2.2309 at the same wavelength. 0 34| As used herein, “optic nonlinearity” refers to the behavior of light in nonlinear media, that is, media in which the polarization density P responds non-linearly to the electric field (E) of the light. The non-linearity is typically observed only at very high light intensities (when the electric field of the light is >108 V / m and thus comparable to the atomic electric field of -1011V / m) such as those provided by lasers. A dielectric material or structure is any nonlinear media that can be polarized by an applied electric field (E). When a dielectric material is placed in an electric field pr am electric field is generated within a dielectric structure, electric charges of the dielectric shift, only slightly, from their average equilibrium positions, causing dielectric polarization. Because of dielectric polarization, positive charges are displaced in the direction of the field and negative charges shift in the direction opposite to the field. (For example, if the field is oriented parallel to the positive x axis, the negative charges will shift in the negative x direction.) This creates an internal electric field that reduces the overall field within the dielectric material or structure itself. Optic sensing applications based on induced fluorescence as described herein may include non-parametric nonlinear optical processes in which the quantum state of the medium, e g., a fluorescing species, is changed such that the input photon energy of incident light is converted into an output photon with a frequency which is detuned from the input photon frequency by an amount determined by the photon energy. By way of example, an excitation light at 488 nm (blue) may generate an optic field response and emission signal at ~610nm (red).(0035) As used herein, the term “cavity” in the context of nanoantennae disclosed herein refers to an optical structure with a hollow morphology that supports resonant modes. As used herein, the quality factor of a cavity, or its Q, measures how ideal or lossless a cavity resonator is. An ideal lossless cavity resonator will sustain free oscillations forever, while most resonators sustain free oscillations for a finite time. This is because of losses coming from radiation, dissipation in the dielectric material filling the cavity, or resistive loss of the metallic part of the cavity. The Q of a cavity is defined as the number of free oscillations in radians (rather than cycles) that the field undergoes before the energy stored decreases to 1 / e of its original value. A higher Q factor will lead to stronger cavity effects. The mode volume (V) of a cavity is a measure of the spatial confinement of electromagnetic radiation inside the cavity. Mathematically, the mode volume may be defined as the volume integral of the electric field intensity in and around the cavity, normalized to the maximum field intensity. A smaller mode volume leads to stronger cavity effects.(0036] As used herein, the amplification factor engendered by a resonant system may be determined based on the ratio of the peak dynamic displacement imparted on a system by an oscillating force with a given peak magnitude compared with the displacement imparted by astatic force of the same magnitude. Acceptance bandwidth is the range of propagation angles for which critical phase matching can be achieved. Generally, the amplification factor and acceptance bandwidth have an inverse relation such that the narrower the acceptance bandwidth the stronger the amplification. 0037| As used herein, an “optic signal,” “output signal,” “emission signal,” or “signal” refers to a detectable event such as an emission, such as light emission, for example, in an image. Thus, in some implementations, a signal may represent any detectable light emission that is captured in an image (i.e., a “spot”) from a fluorescing analyte (as a “point source”). Thus, as used herein, “signal” may refer to both an actual emission from an analyte of the specimen and may refer to a spurious emission that does not correlate to an actual analyte. Thus, a signal could arise from noise and could be later discarded as not representative of an actual analyte of a specimen. A signal consistent with the disclosure includes, for example, fluorescent, luminescent, scatter, or absorption signals. Signals can be detected in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 391 to 770 nm), infrared (IR) range (about 0.771 to 25 microns), or other ranges of the electromagnetic spectrum. Signals may be detected in a way that excludes all or part of one or more of these ranges.|0038| A signal may include light emissions from conjugate fluorescent species of a biological material or chemical reagent. A signal may also include transmitted light refracted and / or reflected by optical substrates. Optical signals, including excitation radiation that is incident upon the sample and light emissions that are provided by the sample, may have one or more spectral patterns. For example, more than one type of fluorescent species may be excited in an imaging session. In such cases, the different types of fluorescent species may be excited by a common excitation light source or may be excited by different excitation light sources that simultaneously provide incident light. Each type of fluorescent species may emit optical signals having a spectral pattern that is different from the spectral pattern of other labels. For example, the spectral patterns may have different emission spectra. The light emissions may be filtered to separately detect the optical signals from other emission spectra. As used herein, when the term “different” is used with respect to emission spectra, the emission spectra may have wavelength ranges that at least partially overlap so long as at least a portion of one emission spectrum does not completely overlap the other emission spectrum. Different emission spectra may have othercharacteristics that do not overlap, such as emission anisotropy or fluorescence lifetime. When the light emissions are filtered, the wavelength ranges of the emission spectra may be narrowed. |0039| As used herein, the term “signal level” is intended to mean an amount or quantity of detected energy or coded information that has a desired or predefined characteristic. For example, an optical signal may be quantified by one or more of intensity, SNR, wavelength, energy, frequency, power, luminance, or the like. Other signals may be quantified according to characteristics such as voltage, current, electric field strength, magnetic field strength, frequency, power, or temperature. Absence of signal may refer to a signal level of zero or a signal level that is not meaningfully distinguished from noise.Optical Detection Systems O040 The nanoantennae technology described herein may applied in the context of a variety of optical detection systems. Such systems and methods herein may be useful for processing optic signals obtained for a field of view in which target analytes being viewed are in states of fluorescence that differ relative to each other in the field of view. The same or other systems and methods described herein may be useful for processing optic signals obtained for a field of view in which individual targets analyte under fluorescence have emission characteristics that differ when viewed at different times, e.g., differing wavelengths (i.e., color) or signal intensity.In certain system embodiments, the optical signals may be directed through an optical train having a plurality of optical components. The optical signals may be directed to a detector (e g., image sensor). In particular embodiments, the optical components of the optical train may be selectively moveable. As used herein, when the term “selectively” is used in conjunction with “moving” and similar terms, the phrase means that the position of the optical component may be changed in a desired manner. For example, at least one of the location and the orientation of the optical component may be changed. The phrase “selectively moving” includes removing the optical component from the optical path, adjusting an orientation of the optical component in the optical path (e.g., rotating the optical component), or moving the optical component such that the orientation does not change, but the location of the optical component does change. In particular embodiments, the optical components may be selectively moved between imaging sessions. However, in other embodiments, the optical components may be selectively moved during an imaging session. As used herein, the term “xy coordinates” is intended to mean information that specifies location, size, shape, and / or orientation in an xy plane. The information may be, forexample, numerical coordinates in a Cartesian system. The coordinates may be provided relative to one or both of the x and y axes or can be provided relative to another location in the xy plane. For example, coordinates of an analyte of an object may specify the location of the analyte relative to location of a fiducial or other analyte of the object. O042| As used herein, the term “z coordinate” is intended to mean information that specifies the location of a point, line or area along an axis that is orthogonal to an xy plane. In particular implementations, the z axis may be orthogonal to an area of an object that is observed by a detector. For example, the direction of focus for an optical system may be specified along the z axis. 0043 As used herein, the term “fiducial” is intended to mean a distinguishable point of reference in or on an object. The point of reference may be, for example, a mark, second object, shape, edge, area, irregularity, channel, pit, post, or the like. The point of reference may be present in an image of the object or in another data set derived from detecting the object. The point of reference may be specified by an x and / or y coordinate in a plane of the object. Alternatively, or additionally, the point of reference may be specified by a z coordinate that is orthogonal to the xy plane, for example, being defined by the relative locations of the object and a detector. One or more coordinates for a point of reference may be specified relative to one or more other analytes of an object or of an image or other data set derived from the object. (IO44| In some implementations, acquired signal data may be transformed using an affine transformation. In some such implementations, template generation may make use of the fact that the affine transforms between color channels are consistent between runs. Because of this consistency, a set of default offsets may be used when determining the coordinates of the analytes in a specimen. For example, a default offsets file may contain the relative transformation (shift, scale, skew) for the different channels relative to one channel, such as the A channel. In other implementations, however, the offsets between color channels may drift during a run and / or between runs, making offset-driven template generation difficult. In such implementations, the methods and systems provided herein may utilize offset-less template generation, which is described further below.(0045] Various types of fluorescence microscopy may be used with system embodiments described herein. Fluorescence microscopy may be performed using an optical detection system that includes a light source (e g., lasers, light emitting diodes (LEDs)) tuned to wavelengths oflight that induce excitation in the fluorescent dyes used for labelling a sample biological material or probe; one or more optical instruments, such as cameras, lenses, sensors, to capture signals emitted through induced excitation, and one or more processors for developing composite images from captured signals emitted from labelled targets within the optical elements’ field of view (tile) in a given sequencing assay. For example, embodiments may be configured to perform at least one of conventional fluorescent imaging, epifluorescence imaging, total-internal- reflectance-fluorescence (TIRF) imaging, a time-delay integration (TDI) imaging (CCD-TDI or CMOS-TDI), or Super Resolution imaging, e.g., Structured Illumination Microscopy (SIM). Furthermore, the imaging sessions may include line scanning one or more samples such that a linear focal region of light is scanned across the sample(s). Imaging sessions may also include moving a point focal region of light in a raster pattern across the sample(s). Alternatively, one or more regions of the sample(s) may be illuminated at one time in a step and shoot manner. |0046j In some embodiments, an optical detection system may include high-resolution optical components. Generally, an optical detection system may be limited by the optical resolution of the data capable of being detected by the optical components of the system. In microscopy, optical resolution is the shortest distance between two separate points in a microscope’s field of view that can still be distinguished as distinct entities, i.e., the Rayleigh limit. For example, the optical resolution of such objects may be expressed as a function of a wavelength ( ) of light in the optical sequencing system, in which shorter wavelengths yield higher resolution, and an objective, or optical element (e.g., lens or lenses) used to gather the light from the target objects, which may be measured by a numerical aperture (NA). NA of an objective lens may be given by the formula nsine 9. where n is the index of refraction of the medium in which the lens is working (nair » 1), and 0 is the half-angle of the maximum cone of light that can enter or exit the lens.(0047] In one example, high-resolution images may be obtained through an optical detection system employing a high NA objective lens. An optical sequencing system using an objective lens having a relatively high NA is capable of resolving more closely adjacent point sources compared to a system characterized by a relatively lower NA. NA thus may determine the resolving power of an objective lens of an optical sequencing system. The higher the NA of the total system, the better the resolution. Higher quality detection lenses and other detection optical elements thus may be used to improve the optical resolution of the optical sequencing systems.0048| In another example, high-resolution images may be obtained through implementation of a subpixel imaging system, e.g., TDI using CCD- or CMOS-based sensors. Subpixel imaging is based on increasing a sample rate to raise the Nyquist frequency, which limits the highest frequency the optical sequencing system can reliably measure (e.g., translate digitally) to one half the sample rate at which the equipment operates. Subpixel imaging may be performed by staggering TDI sensors by a subpixel offset and subsampling a given collection area, which effectively doubles the Nyquist frequency along the offset-axis.|0049| In yet another example, high-resolution images may be obtained through SIM or other SR techniques in connection, e.g., with optimized diffraction-limited imaging. SIM may be implemented by an optical sequencing system to take multiple images of a target object, with varying angles and phase displacements of structured illumination to generate a computational transform (Fourier transform) that is then used to reconstruct closely spaced, otherwise unresolvably high spatial frequency features, into lower frequency signals that may be sensed by an optical system without violating the Abbe diffraction limit. In that manner, captured raw images (e.g., six or nine images) of a same point source or point sources within a same tile may be assembled into a single image having an extended spatial frequency bandwidth, which may be transformed into real space to generate an image having a higher resolution than one captured by other imaging systems. Other apt SR microscopy systems include, e.g., direct stochastic optical reconstruction microscopy (dSTORM)); photo-activated localization microscopy (PALM)) and stimulated emission depletion microscopy (STED).[0050J In certain embodiments, optical detection systems may process signals into spectral data through spectroscopy, using various techniques known in the art, including, e.g., Raman spectroscopy, including surface-enhanced Raman spectroscopy (SERS), and up-conversion spectroscopy. SERS is a spectroscopic technique that can enhance the otherwise feeble Raman scattering effect with the help of plasmonic or dielectric nanoantennae. The enhancement effect originates due to an increase in the local electric field magnitude. SERS allows for the structural fingerprinting of low-concentration analytes through the plasmon-mediated amplification of electrical fields. j 0051 ] Various assay protocols involve performing a large number of controlled reactions on local support surfaces or within predefined reaction chambers. The occurrence of desired reactions may then be sensed or detected, and subsequent analysis may help identify or revealproperties of the kinetics and chemical(s) involved in the reaction. Many such protocols involve signal processing using fluorescence-based assays, which are among the most widely applied sensing techniques due to their high sensitivity, specificity, efficient operation, and the availability of diverse types of fluorophores that absorb and emit light covering a broad spectrum of wavelengths from ultraviolet to infrared. The nanoantennae array technology herein may be utilized with any such fluorescence-based assay that involves the processing of optic information imparted in signals emitted by a biological material under fluorescence and captured by a sensor at different time points, spatial locations, or other temporal or physical perspectives as images and / or spectral data.|0 52| The term “biological material” herein refers to a sample, typically derived from a biological fluid, cell, tissue, organ, or organism containing a nucleic acid or a mixture of nucleic acids containing at least one nucleic acid sequence that is to be sequenced and / or phased. Such samples include, but are not limited to sputum / oral fluid, amniotic fluid, blood, a blood fraction, fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.), urine, peritoneal fluid, pleural fluid, tissue explant, organ culture and any other tissue or cell preparation, or fraction or derivative thereof or isolated therefrom. Although the material is often taken from a human subject (e.g., patient), materials may be taken from any organism having chromosomes, including, but not limited to dogs, cats, horses, goats, sheep, cattle, pigs, etc. The material may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. For example, such pretreatment may include preparing plasma from blood, diluting viscous fluids and so forth. Methods of pretreatment may also involve, but are not limited to, filtration, precipitation, dilution, distillation, mixing, centrifugation, freezing, lyophilization, concentration, amplification, nucleic acid fragmentation, inactivation of interfering components, the addition of reagents, lysing, etc.|0053] The nanoantennae array technology presently disclosed may be utilized for the optical detection, characterization, and / or identification of a variety of biological materials or other targeted analytes including, but are not limited to, nucleic acid materials (e.g., DNA, RNA, or analogs thereof), peptides, proteins, polysaccharides, cells, antibodies, epitopes, receptors, ligands, enzymes (e.g., kinases, phosphatases, or polymerases), small molecule drug candidates, cells, viruses, organisms, and the like.0 54| A biological material herein may include nucleic acid materials as target analytes, primers, templates, or probes. Nucleic acid materials may be referred to herein as “nucleic acids,” “nucleic acid molecules,” “nucleic acid materials,” “nucleic acid sequences,” “polynucleotides,” or “oligonucleotides,” and can comprise a polymeric form of nucleotides of any length, can comprise DNA and / or RNA, and can be single-stranded, double- stranded, or multiple stranded. One strand of a nucleic acid also refers to its complement. Nucleic acid analytes may be gDNA, including DNA variants (e.g., alleles, polymorphs, missense), mtDNA, mRNA, cDNA transcribed from mRNA, non-coding RNA, and small RNA. Nucleic acid materials herein may also include polynucleotide analogues, amplicons, conjugates, and substitutions, crosslinked polynucleotides, polynucleotide complexes, and non-natural polynucleotides, including, but not limited to, dideoxynucleotides, or biotinylated, aminated, deaminated, alkylated, benzylated, flourophor-labeled polynucleotides.|0055j Nucleic acids in certain implementations may include, for instance, linear polymers of deoxyribonucleotides in 3 '-5' phosphodiester or other linkages, such as DNA, for example, single- and double-stranded DNA, genomic DNA, copy DNA or complementary DNA (cDNA), recombinant DNA, or any form of synthetic or modified DNA. In other implementations, nucleic acids include for instance, linear polymers of ribonucleotides in 3 '-5' phosphodiester or other linkages such as ribonucleic acids (RNA), for example, single- and double-stranded RNA, messenger (mRNA), copy RNA or complementary RNA (cRNA), alternatively spliced mRNA, ribosomal RNA, small nucleolar RNA (snoRNA), microRNAs (miRNA), small interfering RNAs (sRNA), piwi RNAs (piRNA), or any form of synthetic or modified RNA. Nucleic acids used in the compositions and methods of the present invention may vary in length and may be intact or full-length molecules or fragments or smaller parts of larger nucleic acid molecules. In particular implementations, a nucleic acid may have one or more detectable labels, as described elsewhere herein.|0056 In various implementations, nucleic acids may be used as templates as provided herein (e.g., a nucleic acid template, or a nucleic acid complement that is complementary to a nucleic acid nucleic acid template) for particular types of nucleic acid analysis, including but not limited to nucleic acid amplification, nucleic acid expression analysis, and / or nucleic acid sequence determination or suitable combinations thereof.0 57| In some implementations, the nucleic acid may comprise a plurality of copies of template nucleic acid and / or complements thereof, attached via their 5' termini to the solid support. Such nucleic acid materials may be referred to “clusters” “colonies,” or “clonal populations.” The copies of nucleic acid strands making up the nucleic acid clusters may be in a single or double stranded form. Copies of a nucleic acid template that are present in a cluster can have nucleotides at corresponding positions that differ from each other, for example, due to presence of a label moiety. The corresponding positions can also contain analog structures having different chemical structure but similar Watson-Crick base-pairing properties, such as is the case for uracil and thymine. Nucleic acid clusters can optionally be created on solid supports by amplification, including, e.g., bridge amplification or exclusion amplification (ExAmp) techniques. Multiple repeats of a target sequence can be present in a single nucleic acid molecule, such as a concatemer created using a rolling circle amplification procedure. Such clusters may be characterized by a degree or ratio of monoclonality, or polyclonality.|0058 A biological material herein may also include polypeptides, e.g., as analytes or reagent enzymes. Polypeptide analytes may include functional polypeptides acting, e.g., as effectors, inhibitors, modulators, mediators, transporters, or stimulators in connection with a specific activity affected by a target molecule. Reagent enzymes may include polypeptides involved in nucleic acid synthesis, extension, fragmentation, amplification, or ligation.|0 59j As used herein, the term “analyte” is intended to mean a point or area in a pattern that can be distinguished from other points or areas according to relative location. An individual analyte can include one or more sample cells, cellular constituents, or molecules of a particular type. For example, an analyte can include a single target nucleic acid molecule having a particular sequence or an analyte can include several nucleic acid molecules having the same sequence (and / or complementary sequence, thereof). Different molecules that are at different analytes of a pattern can be differentiated from each other according to the locations of the analytes in the pattern. Example analytes include without limitation, cavities or wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate, pads of gel material on a substrate, or channels in a substrate. O06O As used herein, the term “fluorescent species” or “fluorescing species” refers to any target analyte, analyte conjugate, or other moiety that can be detected based on an optical field response to excitation. Fluorescing species may include a target analyte with inherentfluorescence (e.g., peptide tracers) or detectible label moieties. Exemplary labels for use consistent with various embodiments, for example, include chromophores; luminophores; fluorophores; optically encoded nanoparticles; particles encoded with a diffraction-grating; electrochemiluminescent labels such as Ru(bpy).sup.32+; or other species capable of detection based on an optical characteristic. Fluorophores that may be useful include, for example, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, alexa dyes, phycoerythin, bodipy, and others known in the art.|0 611 The distances between analytes may be described in any number of ways. In some implementations, the distances between analytes may be described from the center of one analyte to the center of another analyte. In other implementations, the distances may be described from the edge of one analyte to the edge of another analyte, or between the outer-most identifiable points of each analyte. The edge of an analyte may be described as the theoretical or actual physical boundary on a chip, or some point inside the boundary of the analyte. In other implementations, the distances may be described in relation to a fixed point on the specimen or in the image of the specimen.[006 | The size of an analyte on an array (or other object used in a method or system herein) may be selected to suit a particular application. For example, in some implementations, an analyte of an array may have a size that accommodates only a single nucleic acid molecule. A surface having a plurality of analytes in this size range is useful for constructing an array of molecules for detection at single molecule resolution. Analytes in this size range are also useful for use in arrays having analytes that each contain a colony of nucleic acid molecules. Thus, the analytes of an array may each have an area that is no larger than about 1 mm2, no larger than about 500 pm2, no larger than about 100 pm2, no larger than about 10 pm2, no larger than about 1 pm2, no larger than about 500 nm2, or no larger than about 100 nm2, no larger than about 10 nm2, no larger than about 5 nm2, or no larger than about 1 nm2. Alternatively, or additionally, the analytes of an array will be no smaller than about 1 mm2, no smaller than about 500 pm2, no smaller than about 100 pm2, no smaller than about 10 pm2, no smaller than about 1 pm2, no smaller than about 500 nm2, no smaller than about 100 nm2, no smaller than about 10 nm2, no smaller than about 5 nm2, or no smaller than about 1 nm2. Indeed, an analyte may have a size thatis in a range between an upper and lower limit selected from those exemplified above. Although several size ranges for analytes of a surface have been exemplified with respect to nucleic acids and on the scale of nucleic acids, it will be understood that analytes in these size ranges may be used for applications that do not include nucleic acids. It will be further understood that the size of the analytes need not necessarily be confined to a scale used for nucleic acid applications. (HI63| For implementations that include an object having a plurality of analytes, such as an array of analytes, the analytes may be discrete, being separated with spaces between each other. An array useful in the context of the present technology may have analytes that are separated by edge-to-edge distance of at most 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, 0.5 pm, or less. Alternatively, or additionally, an array may have analytes that are separated by an edge-to-edge distance of at least 0.5 pm, 1 pm, 5 pm, 10 p

[0064] The average pitch in a regular pattern may be at most 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, 0.5 pm, or less. Alternatively, or additionally, the average pitch in a regular pattern may be at least 0.5 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm, or more. These ranges may apply to the maximum or minimum pitch for a regular pattern as well. For example, the maximum analyte pitch for a regular pattern may be at most 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, 0.5 pm, or less; and / or the minimum analyte pitch in a regular pattern may be at least 0.5 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm, or more.|O065j The density of analytes in an array may also be understood in terms of the number of analytes present per unit area. For example, the average density of analytes for an array may be at least about IxlO3analytes / mm2, IxlO4analytes / mm2, IxlO5analytes / mm2, IxlO6analytes / mm2, IxlO7analytes / mm2, IxlO8analytes / mm2, or IxlO9analytes / mm2, or higher. Alternatively, or additionally the average density of analytes for an array may be at most about IxlO9analytes / mm2, IxlO8analytes / mm2, IxlO7analytes / mm2, IxlO6analytes / mm2, IxlO5analytes / mm2, IxlO4analytes / mm2, or IxlO3analytes / mm2, or less.|0066| The size and shape of analytes in a pattern may be determined by the size and shape of nanostructures in an array. For example, when observed in a two-dimensional plane, such as on the surface of an array, the analytes may appear rounded, circular, oval, rectangular, square, symmetric, asymmetric, triangular, polygonal, or the like. The analytes may be arranged in a regular repeating pattern including, for example, a hexagonal or rectilinear pattern. A pattern may be selected to achieve a desired level of packing. For example, round analytes are optimallypacked in a hexagonal arrangement. Of course, other packing arrangements may also be used for round analytes and vice versa.|0067| A pattern may be characterized in terms of the number of analytes that are present in a subset that forms the smallest geometric unit of the pattern. The subset may include, for example, at least about 2, 3, 4, 5, 6, 10 or more analytes. Depending upon the size and density of the analytes the geometric unit may occupy an area of less than 1 mm2, 500 pm2, 100 pm2, 50 pm2, 10 pm2, 1 pm2, 500 nm2, 100 nm2, 50 nm2, 10 nm2, or less. Alternatively, or additionally, the geometric unit may occupy an area of greater than 10 nm2, 50 nm2, 100 nm2, 500 nm2, 1 pm2, 10 pm2, 50 pm2, 100 pm2, 500 pm2, 1 mm2, or more. Characteristics of the analytes in a geometric unit, such as shape, size, pitch, and the like, may be selected from those set forth herein more generally with regard to analytes in an array or pattern.

[0068] An array having a regular pattern of analytes may be ordered with respect to the relative locations of the analytes but random with respect to one or more other characteristic of each analyte. For example, in the case of a nucleic acid array, the nuclei acid analytes may be ordered with respect to their relative locations but random with respect to one’s knowledge of the sequence for the nucleic acid species present at any particular analyte. As a more specific example, nucleic acid arrays formed by seeding a repeating pattern of analytes with template nucleic acids and amplifying the template at each analyte to form copies of the template at the analyte (e.g, via cluster amplification, bridge amplification, or exclusion amplification (ExAmp)) will have a regular pattern of nucleic acid analytes but will be random with regard to the distribution of sequences of the nucleic acids across the array. Thus, detection of the presence of nucleic acid material generally on the array may yield a repeating pattern of analytes, whereas sequence specific detection may yield non-repeating distribution of signals across the array.{0069] The nanoantennae array technology described herein may be used in conjunction with a variety of nucleic acid sequencing techniques. Particularly applicable techniques are those in which nucleic acids are attached at fixed locations in an array such that their relative positions do not change and wherein the array is repeatedly imaged. Embodiments in which images are obtained in different color channels, for example, coinciding with different labels used to distinguish one nucleotide base type from another are also applicable.|0070] The term “sequence” in this context includes or represents a strand of nucleotides coupled to each other. The nucleotides may be based on DNA or RNA. It should be understoodthat one sequence may include multiple sub-sequences. For example, a single sequence (e.g., of a PCR amplicon) may have 350 nucleotides. The sample read may include multiple sub-sequences within these 350 nucleotides. For instance, the sample read may include first and second flanking subsequences having, for example, 20-50 nucleotides. The first and second flanking subsequences may be located on either side of a repetitive segment having a corresponding subsequence (e.g., 40-100 nucleotides). Each of the flanking sub-sequences may include (or include portions of) a primer sub-sequence (e.g., 10-30 nucleotides). For ease of reading, the term “subsequence” will be referred to as “sequence,” but it is understood that two sequences are not necessarily separate from each other on a common strand. To differentiate the various sequences described herein, the sequences may be given different labels (e.g., target sequence, primer sequence, flanking sequence, reference sequence, and the like). Other terms, such as “allele,” may be given different labels to differentiate between like objects.

[0071] The term “read” refers to a collection of sequence data that describes a fragment of a nucleotide sample or reference. The term “read” may refer to a sample read and / or a reference read. Typically, though not necessarily, a read represents a short sequence of contiguous base pairs in the sample or reference. The read may be represented symbolically by the base pair sequence (in ATCG) of the sample or reference fragment. It may be stored in a memory device and processed as appropriate to determine whether the read matches a reference sequence or meets other criteria. A read may be obtained directly from a sequencing apparatus or indirectly from stored sequence information concerning the sample. In some cases, a read is a DNA sequence of sufficient length (e. ., at least about 25 bp) that may be used to identify a larger sequence or region, e.g., that may be aligned and specifically assigned to a chromosome or genomic region or gene. 0072 In some embodiments, the process to determine the nucleotide sequence of a target nucleic acid may be an automated process using a sequencing-by-synthesis (“SBS”) technique. SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleoside monomers against a template strand. SBS in general involves the enzymatic extension of a nascent copy strand through iterative addition and simultaneous detection of nucleoside monomers against a template strands. For each iteration (or cycle), labeled nucleoside monomers are detected through induced fluorescence as each monomer is added to the copy strand then replaced in a reverse-terminator process with non-labeled analogues before the start of a subsequent cycle. According to the example method, at a first cycle, (1) a buffer solution containing a mixture of four different labeled nucleoside derivatives (one for each of the four DNA nucleobases (A, G, C, T)) is provided to a cluster of template strands in the presence of polymerase; (2) a complementary nucleoside derivative is added to a nascent copy strand hybridized to each template strand via polymerase primer extension; (3) the added nucleoside derivatives are irradiated with incident light via an excitation source to induce fluorescence and emission of an output signal from the cluster; (4) the output signal is detected by one or more optical sensors through imaging of the signal as a point source at an addressed location on the optic substrate; and (5) information imparted in the imaged output signal is processed by a signal processor to record the nucleobase of the added nucleoside derivative and an address of the cluster on the optic substrate. Steps (l)-(5) are performed simultaneously for a plurality of clusters with a given frame (or tile) of the one or more optic sensors. Steps (l)-(5) are then repeated in subsequent cycles to n number of total cycles, where n is equal to the read length of the template strands in base pairs (bp). 0073 Reads in the range of 50-100 bp may be obtained using an SBS-based technique paired with a single end sequencing chemistry, in which template strands are sequenced in one direction. Larger reads in the range of -300 to 800 bp may be obtained using an SBS-based technique with a paired-end sequencing chemistry to generate paired-end reads of each fragment in both forward and reverse directions. Thus, for example, continuous reads may be generated for 300 bp fragments using a 150 bp cycle kit, for 600 bp fragments using a 300 bp cycle kit, and so on. Still larger reads may be generated through computational leveraging. For example, reads may be generated for 800 bp fragments using a 300 bp cycle kit by inserting a known length between the paired ends (for simplicity, a 200 bp insert corresponding to the delta between the 800 bp fragment and the 2x 300 bp paired end reads) and inferring the sequence of the insert from the intersection of aligned read data in a pileup format. In one example, long insert paired- end reads are generated in combination with short insert paired reads sequenced at higher depth to infer long insert sequences. 0 74| Still larger reads in the range of several kilobases may be obtained using an SBS- based technique paired with mate pair sequencing chemistry such as Illumina Complete Long Reads (ICLR). Here, the sample gDNA may first be tagmented at desired fragment lengths with a Mate Pair Tagment Enzyme, which attaches a biotinylated junction adapter to each end of thetagmented molecule. The tagmented DNA molecules may then be circularized and the ends of the genomic fragment linked by the respective biotin junction adapters. Circularized molecules may then be re-fragmented yielding smaller fragments suitable for amplification and sequencing. Sub-fragments containing the original junction may then be enriched via the biotin tag in the junction adapter. After End Repair and A-tailing, TruSeq DNA adapters are then added, enabling amplification and sequencing. The short, fragmented reads may then be aligned to yield a long read for the tagmented fragment.|0075j SBS may utilize nucleotide monomers that have a terminator moiety or those that lack any terminator moi eties. Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using y-phosphate-labeled nucleotides, as set forth in further detail below. In methods using nucleotide monomers lacking terminators, the number of nucleotides added in each cycle is generally variable and dependent upon the template sequence and the mode of nucleotide delivery. For SBS techniques that utilize nucleotide monomers having a terminator moiety, the terminator may be effectively irreversible under the sequencing conditions used as is the case for traditional Sanger sequencing which utilizes dideoxynucleotides, or the terminator may be reversible.|0076j Devices, assays, methods, and systems herein may also utilize probe-grafted arrays for screening biological molecules, such as nucleic acids and polypeptides, for a locus of interest. Such microarrays may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) capture probes, which are specific for nucleotide sequences present in humans and other organisms. In certain applications, for example, individual DNA or RNA probes may be grafted at addressable reaction sites on an array surface. A test sample, such as from a known person or organism, can be exposed to the array, such that target nucleic acids hybridize to complementary probes grafted on the array. The probes can be labeled in a target specific process (e.g., due to labels present on the target nucleic acids or due to enzymatic labeling of the probes or targets that are present in hybridized form). The array may then be examined by scanning specific frequencies of light over the analytes to identify which target nucleic acids are present in the sample. 0077| By way of example, a genotyping application as contemplated may be implemented to screen for the presence of a genetic locus of interest in a target nucleic acid sample. A locus of interest in a typical genotyping protocol, and as disclosed herein, may include, without limitation, polymorphs (e.g., single nucleotide polymorphs (SNPs), indels), short tandem repeats(STR), copy number variants (CNV), germline variants, methylation sites (e.g., CpG islands), and exogenous sequences (e.g., virus). Target nucleic acid samples herein may include polynucleotides of any length and may be derived from any number of genetic sources including from human or non-human organisms, and from individual organisms or organism populations. Samples herein may be obtained from a wide variety of genetic materials — e g., gDNA, mtDNA, mRNA, cDNA transcribed from mRNA, non-coding RNA, and small RNA, polynucleotide conjugates, analogues, and amplicons.|0078j Image-generating chip arrays provide a convenient format for assaying SNPs, particularly at commercial scale. An example workflow may begin with accession and extraction of a DNA sample, either from single cell source or a tissue sample. The extracted DNA sample may be amplified, usually off-chip in solution, and the amplicon output is then subjected to controlled enzymatic fragmentation. The processed DNA sample is loaded onto the imagegenerating chip and subjected to hybridization using locus specific oligo probes functionalized on the chip substrate. Allelic specificity of hybridized DNA is conferred by enzymatic base extension at the 3' end of the probe. Base extensions are applied fluorescent labels, imaged under excitation, and allele signal intensity data is used to perform genotype calling. An array may be functionalized with an individual probe or a population of probes. In the latter case, the population of probes at each analyte is typically homogenous having a single species of probe. For example, in the case of a nucleic acid array, each locus specific probe may be amplified to yield multiple nucleic acid molecules each having a common sequence. However, in some implementations the population of probes at a given reaction site of an array can be heterogeneous. Similarly, protein arrays can be functionalized with a single protein probe or a population of protein probes typically, but not always, having the same amino acid sequence. The probes can be attached to the surface of an array for example, via covalent linkage of the probes to the surface or via non-covalent interaction(s) of the probes with the surface.|0079 In certain other implementations, the devices, methods, assays, and / or systems of the present disclosure may support a variety of fluorescence-based assays involving the detection and / or interaction of polypeptide materials, including, e.g., proteins, antibodies, epitopes, receptors, ligands, ligases, and enzymes (e.g., kinases, phosphatases, or polymerases). Polypeptide materials may include bioactive screening libraries containing collections of inhibitors, antagonists and agonists organized according to signaling pathways, including, e.g.,DNA Damage / DNA Repair, Cell Cycle / Checkpoint, JAK / STAT Signaling Pathway, MAPK Signaling Pathway, GPCR / G protein, Angiogenesis, Immunol ogy / Infl animation, ubiquitination, and proteolysis, among others.|0080 In certain other implementations, nanostructured substrates of the present disclosure may support a variety of fluorescence-based high throughput screening (HTS) assays involving the detection, molecular interactions and / or activity of polypeptide materials, including, e.g., proteins, antibodies, epitopes, receptors, ligands, ligases, and enzymes (e.g., kinases, phosphatases, or polymerases). By way of example, a nanostructured substrates herein may support fluorescence polarization (FP)-based assay protocols, in which alterations in the apparent molecular weight of a fluorescent probe (or tracer) in solution are indicated by changes in the polarization of the sample’s emitted light, for measuring ligand efficiency. The ability of FP assays to report on changes in molecular weight may be used as an investigative tool for variety of biological processes involving both molecular interactions (protein-protein, protein-peptide, protein-nucleic acid, protein-small molecule) and enzymatic activity (including substrate depletion and product formation). In one example, a nanostructured substrate in a microplate format may be used to support interrogation of candidate compounds selected, e.g., from a medicinal chemistry program a profile drug library for ligand efficiency using an FP-based assay. According to an example method, a ligase of interest with one or more defined ligand binding domains may be contacted with a known small-molecule peptide binder to form a binder-ligase complex formed via the one or more binding domains. The binder may be a tracer with inherent fluorescence, or the binder may be conjugated with a fluorescent species, e.g., a fluorophore. The complex solution is introduced to a nanostructured substrate constructed and arranged in accordance with any number of the examples provided herein such that the complex is immobilized across a nanoantennae array imprinted in the substrate. A baseline measure of fluorescence is first taken, and then a set of candidate compounds in solution is brought into contact with immobilized complexes across the array. The basis of the assay is that FP will increase when the fluorescent small molecule binder is bound to the protein. Equilibrium displacement of the protein-bound binder by a candidate compound will result in reduced FP, due to the faster rotation of the displaced binder, and no measurable change in fluorescence. If, on the other hand, the compound cannot bind the domain and the binder is not displaced,fluorescence will increase over time. The FP-based assay may be further utilized to quantitate substrate recruitment. jOOSl j As another example, nanoantennae array technology herein may support fluorescence resonance energy transfer (FRET)-based assay protocols or Time-resolved FRET (TR-FRET) protocols to determine protein-protein interactions, e.g., ligand-receptor interaction. During FRET, a donor fluorophore excited by a light source may transfer its energy to a nearby acceptor fluorophore. The acceptor fluorophore absorbs the energy to produce a detectable light emission signal. This process results in the loss of fluorescence of the donor and the gain of fluorescence of the acceptor, both of which can be measured. Interactions between conjugated proteins of each respective fluorophore are determined based on FRET efficiency (E) which is based on donor and acceptor fluorophore proximity and is given by the formula E = Ro6 / (Ro6+ r6), where Ro is the Forster radius, and r is the actual distance between the two fluorophores. The Forster radius is the distance at which 50% of the excitation energy is transferred from the donor to the acceptor, and the Ro value usually lies between 10-lOOA (l-10nm). FRET pairs with an Ro value towards the higher end of this range are often preferred due to the increased likelihood of FRET occurrence. By way of example, FRET-based assays contemplated herein may be used to investigate enzyme-mediated ubiquitination pathways, inhibitors of protein: protein complex formation, protein dysregulation, and the like. FRET-based detection methods may also be used to sequence nucleic acid materials, e.g., detection of FRET interactions between a fluorophore- bearing polymerase and y-phosphate-labeled nucleotides during SBS.

[0082] Similar to FRET, TR-FRET is based on the proximity of a donor label and an acceptor label, which have been brought together by a specific binding reaction. However, TR- FRET utilizes fluorescent lanthanide chelates (e.g., europium chelate, terbium chelate, fluorescein, europium cryptate) to avoid interference caused by short-lived emission from acceptor molecules excited directly, rather than by energy transfer.

[0083] Other applicable methodologies include, e.g., bimolecular fluorescence complementation (BiFC), green fluorescent protein GFP fluorescence, fluorescence activated cell sorting (FACS) and fluorescence intensity (FLINT) / Fluorescence Intensity Ratio (FIR). |0084] As used herein, the term “optic solid support” or simply “solid support” refers to a structure upon which various chemistry (e.g., polymeric hydrogel, primers, etc.) may be added in connection with molecular analysis contemplated herein. The substrate may be a wafer, a panel,a rectangular sheet, a die, or any other suitable configuration. The substrate may generally be rigid and insoluble in an aqueous liquid. The substrate may be a single layer structure, or a multi-layered structure (e.g., including a support and a patterned material on the support). Examples of suitable substrates will be described further herein.|O085| Various example embodiments described herein include methods and compositions for flow-cell based sequencing, e.g., of clonal populations of a nucleic acid library clustered on an array of solid support structures, orthogonal reagents, and complimentary chemistry for functionalization of a flow cell surface for selective capture, in situ enrichment, imaging, and traceless release of a nucleic acid sample in a sequencing cycle. As used herein, a flow cell is a vessel having a flow channel that is in fluid communication with at least one unmodified surface or at least one surface modified with a first member of a transition metal complex binding pair. The unmodified or modified surface is capable of attaching surface chemistry that to be used in during a nucleic acid analysis and is capable of releasing the surface chemistry either electrochemically or upon exposure to visible light. The flow cell may also include an inlet for delivering reagent(s) to the flow channel and an outlet for removing reagent(s) from the flow channel. The flow cell enables the detection of the reactions involving the surface chemistry. For example, the flow cell may include one or more transparent surfaces, which allow for the optical detection of arrays, optically labeled molecules, or the like within the flow channel.|0O86j As used herein, a “flow channel” or “channel” may be an area defined between two bonded components, which can selectively receive a liquid sample. In some examples, the flow channel may be defined between a patterned or nonpatterned structure and a lid. In other examples, the flow channel may be defined between two patterned or non-pattemed structures that are bonded together.|0fl87| Any of a variety of array configurations (also referred to as “microarrays” or “microarray chips”) known in the art can be used in a system, method or device set forth herein, including, e.g., with assay workflows for SNP genotyping, epigenetics, genotyping, biomarker profiling, translation profiling, pathway identification mutations, allele specific primer extension (APSE), and gene expression profiling.(0088] By way of example, nanostructured substrates herein may be implemented on microarrays useful, e.g., in connection with genotyping assays, systems and platforms. Microarrays typically include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) probes.These are specific for nucleotide sequences present in humans and other organisms. In certain applications, for example, individual DNA or RNA probes can be attached to individual analytes of an array. A test sample, such as from a known person or organism, can be exposed to the array, such that target nucleic acids (e.g., gene fragments, mRNA, or amplicons thereof) hybridize to complementary probes at respective analytes in the array. The probes can be labeled in a target specific process (e.g., due to labels present on the target nucleic acids or due to enzymatic labeling of the probes or targets that are present in hybridized form at the analytes). The array can then be examined by scanning specific frequencies of light over the analytes to identify which target nucleic acids are present in the sample.|0 89| Microarrays may also be used for genetic sequencing and similar applications. In general, genetic sequencing comprises determining the order of nucleotides in a length of target nucleic acid, such as a fragment of DNA or RNA. Relatively short sequences are typically sequenced at each analyte, and the resulting sequence information may be used in various bioinformatics methods to logically fit the sequence fragments together so as to reliably determine the sequence of much more extensive lengths of genetic material from which the fragments were derived. Automated, computer-based algorithms for characteristic fragments have been developed, and have been used more recently in genome mapping, identification of genes and their function, and so forth. Microarrays are particularly useful for characterizing genomic content because a large number of variants are present, and this supplants the alternative of performing many experiments on individual probes and targets. The microarray is an ideal format for performing such investigations in a practical manner.

[0090] The term “threshold” herein refers to a numeric or non-numeric value that is used as a cutoff to characterize a sample, a nucleic acid, or portion thereof (e.g., a read). A threshold may be varied based upon empirical analysis. The threshold may be compared to a measured or calculated value to determine whether the source giving rise to such value suggests should be classified in a particular manner. Threshold values can be identified empirically or analytically. The choice of a threshold is dependent on the level of confidence that the user wishes to have to make the classification. The threshold may be chosen for a particular purpose (e.g., to balance sensitivity and selectivity). As used herein, the term “threshold” indicates a point at which a course of analysis may be changed and / or a point at which an action may be triggered. A threshold is not required to be a predetermined number. Instead, the threshold may be, forinstance, a function that is based on a plurality of factors. The threshold may be adaptive to the circumstances. Moreover, a threshold may indicate an upper limit, a lower limit, or a range between limits. As an example, threshold reliability of sequencing (or base call) data generated in a nucleic acid sequencing operation may be based on a percent passing fdter (%PF), which may be calculated based on the computational application of a chastity fdter to a point source (e.g., a cluster), where chastity is defined as the ratio of the brightest base intensity divided by the sum of the brightest and second brightest base intensities. A point source “passes filter” if no more than a defined number of base calls has a chastity value below a set threshold.(0091 J In some implementations, a metric or score that is assigned to sequencing data may be compared to the threshold. As used herein, the terms “metric” or “score” may include values or results that were determined from the sequencing data or may include functions that are based on the values or results that were determined from the sequencing data. Like a threshold, the metric or score may be adaptive to the circumstances. For instance, the metric or score may be a normalized value. As an example of a score or metric, the quality of a base call determination in a nucleic acid sequencing protocol may be reflected in a “Q-score” based on a PHRED-scaled probability ranging from 0-50 inversely proportional to the probability that a single sequenced base is correct. For example, a thymine (T) base call with Q of 20 is considered likely correct with a probability of 99.99%. Any base call with Q<20 may be considered low quality. (H>92| Optical detection systems herein may perform computation-based image or spectral data analysis using one or more automated subsystems. Subsystems may include a processor; a storage capacity; and a program(s) for image analysis, the program comprising instructions for processing a first data set for storage and the second data set for analysis, wherein the processing comprises acquiring and / or storing the first data set on the storage device and analyzing the second data set when the processor is not acquiring the first data set. In certain aspects, the program includes instructions for identifying at least one instance of a conflict between acquiring and / or storing the first data set and analyzing the second data set; and resolving the conflict in favor of acquiring and / or storing image data such that acquiring and / or storing the first data set is given priority. In certain aspects, the first data set comprises image files obtained from an optical imaging device. In certain aspects, the system further comprises an optical imaging device. In some aspects, the optical imaging device comprises a light source and a detection device.{0 93| Generally, several implementations will be described herein with respect to methods of analysis. It will be understood that systems are also provided for carrying out the methods in an automated or semi-automated way. Accordingly, this disclosure provides computation-based template generation and base calling systems, wherein the systems can include a processor; a storage device; and a program for image analysis, the program including instructions for carrying out one or more of the methods set forth herein. Accordingly, the methods set forth herein can be carried out on a computer, for example, performance of real time analysis of image and sequence data generated during a DNA sequencing operation.(O094| Also provided herein are systems for performing secondary and / or tertiary analysis of molecular analysis performed using devices, methods, assays, and / or systems presently described. For example, computational systems implementing an SV calling pipeline for generation of diploid assemblies and SV call sets from whole genome or whole exome sequencing data using sequencing devices, methods, assays, and systems described herein. Tertiary analysis applications may include comprehensive genomic profiling, Genome-wide Association Studies (GWAS), Variant to Function (V2F), QTL mapping, missense studies, loss of function analysis, gain of function analysis, conservation, depletion, deletion analyses.{0095! The technology disclosed may use neural networks to improve the quality and quantity of nucleic acid sequence information that can be obtained from a nucleic acid sample such as a nucleic acid template or its complement, for instance, a DNA or RNA polynucleotide or other nucleic acid sample. Accordingly, certain implementations of the technology disclosed provide higher throughput polynucleotide sequencing, for instance, higher rates of collection of DNA or RNA sequence data, greater efficiency in sequence data collection, and / or lower costs of obtaining such sequence data, relative to previously available methodologies.|0096| The technology disclosed may utilize neural networks to identify the center of a nucleic acid clusters described herein and to analyze optical signals that are generated during sequencing of such clusters, to discriminate unambiguously between adjacent, abutting or overlapping clusters in order to assign a sequencing signal to a single, discrete source cluster. These and related implementations thus permit retrieval of meaningful information, such as sequence data, from regions of high-density cluster arrays where useful information could not previously be obtained from such regions due to confounding effects of overlapping or veryclosely spaced adjacent clusters, including the effects of overlapping signals (e.g., as used in nucleic acid sequencing) emanating therefrom.|0097| A number of tasks of automated systems described herein may be offloaded from software to configurable hardware, e.g., FGPAs, for acceleration of computational processing. For example, one or more FPGAs may be configured to perform tasks at various stages of secondary analyses performed herein. For instance, FPGA may be parallelized for multithreading, including, e.g., multithreading variant calling by chromosome. FPGAs may be instantiated with graphs or patterns for do novo assembly. And they may be configured for highly repeated processes, such as hidden Markov Models or Smith-Waterman algorithms. |0 98| The illustrated embodiments of the Figures herein are disclosed in the context assay protocols to which the nanoantennae array technology of the present disclosure has application. These include DNA sequencing using sequencing-by-synthesis (SBS) techniques on NGS platforms; screening nucleic acid materials for loci of interest using microarray formats, including, e.g., single nucleotide polymorph (SNP) screens; and high throughput screening (HTS) for proteimprotein interaction investigation. These protocols, while well suited to the devices, methods, assays, and systems of the present disclosure, serve merely to highlight the utility and various advantages for particular embodiments herein. The technology presently disclosed, including as disclosed through the illustrated embodiments, may be utilized in connection with any optics-based assay, including fluorescence-based assay involving the processing of optic information imparted in signals emitted by target analytes under fluorescence and captured by a sensor at different time points, spatial locations, or other temporal or physical perspectives as images and / or spectral data.|0099 Figs. 1A-C illustrates an example nanostructure architecture 10 of the disclosure. The illustrated substrate may be implemented in a variety of support formats. With reference to Fig. 1A, the nanostructure architecture 10' may include an optic support layer 14 supported on a base layer 12. Base layer 12 may be constructed of any suitable support material appropriate to a given format and / or operation. In certain applications, the support layer may be a low- background material, including materials exhibiting both high transmissivity and high fluorescence transparency, particularly for use as multilayer solid supports for high-density applications. Examples of suitable base layer 12 include epoxy siloxane, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene andother materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefins / cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon (polyamides), ceramics / ceramic oxides, silica, fused silica, silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO ), tantalum pentoxide (Ta20s) or other tantalum oxide(s) (TaOx), hafnium oxide (HfO ), carbon, metals, inorganic glasses, or the like. In one example, base layer 12 is a glass, for example, alkaline earth boro-aluminosilicate glass (e.g., EAGLE XG® (Corning, NY)).100100] The optic support layer 14 may be constructed of any suitable material appropriate for use with a variety of known nanoconstruct imprinting techniques in which the material may be imparted with a pre-determined pattern of nanostructures. Such techniques include hard stamping using, e.g., a SiC stamp, Ni Stamp, or SI2N4 stamp; nanoimprint lithography (NIL), focused ion beam (FIB) lithography, laser-interference lithography, holographic lithography, resist-assisted FIB lithography, colloidal lithography, block copolymer self-assembly, soft imprinting, and step and flash lithography. Certain techniques are particularly amenable to production scale batch microfabrication, including, e.g., NIL, which is a relatively simple nanolithography process with low cost, high throughput, and high resolution. Support materials for use with NIL and other imprinting techniques may be selected from any suitable UV-curable resins, including tripropyleneglycol diacrylate (TPGDA resin), polypropyleneglycol diacrylate (PPGDA resin), poly-urethane acrylate (PUA resin), and fluoroacrylates, e.g., perfluoropolyether (PFPE)- urethane methacrylate (MD 700 resin).[001011 By way of example, the optic support layer 14 may be constructed of a cured resin material, e.g., UV-curable material. Cured resin materials consistent for use with systems, methods, assays, and devices herein may have a refraction index (n) from about 1.45 to 2.0 within the wavelength (X) spectrum of 403nm to 848nm. The cured resin material in certain examples may be characterized as having a standard refraction index in the range of 1.45 < n < 1.55 at 403nm < X < 848nm. In one such example, the cured resin material may have a refraction index in the range of l.5 < n < 1.51 at 403nm < X < 848nm, e.g., standard UVK resin. The cured resin in other examples may characterized by a high refractive index, e.g., a refraction index of n > ~1.5 at 403nm < X >848nm, n > -1.6 at 403nm < X >848nm, n > -1.7 at 403nm < >848nm, n > ~1.8 at 403nm < X >848nm, 1.65 < n > 2.0 at 403nm < X >848nm, or 1.70 < n > 1.80 at403nm < X >848nm. In one such example, the optic support layer 14 is constructed of UV Resin #18247 manufactured by NTT Advanced Technology Corp., which has a refraction index of n > -1.75 at 403nm < X >848nm, and which exhibits high transmittance of 95-97% at X > 450nm. [00102 The optic support layer 14 may include an array of nanoantennae 16 imparted in the support material and distributed in one or more patterns, in which the nanoantennae 16 may be separated by interstitial regions 26 of planar or featureless resin surface. A particular surface morphology may be calibrated to the particular operation, support format, or imaging equipment for a given molecular analysis, in which nanoantennae size, density / pitch / porosity, geometry, uniformity, and structural arrangement may be precisely controlled using, e.g., one or more known template-assisted techniques.|00I03] In the example of Fig. 1A, each nanoantenna 16 of the array may include a conically- formed cavity 24 impressed in the optic support layer 14. The cavity 24 may define an interior volume 22 and may include an opening 18 coplanar with the featureless resin surface, a base portion 20a providing a solid support for optic detection of a target analyte, and an inwardly conical wall portion 20b joining the opening and the base portion. A reaction site 30 may be collocated with at least a portion of the solid support, which can be functionalized for interaction with a target analyte. Each nanoantenna 16 may further include a conformal shell of high refractive index medium supporting the interior volume 22 of the cavity, which, through resonant interaction with a resident target analyte under fluorescence, can localize the optic field response of the analyte to the vicinity of the nanoantenna and within a volume (hot spot) roughly equivalent in diameter to the wavelength of incident light.100104] In some embodiments, the opening 18 of each nanoantennae can define a radius (R) and perpendicular axis, and the base portion can be coaxially located on the axis that defines a second radius (r) such that the cavity forms a frustrum. Dimensions of the first second radii may be characterized in terms of a ratio (R:r), which may be anywhere from -10:1 to -1000: 1, depending, e.g., operational requirements of a particular assay protocol, the desired resonance effect, as well as exigent considerations such as the stochastics of efficient analyte and / or reagent distribution across nanoantennae arrays.100105] For certain applications herein, the conformal shell 28 may be constructed of a low- loss dielectric material having a high refractive index of n > -2. Such materials may include metal oxides, in either amorphous or crystalline form, such as, e.g., tantalum pentoxide (Ta2Os),aluminum oxide (A12O3), and titanium oxide (TiO2), hafnium oxide (HfCh), niobium oxide (NbO), cerium oxide (CeO2), and gallium oxide (GaO2), tungsten oxide (WO), zirconium oxide (ZrO2), and tin oxide (SnO2). Such materials may also include silicon nitride (Si3N4), indium phosphide (InP), gallium phosphide (GaP) arsenic phosphide (AsP), and germanium (Ge). According to examples, the conformal shell 18 may have an average thickness of between 10 nm and 60 nm, 20 nm and 50 nm, 30 nm and 40 nm. The dielectric material may be applied to the interior surface 20 to form conformal shell 18 as a uniform fdm or as densely packed nanoparticles.100106] In certain other applications, the conformal shell 28 may be constructed of a metal material, in which case the conformal shell 28 may be adapted as a plasmonic structure appropriate for use, e.g., SERS detection techniques, and in applications where localized heating of target molecules, particles and / or other markers are desired, including, e g., photothermal imaging, drug delivery, cancer therapy (targeted localized heating to kill tumors), biosensing, micro and nanofluidics, chemical catalysis, and assays involving controlled apoptosis of cell structure. Appropriate metals for use as plasmonic structures include, e.g., gold (Au), silver (Ag), and alloys thereof, e.g., AuxAg(i-x). Other noble metals may be used consistent with the disclosure, including, e.g., ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), osmium (Os), iridium (Ir), and platinum (Pt). According to examples, the conformal shell 18 constructed of a metal material may have an average thickness from about 1 nm to about 200 nm, depending on wavelength and extinction coefficient. The metal material may be applied to the interior surface 20 as a uniform film or as densely packed nanoparticles. In certain examples, the conformal shell 28 is a composite of a dielectric interior layer and a metal interface layer, forming a metal-dielectric interface, in which case each nanoantennae 16 may be adapted as gratings resonator.100107] The nanoantennae array 16 may be characterized by the opening 18 geometry when observed in a two-dimensional plane, such as on or above the surface of optic support layer 14. For example, the nanoantennae 16 may be configured with substantially circular openings or with a non-circular geometry, e.g., hexagonal configuration, and may be further defined by a density (number of nanoantennae) or porosity (pore area as a % portion of film area). As shown in the illustration of Fig. IB, the nanoantennae array 16 may also be characterized by the geometric shape of a cross-section of the nanoantennae array 16 taken perpendicular to a two-dimensional plane, such as a plane perpendicular to the surface of optic support layer 14. With reference to Fig. IB, nanoantennae herein may be characterized with respect to pitch (p), opening diameter (£>), radius (R), or area G4), base diameter (d), radius (r), or area (a), a ratio of the opening diameter, radius, or area to the base diameter, radius, or area (D lR: Ra height ( / / ), slant height ( / ), slant angle (9), total surface area (TSA), or volume, and each such value may be characterized as an average value across an array or portion of an array of nanoantennae 16.|OO.1081 For example, the layout or pattern of nanoantennae 16 may be characterized in terms of the average pitch, or the spacing from the center of one nanoantennae 16a to the center of an adjacent nanoantennae 16b (center-to-center spacing) or from the right edge of one nanoantennae 16a to the left edge of an adjacent nanoantennae 16b (edge-to-edge spacing, not shown). The pattern can be regular, such that the coefficient of variation around the average pitch is small, or the pattern can be non-regular in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, about 0.1 pm, about 0.5 pm, about 1 pm, about 5 pm, about 10 pm, about 100 pm, or more or less. The average pitch for a particular pattern can be between one of the lower values and one of the upper values selected from the ranges above. In some embodiments, the nanoantennae 16 have an average pitch (center-to- center spacing) about 250 nm or greater, 300 nm or greater, 350 nm or greater, 400 nm or greater, 450 nm or greater, 500 nm or greater, 550 nm or greater, 600 nm or greater, 650 nm or greater, or 700 nm or greater, or may be in a range between about 250 nm and 800 nm, 300 nm and 750 nm, 350 nm and 700 nm, 400 nm and 650 nm, 450 nm and 600 nm, 500 nm and 550 nm. In an example, the nanoantennae 16 have an average pitch (center-to-center spacing) between about 350 nm and 750 nm.{00109] The average opening diameter (Z>) can be, for example, about 200 nm or greater, 300 nm or greater, 350 nm or greater, 400 nm or greater, 450 nm or greater, 500 nm or greater, 550 nm or greater, 600 nm or greater, 650 nm or greater, or 700 nm or greater, or may be in a range between about 200 nm and 1000 nm, 300 nm and 900 nm, 300 nm and 800 nm, 400 nm and 650 nm, 450 nm and 600 nm, 500 nm and 550 nm. For another example, the opening area (A) can range from about 1 x10-3 pm2 to about 100 pm2, e.g., about 1 x10-2 pm2, about 0.1 pm2, about 1 pm2, at least about 10 pm2, or more, or less. In another example, the volume can range from about 1 x 10-3 pm3 to about 100 pm3, e.g., about 1 x 10-2 pm3, about 0.1 pm3, about 1 pm3,about 10 pm3, or more, or less. For still another example, the depth can range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less.|00110 J The average base diameter (d) may be characterized in terms of a ratio of the opening diameter / base diameter (D.d). For example the ratio D:d may be > 10: 1, > 20: 1, > 30:1, > 40: 1,> 50: 1, > 60: 1, > 100: 1, > 150: 1, > 200: 1, > 250:1, > 300: 1, > 400:1, > 500: 1, > 600: 1, > 700: 1,> 800: 1, > 900: 1, > 1000: 1, or may be in the range of between about 10: 1 and 20: 1, about 10: 1 and 50: 1, about 10: 1 and 100:1, about 20: 1 and 100: 1 about 20: 1 and 200: 1, about 50:1 and 100: 1, about 50: 1 and 200:1, about 100: 1 and 150: 1, about 200: 1 and 500: 1 about 500: 1 and 1000: 1. The same ranges may be characterize as the ratio of the opening radius to the base radius 00111] The height (H) may range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. In certain examples, the height may range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. In another example, the average height is 150 nm or greater, 200 nm or greater, 250 nm or greater, 300 nm or greater, 350 nm or greater, or 400 nm or greater, or may be in a range between about 150 nm and 500 nm, 200 nm and 450 nm, or 250 nm and 400 nm., or 300 nm and 350 nm.|00112| The slant angle (0) may range from about 3° to about 80°. In certain examples, the height may be about 3° or greater, 10° or greater, 20° or greater, 30° or greater, 40° or greater, 50° or greater, 60° or greater, or may be in a range between about 5° and about 70°, about 10° and about 60°, about 15° and about 50°, about 20° and about 40°, about 25° and about 40°, about 30° or about 40°. And the slant height ( / ) may be given by the formula:I = H / cos 0Where the height H) and slant angle (ff) may each be any value within the respective ranges for same as set forth in the disclosure.|00113] The TSA of nanoantennae 16 may be given by the formula: [(R + r) + r2+ I?2] and volume may be given by the formula:1-nH R2+ r2+ Rr) where, for both TSA and volume, the opening radius (R) and base radius (r) may be 'A any common value within the respective ranges for D and d as set forth in the disclosure.00114] While the foregoing example values have been provided, it is to be understood that other values may be used. To that end, a particular surface morphology may be calibrated to the particular operation, support format, or imaging equipment for a given molecular and / or cellular analysis, in which nanoantennae geometry may be precisely controlled using, e.g., one or more known template-assisted techniques. Similarly, nanoantennae geometry and refractive index gradients at interfaces may be tuned to particular resonant emission spectra of a given operation guided, e.g., by numerical solutions to the fundamental Maxwell equations using the finite- difference time-domain (FDTD) method (e.g., Ansys Lumerical FDTD software).{00115] Many different layouts of the nanoantennae 16 may be envisaged, including regular, repeating, and non-regular patterns. In an example, the nanoantennae 16 are disposed in a hexagonal grid for close packing and improved density. Other layouts may include, for example, rectilinear (i.e., rectangular) layouts (e.g., lines or trenches), triangular layouts, and so forth. In some examples, the layout or pattern can be an x-y format of nanoantennae 16 that are in rows and columns. In some other examples, the layout or pattern can be a repeating arrangement of nanoantennae 16 and / or interstitial regions 26. In still other examples, the layout or pattern can be a random arrangement of nanoantennae 16 and / or interstitial regions 26. The pattern may include stripes / lines, swirls, triangles, rectangles, circles, arcs, diagonals, arrows, squares, and / or cross-hatches.106116] The nanoantennae array may also be characterized with respect to the density (number) or porosity (pore (opening 20) area as a % portion of substrate area). For example, the nanoantennae 16 may be present at a density of approximately 2 million per mm2. The density may be tuned to different densities including, for example, a density of about 100 per mm2, about 1,000 per mm2, about 0.1 million per mm2, about 1 million per mm2, about 2 million per mm2, about 5 million per mm2, about 10 million per mm2, about 50 million per mm2, or more, or less. As examples, a high-density array may be characterized as having the nanoantennae 16 separated by less than about 100 nm, a medium density array may be characterized as having the nanoantennae 16 separated by about 400 nm to about 1 pm, and a low-density array may be characterized as having the nanoantennae 16 separated by greater than about 1 pm. Density may also be expressed as a pitch ratio of nanowell diameter to shortest (or nearest neighbor) distance between adjacent nanowells. For example, patterned flow cells can support a nanoantennae 16 array having up to around a 1 : 1 pitch ratio. It is to be further understood that the density can bebetween one of the lower values and one of the upper values selected from the ranges above, or that other densities (outside of the given ranges) may be used. To that end, a particular surface morphology may be calibrated to the particular operation, support format, or imaging equipment for a given molecular analysis, in which density may be precisely controlled using, e.g., one or more known template-assisted techniques.J00117] As illustrated in Fig. 1C, the base portion 20a of the interior surface 20 may serve as a solid support for analyzing a biological material in the form of an analyte or clonal population of analytes, in which the solid support is functionalized as reaction site 30 for chemical interaction with the analyte or analyte population. In a contemplated operation based on the processing of optic information imparted in signals emitted through induced fluorescence into an image, i.e., fluorescence microscopy, an immobilized analyte or analyte population may be or may become conjugated with a fluorescing species (such as, for example, during a nucleic acid sequencing operation in which a fluorophore-conjugated nucleoside is incorporated onto a nucleic acid fragment or template). jOOLlS] In the example illustration of Fig. ID (A), an excitation source 36 exposes the fluorescing species interacting with a target analyte to an incident light 34, the spectra of which is calibrated to induce fluorescence. The incident light changes the quantum state of the fluorescing species, which, as illustrated in Fig. 1C (B), converts the input photon energy of incident light to an optic field of output photons within cavity 24 having a wavelength longer than that of the incident light spectra. Cavity 24 promotes strong resonant field coupling between optical modes of the conformal shell 28 and the fluorescing species, which, in turn, enhances the local density of states (LDOS) at the emitter position (i.e., in the vicinity of the reaction site 30. In operation, conformal shell 28 of high refractive index medium induces resonance enhancement of the emission field limited to the LDOS and a reduction in transmission efficiency at the resonant frequency. In that manner, cavity 24 may induce resonance enhancement of the emission field limited to the LDOS and a reduction in transmission efficiency at the resonant frequency, resulting in an intensified emission field 32 confined to a hot spot within the interior volume 22 of cavity 24. Nanoantennae 16 thus provide maximalization of signal strength and minimalization of parasitic autofluorescence in the interstitial substrate.|00119| As a result, the output signal 38 detected by sensor 40 are amplified well above the noise floor at exceedingly high Signal-to-Noise ratio (SNR). For example, comparing intensityprofiles using FDTD method of modeled conventional cylindrical nanowells versus modeled nanoantennae of the disclosure (in X-Z projection) as shown in Figs. 2A and 2B, respectively, a 140X enhancement of signal strength over conventional nanowell technology was calculated at 488 nm pump wavelength. The nanoantennae of Fig. 2B were modeled based on 375 nm pitch conical cavities with n = 1.8 resin and a 40 nm conformal tantalum pentoxide (Ta20s) shell. As shown in Fig. 2A, whereas the optical field of conventional cylindrical nanowells is homogeneously distributed across the nanowells and interstitial regions, modeled nanoantennae of Fig. 2B are characterized by strong localization of the excitation field within the cavity volume.|OO120] Flow cells provide a convenient format for high throughput sequencing operations involving multiple cycles of repeated chemical delivery and image capture and, in that regard, may be an appropriate, but not exclusive, format for use with a number of applications described herein. One example of a flow cell format supporting the nanostructure architecture 10 of Fig. 1A-B is flow cell 50 as shown in Figs. 3A and 3B. Generally, flow cell 50 may include a patterned structure, e.g., an array of nanoantennae 16' and interstitial regions 26' of nonfunctionalized, and substantially planar and featureless surface. The patterned structure may be organized into lanes, each separated by non-patterned, non-functionalized interstitial regions, which may be bonded to a lid (not shown) to form flow channels 40 along each lane of patterned structure. The example shown in FIG. 3A includes eight flow channels 40. While eight flow channels 40 are shown, it is to be understood that any number of flow channels 40 may be included in the flow cell 50 (e.g., a single flow channel 40, four flow channels 40, etc.). Each flow channel 40 may be isolated from another flow channel 40 so that fluid introduced into a flow channel 40 does not flow into adjacent flow channel(s) 40. Some examples of the fluids introduced into the flow channel 40 may introduce sample and reaction components (for NGS: e.g., DNA sample, polymerases, sequencing primers, nucleotides, etc.), washing solutions, deblocking agents, etc.(001211 As illustrated in FIG. 3B, the flow channel 40 may include a multi-layered nanostructure architecture 10', which includes a base layer 12' and optic support layer 14'. The base layer 12' may be any suitable low-background material, including materials exhibiting both high transmissivity and high fluorescence transparency, particularly for use as supports for multilayer imaging implementations. Examples of suitable base layers 12' include epoxy siloxane,glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefins / cyclo-olefm polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon (polyamides), ceramics / ceramic oxides, silica, fused silica, silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p+ silicon), silicon nitride (SisN4), silicon oxide (SiO2), tantalum pentoxide (Ta20s) or other tantalum oxide(s) (TaOx), hafnium oxide (HfCh), carbon, metals, inorganic glasses, or the like. In one example, the base layer is a glass, for example, alkaline earth boro-aluminosilicate glass (e.g., EAGLE XG® (Corning, NY)).|00122] The optic support layer 14' may have thickness sufficient to accommodate the depth(or height (A)) of nanoantennae 16', which can range, for example, between .01 nm to 450 nm depending on the application. In one example, as illustrated in Fig. 3B, the thickness of the optic support layer 14' may accommodate the entire depth of nanoantennae 16', such the entire inner surface of nanoantennae 16' is formed in the material of the optic support layer 14'. Alternatively, the thickness of the optic support layer 14' may be coterminous with the depth of nanoantennae 16', such that a base portion of the inner surface of nanoantennae 16' exposes a surface of the base layer 12', in which case the exposed surface of the base layer 12' may serve as a solid support.100123] The optic support layer 14' may be constructed of any suitable material appropriate for use with a variety of known nanoconstruct imprinting techniques in which the material may be imparted with a pre-determined pattern of nanostructures. Such techniques include hard stamping using, e.g., a SiC stamp, Ni Stamp, or SI2N4 stamp; nanoimprint lithography (NIL), focused ion beam (FIB) lithography, laser-interference lithography, holographic lithography, resist-assisted FIB lithography, colloidal lithography, block copolymer self-assembly, soft imprinting, and step and flash lithography. Certain techniques are particularly amenable to production scale batch microfabrication, including, e.g., NIL, which is a relatively simple nanolithography process with low cost, high throughput, and high resolution. Support materials for use with NIL and other imprinting techniques may be selected from any suitable UV-curable resins, including tripropyleneglycol diacrylate (TPGDA resin), polypropyleneglycol diacrylate (PPGDA resin), poly-urethane acrylate (PUA resin), and fluoroacrylates, e.g., perfluoropolyether (PFPE)-urethane methacrylate (MD 700 resin).(00124) In certain examples, the optic support layer 14' is constructed of a UV-curable resin characterized by a high refractive index, e.g., a refraction index of n > ~1.5 at 403 nm < >848nm, n > ~1.6 at 403nm < >848nm, n > ~1.7 at 403nm < >848nm, n > -1.8 at 403nm < X >848nm, 1.65 < n > 2.0 at 403nm < X >848nm, or 1.70 < n > 1.80 at 403nm < X >848nm. In one example, the optic support layer 14' is constructed of UV Resin #18247 manufactured by NTT Advanced Technology Corp., which has a refraction index of n > -1.75 at 403 nm < X >848nm, and which exhibits high transmittance of 95-97% at X > 450nm.[001251 With reference to Fig. 3B, each nanoantenna 16' of the Figs. 3 A and B flow cell example may include a conformal shell 28' of high refractive index medium applied to the interior surface of the nanoantenna. For certain applications herein, the conformal shell 28' may be constructed of a low-loss dielectric material having a refractive index of n > -2. Such materials may include metal oxides such as, e.g., tantalum pentoxide (Ta2Os), aluminum oxide (AO), and titanium oxide (TiO), hafnium oxide (HfCh), niobium oxide (NbO), cerium oxide (CeO), and gallium oxide (GaO), tungsten oxide (WO), zirconium oxide (ZrO), and tin oxide (SnO). Such materials may also include silicon nitride (SisN4). In some implementations, the metal oxide is applied as a uniform layer. According to examples, the conformal shell 28' may have an average thickness of between 10 nm and 60 nm, 20 nm and 50 nm, 30 nm and 40 nm. The dielectric material may be applied to the interior surface 20' to form conformal shell 28' as a uniform fdm or as densely packed nanoparticles.(00126) In certain other applications, the conformal shell 28' may be constructed of a metal material, in which case the conformal shell 28' may be adapted as a plasmonic structure and each nanoantennae a plasmonic resonator appropriate for use, e.g., SERS detection techniques. Appropriate metals for use as plasmonic structures include, e.g., gold (Au), silver (Ag), and alloys thereof, e.g., AuxAg(l x). Other noble metals may be used consistent with the disclosure, including, e.g., ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), osmium (Os), iridium (Ir), and platinum (Pt). According to examples, the conformal shell 28' constructed of a metal material may have an average thickness of between 1 nm and 200 nm, depending on wavelength and extinction coefficient. The metal material may be applied to the interior surface as a uniform film or as densely packed nanoparticles. In certain examples, the conformal shell 28' is a composite of a dielectric interior layer and a metal interface layer, in which case each nanoantennae 16' may be adapted as gratings resonator.00127] The array of nanoantennae 16' may be characterized by the opening 18' geometry when observed in a two-dimensional plane, such as on or above the surface of optic support layer 14'. For example, the nanoantennae 16' may be configured with substantially circular openings, as shown in the illustrated embodiment of Fig. 3B, or with a non-circular geometry, e.g., hexagonal configuration, and may be further defined by a density (number of nanoantennae) or porosity (pore area as a % portion of film area). As shown in the illustration of Fig. 3B, the nanoantennae 16' array may also be characterized by the geometric shape of a cross-section of the nanoantennae 16' array taken perpendicular to a two-dimensional plane, such as a plane perpendicular to the surface of optic support layer 14'. Referring back to Fig. IB, nanoantennae 16' may be characterized with respect to pitch (p opening diameter D radius (7?), or area (A), base diameter (d), radius (r), or area (rz), a ratio of the opening diameter, radius, or area to the base diameter, radius, or area p-.dlR:vl A:a), height (A), slant height ( / ), slant angle (9), total surface area (TSA), or volume, and each such value may be characterized as an average value across an array or portion of an array of nanoantennae 16'.{00128] For example, the nanoantennae 16' may be present at a density of approximately 2 million per mm2. The density may be tuned to different densities including, for example, a density of about 100 per mm2, about 1,000 per mm2, about 0.1 million per mm2, about 1 million per mm2, about 2 million per mm2, about 5 million per mm2, about 10 million per mm2, about 50 million per mm2, or more, or less. It is to be further understood that the density can be between one of the lower values and one of the upper values selected from the ranges above, or that other densities (outside of the given ranges) may be used. As examples, a high-density array may be characterized as having the nanoantennae 16' separated by less than about 100 nm, a medium density array may be characterized as having the nanoantennae 16' separated by about 400 nm to about 1 pm, and a low-density array may be characterized as having the nanoantennae 16' separated by greater than about 1 pm.[001291 The layout or pattern of the nanoantennae 16' may also or alternatively be characterized in terms of the average pitch (p), or the spacing from the center of one nanoantennae 16a' to the center of an adjacent nanoantennae 16b' (center-to-center spacing) or from the right edge of the one nanoantennae 16a' to the left edge of the adjacent nanoantennae 16b' (edge-to-edge spacing). The pattern can be regular, such that the coefficient of variation around the average pitch is small, or the pattern can be non-regular in which case the coefficient of variation can be relatively large.In either case, the average pitch can be, for example, about 50 nm, about 0.1 pm, about 0.5 pm, about 1 pm, about 5 pm, about 10 pm, about 100 pm, or more or less. The average pitch for a particular pattern can be between one of the lower values and one of the upper values selected from the ranges above. In some embodiments, the nanoantennae 16' are nanowells and have an average pitch (center-to-center spacing) about 250 nm or greater, 300 nm or greater, 350 nm or greater, 400 nm or greater, 450 nm or greater, 500 nm or greater, 550 nm or greater, 600 nm or greater, 650 nm or greater, or 700 nm or greater, or may be in a range between about 250 nm and 800 nm, 300 nm and 750 nm, 350 nm and 700 nm, 400 nm and 650 nm, 450 nm and 600 nm, 500 nm and 550 nm. In an example, the nanoantennae 16' are nanowells and have an average pitch (center-to-center spacing) between about 350 nm and 750 nm. While example average pitch values have been provided, it is to be understood that other average pitch values may be used.|00 0| For another example, the opening 18 area (A) can range from about R I O3pm2to about 100 pm2, e.g., about R I O2pm2, about 0.1 pm2, about 1 pm2, at least about 10 pm2, or more, or less. In another example, the volume can range from about l >< 103pm3to about 100 pm3, e.g., about R I O2pm3, about 0.1 pm3, about 1 pm3, about 10 pm3, or more, or less. For still another example, the depth can range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. For another example, the depth can range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. For yet another example, the diameter or each of the length and width can range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. In another example, the nanoantennae 16’ are nanowells and the average depth is 150 nm or greater, 200 nm or greater, 250 nm or greater, 300 nm or greater, 350 nm or greater, or 400 nm or greater, or may be in a range between about 150 nm and 500 nm, 200 nm and 450 nm, or 250 nm and 400 nm., or 300 nm and 350 nm.J00131 J For sequencing operations implementing, e.g., SBS, each nanoantennae 16' may have any interior volume 22' that is capable of confining a reagent fluid. The minimum or maximum volume can be selected, for example, to accommodate the throughput (e.g., multiplexity), resolution, nucleotides, or analyte reactivity expected for downstream uses of the flow cell. For example, the volume can be at least about 1x10-3 pm3, about 1 * 10-2 pm3, about 0.1 pm3, about 1 pm3, about 10 pm3, about 100 pm3, or more, or less. It is to be understood that the polymeric hydrogel can fill all or part of the volume of a nanoantennae 16'.(00132] The area (A) occupied by each opening 18' can be selected based upon similar criteria as those set forth above for well volume. Moreover, preparation of monoclonal clusters within each nanoantennae 16' substrate ideally requires a 1 : 1 template seeding ratio to ensure the cluster results from amplification of only a single template fragment. In reality, however, a 1 : 1 seeding ratio is difficult to achieve because the variance of the number of seeded fragments per bead is stochastic and, thus, seeding events tend to occur along a Poisson distribution. In that regard, the area ( ) should be calibrated to optimize template distribution. For example, the area for each opening 32 can be at least about 1 x 10-3 pm2, about 1 x 10-2 pm2, about 0.1 pm2, about 1 pm2, about 10 pm2, about 100 pm2, or more, or less. The area occupied by each cavity opening can be greater than, less than or between the values specified above.(00133] The depth of each nanoantennae 16' can be large enough to house some of the polymeric hydrogel. In an example, the depth may be about 0.1 pm, about 0.5 pm, about 1 pm, about 10 pm, about 100 pm, or more, or less. In some examples, the depth is about 0.4 pm. The depth of each nanoantennae 16'may be greater than, less than or between the values specified above.(00134] In some instances, the diameter or length and width of each nanoantenna 38 can be about 50 nm, about 0.1 pm, about 0.5 pm, about 1 pm, about 10 pm, about 100 pm, or more, or less. The diameter or length and width of each nanoantennae 16' can be greater than, less than or between the values specified above.(00135] The nucleic acid clusters of the invention can have different shapes, sizes and densities depending on the conditions used. For example, clusters can have a shape and size that conforms to solid support structures herein. The diameter of a nucleic acid cluster can be designed to be from about 0.2 pm to about 6 pm, about 0.3 pm to about 4 pm, about 0.4 pm to about 3 pm, about 0.5 pm to about 2 pm, about 0.75 pm to about 1.5 pm, or any intervening diameter. In a particular implementation, the diameter of a nucleic acid cluster is about 0.5 pm, about 1 pm, about 1.5 pm, about 2 pm, about 2.5 pm, about 3 pm, about 4 pm, about 5 pm, or about 6 pm. The diameter of a nucleic acid cluster may be influenced by a number of parameters, including, but not limited to the number of amplification cycles performed in producing the cluster, the length of the nucleic acid template or the density of primers attached to the surface upon which clusters are formed. The density of nucleic acid clusters can be designed to typically be in the range of O. l / mm2, 1 / mm2, 10 / mm2, 100 / mm2, 1,000 / mm2, 10,000 / mm2to 100,000 / mm2.The present invention further contemplates, in part, higher density nucleic acid clusters, for example, 100,000 / mm2to 1,000,000 / mm2and 1,000,000 / mm2to 10,000,000 / mm2.

[0136] In one example, in addition to a base layer 12' and optic support layer 14', the multilayered nanostructure architecture 10' includes a polymeric resin coating applied to the base portion 20a' of each nanoantennae 16', which may serve as a solid support surface. Some examples of suitable resins include a polyhedral oligomeric silsesquioxane-based resin, a non-polyhedral oligomeric silsesquioxane epoxy resin, a poly(ethylene glycol) resin, a polyether resin (e.g., ring opened epoxies), an acrylic resin, an acrylate resin, a methacrylate resin, an amorphous fluoropolymer resin (e.g., CYTOP® from Bellex), and combinations thereof.J00137] The base layer 12' may be a circular sheet, a panel, a wafer, a die etc. having a diameter ranging from about 2 mm to about 300 mm, e.g., from about 200 mm to about 300 mm, or may be a rectangular sheet, panel, wafer, die etc. having its largest dimension up to about 10 feet (~3 meters). For example, a die may have a width ranging from about 0.1 mm to about 10 mm. While example dimensions have been provided, it is to be understood that a base layer 12' any suitable dimensions may be used.(001381 In an example, the flow channel 50 has a rectangular configuration. The length and width of the flow channel 50 may be selected so a portion of the base layer 12' or an outermost layer of the multi-layer nanostructure architecture 10' surrounds the flow channel 40 and is available for attachment to a lid (not shown) or another patterned or non-pattemed structure. The surrounding portions are the bonding regions (not shown).[001391 The depth of the flow channel 40 may be as small as a monolayer thick when microcontact, aerosol, or inkjet printing is used to deposit a separate material over the bonding region that defines the flow channel 40 walls. In other examples, a thicker spacer layer may be applied to bonding region so that the spacer layer defines at least a portion of the walls of the flow channel 40. As one example, the spacer layer may be a radiation-absorbing material that aids in bonding. In these examples, the depth of the flow channel 40 can be about 1 pm, about 10 pm, about 50 pm, about 100 pm, or more. In an example, the depth may range from about 10 pm to about 100 pm. In another example, the depth may range from about 10 pm to about 30 pm. In still another example, the depth is about 5 pm or less. It is to be understood that the depth of the flow channel 33 may be greater than, less than or between the values specified above.J00140] Each nanoantennae 16' of the illustrated flow cell format of Fig. 3B includes a reaction site 30' defined on at least a portion of the solid support surface of the base portion 20a' of interior surface 20'. In one example, the reaction sites 30' are coextensive with the solid support surface of the base portion 20a'. In another example, the reaction sites 30' overlays a portion of the solid support surface of the base portion 20a'.{00141] For the patterned structure, many different layouts of the nanoantennae 16' or reaction sites 30' may be envisaged, including regular, repeating, and non-regular patterns. In an example, the nanoantennae 16’ or reaction sites 30' are disposed in self-ordered hexagonal grid. As discussed in greater detail herein, other layouts may be engineered by pre-patterning processing using, for example, imprinting techniques, masking, photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, microetching techniques, or a combination thereof, for example, rectilinear (rectangular) layouts, triangular layouts, and so forth. In some examples, the layout or pattern can be an x-y lattice format in rows and columns. In other examples, the layout or pattern can be a repeating arrangement of nanoantennae 16' or reaction sites 30' and the interstitial regions 26'. In still other examples, the layout or pattern can be a random arrangement of the nanoantennae 16' within the interstitial regions 26'.|00142| The layout or pattern may be characterized with respect to the density (number) of the nanoantennae 16' or reaction sites 30' in a defined area. For example, the nanoantennae 16' or reaction sites 30' may be present at a density of approximately 2 million per mm2. The density may be tuned to different densities including, for example, a density of about 100 per mm2, about 1,000 per mm2, about 0.1 million per mm2, about 1 million per mm2, about 2 million per mm2, about 5 million per mm2, about 10 million per mm2, about 50 million per mm2, or more, or less. It is to be further understood that the density can be between one of the lower values and one of the upper values selected from the ranges above, or that other densities (outside of the given ranges) may be used. As examples, a high-density array may be characterized as having the nanoantennae 16' or reaction sites 30' separated by less than about 100 nm, a medium density array may be characterized as having the nanoantennae 16' or reaction sites 30' separated by about 400 nm to about 1 pm, and a low-density array may be characterized as having the nanoantennae 16' or reaction sites 30' separated by greater than about 1 pm.J00143] The layout or pattern of the nanoantennae 16' or reaction sites 30' may also or alternatively be characterized in terms of the average pitch, or the spacing from the center of one nanoantenna 16' or reaction site 30' to the center of an adjacent nanoantenna 16' or reaction site 30' (center-to-center spacing) or from the right edge of one nanoantenna 16' or reaction site 30’ to the left edge of an adjacent nanoantenna 16' or reaction site 30' (edge-to-edge spacing). The pattern can be regular, such that the coefficient of variation around the average pitch is small, or the pattern can be non-regular in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, about 50 nm, about 0.1 pm, about 0.5 pm, about 1 pm, about 5 pm, about 10 pm, about 100 pm, or more or less. The average pitch for a particular pattern of can be between one of the lower values and one of the upper values selected from the ranges above. In an example, the nanoantennae 16' have a pitch (center-to-center spacing) of about 1.5 pm. While example average pitch values have been provided, it is to be understood that other average pitch values may be used.1001 4] The size of each nanoantenna 16' may be characterized by its volume, opening area, depth, and / or diameter or length and width. For example, the volume can range from about 1 x 10-3 pm3 to about 100 pm3, e.g., about 1x 10-2 pm3, about 0.1 pm3, about 1 pm3, about 10 pm3, or more, or less. For another example, the opening area can range from about 1 x 10-3 pm2to about 100 pm2, e.g., about 1 x 10-2 pm2, about 0.1 pm2, about 1 pm2, at least about 10 pm2, or more, or less. For still another example, the depth can range from about 0.1 pm to about 100 pm, e g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. For another example, the depth can range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. For yet another example, the diameter or each of the length and width can range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less.100145] The size of each reaction site 30' may be characterized by its top surface area, height, and / or diameter or length and width. By way of example, in an SBS-based sequencing protocol, the top surface area of each reaction site 30’ may correspond to, or be sized to accommodate, cluster surface areas disclosed herein. Other considerations in sizing each reaction site include, e.g., stoichiometry in efficient distribution and immobilization of target analytes across nanoantennae array 16'. In an example, the top surface area can range from about 1 x 10-3 pm2to about 100 pm2, e g., about 1 x 10-2 pm2, about 0.1 pm2, about 1 pm2, at least about 10 pm2, ormore, or less. For still another example, the height can range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. For yet another example, the diameter or each of the length and width can range from about 0.1 pm to about 100 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less.[00146J Each reaction site 30' provides a functionalized solid support for staging molecular analyses herein. The reaction site 30' includes the polymeric hydrogel (not shown) and primers 42. The polymeric hydrogel may be any gel material that can swell when liquid is taken up and can contract when liquid is removed, e.g., by drying. In an example, the polymeric hydrogel includes an acrylamide copolymer, such as poly(N-(5-azidoacetamidylpentyl) acrylamide-co- acrylamide, PAZAM or other forms of the acrylamide copolymer. In some examples, PAZAM and other forms of the acrylamide copolymer are linear polymers. In some other examples, PAZAM and other forms of the acrylamide copolymer are lightly cross-linked polymers. In other examples, the gel material may be a variation of the structure (I). In one example, the acrylamide unit may be replaced with N,N-dimethylacrylamide.{00147] It is to be understood that other polymeric hydrogels may be used, as long as they are functionalized to graft oligonucleotide primers 42 thereto. Some examples of suitable the polymeric hydrogel include functionalized polysilanes, such as norbornene silane, azido silane, alkyne functionalized silane, amine functionalized silane, maleimide silane, or any other polysilane having functional groups that can attach the desired set of primers 42. Other examples of suitable polymeric hydrogel include those having a colloidal structure, such as agarose; or a polymer mesh structure, such as gelatin; or a cross-linked polymer structure, such as polyacrylamide polymers and copolymers, silane free acrylamide (SFA), or an azidolyzed version of SFA. Examples of suitable polyacrylamide polymers may be synthesized from acrylamide and an acrylic acid or an acrylic acid containing a vinyl group, or from monomers that form [2+2] photo-cycloaddition reactions. Still other examples of suitable polymeric hydrogels include mixed copolymers of acrylamides and acrylates. A variety of polymer architectures containing acrylic monomers (e.g., acrylam ides, acrylates etc.) may be utilized in the examples disclosed herein, such as branched polymers, including dendrimers, and the like. For example, the monomers (e.g., acrylamide, etc.) may be incorporated, either randomly or in block, into the branches (arms) of a dendrimer.J00148] The polymeric hydrogel may be formed using any suitable copolymerization process. The polymeric hydrogel may be deposited using any of the methods disclosed herein. For at least some of the deposition techniques, the polymeric hydrogel may be incorporated into a mixture, e.g., with water or with ethanol and water, and then applied. The attachment of the polymeric hydrogel to the underlying base layer 12' may be through covalent bonding. In some instances, the underlying base layer 12' may first be activated, e.g., through silanization or plasma ashing. As discussed in greater detail herein, with respect to certain embodiments herein, each of the architectures also includes the primer 42 attached to the polymeric hydrogel.|00149] An exemplary process for making a flow channel supporting a substrate layer having a patterned structure of well-ordered nanostructures is shown in Figs. 4A-H. The example process is adapted for automated manufacture of flow cells herein at production scale. However, the process can also be adapted, e.g., for laboratory production of flow cells, which is also contemplated herein.100150] Referring back to Fig. 3B, the flow cell prepared according to the example method includes a multi-layered nanostructure architecture 10', which includes a base layer 12' and optic support layer 14'. The surface of the optic support layer 14' is imparted with an array of nanoantennae 16', each nanoantenna 16' a having base portion covered, at least in part, by a reaction site 30'. In some examples, the thickness of the optic support layer 14' may accommodate the entire depth of nanoantennae 16', such the entire inner surface of nanoantennae 16' is formed in the material of the optic support layer 14'. Alternatively, the thickness of the optic support layer 14' may be coterminous with the depth of the nanoantennae 16' such that a base portion of the inner surface of each nanoantenna 16' exposes a surface of the base layer 12', which may serve as a low-background solid phase support for molecular analysis.|O0151] Examples of suitable base layers 12' include epoxy siloxane, glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefins / cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon (polyamides), ceramics / ceramic oxides, silica, fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p+ silicon), silicon nitride (SisN4), silicon oxide (SiO2), tantalum pentoxide (Ta2Os) or other tantalum oxide(s) (TaOx), hafnium oxide (HfO ), carbon,metals, inorganic glasses, or the like. The base layer 12' may also be glass or silicon, with a coating layer of tantalum oxide or a ceramic oxide at the surface. Some examples of the base layer 12' may have a surface-bound epoxy silane attached thereto, which can interact with the other resin composition components to form the cured resin composition on the base layer 12'. J00152] In an example, the base layer 12' may have a diameter ranging from about 2 mm to about 300 mm, or a rectangular sheet or panel having its largest dimension up to about 10 feet (~3 meters). In an example, the base layer 12' is a wafer having a diameter ranging from about 200 mm to about 300 mm. In another example, the base layer 12' is a die having a width ranging from about 0.1 mm to about 10 mm. While example dimensions have been provided, it is to be understood that a base layer 12' with any suitable dimensions may be used. For another example, a panel may be used that is a rectangular base layer 12', which has a greater surface area than a 300 mm round wafer.|00153 In the examples disclosed herein, the optic support layer 14' may be constructed of a UV-curable resin composition characterized by a high refractive index, e.g., a refraction index of n > -1.5 at 403nm < X >848nm, n > -1.6 at 403nm < X >848nm, n > -1.7 at 403nm < >848nm, n > -1.8 at 403nm < X >848nm, 1.65 < n > 2.0 at 403nm < >848nm, or 1.70 < n > 1.80 at 403nm < X >848nm. In one example, the optic support layer 14' is constructed of UV Resin #18247 manufactured by NTT Advanced Technology Corp., which has a refraction index of n > -1.75 at 403nm < >848nm, and which exhibits high transmittance of 95-97% at X > 450nm.100154] In the same or other examples, the resin composition for constructing the optic support layer 14' may include at least one epoxy material. Any suitable epoxy monomer or crosslinkable epoxy copolymer may be used as the epoxy material. The epoxy material may be selected from the group consisting of: i) an epoxy functionalized silsesquioxane (described further hereinbelow); ii) trimethylolpropane triglycidyl ether; iii) tetrakis(epoxycyclohexyl ethyl)tetramethyl cyclotetrasiloxane; iv) a copolymer of (epoxycyclohexylethyl)methylsiloxane and dimethylsiloxane (wherein a ratio of m:n ranges from 8:92 to 10:90); v) 1 ,3 -bis[2-(3 ,4- epoxycyclohexyl) ethyl] tetramethyl disiloxane; vi) l,3-bis(glycidoxypropyl)tetramethyl disiloxane; vii) 3,4-epoxycyclohexylmethyl-3,4-epoxycyclo-hexanecarboxylate; viii) bis((3,4- epoxycyclohexyl)methyl) adipate; ix) 4-vinyl-l -cyclohexene 1,2-epoxide; x) vinylcyclohexene dioxide; xi) 4,5-epoxytetrahydrophthalic acid diglycidylester; xii) l,2-epoxy-3-phenoxypropane; xiii) glycidyl methacrylate; xiv) 1,2-epoxyhexadecane; xv) poly(ethylene glycol) diglycidylether(wherein n ranges from 1 to 100); xvi) pentaerythritol glycidyl ether; xvii) diglycidyl 1,2- cyclohexanedicarboxylate; xviii) tetrahydrophthalic acid diglycidyl ester; and xix) combinations thereof.|00155] When combinations are used, it is to be understood that any number of the listed epoxy resin materials may be used together in the resin composition. In an example, the resin composition does not include any other monomers or co-polymers.(001561 The epoxy functionalized silsesquioxane includes a silsesquioxane core that is functionalized with epoxy groups. As used herein, the term “silsesquioxane” refers to a chemical composition that is a hybrid intermediate (RSiOi s) between that of silica (SiCh) and silicone (R2SiO). An example silsesquioxane includes a polyhedral oligomeric silsesquioxane with the chemical formula [RSiO3 / 2]n, where n is an even integer ranging from 6 to 14 and at least some of the R groups are epoxy groups. The resin composition disclosed herein may include one or more different cage or core silsesquioxane structures as monomeric units.(00157] In some examples, all of the R groups of the polyhedral oligomeric silsesquioxane may be epoxy groups. An example of this type of epoxy functionalized silsesquioxane is glycidyl polyhedral oligomeric silsesquioxane. Another example of this type of epoxy functionalized silsesquioxane is epoxycyclohexyl ethyl functionalized polyhedral oligomeric silsesquioxane.100158] One example of the epoxy resin matrix disclosed herein includes the epoxy functionalized polyhedral oligomeric silsesquioxane, where the epoxy functionalized polyhedral oligomeric silsesquioxane may be selected from the group consisting of a glycidyl functionalized polyhedral oligomeric silsesquioxane, an epoxycyclohexyl ethyl functionalized polyhedral oligomeric silsesquioxane, and combinations thereof. This example may include the epoxy silsesquioxane material(s) alone, or in combination with an additional epoxy material selected from the group consisting of trimethylolpropane triglycidyl ether; tetrakis(epoxycyclohexyl ethyl)tetramethyl cyclotetrasiloxane; a copolymer of (epoxycyclohexylethyl)methylsiloxane and dimethylsiloxane; l,3-bis[2-(3,4-epoxycyclohexyl) ethyl] tetramethyl disiloxane; 1,3- bis(glycidoxypropyl)tetramethyl disiloxane; 3, 4-epoxycyclohexylmethyl-3,4-epoxy cyclohexanecarboxylate; bis((3,4-epoxycyclohexyl)methyl) adipate; 4-vinyl-l -cyclohexene 1,2- epoxide; vinylcyclohexene dioxide; 4,5-epoxytetrahydrophthalic acid diglycidylester; 1,2-epoxy- 3-phenoxypropane; glycidyl methacrylate; 1,2-epoxyhexadecane; poly(ethylene glycol)diglycidylether; pentaerythritol glycidyl ether; diglycidyl 1,2-cyclohexanedicarboxylate; tetrahydrophthalic acid diglycidyl ester; and combinations thereof.(001591 In other silsesqui oxane examples, a majority of the R groups may be epoxy groups.For example, 6 of the 8 R groups are epoxy groups and 2 of the 8 R groups are non-epoxy functional groups, which may be selected from the group consisting of an azide / azido, a thiol, a poly(ethylene glycol), a norbomene, and a tetrazine, or further, for example, alkyl, aryl, alkoxy, and haloalkyl groups. In some aspects, the non-epoxy functional group is selected to increase the surface energy of the resin. In these other examples, the ratio of epoxy groups to non-epoxy groups ranges, for example, from 7: 1 to 5:3, or 9: 1 to 6:4, or 11 : 1 to 7:5.(00160] With any of the example epoxy materials disclosed herein, it is to be understood that the epoxy group(s) allow the monomeric units and / or the copolymer to polymerize and / or crosslink into a cross-linked matrix upon initiation using ultraviolet (UV) light and acid(s) (generated by a photoacid generator, which is described below).(00161] The suitable extent of cure and increased hardness may be determined to ensure that other desirable properties of the cured resin, such as low autofluorescence, are not deleteriously affected in the process. Some cured resins exhibit undesirable levels of autofluorescence at excitation wavelengths of interest (e.g., violet excitation wavelengths ranging from about 380 nm to about 450 nm, or blue excitation wavelengths ranging from about 450 nm to about 495 nm, or green excitation wavelengths ranging from about 495 nm to about 570 nm). Fluorescence from the cured resin may increase the background noise when imaging optical labels of nucleotides that have been incorporated into individual nascent strands formed in the cavity during sequencing. Increased background noise can decrease signal to noise ratios (SNRs) so that signals from individual clusters within individual cavities are more difficult to interpret during sequencing. In certain examples, the cured resin disclosed herein may have minimal absorbance of blue excitation, resulting in relatively low or no blue or longer wavelength autofluorescence when exposed to violet or blue excitation wavelengths. As such, the potential for signal interference during sequencing on a patterned flow cell surface of examples of the cured resin disclosed herein is minimal or non-existent. In the same or other examples, a high-index cured resin material may be used having a refraction index of 1.70 < n > 1.80 at 403nm < >848nm. (001621 Because the resin material may include one or more cationically curable species, the resin composition also includes an initiating system, such as a direct photoacid generator or acombination of a photoinitiator and a photoacid generator to initiate curing of the epoxy resin matrix material(s). The direct photoacid generator does not require a photoinitiator to initiate its decomposition, and thus can directly generate an acid, which, in turn, initiates the polymerization and / or crosslinking of the epoxy resin matrix component(s). Suitable examples of the direct photoacid generator may be selected from the group consisting of: The free radical photoinitiator may be selected from the group consisting of 2-ethyl-9,10-dimethoxyanthracene, 2,2-dimethoxy- 2-phenylacetophenone, 2-ethoxy-2-phenylacetophenone, and a phosphine oxide, or combinations thereof. When combinations are used, it is to be understood that any two or more of the listed free radical photoinitiators may be used together in this example of the resin composition. J00163] In addition to the free radical photoinitiator, these examples of the resin composition also include the photoacid generator (PAG), which is not a direct photoacid generator. It is believed that any suitable photoacid generator that will not undergo undesirable intramolecular interactions with the free radical photoinitiator may be used. Examples of suitable photoacid generators may include benzyl, imino ester, conjugated imino ester, spiropyran, teraylene-based, two-photon, and organometallic PAG systems.(00164] Some examples of the resin composition may also include a surface additive. The surface additive can adjust the surface tension of the resin composition, which can improve the detachability of the resin from an imprinting apparatus (e.g., a working stamp), improve the coatability of the diluted resin composition, promote thin film stability, and / or improve leveling. Examples of surface additives include polyacrylate. The amount of the surface additive may be 3 mass % or less, based on the total mass of the resin composition.(00165] According to certain examples, the resin composition may be carrier or solvent free, e.g., NTT-AT #18247. Alternatively, the resin composition may include a liquid carrier or solvent, e.g., NTT-AT #1820 composition containing propylene glycol monomethyl ether acetate (PGMEA). The resin composition including the liquid carrier may be referred to herein as the diluted resin composition. The liquid carrier may be added to achieve a desired viscosity for the deposition technique being used to apply the resin composition. Examples of the diluted resin composition viscosity (e.g., after the liquid carrier is introduced) ranges from about 1.75 mPa to about 2.2 mPa (measured at 25° C.). Examples of other suitable liquid carriers include toluene, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), etc. In some examples, the liquid carrier is PGMEA. In examples, the total solids concentration of the diluted resin composition may rangefrom about 15 mass % to about 60 mass %, and the amount of liquid carrier may range from about 40 mass % to about 85 mass %.|00166| One example of the diluted resin composition includes the liquid carrier and solids dispersed in the liquid carrier, the solids consisting of an epoxy material selected from the group consisting of an epoxy functionalized silsesquioxane; trimethylolpropane triglycidyl ether; tetrakis(epoxy cyclohexyl ethyl)tetramethyl cyclotetrasiloxane; a copolymer of (epoxycyclohexylethyl)methylsiloxane and dimethylsiloxane; l,3-bis[2-(3,4-epoxycyclohexyl) ethyl] tetramethyl disiloxane; l,3-bis(glycidoxypropyl)tetramethyl disiloxane; 3,4- epoxycyclohexylmethyl-3,4-epoxycyclo-hexanecarboxylate; bis((3,4-epoxycyclohexyl)methyl) adipate; 4-vinyl-l -cyclohexene 1,2-epoxide; vinylcyclohexene dioxide; 4,5- epoxy tetrahydrophthalic acid diglycidylester; l,2-epoxy-3 -phenoxy propane; glycidyl methacrylate; 1,2-epoxyhexadecane; poly(ethylene glycol) diglycidyl ether; pentaerythritol glycidyl ether; diglycidyl 1,2-cyclohexanedicarboxylate; tetrahydrophthalic acid diglycidyl ester; and combinations thereof; functionalized silica nanoparticles; an initiating system selected from the group consisting of a direct photoacid generator and a combination of a photoinitiator and a photoacid generator; and an optional surface additive.]00l671 Any example of the resin composition disclosed herein may be used in the formation of an optic support layer herein. An example of a method 400 for patterning the resin composition to form an optic support layer of a flow cell is shown in Figs. 4A-4H. As shown at Figs. 4A and 4B, one example of the method includes applying the diluted resin composition on a substrate, whereby at least some of the liquid carrier evaporates to form a nanoimprintable resin composition 46 having featureless surface 48. The diluted resin composition may be applied on the base layer 12" using any suitable application technique, which may be manual or automated. As examples, the application of the diluted resin composition may be performed using vapor deposition techniques, coating techniques, grafting techniques, or the like. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, puddle dispensing, aerosol printing, screen printing, microcontact printing, inkjet printing, or the like. In one example, spin coating is used.|00168| The technique used to apply the diluted resin composition may cause at least some of the liquid carrier to evaporate. After the diluted resin composition is applied to the substratesurface, it may be softbaked to remove excess liquid carrier. The resulting film has a desirable thickness and is nanoimprintable. This film is referred to as the nanoimprintable resin composition. When performed, the softbake may take place after the diluted resin composition is deposited and before the working stamp 44 is positioned therein. The softbake may take place at a lower temperature than is used for curing (e.g., ranging from about 50° C. to about 150° C.) and for a time ranging from greater than 0 seconds to about 3 minutes. In an example, the softbake time may range from about 30 seconds to about 2.5 minutes.|00.1691 At Figs. 4C and 4D, the method may include imprinting the nanoimprintable resin composition 46 with a working stamp 44 having a plurality of conically-formed nanofeatures 50; and curing the nanoimprintable resin composition 46 while the working stamp is in place, thereby forming a cured, patterned resin 46' on the substrate to form optic support layer 14". The nanoimprintable resin composition 42' may then be patterned, using any suitable patterning technique. In the example shown in FIG. 4C, nanoimprint lithography (NIL) is used to pattern the resin composition 46. As illustrated in FIG. 4C and D, the working stamp 44 is pressed against the layer of the nanoimprintable resin composition to create an imprint in the nanoimprintable resin composition 46. In other words, the nanoimprintable resin composition 46 is indented or perforated by the conically-formed nanofeatures 54 of the working stamp 44 to form proto-nanoantennae 38 and interstitial regions 26". The patterned resin 46' may then be cured with the working stamp 44 in place.100170] During imprinting, one or more long range macroscopic features may be formed (e g., unpatterned interstitial areas separating patterned lanes, registration fiducials, alignment features). A fiducial may be a distinguishable point of reference in or on an object, where, e.g., the point of reference may be present in an image of the object and / or present in a data set derived from detecting the object; or the point of reference may be any other representation of the object suitable to express information about the point of reference with respect to the object. The point of reference may specifiable by an x and / or y coordinate in a plane of the object. Alternatively, or additionally, the point of reference may be specifiable by a z coordinate that is orthogonal to the x-y plane, e.g., being defined by relative locations of the object and a detector. One or more coordinates for a point of reference may be specifiable relative to one or more other features of an object or of an image or other data set derived from the object.100171 ] For the patterned resin 46' disclosed herein, curing may be accomplished by exposing the applied and nanoimprinted resin composition 10 to the incident light at an energy dose ranging from about 0.5 J to about 10 J for 30 seconds or less. The incident light may be actinic radiation, such as ultraviolet (UV) radiation. In one example, the majority of the UV radiation emitted may have a wavelength of about 365 nm. jOOI 72] In some examples disclosed herein, the energy exposure promotes decomposition of the direct photoacid generator into an acid that initiates polymerization and / or cross-linking of the epoxy resin matrix. In some instances, the incident light exposure time may be 30 seconds or less. In other instances, the incident light exposure time may be 20 seconds or less. In still other instances, the incident light exposure time may be about 5 seconds.100173] In other examples disclosed herein, the energy exposure causes the photoinitiator to generate free radicals, which promote decomposition of the photoacid generator into an acid that initiates polymerization and / or cross-linking of the epoxy resin matrix. With the effective extent of curing brought on by this mechanism, the incident light exposure time may be 30 seconds or less. In some instances, the incident light exposure time may be 20 seconds or less. In still other instances, the incident light exposure time may be about 5 seconds. The curing process may include a single UV exposure stage. After curing and the release of the working stamp 44, nanostructures and / or other topographical features are defined in the cured resin composition. ] 0174] At Fig. 4E, once the optic support layer 14" a conformal coating of a dielectric material having a refractive index of n > ~2 is deposited on interior surface of the nanoantennae 16’ to form a conformal shell 28" in each nanoantennae 16". The thickness of the deposited dielectric material is selected for resonance to particular incident spectra. For example, referring back to Fig 2B, the intensity profile of example nanoantennae 16" of the disclosure were modeled based on 40 nm conformal tantalum pentoxide (Ta2Os) shell at a 488 nm pump wavelength. Suitable deposition techniques may include, e.g., physical vapor deposition methods -thermal evaporation, RF sputtering, chemical vapor deposition, chemical vapor deposition. In example, prior to deposition, a sacrificial layer (not shown) may be applied to the interstitial regions 26". After deposition, the sacrificial layer is then removed and the conformal shell 28" may be cleaned ultrasonically and / or with organic solvent to remove grease residues from its surface. As shown in FIG. 4E, the resulting cured optic support layer 14" includes thenanoantennae 16" defined therein, and interstitial regions 26" separating adjacent nanoantennae 38.|00175| The attachment of the silane or silane derivative forms a pre-treated (e.g., silanized) optic support layer 14", which includes silanized cavities and silanized interstitial regions. f 00176) In other examples, the optic support layer 14" may not be exposed to silanization.Rather, the optic support layer 14" may be exposed to plasma ashing, and then the polymeric hydrogel may be directly spin coated (or otherwise deposited) on the plasma ashed optic support layer 14". In this example, plasma ashing may generate surface-activating agent(s) (e.g., hydroxyl (C — OH or Si — OH) and / or carboxyl groups) that can adhere the polymeric hydrogel to the optic support layer 14". In these examples, the polymeric hydrogel is selected so that it reacts with the surface groups generated by plasma ashing.[001771 Silanization may be accomplished using any silane or silane derivative. The selection of the silane or silane derivative may depend, in part, upon the material that is to be used to form the polymeric hydrogel, as it may be desirable to form a covalent bond between the silane or silane derivative and the polymer hydrogel. The method used to attach the silane or silane derivative to the optic support layer 14" may vary depending upon the silane or silane derivative that is being used. Several examples are set forth herein. f00178| Examples of suitable silanization methods include vapor deposition (e.g., a YES method), spin coating, or other deposition methods. Some examples of methods and materials that may be used to silanized the optic support layer 14" are described herein, although it is to be understood that other methods and materials may be used.|00179| In an example utilizing the YES CVD oven, the optic support layer 14" on the base layer 12" is placed in the CVD oven. The chamber may be vented and then the silanization cycle started. During cycling, the silane or silane derivative vessel may be maintained at a suitable temperature (e.g., about 120° C. for norbornene silane), the silane or silane derivative vapor lines be maintained at a suitable temperature (e.g., about 125° C. for norbomene silane), and the vacuum lines be maintained at a suitable temperature (e.g., about 145° C ).100180] In another example, the silane or silane derivative (e.g., liquid norbornene silane) may be deposited inside a glass vial and placed inside a glass vacuum desiccator with the base layer 12" having the optic support layer 14" thereon. The desiccator can then be evacuated to a pressure ranging from about 15 mTorr to about 30 mTorr and placed inside an oven at atemperature ranging from about 60° C. to about 125° C. Silanization is allowed to proceed, and then the desiccator is removed from the oven, cooled and vented in air.[001811 Vapor deposition, the YES method and / or the vacuum desiccator may be used with a variety of silane or silane derivatives, such as those silane or silane derivative including a cycloalkene unsaturated moiety, such as norbornene, a norbomene derivative (e.g., a (hetero)norbornene including an oxygen or nitrogen in place of one of the carbon atoms), transcyclooctene, transcyclooctene derivatives, transcyclopentene, transcycloheptene, transcyclononene, bicyclo[3.3.1]non-l-ene, bicyclo[4.3.1]dec-l (9)-ene, bicyclo [4.2.1]non-l(8)-ene, and bicyclo[4.2.1]non-l-ene. Any of these cycloalkenes can be substituted, for example, with an R group, such as hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicycle, aralkyl, or (heteroalicyclel)alkyl. An example of the norbomene derivative includes [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. As other examples, these methods may be used when the silane or silane derivative includes a cycloalkyne unsaturated moiety, such as cyclooctyne, a cyclooctyne derivative, or bicyclononynes (e.g., bicyclo[6.1 ,0]non-4-yne or derivatives thereof, bicyclo[6.1 ,0]non-2-yne, or bicyclo[6.1 ,0]non-3- yne). These cycloalkynes can be substituted with any of the R groups described herein.|00l821 In still other examples, the optic support layer 14" may include unreacted epoxy groups and thus may not be exposed to silanization because the unreacted epoxy groups can react directly with amino functional groups of the polymeric hydrogel. In this example, plasma ashing may be performed, e.g., if it is desirable to clean the surface of potential contaminants.[001831 After completion nanoconstruction within the substrate layer, the substrate layer is put through a traditional flow cell process workflow. For example, as illustrated in 4H the base portion 20a" may provide a surface chemistry including a functionalized coating layer and one or more surface primers covalently bound to the functionalized coating layer to create a reaction site 30" for a biological material. The selective application of the polymer may involve multiple processes, including activation of the interstitial regions 26" and the exposed surfaces in the nanoantennae 16", depositing the polymer on the activated interstitial regions 36 and in the nanoantennae 16", and polishing the interstitial regions 26" to remove polymer from the interstitial regions to limit polymer to nanoantennae 16".[001841 The polymer layer may then be applied as described herein. As examples, the polymer (e.g., PAZAM) may be deposited using spin coating, or dipping or dip coating, or flowof the functionalized molecule under positive or negative pressure, or another suitable technique. The polymer deposited to form the polymer may be present in a mixture. In an example, the mixture includes PAZAM in water or in an ethanol and water mixture.|00185] After being coated, the mixture including the polymer may also be exposed to a curing process to form the polymer across the activated interstitial regions 26" of the patterned resin 46' and in the nanoantennae 16". In an example, curing may take place at a temperature ranging from room temperature (e.g., about 25° C.) to about 95° C. for a time ranging from about 1 millisecond to about several days. In another example, the time may range from 10 seconds to at least 24 hours. In still another example, the time may range from about 5 minutes to about 2 hours. 00186] The attachment of the polymer to the activated (in this example silanized) surfaces may be through covalent bonding. Covalent linking is helpful for maintaining at least the first primer set in the nanoantennae 16" throughout the lifetime of the ultimately formed flow cell during a variety of uses. The following are some examples of reactions that can take place between the activated (e.g., silanized) surfaces and the polymer.(00.187] When the silane or silane derivative includes norbornene or a norbornene derivative as the unsaturated moiety, the norbornene or a norbomene derivative can: i) undergo a 1,3- dipolar cycloaddition reaction with an azide / azido group of PAZAM; ii) undergo a coupling reaction with a tetrazine group attached to PAZAM; undergo a cycloaddition reaction with a hydrazone group attached to PAZAM; undergo a photo-click reaction with a tetrazole group attached to PAZAM; or undergo a cycloaddition with a nitrile oxide group attached to PAZAM. 100188] When the silane or silane derivative includes cyclooctyne or a cyclooctyne derivative as the unsaturated moiety, the cyclooctyne or cyclooctyne derivative can: i) undergo a strain- promoted azide-alkyne 1,3-cycloaddition (SPAAC) reaction with an azide / azido of PAZAM, or ii) undergo a strain-promoted alkyne-nitrile oxide cycloaddition reaction with a nitrile oxide group attached to PAZAM.]00189] When the silane or silane derivative includes a bicyclononyne as the unsaturated moiety, the bicyclononyne can undergo similar SPAAC alkyne cycloaddition with azides or nitrile oxides attached to PAZAM due to the strain in the bicyclic ring system.(00190] In other examples, plasma ashing rather than silanization may be used to activate the interstitial regions 26" and the exposed surfaces of the base portions 20a" of the nanoantennae16". After plasma ashing, the mixture containing the polymer may be directly spin coated (or otherwise deposited) on the plasma ashed surfaces and then cured to form the polymer layer. In this example, plasma ashing may generate surface-activating agent(s) (e.g., hydroxyl (C — OH or Si — OH) and / or carboxyl groups) that can adhere the polymer to the interstitial regions 26" and the exposed surfaces of the base portions 20a" in the nanoantennae 16". In these examples, the polymer is selected so that it reacts with the surface groups generated by plasma ashing.(001911 In some sequencing operations involving target nucleic acid materials, the primers may be an oligonucleotide (i.e., a surface oligo) having a complementary sequence to the target nucleic acid material, in which case the target nucleic acid material may be captured or immobilized at the reaction site through hybridization to a primer under stringent conditions. A primer may be functionalized with a capture agent, e.g., streptavidin, and the nucleic acid sequence is ligated with a capture partner, e.g., biotin, (or vice versa) in which case the target nucleic acid material may be captured at the reaction site through, e g., formation of a streptavidin-biotin complex. The capture agent may also be ligated to the functionalized coating layer and capture takes place without the need for a primer. The target nucleic acid material may also be captured through a reaction of clickable groups conjugating either the nucleic acid material and a primer or nucleic acid material and a functionalized coating layer.1001921 It may be desirable for the primers 42' to be immobilized to the polymeric hydrogel. In some examples, immobilization may be by single point covalent attachment to the polymeric hydrogel at the 5' end of the respective primers 42'. Any suitable covalent attachment means known in the art may be used. In some examples, immobilization may be by strong non-covalent attachment.{00193 J Examples of terminated primers that may be used include an alkyne terminated primer, a tetrazine terminated primer, an azido terminated primer, an amino terminated primer, an epoxy or glycidyl terminated primer, a thiophosphate terminated primer, a thiol terminated primer, an aldehyde terminated primer, a hydrazine terminated primer, a phosphorami di te terminated primer, a triazolinedione terminated primer, and a biotin-terminated primer. In some specific examples, a succinimidyl (NHS) ester terminated primer may be reacted with an amine at a surface of the polymeric hydrogel, an aldehyde terminated primer may be reacted with a hydrazine at a surface of the polymeric hydrogel, or an alkyne terminated primer may be reacted with an azide at a surface of the polymeric hydrogel, or an azide terminated primer may bereacted with an alkyne or DBCO (dibenzocyclooctyne) at a surface of the polymeric hydrogel, or an amino terminated primer may be reacted with an activated carboxylate group or NHS ester at a surface of the polymeric hydrogel, or a thiol terminated primer may be reacted with an alkylating reactant (e.g., iodoacetamine or maleimide) at a surface of the polymeric hydrogel, a phosphoramidite terminated primer may be reacted with a thioether at a surface of the polymeric hydrogel, or a biotin-modified primer may be reacted with streptavidin at a surface of the polymeric hydrogel.[001941 Each of the primers 42' has a universal sequence for capture and / or amplification purposes. Examples of the primers 42' include P5 and P7 primers, examples of which are used on the surface of commercial flow cells sold by Illumina Inc. for sequencing, for example, on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, ISEQ™, GENOME ANALYZER™, and other instrument platforms. 00195] For sequential paired end sequencing, each of these primers 42' may also include a cleavage site. The cleavage sites of the primers 42' may be different from each other so that cleavage of the primers 42' does not take place at the same time. Examples of suitable cleavage sites include enzymatically cleavable nucleobases or chemically cleavable nucleobases, modified nucleobases, or linkers (e.g., between nucleobases). The enzymatically cleavable nucleobase may be susceptible to cleavage by reaction with a glycosylase and an endonuclease, or with an exonuclease. One specific example of the cleavable nucleobase is deoxyuracil (dU), which can be targeted by the USER enzyme. In an example, the uracil base may be incorporated at the 7th base position from the 3' end of the P5 primer (P5U) or of the P7 primer (P7U). Other abasic sites may also be used. Examples of the chemically cleavable nucleobases, modified nucleobases, or linkers include 8-oxoguanine, a vicinal diol, a disulfide, a silane, an azobenzene, a photocleavable group, allyl T (a thymine nucleotide analog having an allyl functionality), allyl ethers, or an azido functional ether.

[0196] In an example, grafting may be accomplished by flow through deposition (e.g., using a temporarily bound lid), dunk coating, spray coating, puddle dispensing, or by another suitable method that will attach the primer(s) 42' to the polymeric hydrogel. Each of these example techniques may utilize a primer solution or mixture, which may include the primer(s) 42', water, a buffer, and a catalyst.{00197] Dunk coating may involve submerging the optic support layer 14' into a series of temperature-controlled baths. The baths may also be flow controlled and / or covered with a nitrogen blanket. The baths may include the primer solution or mixture. Throughout the various baths, the primer(s) 42' will attach to the primer-grafting functional group(s) of the polymeric hydrogel in at least some of the nanoantennae 16'. In an example, the flow cell surface precursor 30 will be introduced into a first bath including the primer solution or mixture where a reaction takes place to attach the primer(s) 42', and then moved to additional baths for washing. Movement from bath to bath may involve a robotic arm or may be performed manually. A drying system may also be used in dunk coating.{00198] Spray coating may be accomplished by spraying the primer solution or mixture directly onto the optic support layer 14". The spray coated wafer may be incubated for a time ranging from about 4 minutes to about 60 minutes at a temperature ranging from about 0° C. to about 70° C. After incubation, the primer solution or mixture may be diluted and removed using, for example, a spin coater.{001 9] Puddle dispensing may be performed according to a pool and spin off method, and thus may be accomplished with a spin coater. The primer solution or mixture may be applied (manually or via an automated process) to the optic support layer 14". The applied primer solution or mixture may be applied to or spread across the entire surface of the optic support layer 42. The primer coated optic support layer 14' may be incubated for a time ranging from about 2 minutes to about 60 minutes at a temperature ranging from about 0° C. to about 80° C. After incubation, the primer solution or mixture may be diluted and removed using, for example, the spin coater.{00200] The flow cell of Figs. 3A-B and 4A-H may be used in a variety of sequencing approaches or technologies, including SBS. As one example, SBS reaction may be run on a system such as the HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NOVASEQ™, ISEQ™, NEXTSEQDX™, orNEXTSEQ™ sequencer systems from Illumina (San Diego, Calif.). In SBS, extension of a nucleic acid primer (e.g., a sequencing primer) along a nucleic acid template (i.e., the sequencing template) is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase enzyme) or ligation (e.g., catalyzed by a ligase enzyme). In a particular polymerase-based SBS process, fluorescently labeled nucleotides are added to thesequencing primer (thereby extending the sequencing primer) in a template dependent fashion such that detection of the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template.(002011 Sequencing methodologies for nucleic acid samples on NGS platforms commonly deploy DNA libraries in which a DNA target (e g., genomic DNA (gDNA), or complimentary DNA (cDNA)) is processed into fragments and ligated with technology-specific adaptors. Fragments may also be ligated with sample source-specific barcoding in multiplexed operations. NGS workflow using SBS involves loading a DNA library onto a flow cell and hybridizing individual DNA fragments to adapter-specific complimentary oligonucleotides (oligos) covalently bound to the flow cell surface (planar or pattemed)(here, primers 42). In one example of cluster generation, the library fragments are copied from the hybridized primers 42 by 3' extension using a high-fidelity DNA polymerase. The original library fragments are denatured, leaving the copies immobilized. Isothermal bridge amplification may be used to amplify the immobilized copies. For example, the copied templates loop over to hybridize to an adjacent, complementary primer 421and a polymerase copies the copied templates to form double stranded bridges, which are denatured to form two single stranded strands. These two strands loop over and hybridize to adjacent, complementary primers 42' and are extended again to form two new double stranded loops. The process is repeated on each template copy by cycles of isothermal denaturation and amplification to create dense clonal clusters. Each cluster of double stranded bridges is denatured. In an example, the reverse strand is removed by specific base cleavage, leaving forward template polynucleotide strands. Clustering results in the formation of several template polynucleotide strands (or amplicons) on the reactions sites 30' of the array of nanoantennae 38.(00202 ( Amplification may also be performed using an exclusion amplification (ExAmp) technique. ExAmp cluster generation is particularly appropriate for optimizing monoclonality of amplicon populations on a given reaction site 30'. ExAmp chemistry carries out seeding and cluster amplification steps simultaneously such that amplification of a first seeding event on a given reaction site 30' occurs nearly instantaneously and the amplification rate far exceeds the reaction rate for seeding. In that manner, the ExAmp amplification of a fragment seeded to a reaction site prevents further seeding by other fragments, thus reducing the occurrence of undesirable polyclonal clustering.(OO203J A sequencing primer may be introduced that hybridizes to a complementary sequence on the template strand. This sequencing primer renders the template polynucleotide strand ready for sequencing. The 3 '-ends of the templates and any flow cell-bound primers 42 (not attached to the copy) may be blocked to prevent interference with the sequencing reaction, and in particular, to prevent undesirable priming.(00204] To initiate sequencing, an incorporation mix may be added to the flow cell. In one example, the incorporation mix may include a liquid carrier, fluorescently labeled nucleoside triphosphates (NTPs), and one or more replication enzymes (e.g., polymerase). The fluorescently labeled NTPs may include a 3' OH blocking group. When the incorporation mix is introduced into the flow cell in a given cycle, the fluid enters a flow channel and flows into nanoantennae 16’ to contact the reaction sites 30' hosting the clustered template strands.|00205| The fluorescently labeled nucleotides may be added to the sequencing primer one nucleotide per cycle in a template dependent fashion such that detection of the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template. More particularly, one of the nucleotides is incorporated, by a respective polymerase, into a nascent strand that extends the sequencing primer and that is complementary to the template polynucleotide strand.In certain sequencing protocols herein, e.g., SBS, each image may include color signals, wherein a different color corresponds to a different nucleotide base. As illustrated in Fig. 5, in a four-channel chemistry, each image of the set of images comprises signals may have a single color selected from at least four different colors. For instance, each image in the set of images comprises signals having a single color selected from four different colors. In some of the systems described herein, nucleic acids can be sequenced by providing four different labeled nucleotide bases (for DNA: A, G, T, and C) to the array of molecules so as to produce four different images, each image comprising signals having a single color, wherein the signal color is different for each of the four different images, thereby producing a cycle of four-color images that corresponds to the four possible nucleotides present at a particular position in the nucleic acid (four-channel chemistry). A 2-channel chemistry technique uses two channels per sequencing cycle and decodes results from the two channels into individual base identifications. For example, imaging is performed at each of two wavelengths, e.g., blue and green, and decoded. Labeled nucleotide bases (or clusters) seen in blue or green images are interpreted as Cand T bases, respectively. Bases observed in both blue and green images are flagged as A bases, while unlabeled clusters are identified as G bases. A 1-channel chemistry technique uses one channel two times separated by an intermediate chemistry step for each sequencing cycle. For example, imaging is performed at a particular wavelength, the intermediate chemistry step is performed, and then imaging is performed again at the particular wavelength. In certain aspects, the system comprises a flow cell that is configured to deliver additional labeled nucleotide bases to the array of molecules, thereby producing a plurality of cycles of color images.[002071 Image data of nucleotides incorporated during a given cycle may be collected by an imager, e.g., in a fluorescence microscopy system using a point, area, or line imaging technique. Image data may be taken for a collection area, such as corresponding to a tile of a flow cell. Some implementations of line imaging correspond to discrete point-and-shoot operation. Point imaging collects point-images as relatively smaller collections of one or more pixels, collecting image data for larger areas by progressing left to right along an x axis of the flow cell surface, then up along a y axis, then again left to right, and so forth, via relative movement of the imager and the flow cell, such as in discrete point-and-shoot operation. Area imaging collects areaimages as relatively larger collections of pixels, such as in a rectangular (e.g., square) shape. Area image collection progresses similarly to that of point imaging, by progressing from left to right, then up, then again left to right, and so forth, via relative movement of the imager and the flow cell, such as in discrete point-and-shoot operation. Line imaging collects line-images as collections of pixels corresponding to a rectangular region of a relatively high aspect ratio, such as a single pixel high and several pixels wide corresponding to the collection area width. Line image collection progresses a line at a time in a direction orthogonal to the line, via relative movement of the imager and flow cell. Some implementations of line imaging correspond to continuous scanning operation, e g., the imager and the flow cell are in continuous movement with respect to each other and image capture is performed during the movement. Some implementations of continuous scanning operation are performed using Time Delay Integration (TDI).[00208) In some examples, the nucleotides may further include a reversible termination property (e.g., the 3' OH blocking group) that terminates further primer extension once a nucleotide has been added to the sequencing primer. For example, a nucleotide analog having a reversible terminator moiety may be added to the sequencing primer such that subsequentextension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for examples that use reversible termination, a deblocking reagent can be delivered to the flow cell after detection occurs.(00209| Wash(es) may take place between the various fluid delivery steps. The SBS cycle may then be repeated n times to extend the sequencing primer by n nucleotides, thereby detecting a sequence of length. In some examples, the forward strands may be sequenced and removed, and then reverse strands are constructed and sequenced as described herein.(0021 1 Base call outputs generated from NGS and other sequencing methodologies may be output to a FASTA text file or binary counterpart. A FASTA file may include a text file that contains sequence data from clusters that pass filter on a flow cell. A FASTA file may also include corresponding quality scores of the sequence.(002111 In one example, a method for optical detection of a nucleic acid sample in a sequencing protocol is provided. Referring back to Figs. 3A-B, according to the example method, a DNA library may be loaded onto optic support layer 14' of flow cell device 50, where constituent fragment strands are flowed across an array of nanoantennae 16' impressed in the optic support layer 14' and individual strands are absorbed by nanoantennae 16' of the array at a substantially 1 : 1 basis, and each absorbed strand is immobilized at a reaction site 30' of respective nanoantennae through interaction between the strand adaptor and a capture primer 42 covalently bound to the surface of the reaction site. Each immobilized strand is then amplified (e g., using bridge or exclusion amplification) to yield a substantially monoclonal cluster of template strands within each respective nanoantennae.|00212| Each of the resulting clusters is then sequenced using, e.g., a sequencing-by-synthesis (SBS) technique. According to the example method, in a first cycle, (1) a buffer solution containing a mixture of four different labeled nucleoside derivatives (one for each of the four DNA nucleobases (A, G, C, T)) may be provided to a cluster of template strands in the presence of polymerase; (2) a complementary nucleoside derivative may be added to a nascent copy strand hybridized to each template strand via polymerase primer extension; (3) the added nucleoside derivatives may be irradiated with incident light via an excitation source to induce fluorescence and emission of an output signal from the cluster; (4) the output signal may be detected by one or more optical sensors through imaging of the signal as a point source at an addressed location on the optic substrate; and (5) information imparted in the imaged output signal may be processedby a signal processor to record the nucleobase of the added nucleoside derivative and an address of the cluster on the optic substrate. Steps (l)-(5) may be performed simultaneously for a plurality of clusters with a given frame (or tile) of the one or more optic sensors. Steps ( 1 )-(5) may then be repeated in subsequent cycles to n number of total cycles, where n is equal to the size of the template strands in base pairs (bp).J00213] Photon budget requirements for sequencing methodologies on NGS platforms, particularly with SBS-based processes, are relatively high. In each cycle, a plurality of clusters is exposed to excitation power. The total number of cycles corresponds to the read length of bases on the template strands of each of the clusters. Example read lengths may be 50, 75, 150, and 300 base pairs, which correspond to a respective number of total cycles. Moreover, the fluorescence chemistry of NGS requires as many as four images per cycle to capture fluorescence of each of the four base types added in a given round. As just one example, a typical step and shoot sequencing operation using a flow cell format and 300 cycle SBS chemistry may obtain two images per sequencing cycle per tile (using two-channel chemistry), where the total number of cycles is equal to the length in bp of the DNA template strand, viz., 300 bp, and the total number of tiles image may be 130 or greater, resulting in a total number of imaging steps that can exceed 78,000 (130x300x2).|00214| Resonant enhancement associated with nanoantennae arrays herein results in increased power density and confinement of the excitation radiation within the individual nanoantenna cavity, resulting in an increased intensity in output or emission signal with an SNR value in excess of operating requirements for conventional optical detection systems, which, in turn, enables the utilization of more efficient and compact light sources. In that regard, an output signal of sufficient intensity to pass filter may be generated using lower excitation power than conventional optic solid supports. Further, the contribution of the shot noise to the intensity distribution of an output signal will generally diminish with an increase in average intensity. Given that, the increased signal intensity engendered by nanoantennae herein may improve detection of intensity clouds associated with adjacent analytes as distinct point sources, which may improve quality measures of the information imparted in the signal.

Claims

CLAIMS1. A flow cell, comprising: a base layer; an optic support layer of cured resin material adhered to the support layer, wherein the optic support layer comprises a nanoantennae array separated by interstitial regions of featureless resin surface, each nanoantennae comprising a conically formed cavity impressed in the optic support layer, wherein the conically formed cavity defines an opening coplanar with the featureless resin surface, the conically formed cavity comprising a base portion providing a solid support for optic detection of a target analyte, and an inwardly conical wall portion joining the opening and the base portion; a reaction site collocated with at least a portion of the solid support; a conformal shell of high refractive index medium supporting the interior volume of the cavity.

2. The flow cell of claim 1, wherein an average pitch of the nanoantennae array is equal to or less than 400 nm.

3. The flow cell of claim 2, wherein an average pitch of the nanoantennae array is equal to or less than 300 nm.

4. The flow cell of claim 1, wherein the optic support layer has a refractive index of 1.5 < n > 2.0.

5. The flow cell of claim 5, wherein the optic support layer has a refractive index of n = 1.8.

6. The flow cell of claim 1, wherein the high refractive index medium is a dielectric material.

7. The flow cell of claim 6. comprises a metal oxide material.

8. The flow cell of claim 7, wherein the metal oxide material is one of tantalum pentoxide (Ta2O5), aluminum oxide (A12O3), and titanium oxide (TiO2), hafnium oxide (HfO2), niobium oxide (NbO), cerium oxide (CeO2), and gallium oxide (GaO2), tungsten oxide (WO), zirconium oxide (ZrO2), and tin oxide (SnO2).

9. The flow cell of claim 6, wherein the dielectric material is one of silicon nitride (Si3N4), indium phosphide (InP), gallium phosphide (GaP) arsenic phosphide (AsP), and germanium (Ge)10. The flow cell of claim 1, wherein the conformal shell of high refractive index medium has an average thickness of 40 nm.

11. The flow cell of claim 1, wherein the optic support layer has a length along a y axis and a width along an x axis, wherein the nanoantennae array is arranged into one or more lanes along the y axis, and wherein each of the one or more lanes defines a flow channel for flowing a solution containing a biological material over the nanoantennae array.

12. A biological assay for optical detection of a biological material, the biological assay comprising a flow cell functionalized with a surface chemistry, the flow cell comprising a base layer, an optic support layer of cured resin material adhered to the support layer, wherein the optic support layer comprises a nanoantennae array separated by interstitial regions of featureless resin surface, each nanoantennae comprising a conically formed cavity impressed in the optic support layer, wherein the conically formed cavity defines an opening coplanar with the featureless resin surface, the conically formed cavity comprising a base portion providing a solid support for optic detection of a target analyte, and an inwardly conical wall portion joining the opening and the base portion, a reaction site collocated with at least a portion of the solid support,a conformal shell of high refractive index medium supporting the interior volume of the cavity, wherein the surface chemistry is localized to the reaction site and comprises a capture agent configured to interact with a constituent analyte of the biological material; and a biological material comprising a solution of constituent analytes, wherein at least one constituent analyte is immobilized on each reaction site of at least a portion of the nanoantennae array via interaction with a respective capture agent.

13. The biological assay of claim 12, wherein the optic support layer of the flow cell has a refractive index of l.5 < n > 2.0.

14. The biological assay of claim 13, wherein the optic support layer has a refractive index of n = 1.8.

15. The biological assay of claim 12, wherein the high refractive index medium is a dielectric material.

16. The biological assay of claim 15, wherein the dielectric material comprises a metal oxide material.

17. The biological assay of claim 16, wherein the metal oxide material is one of tantalum pentoxide (Ta2O5), aluminum oxide (A12O3), and titanium oxide (TiO2), hafnium oxide (HfO2), niobium oxide (NbO), cerium oxide (CeO2), and gallium oxide (Ga02), tungsten oxide (WO), zirconium oxide (ZrO2), and tin oxide (SnO2).

18. The biological assay of claim 15, wherein the dielectric material is one of silicon nitride (Si3N4), indium phosphide (InP), gallium phosphide (GaP) arsenic phosphide (AsP), and germanium (Ge)19. The biological assay of claim 12, wherein the conformal shell of high refractive index medium of each nanoantenna has an average thickness of 40 nm.

20. The biological assay of claim 12, wherein the biological material is a nucleic acid material.

21. The biological assay of claim 12, wherein the nucleic acid material is DNA and the constituent analytes are DNA fragments.

22. The biological assay of claim 12, wherein each DNA fragment comprises an adapter sequence of nucleotides and the capture agent comprises a complementary sequence of nucleotides, wherein interaction comprises hybridization of the adapter sequence of an immobilized DNA fragment to the complementary sequence of a respective capture agent.

23. The biological assay of claim 12, wherein the biological material is a protein library and each constituent analyte is a candidate protein.

24. A method of optical detection of a biological material, method comprising: providing a biological assay, wherein the biological assay comprises a flow cell functionalized with a surface chemistry, the flow cell comprising a base layer, an optic support layer of cured resin material adhered to the support layer, wherein the optic support layer comprises a nanoantennae array separated by interstitial regions of featureless resin surface, each nanoantennae comprising a conically formed cavity impressed in the optic support layer, wherein the conically formed cavity defines an opening coplanar with the featureless resin surface, the conically formed cavity comprising a base portion providing a solid support for optic detection of a target analyte, and an inwardly conical wall portion joining the opening and the base portion, a reaction site collocated with at least a portion of the solid support, a conformal shell of high refractive index medium supporting the interior volume of the cavity,wherein the surface chemistry is localized to the reaction site and comprises a capture agent configured to interact with a constituent analyte of the biological material; and a biological material comprising a solution of constituent analytes, wherein at least one constituent analytes is immobilized on each reaction site of at least a portion of the nanoantennae array via interaction with a respective capture agent, and wherein one or more of the immobilized constituent analyte is associated with a respective label moiety; irradiating, via an excitation source, at least a portion of the one or more immobilized constituent analytes with an incident light to induce excitation radiation and an output signal from the irradiated constituent analytes, wherein the conformal shell of high refractive index medium promotes resonant field coupling between respective optical modes of the nanoantenna and the irradiated constituent analytes; and wherein the resonant field coupling causes confinement of the excitation radiation to the nanoantenna cavity and within a volume substantially equal in diameter to the wavelength of the incident light; detecting, via one or more sensors, the output signals of the one or more immobilized labeled constituent analytes; and deriving, via a signal processor, information imparted in each of the detected output signals.

25. The method of claim 24, wherein the optic support layer of the flow cell has a refractive index of 1.5 < n > 2.0.

26. The method of claim 25, wherein the optic support layer has a refractive index of n = 1.8.

27. The method of claim 24, wherein the high refractive index medium is a dielectric material.

28. The method of claim 27, wherein the dielectric material comprises a metal oxide material.

29. The method of claim 28, wherein the metal oxide material is one of tantalum pentoxide (Ta2O5), aluminum oxide (A12O3), and titanium oxide (TiO2), hafnium oxide (HfO2), niobiumoxide (NbO), cerium oxide (CeO2), and gallium oxide (GaO2), tungsten oxide (WO), zirconium oxide (ZrO2), and tin oxide (SnO2).

30. The method of claim 27, wherein the dielectric material is one of silicon nitride (Si3N4), indium phosphide (InP), gallium phosphide (GaP) arsenic phosphide (AsP), and germanium (Ge)31. The method of claim 24, wherein the conformal shell of high refractive index medium of each nanoantenna has an average thickness of 40 nm.

32. The method of claim 24, wherein the biological material is a nucleic acid material.

33. The method of claim 32, wherein the nucleic acid material is DNA and the immobilized constituent analytes are clusters of DNA templates cloned from individual constituent fragments of the DNA.

34. The biological assay of claim 24, wherein the biological material is a protein library and each constituent analyte is a candidate protein.

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