Devices and compositions for use in luminescence imaging and methods of using the same

Abstract

The present application relates to devices and compositions used in luminescence imaging and methods of using the same. A device for use in luminescence imaging may include an imaging pixel array, each imaging pixel in the imaging pixel array is integrated with a first well and a second well arranged above each imaging pixel, and each imaging pixel in the imaging pixel array generates an electrical signal indicating excitation of a first well at a first time and excitation of a second well at a second time.

Description

[0001] This application is a divisional application of an application with an application date of April 21, 2017, application number 201780039083.7, and invention name "Photonic structure-based devices and compositions used in luminescent imaging of multiple sites within a pixel and methods of using the same".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 326,568, filed on April 22, 2016, and entitled “PHOTONIC STRUCTURE-BASEDDEVICES AND COMPOSITIONS FOR USE IN LUMINESCENT IMAGING OF MULTIPLE SITES WITH IN A PIXEL, AND METHODS OF USING THE SAME,” which is incorporated herein by reference in its entirety.

[0004] field

[0005] The present application relates generally to luminescence imaging. background

[0006] Some of the most advanced sequencing tools developed by industry leaders rely on various "synthesis sequencing (SBS)" chemistries to determine polynucleotide sequences, such as DNA or RNA sequences. Sequencing can involve the use of luminescent imaging such as fluorescence microscopy systems to identify nucleotides or localized clusters of identical nucleotides by the emission wavelength of each of their fluorescent labels. Although some SBS chemistries in development may require only one dye, multiple fluorescent dyes (up to 4) are typically used in commercial systems to uniquely identify nucleotides in a polynucleotide, such as A, G, C, and T nucleotides in DNA.

[0007] Overview

[0008] Embodiments of the present invention provide photonic structure-based devices and compositions for use in luminescent imaging of multiple sites within a pixel, and methods of using the same.

[0009] According to one aspect, a device for use in luminescent imaging is provided. The device may include an imaging pixel array and a photonic structure arranged on the imaging pixel array. The device may also include a feature array arranged on the photonic structure. A first feature of the feature array may be arranged on a first pixel of the imaging pixel array, and a second feature of the feature array may be arranged on the first pixel and spatially shifted from the first feature. A first luminophore may be arranged in or on the first feature, and a second luminophore may be arranged in or on the second feature. The device may also include a radiation source configured to generate a first photon having a first characteristic at a first time, and configured to generate a second photon having a second characteristic at a second time. The second characteristic may be different from the first characteristic, and the second time may be different from the first time. The first pixel may selectively receive cold light emitted by the first luminophore in response to the first photon at the first time, and may selectively receive cold light emitted by the second luminophore in response to the second photon at the second time.

[0010] Optionally, a first photon having a first characteristic generates a first resonant pattern in the photonic structure at a first time, and the first resonant pattern selectively excites the first light emitter relative to the second light emitter. Optionally, a second photon having a second characteristic generates a second resonant pattern in the photonic structure at a second time, and the second resonant pattern selectively excites the second light emitter relative to the first light emitter.

[0011] Additionally or alternatively, the imaging pixel array, photonic structure, and feature array are optionally monolithically integrated with one another.

[0012] Additionally or alternatively, the photonic structure optionally comprises a photonic crystal, a photonic superlattice, a microcavity array, or a plasmonic nanoantenna array.

[0013] Additionally or alternatively, the array of features optionally includes a plurality of wells. The first feature may include a first well in which the first luminophore is disposed, and the second feature may include a second well in which the second luminophore is disposed. Alternatively, the array of features optionally includes a plurality of columns. The first feature may include a first column on which the first luminophore is disposed, and the second feature may include a second column on which the second luminophore is disposed.

[0014] Additionally or alternatively, the first and second characteristics are optionally independently selected from the group consisting of wavelength, polarization, and angle. For example, the first characteristic optionally includes a first linear polarization, and the second characteristic optionally includes a second linear polarization different from the first linear polarization. Optionally, the first linear polarization is substantially orthogonal to the second linear polarization, or optionally, the first linear polarization is rotated at an angle between about 15 degrees and about 75 degrees relative to the second linear polarization. Additionally or alternatively, the first characteristic may optionally include a first wavelength, and the second characteristic may optionally include a second wavelength different from the first wavelength.

[0015] Additionally or alternatively, the radiation source optionally includes an optical component. Optionally, the device further includes a controller coupled to the optical component and configured to control the optical component so as to impose a first characteristic on the first photon and to impose a second characteristic on the second photon. Optionally, the optical component includes a birefringent material configured to rotate the first photon to a first linear polarization by the controller in response to a first control signal, and configured to rotate the second photon to a second linear polarization by the controller in response to a second control signal.

[0016] Additionally or alternatively, the first and second photons optionally each illuminate the photonic structure at substantially the same angle as each other. Additionally or alternatively, the first and second photons optionally each illuminate the photonic structure at an angle approximately perpendicular to a major surface of the photonic structure. Additionally or alternatively, the first and second photons optionally each illuminate the photonic structure at an angle approximately parallel to a major surface of the photonic structure.

[0017] Additionally or alternatively, the second feature is optionally laterally displaced from the first feature.

[0018] Additionally or alternatively, a third feature of the feature array is optionally arranged on the first pixel and spatially displaced from each of the first and second features. The device may also optionally include a third luminophore arranged in or on the third feature. The radiation source may optionally be configured to generate a third photon having a third characteristic at a third time. Optionally, the third characteristic may be different from the first and second characteristics, and the third time may be different from the first and second times. Optionally, the first pixel selectively receives luminescence emitted by the third luminophore in response to the third photon at the third time. Additionally or alternatively, a fourth feature of the feature array is optionally arranged on the first pixel and spatially displaced from each of the first, second and third features. The device may optionally also include a fourth luminophore arranged in or on the fourth feature. The radiation source may optionally be configured to generate a fourth photon having a fourth characteristic at a fourth time. Optionally, the fourth characteristic may be different from the first, second and third characteristics, and the fourth time may be different from the first, second and third times. The first pixel selectively receives luminescence emitted by the fourth luminophore in response to the fourth photon at the fourth time. Optionally, a first luminophore is coupled to the first nucleic acid, a second luminophore is coupled to the second nucleic acid, a third luminophore is coupled to the third nucleic acid, and a fourth luminophore is coupled to the fourth nucleic acid.

[0019] Additionally or alternatively, a third feature of the feature array is optionally disposed on a second pixel of the imaging pixel array, and a fourth feature of the feature array is optionally disposed on the second pixel and spatially displaced from the third feature. The device optionally also includes a third luminophore disposed within or above the third feature, and a fourth luminophore disposed within or above the fourth feature. Optionally, the second pixel selectively receives cold light emitted by the third luminophore in response to a first photon at a first time or in response to a second photon at a second time. Optionally, the second pixel selectively receives cold light emitted by the fourth luminophore in response to a first photon at a first time or in response to a second photon at a second time. Optionally, the first luminophore is coupled to a first nucleic acid, the second luminophore is coupled to a second nucleic acid, the third luminophore is coupled to a third nucleic acid, and the fourth luminophore is coupled to a fourth nucleic acid.

[0020] Additionally or alternatively, the first and second features optionally each have a substantially circular cross-section.Additionally or alternatively, the photonic structure optionally comprises a hexagonal lattice, and optionally the imaging pixels are rectangular.

[0021] Additionally or alternatively, the radiation source is optionally configured to flood illuminate the photonic structure with the first and second photons. Additionally or alternatively, the radiation source optionally comprises a laser. Additionally or alternatively, the first and second photons optionally independently have a wavelength between about 300 nm and about 800 nm.

[0022] Additionally or alternatively, the first luminophore is optionally coupled to the first nucleotide, and the second luminophore is optionally coupled to the second nucleic acid. Additionally or alternatively, the device optionally includes at least one microfluidic feature in contact with the feature array and configured to provide a flow of one or more analytes to the first feature and the second feature.

[0023] In addition or alternatively, the first luminophore is optionally coupled to the first polynucleotide to be sequenced, and the second luminophore is optionally coupled to the second polynucleotide to be sequenced. Alternatively, the first polynucleotide is coupled to the first feature, and optionally the second polynucleotide is coupled to the second feature. In addition or alternatively, the device may optionally further include a first polymerase that adds the first nucleic acid to a third polynucleotide that is complementary to and coupled to the first polynucleotide. The first nucleic acid may optionally be coupled to the first luminophore. The device may optionally further include a second polymerase that adds the second nucleic acid to a fourth polynucleotide that is complementary to and coupled to the second polynucleotide. The second nucleic acid may optionally be coupled to the second luminophore. Optionally, the device may further include a channel that allows a first liquid that includes the first and second nucleic acids and the first and second polymerases to flow into or over the first and second features.

[0024] According to another aspect, a method for use in luminescent imaging is provided. The method may include providing an imaging pixel array, and providing a photonic structure arranged on the imaging pixel array. The method may also include providing a feature array arranged on the photonic structure. A first feature of the feature array may be arranged on a first pixel of the imaging pixel array, and a second feature of the feature array may be arranged on the first pixel and spatially shifted from the first feature. The method may also include providing a first luminophore arranged in or on the first feature, and providing a second luminophore arranged in or on the second feature. The method may also include generating, by a radiation source, a first photon having a first characteristic at a first time, and generating, by a radiation source, a second photon having a second characteristic at a second time. The second characteristic may be different from the first characteristic, and the second time may be different from the first time. The method may also include selectively receiving, by a first pixel, luminescent light emitted by a first luminophore in response to a first photon at a first time; and selectively receiving, by a first pixel, luminescent light emitted by a second luminophore in response to a second photon at a second time.

[0025] Optionally, a first photon having a first characteristic generates a first resonant pattern in the photonic structure at a first time, and the first resonant pattern selectively excites the first light emitter relative to the second light emitter. Optionally, a second photon having a second characteristic generates a second resonant pattern in the photonic structure at a second time, and the second resonant pattern selectively excites the second light emitter relative to the first light emitter.

[0026] Additionally or alternatively, the imaging pixel array, photonic structure, and feature array are optionally monolithically integrated with one another.

[0027] Additionally or alternatively, the photonic structure optionally comprises a photonic crystal, a photonic superlattice, a microcavity array, or a plasmonic nanoantenna array.

[0028] Additionally or alternatively, the array of features optionally includes a plurality of wells. The first feature optionally may include a first well in which the first luminophore is disposed, and the second feature optionally may include a second well in which the second luminophore is disposed. Alternatively, the array of features may include a plurality of columns. The first feature optionally may include a first column on which the first luminophore is disposed, and the second feature optionally may include a second column on which the second luminophore is disposed.

[0029] Additionally or alternatively, the first and second characteristics may optionally be independently selected from the group consisting of wavelength, polarization, and angle. For example, the first characteristic may optionally include a first linear polarization, and the second characteristic may optionally include a second linear polarization different from the first linear polarization. Optionally, the first linear polarization may be substantially orthogonal to the second linear polarization, or may be rotated at an angle between about 15 degrees and about 75 degrees relative to the second linear polarization. Additionally or alternatively, the first characteristic may optionally include a first wavelength, and the second characteristic may optionally include a second wavelength different from the first wavelength.

[0030] Additionally or alternatively, the radiation source optionally includes an optical component. The method optionally further includes controlling the optical component so as to impose a first characteristic on the first photon and a second characteristic on the second photon. Optionally, the optical component includes a birefringent material that rotates the first photon to a first linear polarization in response to a first control signal by the controller and rotates the second photon to a second linear polarization in response to a second control signal by the controller.

[0031] Additionally or alternatively, the first and second photons optionally each illuminate the photonic structure at substantially the same angle as each other. Additionally or alternatively, the first and second photons optionally each illuminate the photonic structure at an angle approximately perpendicular to a major surface of the photonic structure, or the first and second photons optionally each illuminate the photonic structure at an angle approximately parallel to a major surface of the photonic structure.

[0032] Additionally or alternatively, the second feature is optionally laterally displaced from the first feature.

[0033] Additionally or alternatively, a third feature of the feature array is optionally disposed on the first pixel and spatially displaced from each of the first and second features. Optionally, the method further includes providing a third luminophore disposed within or above the third feature and generating a third photon having a third characteristic at a third time. The third characteristic may optionally be different from the first and second characteristics, and the third time may optionally be different from the first and second times. The method may further optionally include selectively receiving, by the first pixel, luminescent light emitted by the third luminophore in response to the third photon at the third time. Optionally, a fourth feature of the feature array is disposed on the first pixel and spatially displaced from each of the first, second and third features. The method may further optionally include providing a fourth luminophore disposed within or above the fourth feature and generating a fourth photon having a fourth characteristic at a fourth time. The fourth characteristic may optionally be different from the first, second and third characteristics, and the fourth time may optionally be different from the first, second and third times. The method may further optionally include selectively receiving, by the first pixel, luminescent light emitted by the fourth luminophore in response to the fourth photon at the fourth time. Optionally, a first luminophore is coupled to the first nucleic acid, a second luminophore is coupled to the second nucleic acid, a third luminophore is coupled to the third nucleic acid, and a fourth luminophore is coupled to the fourth nucleic acid.

[0034] Additionally or alternatively, the third feature of the feature array may optionally be arranged on a second pixel of the imaging pixel array, and the fourth feature of the feature array may be arranged on the second pixel and spatially displaced from the third feature. The method may optionally further include providing a third luminophore arranged within or above the third feature, and providing a fourth luminophore arranged within or above the fourth feature. The method may optionally further include selectively receiving, by the second pixel, cold light emitted by the third luminophore in response to a first photon at a first time or in response to a second photon at a second time; and selectively receiving, by the second pixel, cold light emitted by the fourth luminophore in response to a first photon at a first time or in response to a second photon at a second time. Optionally, the first luminophore is coupled to the first nucleic acid, the second luminophore is coupled to the second nucleic acid, the third luminophore is coupled to the third nucleic acid, and the fourth luminophore is coupled to the fourth nucleic acid.

[0035] Additionally or alternatively, the first and second features optionally each have a substantially circular cross-section.Additionally or alternatively, the photonic structure optionally comprises a hexagonal lattice, and the imaging pixels optionally are rectangular.

[0036] Additionally or alternatively, the method optionally includes flooding the photonic structure with the first and second photons. Additionally or alternatively, the method optionally includes generating the first and second photons with a laser. Additionally or alternatively, the first and second photons independently have a wavelength between about 300 nm and about 800 nm.

[0037] Additionally or alternatively, the first luminophore is optionally coupled to the first nucleic acid, and the second luminophore is optionally coupled to the second nucleic acid. Additionally or alternatively, the method optionally includes providing at least one microfluidic feature in contact with the feature array and causing one or more analytes to flow to the first feature and the second feature through the at least one microfluidic feature.

[0038] Additionally or alternatively, the first luminophore is optionally coupled to a first polynucleotide to be sequenced, and the second luminophore is optionally coupled to a second polynucleotide to be sequenced. Alternatively, the first polynucleotide is coupled to a first feature, and the second polynucleotide is optionally coupled to a second feature. Additionally or alternatively, the method optionally includes adding a first nucleic acid to a third polynucleotide complementary to and coupled to the first polynucleotide by a first polymerase. The first nucleic acid may optionally be coupled to the first luminophore. The method optionally also includes adding a second nucleic acid to a fourth polynucleotide complementary to and coupled to the second polynucleotide by a second polymerase. The second nucleic acid may optionally be coupled to a second luminophore. Alternatively, the method also includes flowing a first liquid including the first nucleic acid and the second nucleic acid and the first polymerase and the second polymerase into or over the first feature and the second feature through a channel.

[0039] According to another aspect, a device for use in luminescent imaging is provided. The device may include an imaging pixel array and a photonic structure disposed on the imaging pixel array. The device may also include a feature array disposed on the photonic structure. A first feature of the feature array may be disposed on a first pixel of the imaging pixel array, and a second feature of the feature array may be disposed on the first pixel and spatially displaced from the first feature. The photonic structure may be adjusted to selectively illuminate the first feature with light of a first polarization in contrast to light of a second polarization. The photonic structure may be adjusted to selectively illuminate the second feature with light of a second polarization in contrast to light of the first polarization.

[0040] Optionally, the apparatus further comprises a radiation source configured to generate first photons having a first polarization at a first time and configured to generate second photons having a second polarization at a second time.

[0041] Additionally or alternatively, the device optionally includes a first light emitter disposed within or on the first feature and a second light emitter disposed within or on the second feature.

[0042] In addition or alternatively, the device optionally further comprises a first target analyte disposed in or on the first feature and a second target analyte disposed in or on the second feature. The first target analyte may optionally be different from the second target analyte. The first and second target analytes optionally comprise nucleic acids having different sequences.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A A perspective view of an exemplary photonic structure-based device for use in luminescent imaging of a site within a pixel is schematically shown.

[0045] Figure 1B Schematically shows that in the device array, for example Figure 1A A perspective view of an exemplary array of sites within a device array is shown, where each site corresponds to a pixel.

[0046] Figure 1C Schematically shows an exemplary device such as Figure 1A A cross-sectional view of the device shown.

[0047] Figure 2A Schematically shows Figure 1B A perspective view of an exemplary excitation of an array of sites is shown.

[0048] Figure 2B Schematically shows the response to e.g. Figure 2A The excitation shown in Figure 1A and 1C Simulated exemplary field strengths within an array of devices shown.

[0049] Figure 3A A perspective view of an exemplary array of sites, such as provided herein, is schematically shown, where multiple sites correspond to one pixel.

[0050] Figure 3B A cross-sectional view of a device such as that provided herein is schematically shown, wherein Figure 3A The multiple sites shown correspond to one pixel.

[0051] Figure 4A Schematically illustrates the use of a scanning focused beam illumination pair such as provided herein. Figure 3A A perspective view of exemplary excitation of selected sites of an array of sites is shown.

[0052] Figure 4B Schematically illustrates the use of multiple laser interference illumination as provided herein. Figure 3A A perspective view of exemplary excitation of selected sites of an array of sites is shown.

[0053] Figure 5 Schematically illustrates an exemplary photonic structure such as may be included in, for example, the Figure 3A-3B In the device shown in .

[0054] 6A-6D schematically illustrate the radiation source in the example Figure 5Exemplary simulated field strengths within a photonic structure are shown, with the radiation source generating photons with mutually different properties at different times.

[0055] FIG. 7A schematically shows an example of a method for performing a Figure 3A-3B A plan view of an exemplary photonic structure-based device is shown in , which includes first and second sites (e.g., clusters) per pixel.

[0056] 7B schematically illustrates exemplary simulated field intensities within a device array such as provided herein and shown in FIGS. 7A and 3A-3B for a radiation source that generates photons having a first characteristic that selectively excites a first site at a first time.

[0057] 7C schematically illustrates exemplary simulated field intensities within a device array such as provided herein and shown in FIGS. 7A and 3A-3B for a radiation source that generates photons having a second characteristic that selectively excites a second site at a second time.

[0058] FIG. 7D schematically illustrates exemplary crosstalk terms resulting from selective excitation of first and second sites such as provided herein and illustrated in FIGS. 7B and 7C , respectively.

[0059] Fig. 8A Schematically illustrating an example provided herein and in Figure 3A-3B A plan view of an exemplary photonic structure-based device shown in , the device comprising first, second, and third sites (e.g., clusters) per pixel.

[0060] Figure 8B Schematically shows the radiation source provided here and in Fig. 8A and 3A -Exemplary simulated field strengths within the device array shown in -3B, the radiation source generating photons having a first characteristic that selectively excites a first site at a first time.

[0061] FIG. 8C schematically illustrates a radiation source such as provided herein and Fig. 8A and 3A -Exemplary simulated field strengths within the device array shown in -3B, the radiation source produces photons having a second characteristic that selectively excites a second site at a second time.

[0062] FIG. 8D schematically illustrates a radiation source such as provided herein and Fig. 8A and 3A -Exemplary simulated field strengths within the device array shown in 3B, the radiation source generating photons having a third characteristic that selectively excites a third site at a third time.

[0063] Fig. 8ESchematically illustrates a method according to some embodiments provided by, for example, Figure 8B -Exemplary crosstalk terms resulting from selective excitation of the first, second, and third sites shown in -8D.

[0064] Fig.9A -9D respectively schematically illustrate the use of a radiation source that generates photons with different characteristics at different times, such as provided herein and in Figure 3A-3B A perspective view of exemplary selective excitation of first, second, third, and fourth sites within the device array shown in FIG.

[0065] Fig.10 An exemplary flow diagram of steps in the methods provided herein for use in luminescence imaging is shown.

[0066] Fig.11 An exemplary sequence of steps that can be used to prepare, for example, a device or composition provided herein is shown.

[0067] Fig.12 An exemplary sequence of steps that can be used to prepare, for example, a device or composition provided herein is shown.

[0068] Fig.13 An exemplary apparatus for use in luminescence imaging, such as provided herein, is shown. Detailed Description

[0069] Embodiments of the present invention provide photonic structure-based devices and compositions for use in luminescent imaging of multiple sites within a pixel, and methods of using the same.

[0070] First, some exemplary terms will be defined, followed by a further description of exemplary embodiments of the present photonic structure-based devices and compositions and methods of using the same for use in luminescence imaging.

[0071] As used herein, the term "photonic structure" means a periodic structure comprising one or more optically transparent materials that selectively affects the propagation of radiation having specific characteristics, such as wavelength, angle, and polarization. For example, a photonic structure can selectively cause radiation having such characteristics, such as at such wavelength, such angle, and such polarization, to propagate through the structure or propagate out of the structure at the same angle or at a different angle, and the field intensity of such radiation can have a selected pattern within the photonic structure. In addition, the structure can selectively inhibit radiation having different characteristics, such as at different wavelengths, angles, and / or polarizations, from propagating through the structure or propagating out of the structure at different angles, and / or the field intensity of such radiation can have different selected patterns within the photonic structure. The material of the photonic structure can include features distributed in one or more dimensions, such as in one dimension, in two dimensions, or in three dimensions. The shape, size, and distribution of the features of the photonic structure and the refractive index of the material can be adjusted to select specific radiation characteristics, such as wavelength, angle, or polarization, that can propagate through the photonic structure or propagate out of the photonic structure at an angle, and / or to select a pattern of the field intensity of such radiation within the photonic structure. Exemplary photonic structures include, but are not limited to, photonic crystals, photonic superlattices, microcavity arrays, and plasmonic nanoantenna arrays.

[0072] As used herein, the terms "photonic crystal", "PhC", "photonic lattice", "photonic crystal lattice" and "PhC lattice" mean a photonic structure comprising one or more materials comprising a periodic variation in a refractive index on the order of the wavelength of light. For example, a photonic structure may comprise a material extending in three dimensions, e.g., having a length, a width and a thickness. The material may have two major surfaces, each of which lies in a plane defined by the length and the width and separated from each other by the thickness. The material may be patterned in two or more dimensions so as to define a photonic band structure in which radiation having particular characteristics (e.g., wavelength, angle or polarization) may propagate through the photonic crystal or out of the photonic crystal at an angle, and / or so as to select a pattern of field strengths of such radiation within the photonic crystal. The pattern may comprise, for example, a plurality of features, such as wells or pillars defined within the material, e.g., through one or both major surfaces of the material, with material not present within or between the features, e.g., within the wells or between the pillars. The spaces within or between the features may be filled with one or more additional materials, which may have refractive indices that are different from the refractive index of the material and different from the refractive index of each other. Specific characteristics of radiation that propagates or does not propagate through the photonic structure or propagates or does not propagate outside the photonic structure at an angle, such as wavelength, angle, or polarization, may be based on the refractive index of the material and the refractive index of any additional materials disposed within or between the features, as well as on characteristics of the features, such as the shape, size, and distribution of the features. The features may all be the same shape, size, and / or distribution as one another.

[0073] As used herein, the terms "photonic structure" and "PhC superlattice" mean a photonic structure that selectively affects the propagation of radiation having a first and a second characteristic, such as at a first and a second wavelength, angle, or polarization, as compared to radiation having a third characteristic, such as at a third wavelength, angle, or polarization. For example, the field intensity of the radiation having the first characteristic can have a first pattern, and the field intensity of the radiation having the second characteristic can have a second pattern different from the first pattern. The third wavelength can occur between the first and second wavelengths in the electromagnetic spectrum. For example, the photonic structure can selectively cause radiation having the first and second characteristics, such as at a first and a second wavelength, angle, or polarization, to propagate through the photonic structure or out of the photonic structure at an angle, and the patterns of the field intensity of the radiation having the first and second characteristics can optionally be different from each other. For example, the photonic superlattice can selectively inhibit radiation having the first and second characteristics, such as at a first and a second wavelength, angle, or polarization, from propagating through the photonic superlattice or out of the photonic superlattice at an angle. For example, a photonic superlattice may selectively allow radiation having a third characteristic, such as at a third wavelength, angle, or polarization, to propagate through the photonic superlattice or out of the photonic superlattice at an angle. For example, a photonic superlattice may selectively inhibit radiation having a third characteristic, such as at a third wavelength, angle, or polarization, from propagating through the structure or out of the structure at an angle. A material may include features distributed in one or more dimensions, such as in one dimension, in two dimensions, or in three dimensions. The shape, size, and distribution of the features and the refractive index of the material may be adjusted to select specific characteristics of radiation that may propagate through the photonic superlattice or out of the photonic superlattice at an angle, such as wavelength, angle, or polarization, and patterns of field strength of such characteristics, and to select specific characteristics of radiation that do not substantially propagate through the photonic superlattice or out of the photonic superlattice at an angle.

[0074] Illustratively, a photonic superlattice may include a material extending in three dimensions (e.g., having a length, a width, and a thickness). The material may have two major surfaces, each of which is located in a plane defined by the length and the width and separated from each other by the thickness. The material may be patterned in two or more dimensions to define a photonic band structure that allows radiation having at least a first and a second characteristic, such as a wavelength, an angle, or a polarization, to propagate within a plane defined by the length and the width or to propagate outside the plane at an angle, and inhibits at least radiation having a third characteristic, such as a third wavelength, an angle, or a polarization, from propagating within the material or propagating outside the material at an angle. The pattern may include, for example, a plurality of features, such as a well or a column defined within the material, such as through one or two major surfaces of the material, with material not present within or between the features, such as within the well or between the columns. The space within or between the features may be filled with one or more additional materials, which may have a refractive index different from the refractive index of the material and different from the refractive index of each other, respectively. The specific characteristics of radiation that propagates or does not propagate through the photonic superlattice or propagates or does not propagate outside the photonic superlattice at an angle can be based on the refractive index of the material and the refractive index of any additional material arranged within or between the features, as well as can be based on the characteristics of the features, such as the shape, size, and distribution of the features. Some features can optionally be different from other features in at least one characteristic such as shape, size, or distribution. For additional details about exemplary photonic superlattices that can be used in the devices, compositions, and methods of the present invention, see U.S. Provisional Patent Application 62 / 312,704, filed on March 24, 2016 and entitled "Photonic Superlattice-Based Devices and Compositions for Use in Luminescent Imaging, and Methods of Using the Same", the entire contents of which are incorporated herein by reference.

[0075] As used herein, "microcavity array" means a periodic two-dimensional arrangement of photonic microresonators that supports multiple (e.g., at least two, at least three, or at least four) resonances that can be excited independently of each other by changing the properties of the excitation source (e.g., the wavelength, polarization, or angle of the excitation source). For additional details about exemplary microcavity arrays that can be used in the devices, compositions, and methods of the present invention, see Altug et al., "Polarization control and sensing with twodimensional coupled photonic crystal microcavity arrays" (Opt. Lett. 30:1422-1428 (2011)), the entire contents of which are incorporated herein by reference.

[0076] As used herein, "plasmonic nanoantenna array" means a periodic two-dimensional arrangement of plasmonic nanostructures that support multiple (e.g., at least two, at least three, or at least four) resonances that can be excited independently of each other by changing the properties of the excitation source, such as the wavelength, polarization, or angle of the polarization source. For additional details on exemplary plasmonic nanoantennas that can be used in the devices, compositions, and methods of the present invention, see Regmi et al., "Nanoscale volume confinement and fluorescence enhancement with double nanohole aperture" (Scientific Reports 5: 15852-1-5 (2015)), which is incorporated herein by reference in its entirety.

[0077] One or more materials of the photonic structure may be or include a "dielectric material," meaning a fluid, solid, or semisolid material that is optically transparent and an electrical insulator. Examples of fluid dielectric materials include gases such as air, nitrogen, and argon, and liquids such as water, aqueous solvents, and organic solvents. Examples of solid dielectric materials include glasses (e.g., inorganic glasses such as silica or modified or functionalized glasses) and polymers (e.g., acrylic resins, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TEFLON, etc.). TM , cycloolefins, polyimides or nylons). Examples of semi-solid dielectric materials include gels, such as hydrogels. Additionally or alternatively, one or more materials of the photonic structure may be or include optically transparent solid semiconductor materials.

[0078] As used herein, the term "gel" is intended to mean a semisolid or semirigid material that is permeable to liquids and gases. Typically, gel materials can expand when liquid is drawn in and can shrink when liquid is removed by drying. Exemplary gels include, but are not limited to, those with colloidal structures, such as agarose or hydrogels; polymer mesh structures, such as gelatin; or cross-linked polymer structures, such as polyacrylamide, SFA (see, e.g., US 2011 / 0059865, the entire contents of which are incorporated herein by reference) or PAZAM (see, e.g., US 2014 / 0079923, the entire contents of which are incorporated herein by reference). Particularly useful gel materials will conform to the shape of the well or other concave features in which it is present.

[0079] As used herein, the term "well" means a discrete concave feature in a material having a surface opening (hole) completely surrounded by an interstitial region of the surface. Wells can have characteristics such as size (e.g., volume, diameter, and depth), cross-sectional shape (e.g., circular, elliptical, triangular, square, polygonal, star-shaped (with any suitable number of vertices), irregular, or with concentric wells separated by dielectric material), and distribution (e.g., spatial location of wells within dielectric material, such as regularly spaced or periodic locations, or irregularly spaced or non-periodic locations). The cross-section of a well can be, but need not necessarily be, uniform along the length of the well.

[0080] As used herein, the term "pillar" means a discrete convex feature protruding from the surface of a material and completely surrounded by the inter-vein region of the surface. The pillar can have characteristics such as size (e.g., volume, diameter, and depth), shape (e.g., circular, elliptical, triangular, square, polygonal, star-shaped (with any suitable number of vertices), irregular, or with concentric pillars separated by dielectric material), and distribution (e.g., the spatial location of the pillar protruding from the surface of the dielectric material, such as regularly spaced or periodic locations, or irregularly spaced or non-periodic locations). The cross-section of the pillar can be, but does not necessarily need to be, uniform along the length of the pillar.

[0081] As used herein, the term "surface" means a portion or layer of a material that is in contact with another material.

[0082] As used herein, the term "interpulse region" means a region in a material or in a surface that separates regions of a material or surface. For example, an interpulse region can separate one feature of a photonic structure from another feature of a photonic structure, or an interpulse region can separate one site of an array from another site of an array.

[0083] As used herein, the term "luminescent" means emitting cold body radiation, and the term "luminophore" means an article that emits light. The term "luminescent" is defined to be distinct from incandescence, which is radiation emitted from a material as a result of heat. Generally, cold light is produced when an energy source transfers an electron of an atom from its lowest energy ground state to a higher energy excited state; the electron then returns energy in the form of radiation so it can fall back to its ground state. A particularly useful type of luminescent article is one that emits cold body radiation when energy is provided by excitation radiation. Such articles may be referred to as "photoluminescent". Examples of photoluminescent articles include "fluorescent" articles that emit cold body radiation relatively quickly (e.g., less than 1 millisecond) after excitation radiation and "phosphorescent" articles that emit cold body radiation relatively slowly (e.g., greater than or equal to 1 millisecond) after excitation radiation. Photoluminescence can be perceived as the emission of radiation at one wavelength by an article as a result of irradiating the article at another wavelength. Another useful type of luminescent article is one that emits cold body radiation when energy is provided by a chemical or biological reaction. Such articles may be referred to as "chemiluminescent".

[0084] Any of a variety of signals can be detected in the methods described herein, including, for example, optical signals, such as radiation absorptivity, luminescent emission, luminescent lifetime, luminescent polarization, etc.; Rayleigh scattering and / or Mie scattering; etc. Exemplary labels that can be detected in the methods described herein include, but are not limited to, fluorophores, luminophores, chromophores, nanoparticles (e.g., gold, silver, carbon nanotubes), etc.

[0085] As used herein, the term "feature" means a unique change in the structure or composition of a material, such as a solid support. Optionally, the change is also repeated in the structure or composition of the material. A collection of features can form an array or lattice in or on a material. Exemplary features include, but are not limited to, wells, columns, ridges, channels, sites for carrying analytes, layers of multilayer materials, regions in or on a material having a chemical composition different from the chemical composition of other regions in or on a material, etc. Features can have properties, such as size (e.g., volume, diameter, and depth), shape (e.g., circular, elliptical, triangular, square, polygonal, star-shaped (with any suitable number of vertices), irregular or with concentric features separated by dielectric materials) and distribution (e.g., spatial positions of features in dielectric materials, such as regularly spaced or periodic positions, or irregularly spaced or non-periodic positions). The cross section of a feature can be, but does not necessarily need to be, uniform along the length of the feature.

[0086] As used herein, the term "site" means the position of a particular species of a molecule or cell (or other analyte) in an array. A site may contain only a single molecule (or cell or other analyte) or it may contain a population of several molecules (or cells or other analytes) of the same species. In some embodiments, the site exists on the material before attaching a particular analyte. In other embodiments, the site is created by attaching a molecule or cell (or other analyte) to the material. The sites of an array are typically discrete. Discrete sites may be adjacent or they may have a distance between each other. It should be understood that a site is a feature. Features may function as a component of a lattice, an array, or both.

[0087] As used herein, the term "array" means a population of sites that can be distinguished from one another based on relative position.

[0088] As used herein, the term "spacing" when used with reference to a feature of a lattice (e.g., a photonic structure) or an array is intended to refer to the center-to-center spacing of adjacent features of the lattice or array. The pattern of the features can be characterized in terms of an average spacing. The pattern can be ordered so that the coefficient of variation around the average spacing is small, or the pattern can be random, in which case the coefficient of variation can be relatively large. In either case, the average spacing can be, for example, at least about the order of magnitude of the wavelength of light in one or more regions of the spectrum. For example, the spacing can correspond to a wavelength in one or more of the visible spectrum (about 380-700nm), the UV spectrum (less than about 380nm to about 10nm), and the IR spectrum (greater than about 700nm to about 1mm). In a photonic structure, features can have spacings that are different from each other in different directions. For example, in a photonic superlattice, different types of features can have spacings and patterns that are different from each other. For example, the spacing of one type of feature (e.g., in a first lattice) can be different from the spacing of another type of feature (e.g., in a second lattice).

[0089] As used herein, the term "random" may be used to refer to the spatial distribution, e.g., arrangement, of locations on a surface. For example, one or more features (e.g., wells or pillars) of a photonic structure or photonic superlattice may be randomly spaced such that nearest neighbor features, which may be of the same type or of different types to one another, have variable spacing between one another. Alternatively, the spacing between features of the same type or of different types to one another may be ordered, e.g., forming a regular pattern, such as a rectilinear grid or a hexagonal grid.

[0090] As used in this article, the term "nucleotide" or "nucleic acid" is intended to mean a molecule including a sugar and at least one phosphate group, and optionally also includes a core base. Nucleotides lacking a core base can be referred to as "abasic". Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides and mixtures thereof. The example of nucleotides includes adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (d In some embodiments, the present invention relates to a reversible blocking nucleoside triphosphate (rbNTP), a reversible blocking nucleoside triphosphate (rbNTP), a reversible blocking thymidine triphosphate (rbTTP), a reversible blocking cytidine triphosphate (rbCTP), a reversible blocking guanosine triphosphate (rbGTP), a reversible blocking thymidine diphosphate (rbTMP), a reversible blocking thymidine triphosphate (rbTTP), a reversible blocking cytidine diphosphate (rbCTP), and a reversible blocking guanosine triphosphate (rbGTP). For more details on reversible blocking nucleoside triphosphates (rbNTPs), see U.S. Patent Publication No. 2013 / 0079322, the entire contents of which are incorporated herein by reference.

[0091] The term "nucleotide" or "nucleic acid" is also intended to include any nucleotide analogs, which are a type of nucleotide that includes a modified nucleobase, sugar and / or phosphate moiety. Exemplary modified nucleobases that can be included in polynucleotides, whether or not having a natural backbone or similar structure, include inosine, xanthine (xathanine), hypoxathanine (hypoxathanine), isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine. In some embodiments, the polynucleotides may be nucleotides of the invention, for example, 5-uracil, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-thioadenine or guanine, 8-hydroxyadenine or guanine, 5-halo uracil or cytosine, 7-methyl guanine, 7-methyl adenine, 8-aza guanine, 8-aza adenine, 7-deaza guanine, 7-deaza adenine, 3-deaza guanine, 3-deaza adenine or the like. As known in the art, some nucleotide analogs cannot be incorporated into the polynucleotides, for example nucleotide analogs, for example adenosine 5'-phosphosulfate.

[0092] As used herein, the term "polynucleotide" refers to a molecule comprising a sequence of nucleotides bound to each other. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and analogs thereof. A polynucleotide can be a single-stranded sequence of nucleotides such as RNA or single-stranded DNA, a double-stranded sequence of nucleotides such as double-stranded DNA, or can include a mixture of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA and PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA, and vice versa. The precise sequence of nucleotides in a polynucleotide can be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (e.g., a probe, a primer, an expressed sequence tag (EST) or a serial analysis of gene expression (SAGE) tag), genomic DNA, a genomic DNA fragment, an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, a recombinant polynucleotide, a synthetic polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of any sequence, isolated RNA of any sequence, a nucleic acid probe, a primer, or an amplified copy of any of the foregoing.

[0093] As used herein, "chemical coupling" is intended to mean the attachment between the first member and the second member. In some embodiments, this attachment is generally irreversible under the conditions under which the attached member is used. In other embodiments, this attachment is reversible, but lasts for at least a period of time, during which time it is used for one or more steps of the analysis or preparation techniques set forth herein (e.g., the analysis step of detecting the subunit of a polymer). This attachment can be formed by chemical bonds, such as by covalent bonds, hydrogen bonds, ionic bonds, dipole-dipole bonds, London dispersion forces, or any suitable combination thereof. A covalent bond is just an example of an attachment that can be appropriately used to couple the first member to the second member. Other examples include duplexes, peptide-peptide interactions, and hapten-antibody interactions between oligonucleotides, such as streptavidin-biotin, streptavidin-desthiobiotin, and digoxin-anti-digoxin. In one embodiment, attachment can be formed by hybridizing a first polynucleotide with a second polynucleotide that inhibits the separation of the first polynucleotide from the second polynucleotide. Alternatively, the attachment can be formed using a physical or biological interaction, such as an interaction between a first protein and a second protein, which inhibits separation of the first protein from the second protein. As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind to a primer single-stranded polynucleotide template, and can sequentially add nucleotides to the growing primer to form a polynucleotide having a sequence complementary to that of the template.

[0094] As used herein, the term "approximately" or "about" means within 10% of a stated value.

[0095] Provided herein are compositions and devices including photonic structures, for example, for optionally enhancing the monochromatic or polychromatic luminescent signal from an analyte (e.g., DNA cluster) in multiple excitation and / or cold light emission bands under the normal incidence of excitation. For example, the monolithic integration of a photonic chip and a microfluidic chip on top of a CMOS imaging array can be used to reduce the size of a DNA sequencer, such as miniaturizing a DNA sequencer. The throughput of a sequencing device based on CMOS may be limited by the size of an imaging pixel. For example, a relatively large pixel size may be useful for providing enough signal collection from a single DNA molecule or a cluster of the same molecule. Although pixels may be made smaller to increase throughput, this size reduction may reduce full well capacity, and may increase crosstalk between pixels, thereby reducing the signal-to-noise ratio (SNR) of imaging and sequencing. This method may also, for example, increase the cost of manufacturing an imaging array by increasing the engineering effort of the imaging array and the integration of this imaging array with photons and / or microfluidic components.

[0096] An optional way to increase throughput by providing multiple test sites per device can involve introducing multiple luminescent sites per pixel (e.g., DNA clusters, microarray reaction chambers, etc.). For example, in a particular embodiment, the compositions, devices, and methods of the present invention can use imaging pixels to image multiple sites by using an excitation source to selectively excite different sites at different times from each other and obtain corresponding images at each such time, and each site can include a corresponding analyte. Illustratively, an array of imaging pixels can be provided, and multiple sites can be arranged on each such imaging pixel. Relative to a configuration in which only one site is arranged on each given pixel, the multi-site configuration per pixel of the present invention can significantly increase the number of sites that can be imaged using a given pixel array. However, if all sites arranged on a given imaging pixel are excited simultaneously with each other, the pixel will receive simultaneous luminescence from each such site, thereby hindering the ability to distinguish luminescence from one such site from luminescence from another such site based on an electrical signal, and the pixel generates the electrical signal in response to receiving such luminescence.

[0097] Optical techniques such as those provided herein may be used to selectively excite only a single site of a plurality of sites arranged on a given imaging pixel at a given time, so as to obtain an electrical signal from the pixel in response to the luminescence from only that site at that time, and then to excite a second site of a plurality of sites on the imaging pixel at a second time, so as to obtain a second electrical signal from the pixel in response to the luminescence from the second site. Thus, based on the electrical signals obtained from the imaging pixel at these two times, the luminescence from the two sites can be distinguished from each other. Thus, the compositions, devices, and methods of the present invention can provide luminescent imaging of a greater number of sites than the number of pixels in the imaging array, such as an integer multiple n of the number of pixels, where n is greater than or equal to 2, 3, 4, 5, or greater than 5.

[0098] As provided herein, by selectively directing the excitation photons to the corresponding sites in the sites at different times from each other, different sites arranged on the imaging pixel can be selectively excited. For example, a focused laser beam can be scanned over different sites at different times from each other so as to selectively excite sites in different sites at such times, and the pixel generates an electrical signal in response to the luminescence from the specific site being excited at such a time. As another example, the site can be irradiated with any suitable number of mutually interfering laser beams at a first time so as to generate a first light intensity pattern that selectively excites one of the sites at a first time, and can be irradiated with any suitable number of mutually interfering laser beams at a second time so as to generate a second light intensity pattern that selectively excites another one of the sites at a second time. The pixel can generate corresponding electrical signals at first and second times in response to the luminescence from the corresponding site. As another example, the sites can be arranged on or within a photonic structure, and the photonic structure is arranged on the imaging pixel. The photonic structure can be configured to selectively excite one of the sites on the pixel in response to irradiation with a photon having a first characteristic at a first time, and selectively excite another one of the sites on the pixel in response to irradiation with a photon having a second characteristic at a second time. The pixels may generate corresponding electrical signals at first and second times in response to luminescence from corresponding sites.

[0099] The photonic structure-based devices, compositions and methods of the present invention are compatible with previously known epifluorescence microscopy and microscope scanning systems (such as those in commercially available sequencing platforms produced, for example, by Illumina, Inc. (San Diego, California), which in some cases can use a variety of fluorescent dyes that are excited under normal and imaged at normal incidence in various spectral windows. Such dyes can be coupled to nucleotides to facilitate sequencing of polynucleotides such as DNA. However, it should be recognized that the photonic structure-based devices, compositions and methods of the present invention can be suitably used in any type of luminescent imaging or any other suitable application, and are not limited to use in sequencing of polynucleotides such as DNA.

[0100] Patterning of dielectric substrates has been successfully used previously to control the size and uniformity of polynucleotide clusters, and to increase the density of such clusters, so as to improve the throughput of sequencing. See, for example, U.S. Patent Application Publication No. 2014 / 0243224A1, which is incorporated herein by reference. However, the reduction in cluster size leads to a significant reduction in the number of collected multicolor fluorescent signals. For example, when the number of labeled nucleotides in a DNA cluster is reduced (e.g., reduced to the single molecule level or the resolution limit of an imaging system), the detection of weak multicolor fluorescent signals from a large sampling area may become increasingly difficult. Therefore, significant fluorescence signal enhancement can help promote nucleotide identification and increase the throughput of next-generation SBS systems.

[0101] For example, periodic patterning of a material such as a high-refractive-index dielectric near fluorescently labeled biomolecules can enhance the fluorescence signal by creating a one- or two-dimensional waveguide with a periodic variation in refractive index on the order of the wavelength of light. Such a waveguide, which may be referred to as a photonic crystal (PhC), a photonic lattice, a photonic crystal lattice, or a PhC lattice, can support high-Q resonant modes that can enhance the fluorescence signal by resonantly enhancing fluorophore excitation, fluorescence collection, or both. For examples of monochromatic fluorescence signal enhancement using PhC lattices, see the following references, each of which is incorporated herein by reference in its entirety: U.S. Pat. No. 7,768,640 to Cunningham et al.; “Small volume excitation and enhancement of dye fluorescence on a 2D photonic crystal surface” (Opt. Express 18:3693-3699 (2010)) to Estrada et al.; “Enabling enhanced emission and low-threshold lasing of organic molecules using special Fano resonances of macroscopic photonic crystals” (PNAS 110:13711-13716 (2013)) to Zhen et al.; “Fabrication of two-dimensional Ta2O5 photonic crystal slabs with ultra-low background emission toward highly sensitive fluorescence spectroscopy” (Opt. Express 19:1422-1428 (2011)) to Kaji et al.; and “Photonic crystal enhanced fluorescence using a quartz substrate to reduce limits of detection” (Opt. Express 18:24793-24808(2010)).

[0102] PhC lattices can also be used in multicolor fluorescence signal enhancement. For example, dual excitation fluorescence signal enhancement is achieved using PhC by resonant enhancement of excitation at different wavelengths, which requires adjustment of the incident angle of the excitation source to match the resonance supported by the PhC. For additional details, see U.S. Patent No. 8,344,333 to Lu et al., the entire contents of which are incorporated herein by reference. However, because the signal enhancement scheme described by Lu et al. operates in trans-fluorescence mode by adjusting the illumination angle, such a scheme is inconvenient for imaging or sequencing platforms that rely on multicolor epitaxial illumination at fixed incident angles for all wavelengths of interest (e.g., normal or near-normal incidence angles).

[0103] Figure 1A A perspective view of an exemplary photonic structure-based device for use in luminescent imaging of a site within a pixel is schematically shown. Figure 1A The device shown includes an imaging pixel, such as an image sensor based on a complementary metal oxide semiconductor (CMOS); a photonic structure arranged on the imaging pixel, such as a PhC layer; and a nanowell defined within a third material arranged on the PhC layer. The PhC layer may include a first material (shown in black) having a refractive index of n1 and a regular pattern of wells of uniform shape and size defined within the first material and filled with a second material (shown in white) having a refractive index of n2, wherein n1 and n2 are different from each other. Sites including one or more luminophores, such as one or more analytes, such as one or more nucleotides, respectively coupled to the luminophores, may be arranged within the nanowell. The luminophores may be arranged in the near field of the PhC layer and excited by an excitation wavelength, such as photons having suitable characteristics (at Figure 1A The imaging pixel may be suitably electrically coupled to a detection circuit (not specifically shown) that may be configured to receive and analyze the electrical signal generated by the imaging pixel in response to the luminescence generated by the luminophore. Figure 1A As shown in FIG. 5 as having a dimension of 1.75 μm on each side, it will be appreciated that any suitable sized imaging pixels may be used.

[0104] Alternatively, any suitable number of arrays of such devices may be provided. Figure 1B Schematically shows that in the device array, for example Figure 1A A perspective view of an exemplary array of sites within a device array is shown, where each site (represented as a black circle) corresponds to a pixel (represented as a rectangle). That is, Figure 1B The exemplary array shown includes one site for each pixel. In addition, each such device may include any suitable number and type of materials. For example, Figure 1CSchematically showing, for example Figure 1A A cross-sectional view of an exemplary device is shown. Figures 1A-1C In an exemplary embodiment of the device shown, the PhC layer can be disposed on any suitable imaging pixel, such as known in the art. The PhC layer can include a first material, such as silicon nitride (SiN), that is patterned to define a photonic crystal. A second material, such as tantalum oxide (TaO), can be disposed on the PhC layer. Nanowells can be defined in a third material, such as SiN, and a fourth material, such as TaO, can be disposed on the nanowells. Figures 1A-1C As shown, a single nanowell can be arranged on each imaging pixel. Therefore, each imaging pixel can receive luminescence from the luminophore within the nanowell arranged on the pixel and generate a suitable electrical signal in response to receiving such luminescence.

[0105] For example, Figure 2A Schematically shows Figure 1B 1 is a perspective view of an exemplary excitation of an array of sites shown. Illustratively, the array of sites may be illuminated with uniform (top hat) illumination from a single light source (e.g., a laser). Such illumination may be appropriately applied to such sites (e.g., at Figure 1A and 1C ) below the PhC. For example, Figure 2B Schematically shows the response to e.g. Figure 2A The excitation shown in Figure 1A and 1C Simulated exemplary field strengths within the device array are shown. Characteristics of the PhC can be tuned to provide relatively high field strengths immediately beneath the nanowell 200, thereby selectively exciting the luminophores at sites disposed within the nanowell.

[0106] As provided herein, by selectively exciting different ones of such sites at different times from each other, the number of sites can be increased to an integer multiple n>1 of the number of imaging pixels. For example, Figure 3A Schematically illustrates a perspective view of an exemplary array of sites such as provided herein, where multiple sites correspond to one pixel. Figure 3A In the non-limiting example shown, four sites (represented as circles with different fills from each other) are provided per pixel (represented as rectangles), although it will be appreciated that any suitable number of sites per pixel may be provided, such as 2 or more sites per pixel, 3 or more sites per pixel, 4 or more sites per pixel, or 5 or more sites per pixel. Any suitable features may be used to provide such sites. For example, Figure 3B A cross-sectional view of a device such as provided herein is schematically shown, wherein a plurality of sites correspond to, for example, Figure 3A The pixels shown. Figure 3A-3B In an exemplary embodiment of the device shown, optional photonic structures can be arranged on, for example, any suitable imaging pixel known in the art. The optional photonic crystals can include a first material, such as silicon nitride (SiN), arranged on the imaging pixel; a second material, such as silicon dioxide (SiO2), arranged on the first material; and a photonic crystal comprising a pattern of a third material (e.g., SiN) and a fourth material (e.g., SiO2). A fifth material, such as tantalum oxide (TaO), can be arranged on the PhC layer. Multiple features, such as multiple nanowells, can be defined in a sixth material, such as SiO2, and a seventh material, such as TaO, can be arranged on the multiple nanowells. Figure 3A-3B As shown, multiple features, such as multiple nanowells, can be arranged on each imaging pixel. Thus, each imaging pixel can receive luminescence from a luminophore arranged in or on each such feature, such as in each such nanowell on the pixel, at different times, and generate a suitable electrical signal in response to receiving such luminescence at such different times. The imaging pixels, optional photonic structures, and features can optionally be monolithically integrated with each other.

[0107] It should be recognized that Figure 3B The optional photonic structures shown are intended to be exemplary, not limiting. For example, the photonic structure may include a photonic crystal or a photonic structure or a microcavity array or a plasmonic nanoantenna array.

[0108] For example, reference Figure 3A-3B , any suitable technique may be used to selectively excite sites such as those provided herein. For example, the photonic structure may optionally be omitted, and sites at a given pixel may be selectively excited by directing photons to such sites. Illustratively, a focused laser beam may be scanned over different sites at different times relative to one another to selectively excite sites in different sites at such times that the pixel generates an electrical signal in response to luminescence from the particular site being excited. For example, Figure 4A Schematic diagram of a scanning focused beam illumination system using, for example, the one provided herein Figure 3APerspective view of exemplary excitation of selected sites of the array of sites shown. Illustratively, precise control of the excitation beam can be achieved using high-precision free-space beam steering or by sample manipulation in a manner similar to that described in Hahn et al., "Laser scanning lithography for surface micropatterning on hydrogels" (Adv. Mater. 17:2939-2942 (2005)) or in Brakenhoff et al., "Confocal lightscanning microscopy with high-aperture immersion lenses" (J. Microsc. 117:219-232 (1997)), both of which are incorporated herein by reference in their entirety.

[0109] As another example, the sites can be illuminated at a first time with any suitable number of mutually interfering laser beams to produce a first light intensity pattern that selectively excites one of the sites at the first time, and can be illuminated at a second time with any suitable number of mutually interfering laser beams to produce a second light intensity pattern that selectively excites another of the sites at the second time. The pixels can generate corresponding electrical signals at the first and second times in response to light emission from the corresponding sites. For example, Figure 4B Schematically illustrates the use of multiple laser interference illumination as provided herein. Figure 3A Perspective view of exemplary excitation of selected sites of the array of sites shown. These sites can be selectively illuminated using multi-laser interference illumination similar to that described in van Wolferen et al., "Laser interference lithography" (In Lithography: Principles, Processes and Materials, pp. 133-148, Theodore Hennessy, Ed., Nova Science Publishers, Inc. (2011)) or in He et al., "Polarization control in flexible interference lithography for nano-patterning of different photonic structures with optimized contrast" (Optics Express 11518-11525 (May 4, 2015)), each of which is incorporated herein by reference in its entirety.

[0110] As yet another example, the sites can be arranged on or within a photonic structure that is arranged on an imaging pixel. The photonic structure can be configured to selectively excite one of the sites on the pixel at a first time in response to illumination with photons having a first characteristic, and to selectively excite another of the sites on the pixel at a second time in response to illumination with photons having a second characteristic. The pixel can generate corresponding electrical signals at the first and second times in response to luminescence from the corresponding sites. For example, Figure 5 Schematically illustrates an example that may be included in, for example, the present invention is provided herein and in Figure 3A , 3B An exemplary photonic structure in the device shown in FIG. Figure 5 In the particular embodiment shown, the photonic structure may include a photonic crystal (PhC), but it will be appreciated that the photonic structure may include a photonic superlattice or an array of microcavities or an array of plasmonic nanoantennas. Figure 5 The exemplary PhC shown includes a hexagonal array of features defined within the material, wherein the spacing ΛPhC between the features has an excitation wavelength λ 激发 The order of magnitude of the wavelength.

[0111] Photonic structures, such as PhCs, can be tuned (e.g., features of the PhCs can be selected) so that photons having properties that differ from one another can selectively excite different resonances within the PhCs. Photonic structure design parameters can be computationally tuned to tune the resonances to respective desired locations and / or excitation or emission peaks of emitters, for example, using one or more of finite-difference time-domain (FDTD), rigorous coupled-wave analysis (RCWA), and plane wave expansion (PWE). Design optimization can employ multi-parameter sweeps or self-optimization algorithms to maximize a desired luminescence signal, such as a fluorescence signal, in a desired physical and / or spectral region. For example, the refractive index of the material that the photonic structure will include, the spatial location where high field intensities are desired, and the wavelength for which the photonic structure is desired to selectively support resonances can be computationally defined, and any suitable combination of FDTD, RCWA, PWE, or any other suitable optimization procedure can be used to tune other parameters of the structure, such as the size, shape, and distribution of features within the structure, to explore the design parameter space of the structure and identify a combination of parameters that aligns the spectral and spatial features of the structure with the desired emitter location and / or excitation or emission wavelength.

[0112] For example, FIGS. 6A-6D schematically illustrate the radiation source at, for example, Figure 56A shows an exemplary simulated field intensity within a photonic crystal, the radiation source generating photons with different characteristics at different times. The simulated photonic crystal includes a hexagonal array of air holes in a Ta2O5 film located on top of a SiO2 substrate. More specifically, FIG. 6A shows the field intensity of a photon having a first polarization (e.g., X polarization) at a first time. Figure 5 FIG. 6A-6D shows the simulated field intensity within an exemplary photonic crystal of FIG. 6B ; FIG. 6B shows the simulated field intensity within the photonic crystal for a photon having a second polarization (e.g., Y polarization) at a second time; FIG. 6C shows the simulated field intensity within the photonic crystal for a photon having a third polarization (e.g., RX polarization) at a third time; and FIG. 6D shows the simulated field intensity within the photonic crystal for a photon having a fourth polarization (e.g., RY polarization) at a fourth time. Based on FIG. 6A-6D , it can be understood that by changing the polarization of the photons, different patterns of field intensity within the photonic crystal can be excited. It should be recognized that by changing other properties of the photons, such as the wavelength or angle of the photons, in accordance with different patterns of field intensity, different patterns of field intensity within the photonic crystal can be similarly excited. Similar differences in the pattern of field intensity of any suitable type of photonic structure, such as a photonic crystal, a photonic superlattice, a microcavity array, or a plasma nanoantenna array can be obtained by appropriately adjusting the characteristics of the photonic structure and appropriately changing the properties of the photons that illuminate the structure at different times from each other.

[0113] In some embodiments, the devices, compositions and methods of the present invention may provide multiple luminophores, including sites that overlap spatially with different patterns of field intensities that are excited at different times from one another. For example, FIG. 7A schematically illustrates, for example, a luminophore provided herein and in Figure 3A-3B , a plan view of an exemplary photonic structure-based device is shown in FIG, the device comprising first and second sites (e.g., clusters) per pixel. The device may include an array of imaging pixels, a photonic structure disposed on the array of imaging pixels; and an array of features disposed on the photonic structure. The photonic structure may, for example, include a photonic crystal, a photonic superlattice, a microcavity array, or a plasmonic nanoantenna array. The array of imaging pixels, the photonic structure, and the array of features may optionally be monolithically integrated with each other. In a non-limiting example, the photonic structure may include a hexagonal lattice, and the imaging pixels may be rectangular.

[0114] A first feature of the feature array can be arranged on a first pixel of the imaging pixel array, and a second feature of the feature array can be arranged on the first pixel and spatially shifted from the first feature. For example, in the non-limiting example shown in Figure 7A, a first feature (referred to as "Cluster 1") and a second feature (referred to as "Cluster 2") are arranged on the same pixel as each other. The second feature can be laterally shifted from the first feature in a manner such as shown in Figure 7A. In one example, the first and second features are located at the lower right corner and the upper left corner of the metal light shielding hole above the pixel, respectively. The first light emitter can be arranged in or on the first feature, and the second light emitter can be arranged in or on the second feature. For example, in some embodiments, the feature array may include a plurality of wells; the first feature may include a first well in which the first light emitter is arranged, and the second feature may include a second well in which the second light emitter is arranged, for example in a manner similar to that of Figure 7A. Figure 3B In a similar manner as shown in FIG. 1 , the array of features may include a plurality of pillars; the first feature may include a first pillar on which a first light emitter is disposed, and the second feature may include a second pillar on which a second light emitter is disposed. Illustratively, the first and second features (e.g., wells or pillars) may each have a substantially circular cross-section.

[0115] The device may also include a radiation source configured to generate a first photon having a first characteristic at a first time and configured to generate a second photon having a second characteristic at a second time, the second characteristic being different from the first characteristic, the second time being different from the first time. Figure 4A-4B In contrast to the described embodiments, the radiation source need not necessarily be configured to selectively direct radiation to different locations at different times. Rather, in some embodiments, the radiation source may be configured to flood illuminate the photonic structure with first and second photons at a first time and a second time, respectively, and features of the photonic structure may selectively direct radiation to different locations. Additionally or alternatively, the radiation source may include a laser. Optionally, the first and second photons emitted by the radiation source may be within the optical range of the spectrum, for example, the first and second photons independently have a wavelength between about 300 nm and about 800 nm.

[0116] In some embodiments, the photonic structure can be adjusted to selectively illuminate the first feature with light of the first polarization as opposed to light of the second polarization, and can be adjusted to selectively illuminate the second feature with light of the second polarization as opposed to light of the first polarization. For example, the device may include a first luminophore disposed in or on the first feature and a second luminophore disposed in or on the second feature. Illustratively, the device may include a first target analyte disposed in or on the first feature and a second target analyte disposed in or on the second feature, wherein the first target analyte is different from the second target analyte. Optionally, the first and second target analytes may include nucleic acids having different sequences.

[0117] In some embodiments, a first pixel may selectively receive cold light emitted by a first light emitter in response to a first photon at a first time, and may selectively receive cold light emitted by a second light emitter in response to a second photon at a second time. For example, a first photon having a first characteristic may generate a first resonant pattern within a photonic structure at a first time, and the first resonant pattern selectively excites the first light emitter relative to the second light emitter. Illustratively, FIG. 7B schematically illustrates exemplary simulated field intensities for a radiation source within a device array such as provided herein and shown in FIGS. 7A and 3A-3B, which generates photons having a first characteristic that selectively excites a first point at a first time. It can be seen that the photons having the first characteristic generate a spatial pattern of field intensities that are significantly stronger at the first feature than at the second feature, and thus may selectively excite the first light emitter relative to the second light emitter at a first time. Therefore, an imaging pixel may generate an electrical signal at a first time that substantially corresponds to the selective excitation of a first light emitter disposed within or above the first feature.

[0118] In addition, a second photon having a second characteristic can generate a second resonant pattern within the photonic structure at a second time, the second resonant pattern selectively exciting the second light emitter relative to the first light emitter. Illustratively, FIG. 7C schematically illustrates an exemplary simulated field intensity within an array of devices such as provided herein and shown in FIGS. 7A and 3A-3B for a radiation source that generates photons having a second characteristic that selectively excites a second site at a second time. It can be seen that the photons having the second characteristic generate a spatial pattern of field intensities that are significantly stronger at the second feature than at the first feature, and can therefore selectively excite the second light emitter relative to the first light emitter at the second time. Thus, the imaging pixel can generate an electrical signal at the second time that substantially corresponds to the selective excitation of the second light emitter disposed within or above the second feature. Thus, two or more light emitters within a detection region of a particular pixel can be distinguished from each other using a spatial pattern of excitation light applied to the light emitters at different times. This combination of spatial and temporal separation of excitation events can allow a pixel to distinguish between two or more light emitters within its detection region.

[0119] Note that although the first light emitter can be selectively excited relative to the second light emitter at a first time, such as shown in Figure 7B, the second light emitter can still be excited to a lesser extent than the first light emitter at the first time. Similarly, although the second light emitter can be selectively excited relative to the first light emitter at a second time, such as shown in Figure 7C, the first light emitter can still be excited to a lesser extent than the second light emitter at the second time. This excitation of the second light emitter at the first time and this excitation of the first light emitter at the second time can be referred to as "crosstalk." Figure 7D schematically illustrates an exemplary crosstalk term generated by the selective excitation of the first and second sites, such as provided herein and shown in Figures 7B and 7C, respectively. The photonic structure and / or each of the characteristics of the first and second photons can be adjusted to reduce the crosstalk to a level at which the corresponding emission from the first and second light emitters can be properly distinguished from each other.

[0120] In embodiments such as those shown in FIGS. 7B and 7C , the first and second characteristics of the first and second photons may be independently selected from a group consisting of wavelength, polarization, and angle. Illustratively, the first characteristic may include a first linear polarization, and the second characteristic may include a second linear polarization different from the first linear polarization. As an example, the first linear polarization may be substantially orthogonal to the second linear polarization. Illustratively, a pattern of field intensities shown in FIG. 7B is generated using photons having a first linear polarization (e.g., X polarization), which selectively excites a first luminous body within or above a first feature; and a pattern of field intensities shown in FIG. 7C is generated using photons having a second linear polarization, such as Y polarization, which is substantially orthogonal to the first linear polarization, which selectively excites a second luminous body within or above a second feature. However, it should be recognized that the photons at the first time and the second time may have any suitable polarization. For example, the first linear polarization may be rotated at an angle between about 15 degrees and about 75 degrees relative to the second linear polarization, for example, to produce other patterns of field intensities, such as described herein with reference to FIGS. 6A-6D . As another example, the polarization axis can be rotated 30 degrees clockwise, and the rotated X-polarized beam and Y-polarized beam (RX polarized beam and RY polarized beam) can be used. In addition, it should be appreciated that the photons at the first time and the second time can have any other suitable characteristics, respectively. For example, the first characteristic of the photons at the first time can include a first wavelength, and the second characteristic of the photons at the second time can include a second wavelength different from the first wavelength.

[0121] The characteristics of the first and second photons generated at the first time and the second time can be controlled in any suitable manner. For example, in some embodiments, the radiation source of the device may include an optical component and a controller coupled to the optical component. The controller may be appropriately configured to control the optical component so as to impose the first characteristic on the first photon, and to impose the second characteristic on the second photon. For example, in an embodiment where the corresponding photon characteristics include polarization, the optical component may include a birefringent material that is configured to rotate the first photon to a first linear polarization by the controller in response to a first control signal, and is configured to rotate the second photon to a second linear polarization by the controller in response to a second control signal. In an embodiment where the corresponding photon characteristics include wavelength, the optical component may include an electronically adjustable filter arranged in the path of the photon that can be adjusted to control the wavelength of the photon reaching the photonic structure by the controller in response to the control signal, or may include a portion of the radiation source that can be adjusted to control the wavelength of the photon generated by the radiation source at a given time by the controller in response to the control signal. In embodiments where the corresponding photon property includes an angle, the optical component may include a reflective or transmissive optical device, such as a lens and / or a reflector, that can be adjusted to control the angle of the photons reaching the photonic structure in response to a control signal by the controller. It should be appreciated that more than one photon property may be varied at a time. For example, any suitable combination of two or more of the wavelength, angle, and polarization of the photons may be adjusted to selectively excite a given light emitter disposed on a pixel relative to another light emitter disposed on the pixel.

[0122] In some embodiments, the first and second photons can illuminate the photonic structure at any suitable angle. For example, the first and second photons can each illuminate the photonic structure at substantially the same angle as each other, illustratively at an angle approximately perpendicular to a major surface of the photonic structure, or at an angle approximately parallel to a major surface of the photonic structure.

[0123] In embodiments such as those shown in FIGS. 7A-7D , it will be appreciated that other features of the feature array may be arranged on other pixels. For example, a third feature of the feature array may be arranged on a second pixel of the imaging pixel array, and a fourth feature of the feature array may be arranged on the second pixel and spatially displaced from the third feature. The device may also include a third light emitter arranged within or above the third feature, and a fourth light emitter arranged within or above the fourth feature. For example, if the third light emitter can be excited by a first photon or a second photon, the second pixel may selectively receive cold light emitted by the third light emitter in response to the first photon at a first time or in response to the second photon at a second time. For example, if the fourth light emitter can be excited by a first photon or a second photon, the second pixel may selectively receive cold light emitted by the fourth light emitter in response to the first photon at a first time or in response to the second photon at a second time.

[0124] It will also be appreciated that any suitable number of sites per pixel may be provided. Figure 3A-3B The devices shown in 7A-7D may optionally further include a third feature of the feature array, the third feature being arranged on the first pixel and spatially displaced from each of the first and second features. The device may include a third light emitter arranged within or on the third feature. The radiation source may be configured to generate a third photon having a third characteristic at a third time, the third characteristic being different from the first and second characteristics, and the third time being different from the first and second times. The first pixel may selectively receive cold light emitted by the third light emitter in response to the third photon at the third time. For example, Fig. 8A Schematically illustrated is an example provided herein and in Figure 3A-3B 6A-6D and 7A-7C , the photonic structure can be illuminated with photons having first, second, and third characteristics at first, second, and third times, respectively, so as to excite first, second, and third illuminators at the first, second, and third sites, respectively, at such times.

[0125] For example, Figure 8B Schematically shows the radiation source provided here and in Fig. 8A and 3A-3B, the radiation source generates photons with a first characteristic that selectively excites the first site at a first time. FIG. 8C schematically illustrates an exemplary simulated field intensity within such an array of devices for a radiation source that generates photons with a second characteristic that selectively excites the second site at a second time. FIG. 8D schematically illustrates an exemplary simulated field intensity within such an array of devices for a radiation source that generates photons with a third characteristic that selectively excites the third site at a third time. As an example, the first characteristic may include a first linear polarization, such as a Y polarization, the second characteristic may include a second linear polarization, such as an RY polarization, and the third characteristic may include a third linear polarization, such as an RX polarization. Note that one or more such polarizations may be orthogonal to each other, but need not necessarily be orthogonal to each other. For example, the RX polarization and the RY polarization are orthogonal to each other, and each is at an angle between about 15 degrees and about 75 degrees, such as 45 degrees, with the Y polarization. For example, the first linear polarization may be rotated at an angle between about 15 degrees and about 75 degrees relative to the second linear polarization, for example, in order to generate other patterns of field intensity, as described herein with reference to FIGS. 6A-6D. As another example, the polarization axis can be rotated 30 degrees clockwise, and the rotated X-polarized beam and Y-polarized beam (RX polarized beam and RY polarized beam) can be used. Note also that, in a manner similar to that described above with reference to FIG. 7D , selectively exciting the first site at a first time can also excite the second and / or third sites to a lesser extent, selectively exciting the second site at a second time can also excite the first and / or third sites, and / or selectively exciting the third site at a third time can also excite the first and / or second sites. Fig. 8E Schematically illustrates a method according to some embodiments provided by, for example, Figure 8B -Exemplary crosstalk terms resulting from the selective excitation of the first, second, and third sites shown in FIG8D. The photonic structure and / or the respective properties of the first and second photons can be adjusted to reduce the crosstalk to a level at which the respective cold lights from the first and second luminophores can be properly distinguished from each other.

[0126] The device of the present invention may also include a greater number of sites arranged on each pixel. Figure 3A-3BThe device described in 8A-8E may optionally further include a fourth feature of the feature array, the fourth feature being arranged on the first pixel and spatially displaced from each of the first, second and third features. The device may further include a fourth light emitter arranged within or on the fourth feature. The radiation source may be configured to generate a fourth photon having a fourth characteristic at a fourth time, the fourth characteristic being different from the first, second and third characteristics, and the fourth time being different from the first, second and third times. The first pixel may selectively receive cold light emitted by the fourth light emitter in response to the fourth photon at the fourth time. Illustratively, Fig.9A -9D schematically illustrate the use of radiation source pairs that generate photons with different characteristics at different times, for example, as provided herein and in Figure 3A-3B A perspective view of an exemplary selective excitation of a first, second, third, and fourth site within an array of devices as shown in FIG. Fig.9A In the manner shown, the photonic structure can be illuminated at a first time with photons having a first characteristic (e.g., a first polarization, e.g., X polarization) to selectively excite a first point arranged on each pixel. Fig. 9B In the manner shown, the photonic structure can be irradiated with a photon having a second characteristic (e.g., a second polarization, such as XY polarization) at a second time so as to selectively excite a second site arranged on each pixel. Subsequently, in the manner shown in, for example, FIG. 9C , the photonic structure can be irradiated with a photon having a third characteristic (e.g., a third polarization, such as YX polarization) at a third time so as to selectively excite a third site arranged on each pixel. Subsequently, in the manner shown in, for example, FIG. 9D , the photonic structure can be irradiated with a photon having a fourth characteristic (e.g., a fourth polarization, such as Y polarization) at a fourth time. The pixel can generate electrical signals at a first time, a second time, a third time, and a fourth time, respectively, and based on these electrical signals, the first, second, third, and fourth sites arranged on such a pixel can be distinguished from each other.

[0127] The composition, equipment and method of the present invention can be used appropriately to produce a luminescent image in SBS sequencing fluorescence signal enhancement under normal incident illumination. For example, the device may also include at least one microfluidic feature, which contacts the feature array and is configured to provide a flow of one or more analytes to the first feature and the second feature. In addition or alternatively, the composition, equipment and method of the present invention can use any suitable number of excitation wavelengths to enhance the excitation efficiency of any suitable number of luminophores, such as the excitation efficiency of four different excitation sources under four resonant wavelengths (λ1, λ2, λ3 and λ4) in 4-channel SBS chemical schemes, or the excitation efficiency under two excitation wavelengths (λ1 and λ2) in 2-channel SBS chemical schemes can be enhanced, or the excitation efficiency under one excitation wavelength (λ1) in 1-channel SBS chemical scheme can be enhanced. Exemplary 4-channel, 3-channel, 2-channel or 1-channel SBS schemes are described, for example, in U.S. Patent No. 2013 / 0079232 A1 (incorporated herein by reference), and can be modified to be used for the equipment and methods set forth herein. For example, again referring to the embodiment described in, for example, reference figures 7A-7D, in which the first and second luminophores are arranged on the first pixel, the first luminophore can be coupled to the first nucleic acid, and the second luminophore can be coupled to the second nucleic acid. As another example, referring to the optional embodiment described in, for example, reference figures 7A-7D, in which the first and second luminophores are arranged on the first pixel, and the third and fourth luminophores are arranged on the second pixel, the first luminophore can be coupled to the first nucleic acid, the second luminophore can be coupled to the second nucleic acid, the third luminophore can be coupled to the third nucleic acid, and the fourth luminophore can be coupled to the fourth nucleic acid. As yet another example, again referring to reference figures Fig.9A , 9D, the illustrative embodiment described, the first luminophore can be coupled to the first nucleic acid, the second luminophore can be coupled to the second nucleic acid, the third luminophore can be coupled to the third nucleic acid, and the fourth luminophore can be coupled to the fourth nucleic acid. For example, in a composition for use in sequencing DNA using luminescent imaging, the first luminophore can be coupled to A, the second luminophore can be coupled to G, the third luminophore can be coupled to C, and the fourth luminophore can be coupled to T. As another example, in a composition for use in sequencing RNA using luminescent imaging, the first luminophore can be coupled to A, the second luminophore can be coupled to G, the third luminophore can be coupled to C, and the fourth luminophore can be coupled to U.

[0128] In the examples provided herein, for example, Figure 3A-3B, 7A-7D, 8A-8E or 9A-9D, the first luminophore can be coupled to the first polynucleotide to be sequenced, and the second luminophore can be coupled to the second polynucleotide to be sequenced. For example, the first polynucleotide can be coupled to the first feature, and the second polynucleotide can be coupled to the second feature. The device may also include a first polymerase, which adds the first nucleic acid to the third polynucleotide complementary to and coupled to the first polynucleotide, and the first nucleic acid is coupled to the first luminophore. The device may also include a second polymerase, which adds the second nucleic acid to the fourth polynucleotide complementary to and coupled to the second polynucleotide, and the second nucleic acid is coupled to the second luminophore. The device may also include a channel for flowing a first liquid including the first and second nucleic acids and the first polymerase and the second polymerase into or over the first and second features. For example, the first and second polynucleotides can be coupled to the first and second features arranged on the first pixel and sequenced using a suitable SBS scheme. The first and second luminophores can be coupled to the first and second nucleic acids respectively, for example, using the first and second polymerases, respectively, and incorporated into the first and second polynucleotides. After the SBS step of incorporating the first and second nucleic acids into the first and second polynucleotides, the first and second luminophores can be selectively imaged luminescently at different times from each other, for example, in a manner as provided herein, so as to obtain corresponding electrical signals in response to the presence of the first luminophore at the first polynucleotide (i.e., incorporation of the first nucleic acid into the first polynucleotide) and in response to the presence of the second luminophore at the second polynucleotide (i.e., incorporation of the second nucleic acid into the second polynucleotide).

[0129] It will be appreciated that any suitable method may be used to image a luminophore at multiple locations using a given pixel. For example, Fig.10 An exemplary flow diagram of steps in the methods provided herein for use in luminescence imaging is shown. Fig.10 The illustrated method 1000 may include providing an imaging pixel array (1001). For example, imaging pixel arrays are commercially available. Fig.10 The illustrated method 1000 may also include providing a photonic structure disposed on the imaging pixel array 1002. For example, a photonic crystal, a photonic superlattice, a microcavity array, or a plasmonic nanoantenna array may be disposed on the imaging pixel array using any suitable combination of material fabrication and patterning techniques known in the art.

[0130] Fig.10The method 1000 shown may also include providing an array of features arranged on the photonic structure (1003). For example, an array of wells or columns may be arranged on the photonic structure using any suitable combination of material manufacturing and patterning techniques known in the art. The array of features may be aligned with the photonic crystal and the pixel array so that an integer number n>2 of features is arranged on each pixel. For example, a first feature of the feature array may be arranged on a first pixel of an imaging pixel array, and a second feature of the feature array may be arranged on the first pixel and spatially displaced from the first feature. For example, the second feature may be laterally displaced from the first feature. In a non-limiting example, the photonic structure includes a hexagonal lattice, and the imaging pixel is rectangular. The first and second features, such as the first and second columns or wells, may each optionally have a substantially circular cross-section. Optionally, the imaging pixel array, the photonic structure, and the array of features may be monolithically integrated with each other, for example, may be prepared as a single structure, for example, using a series of CMOS processing steps.

[0131] Fig.10 The illustrated method 1000 may also include providing a first luminophore (1004) disposed within or on a first feature, and providing a second luminophore (1005) disposed within or on a second feature. For example, the feature array may include a plurality of wells. The first feature may include a first well in which the first luminophore is disposed, and the second feature may include a second well in which the second luminophore is disposed. As another example, the feature array may include a plurality of columns. The first feature may include a first column on which the first luminophore is disposed, and the second feature may include a second column on which the second luminophore is disposed. Optionally, the first and second luminophores may be coupled directly or indirectly to the first and second features, respectively. As a non-limiting example, the first and second luminophores may be coupled to the first and second nucleic acids, respectively, and / or may be coupled to the first and second polynucleotides sequenced, for example, in a manner described elsewhere herein.

[0132] Fig.10 The illustrated method 1000 may also include generating, by the radiation source, first photons having a first characteristic at a first time 1006. The first photons having the first characteristic may generate a first resonant pattern within the photonic structure at a first time, the first resonant pattern selectively exciting the first light emitter relative to the second light emitter. Fig.10The method 1000 shown generates a second photon having a second characteristic at a second time by a radiation source (1007). The second characteristic can be different from the first characteristic, and the second time can be different from the first time. In one example, steps 1006 and 1007 can include flooding the photonic structure with the first and second photons, respectively, and / or can include generating the first and second photons with a laser. Optionally, the first and second photons can be in the visible range of the spectrum and / or independently can have a wavelength between about 300 nm and about 800 nm.

[0133] A second photon having a second characteristic can produce a second resonant pattern within the photonic structure at a second time, and the second resonant pattern selectively excites the second light emitter relative to the first light emitter. Exemplary radiation sources, photon characteristics, and resonant patterns are described elsewhere herein. Illustratively, the first and second photon characteristics can be independently selected from a group consisting of wavelength, polarization, and angle. As an example, the first photon characteristic can include a first linear polarization, and the second photon characteristic can include a second linear polarization different from the first linear polarization. Optionally, the first linear polarization is substantially orthogonal to the second linear polarization. Alternatively, the first linear polarization can be rotated at an angle between about 15 degrees and about 75 degrees relative to the second linear polarization. As another example, the first photon characteristic can include a first wavelength, and the second photon characteristic can include a second wavelength different from the first wavelength.

[0134] Optionally, the first and second photons each illuminate the photonic structure at substantially the same angle as each other. For example, the first and second photons can each illuminate the photonic structure at an angle approximately perpendicular to a major surface of the photonic structure. Or for example, the first and second photons can each illuminate the photonic structure at an angle approximately parallel to a major surface of the photonic structure. In some embodiments, the radiation source can include an optical component, and method 1000 can also include controlling the optical component so as to apply a first characteristic to the first photon and be configured to apply a second characteristic to the second photon. Illustratively, the optical component can include a birefringent material that rotates the first photon to a first linear polarization by a controller in response to a first control signal, and rotates the second photon to a second linear polarization by a controller in response to a second control signal. In addition or alternatively, the optical component can control the wavelength or angle of the first and second photons by a controller in response to a control signal.

[0135] Fig.10The method 1000 shown in can also include selectively receiving, by the first pixel, luminescent light emitted by the first light emitter in response to the first photon at the first time (1008), and selectively receiving, by the first pixel, luminescent light emitted by the second light emitter in response to the second photon at the second time (1009). In a manner such as described elsewhere herein, the first pixel can generate corresponding electrical signals at the first and second times, based on which the first and second light emitters can be distinguished from each other.

[0136] Any suitable number of features, such as three or four features, may be used, for example, as referred to herein. Figure 3A-3B , 7A-7D, 8A-8E, or 9A-9D. For example, a third feature of the feature array may optionally be arranged on the first pixel and spatially displaced from each of the first and second features, and method 1000 may further include providing a third light emitter arranged in or on the third feature; generating a third photon having a third characteristic at a third time, the third characteristic being different from the first and second characteristics, and the third time being different from the first and second times; and selectively receiving, by the first pixel, luminescent light emitted by the third light emitter in response to the third photon at the third time. Optionally, a fourth feature of the feature array may be arranged on the first pixel and spatially displaced from each of the first, second, and third features, and method 1000 may further include providing a fourth light emitter arranged in or on the fourth feature; generating a fourth photon having a fourth characteristic at a fourth time, the fourth characteristic being different from the first, second, and third characteristics, and the fourth time being different from the first, second, and third times; and selectively receiving, by the first pixel, luminescent light emitted by the fourth light emitter in response to the fourth photon at the fourth time. In one non-limiting example, a first luminophore can be coupled to a first nucleic acid, a second luminophore can be coupled to a second nucleic acid, a third luminophore can be coupled to a third nucleic acid, and a fourth luminophore can be coupled to a fourth nucleic acid.

[0137] Additionally or alternatively, any suitable number of features, such as two, three, four, or more than four features, may be arranged on the second pixel. For example, a third feature of the feature array may be arranged on the second pixel of the imaging pixel array; a fourth feature of the feature array may be arranged on the second pixel and spatially displaced from the third feature; method 1000 may also include providing a third luminophore arranged within or on the third feature; and providing a fourth luminophore arranged within or on the fourth feature. Method 1000 may also include selectively receiving, by the second pixel, luminescent light emitted by the third luminophore in response to a first photon at a first time or in response to a second photon at a second time; and selectively receiving, by the second pixel, luminescent light emitted by the fourth luminophore in response to a first photon at a first time or in response to a second photon at a second time. In a non-limiting example, the first luminophore may be coupled to a first nucleic acid, the second luminophore may be coupled to a second nucleic acid, the third luminophore may be coupled to a third nucleic acid, and the fourth luminophore may be coupled to a fourth nucleic acid.

[0138] Method 1000 may be suitable for luminescent imaging in an SBS scheme. For example, method 1000 may include providing at least one microfluidic feature in contact with a feature array, and causing one or more analytes to flow to first and second features through at least one microfluidic feature. As another example, a first luminophore may be coupled to a first nucleotide, and a second luminophore may be coupled to a second nucleotide. Additionally or alternatively, a first luminophore may be coupled to a first polynucleotide to be sequenced, and a second luminophore may be coupled to a second polynucleotide to be sequenced. A first polynucleotide may be coupled to a first feature, and a second polynucleotide may be coupled to a second feature. Method 1000 may also include adding a first nucleotide to a third polynucleotide complementary to and coupled to the first polynucleotide by a first polymerase, the first nucleotide being coupled to the first luminophore. Method 1000 may also include adding a second nucleotide to a fourth polynucleotide complementary to and coupled to the second polynucleotide by a second polymerase, the second nucleotide being coupled to a second luminophore. Method 1000 may also include flowing a first liquid including first and second nucleotides and first and second polymerases into or over the first and second features through a channel.

[0139] As mentioned elsewhere herein, any suitable combination of material processing and patterning techniques may be used to prepare the devices and methods of the present invention. For example, Fig.11 An exemplary sequence of steps that can be used to prepare, for example, a device or composition provided herein is shown. Illustratively, two nanoimprint lithography steps followed by a conformal dielectric deposition step can be used to prepare, for example, a device or composition provided herein. Figure 3A-3B For example, in Fig.11In step (a), a first optically transparent material such as a dielectric or a semiconductor such as a polymer (such as a resin) may be disposed on a substrate such as a glass substrate. Fig.11 In step (b), nanoimprint lithography may be used to pattern the first material, for example to define a plurality of features, such as wells or pillars. Fig.11 In step (c), a third optically transparent material, such as a dielectric or semiconductor material having a higher refractive index than the first material, may be disposed (e.g., conformally coated) over the features defined in the first material. Fig.11 In step (d), a fourth optically transparent material such as a dielectric or semiconductor such as a polymer (such as a resin) may be disposed on the third material. Fig.11 In step (e), nanoimprint lithography may be used to pattern the fourth material, for example to define a plurality of wells or nanowells. The fourth material may optionally fill, for example, Fig.11 The space within the photonic structure shown in step (e) of the embodiment of the present invention. A second material (not specifically shown) including one or more luminophores may be arranged in a well or nanowell, for example, on the photonic structure. Fig.11 The preparation of a single well per pixel is shown, but it will be appreciated that multiple wells per pixel may be readily prepared, for example by varying the distribution and size of the features formed in the second nanoimprint step.

[0140] As another example, a combination of two photolithography steps, two RIE steps, dielectric deposition, and CMP may be used to prepare a substrate such as that described herein. Figure 3A-3B For example, Fig.12 Another exemplary sequence of steps that can be used to prepare, for example, a device or composition provided herein is shown. Fig.12 In step (a), a first optically transparent material such as a dielectric or a semiconductor such as a polymer (such as a resin) may be disposed on a substrate such as a glass substrate. Fig.12 In step (b), the first material may be patterned using photolithography followed by reactive ion etching (RIE), for example to define a plurality of features, such as wells or pillars. Fig.12 In step (c), a third optically transparent material, such as a dielectric or semiconductor material having a higher refractive index than the first material, may be disposed (e.g., conformally coated) over the features defined in the first material. Fig.12 In step (d), a fourth optically transparent material such as a dielectric or semiconductor such as a polymer (such as a resin) may be disposed on the third material. Fig.12 In step (e), the fourth material may be planarized, for example, using chemical mechanical polishing (CMP). Fig.12In step (f), the fourth material may be patterned using photolithography followed by RIE, for example to define a plurality of wells or nanowells. The fourth material may optionally fill, for example, Fig.12 The space within the photonic structure shown in step (f) of the embodiment of the present invention. A second material (not specifically shown) including one or more luminophores may be arranged in a well or nanowell, for example, on the photonic structure. Fig.12 The fabrication of a single well per pixel is shown, but it will be appreciated that multiple wells per pixel may be readily fabricated, for example by varying the distribution and size of the features formed in the second lithography and RIE steps.

[0141] It will be appreciated that the apparatus of the present invention may be suitably used in any of a variety of applications, such as for luminescence imaging. Fig.13 An exemplary apparatus for use in luminescence imaging, such as provided herein, is shown. Fig.13 An exemplary device including a photonic structure 1310, an optical component 1330, an imaging pixel 1350, and a detection circuit 1340 is shown. The photonic structure 1310 includes a first material having a first refractive index (indicated by a diagonal pattern) and a second material having a second refractive index different from the first refractive index (indicated by a horizontally crossed pattern). The first material may include first and second major surfaces 1311, 1312 and a first and second plurality of features, such as wells 1313, 1314, defined by at least one of the first and second major surfaces. Features (e.g., wells) in the first plurality of features 1313 may differ from features (e.g., wells) in the second plurality of features 1314 in at least one characteristic, such as may differ in shape, size, or distribution. For example, in Fig.13 In the illustrated exemplary photonic structure 1310, features (e.g., wells) in the first plurality of features 1313 are different in size (e.g., width) and in distribution (e.g., spacing) than features in the second plurality of features 1314. Fig.13 In the non-limiting example shown, the second material can be disposed within or between the first and second plurality of features, such as wells 1313, 1314, and can include first and second luminophores 1321, 1322. For example, some of the first and second luminophores 1321, 1322 can be located within or between the first plurality of features (e.g., well 1313), while other of the first and second luminophores 1321, 1322 can be located within or between the second plurality of features (e.g., well 1314). Figure 3A-3BIn other embodiments discussed, a second material can be disposed on the first and second plurality of features. Illustratively, the first material can include a polymer or glass or other suitable material, or the second material can include a fluid or gel or other suitable material. Optionally, the photonic structure 1310 also includes a third material having a third refractive index different from the first and second refractive indices, such as described herein with reference to Figure 3A-3B In the manner described, the third material is disposed on at least one of the first and second plurality of features, and the second material is disposed on the third material. Optionally, the first luminophore 1321 can be coupled to a first nucleic acid, and the second luminophore 1322 can be coupled to a second nucleic acid different from the first nucleic acid.

[0142] The photonic structure 1310 can selectively support a first resonant pattern in response to being illuminated with photons having a first characteristic at a first time, and the first light emitter 1321 can emit a first wavelength λ1 in response to the illumination. The photonic structure 1310 can selectively support a second resonant pattern in response to being illuminated with photons having a second characteristic at a second time, and the second light emitter 1322 can emit a second wavelength λ2 in response to the illumination. The first and second wavelengths can optionally be different from each other, for example, can optionally be separated from each other by a first non-propagating wavelength that does not selectively resonate within the photonic structure. The optical component 1330 can be arranged on one of the first major surface 1311 and the second major surface 1312 of the first material, for example, above the first major surface 1311 and optionally at a distance spaced apart from the first major surface 1311. The optical component 1330 can be configured to illuminate the photonic structure 1310 with the first photons at a first time, and illuminate the photonic structure 1310 with the second photons at a second time. In Fig.13 In the exemplary apparatus shown, the photonic structure 1310 is illuminated with first and second photons at an angle approximately perpendicular to the first major surface 1311 of the first material, but it will be appreciated that any other angles, such as those disclosed herein, may be suitably used.

[0143] The photonic structure 1310 can be arranged on an imaging pixel 1350, which can include an image sensor configured to image the received first wavelength λ1 and second wavelength λ2 at a first time and a second time, respectively. The pixel 1350 can be spaced apart from the photonic structure 1310, or can be in contact with the photonic structure 1310, for example, it can be arranged to contact the second main surface 1312. Illustratively, the pixel 1350 can include an image sensor based on a complementary metal oxide semiconductor (CMOS) in contact with the photonic structure 1310. The detection circuit 1340, which can be appropriately electrically coupled to the pixel 1350, can be configured to receive and analyze electrical signals from the pixel 1350 at the first and second times. In a non-limiting example in which the first and second luminophores are coupled to the first and second nucleic acids, respectively, the detection circuit 1340 can be configured to identify which of the first and second nucleic acids is coupled to a specific polynucleotide, which is coupled to the photonic structure, based on the electrical signals at the first and second times, for example, in a manner described elsewhere herein. Other imaging pixels can be used, such as pixels of a CCD camera. Exemplary detectors are described in PCT Publication Nos. WO 91 / 06678, WO 04 / 018497, or WO 07 / 123744, U.S. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082 to Bentley et al. (Nature 456:53-59 (2008)), the entire contents of each of which are incorporated herein by reference.

[0144] For example, the devices provided herein can also transmit radiation to the photonic structure to appropriately excite the luminophore therein. For example, the device 1300 can also include a broadband excitation source such as a light emitting diode (LED) or a narrowband excitation source such as a laser configured to generate radiation transmitted by the optical component 1330 to the photonic structure.

[0145] Note that the device of the present invention is, for example, Fig.13 The device 1300 shown in the figure may optionally include one or more microfluidic features, such as those described elsewhere herein. For example, the device 1300 may optionally include at least one microfluidic feature that contacts the photonic structure and is configured to provide a flow of one or more analytes within, between, or on a first and second plurality of features disposed on the pixel. Such analytes may optionally include one or more reagents, such as nucleotides, nucleic acids, or polymerases, for nucleic acid sequencing.

[0146] Thus, provided herein are devices, compositions and methods including photonic structures that can provide monochromatic or multicolor luminescent signal enhancement at a greater number of sites than the number of pixels used in luminescent imaging, such as the devices, compositions and methods compatible with previously known fluorescence microscope scanning systems, such as sequencing platforms available on the market from, for example, Illumina, Inc. For example, some embodiments of the devices, compositions and methods of the present invention can generate excitation "hot spots" separated by distances on the order of the wavelength of light. The spatial distribution of these high-intensity resonant features (e.g., Fano or guided mode resonances) can be adjusted, for example, by appropriately selecting photonic structure lattice features (e.g., symmetry) and / or wavelengths, angles and / or polarization states of the excitation beam. Placing a luminophore (e.g., a biomolecule coupled to such a luminophore) near such a photonic structure can enhance the luminescent signal, but resonantly enhances luminophore excitation, luminescent collection or both. Therefore, the photonic structure is an attractive platform for achieving luminescent signal enhancement from multiple imaging sites on a single pixel, for example, using uniform illumination, wherein selective imaging site excitation can be achieved by controlling the characteristics of the excitation beam at different times. The photonic structure may be adjusted to reduce crosstalk terms such as described herein with reference to Figures 7D and 8E. Alternatively, the photonic structure may be omitted and the excitation beam may be directed to selected ones of the imaging sites, for example using free space optics or multi-laser interferometry.

[0147] Other optional embodiments

[0148] Although various illustrative examples of the present invention have been described above, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the present invention. For example, although certain compositions, systems and methods have been discussed above with reference to luminescent imaging associated with sequencing polynucleotides (e.g., DNA or RNA), it should be understood that the compositions, systems and methods herein may be appropriately adapted for luminescent imaging associated with any suitable object. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the present invention.

Claims

1. A device for use in luminescence imaging, the device comprising: An imaging pixel array, each imaging pixel in the imaging pixel array is integrated with a first well and a second well arranged above each imaging pixel, wherein the first well and the second well are selectively excited at different times from each other, Each imaging pixel in the imaging pixel array generates an electrical signal at the different times in response to emission of light from a corresponding well being excited.

2. The device according to claim 1, wherein The first well and the second well have different shapes.

3. The device according to claim 1, wherein: The first well and the second well have different sizes.

4. The device according to claim 1, wherein: The first well and the second well of each pixel are excited by a radiation source at the different times.

5. The device according to claim 4, wherein: The radiation source comprises a scanning focused beam illumination source.

6. The device according to claim 4, wherein: The radiation source includes a multi-laser interference source.

7. The device according to claim 4, wherein: The radiation source comprises a polarized beam source.

8. The device according to claim 1, wherein: Each of the first well and the second well contains one or more analytes.

9. The device according to claim 8, wherein: The one or more analytes are polynucleotides.

10. The device according to claim 8, wherein: The one or more analytes are coupled to one or more luminophores.

11. A device for use in luminescence imaging, the device comprising: A CMOS imaging array, the CMOS imaging array comprising an imaging pixel array; as well as A microfluidic chip, the microfluidic chip is integrated into the CMOS imaging array, the microfluidic chip comprises a plurality of wells, wherein a first well and a second well are arranged above each imaging pixel in the imaging pixel array, each imaging pixel is integrated with the first well and the second well, and wherein the first well and the second well are selectively excited at different times from each other; Each imaging pixel in the imaging pixel array generates an electrical signal at the different times in response to emission of light from a corresponding well being excited.

12. The device according to claim 11, wherein The first well and the second well have different shapes.

13. The device according to claim 11, wherein: The first well and the second well have different sizes.

14. The device according to claim 11, wherein: The first well and the second well of each pixel are excited by a radiation source at the different times.

15. The device according to claim 14, wherein: The radiation source comprises a scanning focused beam illumination source.

16. The apparatus according to claim 14, wherein: The radiation source includes a multi-laser interference source.

17. The apparatus according to claim 14, wherein: The radiation source comprises a polarized beam source.

18. The apparatus according to claim 11, wherein: Each of the first well and the second well contains one or more analytes.

19. The apparatus according to claim 18, wherein: The one or more analytes are polynucleotides.

20. The apparatus of claim 18, wherein: The one or more analytes are coupled to one or more luminophores.

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