HK40003934B - Photonic stucture-based devices and compositions for use in luminescent imaging of multiple sites within a pixel, and methods of using the same - Google Patents

Description

Devices and compositions based on photonic structures for use in luminescent imaging of multiple sites within a pixel, and methods of using the same

CROSS-REFERENCE TO RELATED APPLICATIONS

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

field

This application relates generally to luminescence imaging.

background

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.

Overview

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.

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 displaced from the first feature. A first light emitter may be arranged within or above the first feature, and a second light emitter may be arranged within or above 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 luminescence emitted by the first light emitter in response to the first photon at the first time, and may selectively receive luminescence emitted by the second light emitter in response to the second photon at the second time.

Optionally, a first photon having a first characteristic generates a first resonant pattern within 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 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.

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

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

Additionally or alternatively, the feature array may optionally include a plurality of wells. The first feature may include a first well in which the first light emitter is disposed, and the second feature may include a second well in which the second light emitter is disposed. Alternatively, the feature array may optionally include a plurality of pillars. The first feature may include a first pillar on which the first light emitter is disposed, and the second feature may include a second pillar on which the second light emitter is disposed.

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 is substantially orthogonal to the second linear polarization, or alternatively, the first linear polarization is rotated relative to the second linear polarization by an angle between about 15 degrees and about 75 degrees. 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.

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

Additionally or alternatively, the first and second photons optionally each strike the photonic structure at substantially the same angle as one another. Additionally or alternatively, the first and second photons optionally each strike 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 strike the photonic structure at an angle approximately parallel to a major surface of the photonic structure.

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

Additionally or alternatively, a third feature of the array of features is optionally disposed on the first pixel and spatially displaced from each of the first and second features. The device may further optionally include a third light emitter disposed within or above 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 light emitter in response to the third photon at the third time. Additionally or alternatively, a fourth feature of the array of features is optionally disposed 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 disposed within or above 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 light emitter 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.

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 further comprises 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 the first photon at a first time or in response to the second photon at a second time. Optionally, the second pixel selectively receives cold light emitted by the fourth luminophore in response to the first photon at a first time or in response to the 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.

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.

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.

Additionally or alternatively, a first luminophore is optionally coupled to the first nucleotide, and a 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.

Additionally or alternatively, a first luminophore is optionally coupled to a first polynucleotide to be sequenced, and a second luminophore is optionally coupled to a second polynucleotide to be sequenced. Alternatively, the first polynucleotide is coupled to a first feature, and optionally the second polynucleotide is coupled to a second feature. Additionally or alternatively, the apparatus may further comprise a first polymerase that adds a 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 apparatus may further comprise a second polymerase that adds a 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. Alternatively, the apparatus may further comprise a channel for flowing a first liquid comprising the first and second nucleic acids and the first and second polymerases into or over the first and second features.

According to another aspect, a method for use in luminescence imaging is provided. The method may include providing an imaging pixel array and providing a photonic structure disposed on the imaging pixel array. The method may also include providing an array of features disposed on the photonic structure. A first feature of the array of features may be disposed on a first pixel in the imaging pixel array, and a second feature of the array of features may be disposed on the first pixel and spatially displaced from the first feature. The method may also include providing a first luminophore disposed within or above the first feature, and providing a second luminophore disposed within or above 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 the 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 the first pixel, luminescence emitted by the first luminophore in response to the first photon at the first time; and selectively receiving, by the first pixel, luminescence emitted by the second luminophore in response to the second photon at the second time.

Optionally, a first photon having a first characteristic generates a first resonant pattern within 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 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.

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

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

Additionally or alternatively, the feature array may optionally include a plurality of wells. The first feature may optionally include a first well in which the first light emitter is disposed, and the second feature may optionally include a second well in which the second light emitter is disposed. Alternatively, the feature array may include a plurality of pillars. The first feature may optionally include a first pillar on which the first light emitter is disposed, and the second feature may optionally include a second pillar on which the second light emitter is disposed.

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. Alternatively, the first linear polarization may be substantially orthogonal to the second linear polarization, or may be rotated relative to the second linear polarization by an angle between about 15 degrees and about 75 degrees. 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.

Additionally or alternatively, the radiation source optionally includes an optical component. The method optionally further includes controlling the optical component to impart a first characteristic to the first photon and a second characteristic to 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.

Additionally or alternatively, the first and second photons optionally each strike the photonic structure at substantially the same angle to one another. Additionally or alternatively, the first and second photons optionally each strike the photonic structure at an angle approximately perpendicular to a major surface of the photonic structure, or the first and second photons optionally each strike the photonic structure at an angle approximately parallel to a major surface of the photonic structure.

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

Additionally or alternatively, a third feature of the array of features 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 light emitter 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 include selectively receiving, by the first pixel, luminescent light emitted by the third light emitter at the third time in response to the third photon. Optionally, a fourth feature of the array of features is disposed on the first pixel and spatially displaced from each of the first, second, and third features. The method may further include providing a fourth light emitter 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 include selectively receiving, by the first pixel, luminescent light emitted by the fourth light emitter at the fourth time in response to the fourth photon. 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.

In addition or alternatively, the third feature of the feature array can optionally be arranged on the second pixel of the imaging pixel array, and the fourth feature of the feature array is arranged on the second pixel and spatially shifted from the third feature. The method optionally also includes providing a third luminophore arranged in or on the third feature, and providing a fourth luminophore arranged in or on the fourth feature. The method optionally also includes selectively receiving, by the second pixel, luminescent light emitted by the third luminophore in response to the first photon at a first time or in response to the second photon at a second time; and selectively receiving, by the second pixel, luminescent light emitted by the fourth luminophore in response to the first photon at a first time or in response to the 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.

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.

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.

Additionally or alternatively, a first luminophore is optionally coupled to the first nucleic acid, and a 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 flowing one or more analytes to the first feature and the second feature through the at least one microfluidic feature.

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 that is 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 that is 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 further includes flowing a first liquid comprising the first and second nucleic acids and the first and second polymerases into or over the first and second features through a channel.

According to another aspect, a device for use in luminescence 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 as opposed to light of a second polarization. The photonic structure may be adjusted to selectively illuminate the second feature with light of a second polarization as opposed to light of the first polarization.

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

Additionally or alternatively, the apparatus optionally further comprises a first light emitter disposed within or on the first feature and a second light emitter disposed within or on the second feature.

In addition or alternatively, the device optionally further comprises a first target analyte disposed within or on the first feature and a second target analyte disposed within 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1A schematically illustrates a perspective view of an exemplary photonic structure-based device for use in luminescent imaging of a site within a pixel.

FIG. 1B schematically illustrates a perspective view of an exemplary array of sites within a device array, such as the device array shown in FIG. 1A , where each site corresponds to a pixel.

FIG. 1C schematically illustrates a cross-sectional view of an exemplary device, such as the device shown in FIG. 1A .

FIG. 2A schematically illustrates a perspective view of an exemplary excitation of the array of sites shown in FIG. 1B .

2B schematically illustrates simulated exemplary field strengths within an array of devices such as shown in FIGs. 1A and 1C in response to an excitation such as shown in FIG. 2A .

FIG3A schematically illustrates a perspective view of an exemplary array of sites, such as provided herein, where multiple sites correspond to one pixel.

FIG3B schematically illustrates a cross-sectional view of a device such as provided herein, wherein a plurality of sites corresponds to one pixel as shown in FIG3A .

4A schematically illustrates a perspective view of exemplary excitation of selected sites of the array of sites shown in FIG. 3A using scanning focused beam illumination, such as provided herein.

4B schematically illustrates a perspective view of exemplary excitation of selected sites of the array of sites shown in FIG. 3A using multi-laser interference illumination, such as provided herein.

FIG5 schematically illustrates an exemplary photonic structure such as may be included in devices such as provided herein and shown in FIG3A-3B.

6A-6D schematically illustrate exemplary simulated field intensities within a photonic structure such as that shown in FIG. 5 for a radiation source that generates photons having mutually different characteristics at different times.

7A schematically illustrates a plan view of an exemplary photonic structure-based device, such as provided herein and shown in FIGs. 3A-3B, comprising first and second sites (eg, clusters) per pixel.

7B schematically illustrates exemplary simulated field strengths 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 first characteristic that selectively excites a first site at a first time.

7C schematically illustrates exemplary simulated field intensities 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.

FIG. 7D schematically illustrates exemplary crosstalk terms resulting from selective excitation of first and second sites, such as provided herein and shown in FIGs. 7B and 7C, respectively.

8A schematically illustrates a plan view of an exemplary photonic structure-based device, such as provided herein and shown in FIGs. 3A-3B, comprising first, second, and third sites (eg, clusters) per pixel.

8B schematically illustrates exemplary simulated field strengths within an array of devices such as provided herein and shown in FIGs. 8A and 3A-3B for a radiation source that generates photons having a first characteristic that selectively excites a first site at a first time.

8C schematically illustrates exemplary simulated field intensities within an array of devices, such as provided herein and shown in FIGs. 8A and 3A-3B, for a radiation source that generates photons having a second characteristic that selectively excites a second site at a second time.

8D schematically illustrates exemplary simulated field intensities within an array of devices, such as provided herein and shown in FIGs. 8A and 3A-3B, for a radiation source that generates photons having a third characteristic that selectively excites a third site at a third time.

8E schematically illustrates exemplary crosstalk terms resulting from selective excitation of first, second, and third sites, such as provided herein and shown in FIGs. 8B-8D, respectively, according to some embodiments.

9A-9D schematically illustrate perspective views of exemplary selective excitation of first, second, third, and fourth sites, respectively, within a device array such as provided herein and shown in FIGs. 3A-3B using a radiation source that produces photons having different characteristics at different times.

FIG10 shows an exemplary flow diagram of steps in the methods provided herein for use in luminescence imaging.

FIG. 11 illustrates an exemplary sequence of steps that can be used to prepare, for example, a device or composition provided herein.

FIG12 illustrates an exemplary sequence of steps that can be used to prepare, for example, a device or composition provided herein.

FIG. 13 illustrates an exemplary apparatus for use in luminescence imaging, such as provided herein.

Detailed description

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.

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

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 allow radiation having such characteristics, such as at a certain wavelength, angle, and polarization, to propagate through the structure or to exit the structure at the same angle or at different angles, and the field intensity of such radiation can have a selected pattern within the photonic structure. Furthermore, the structure can selectively inhibit radiation having different characteristics, such as at different wavelengths, angles, and/or polarizations, from propagating through the structure or from 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, two, or three dimensions. The shape, size, and distribution of the features of the photonic structure, as well as 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 out of the photonic structure at a certain angle, and/or to select the pattern of 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.

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 lying 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 specific 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 can be filled with one or more additional materials, which can each have a refractive index that is different from the refractive index of the material and different from the refractive index of each other. The specific characteristics of radiation that propagates or does not propagate through the photonic structure or propagates or does not propagate out of the photonic structure at a certain angle, such as wavelength, angle, or polarization, can be based on the refractive index of the material and the refractive index of any additional materials arranged within or between the features, as well as on the characteristics of the features, such as the shape, size, and distribution of the features. The features can all have the same shape, size, and/or distribution as each other.

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 that is 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 can 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 can 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. The material 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, as well as the refractive index of the material, can be adjusted to select patterns of specific characteristics of radiation, such as wavelength, angle, or polarization, and field strengths of such characteristics, that can propagate through the photonic superlattice or out of the photonic superlattice at an angle, 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.

Illustratively, a photonic superlattice can include a material extending in three dimensions (e.g., having a length, width, and thickness). The material can have two major surfaces, each of which lies in a plane defined by the length and width and separated from each other by the thickness. The material can 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, angle, or polarization, to propagate within the plane defined by the length and width or to propagate outside the plane at an angle, and inhibits at least radiation having a third characteristic, such as a third wavelength, angle, or polarization, from propagating within the material or to propagate outside the material at an angle. The pattern can include, for example, a plurality of features, such as wells or pillars defined within the material, such as through one or both major surfaces of the material, with material not present within or between the features, such as within the wells or between the pillars. The spaces within or between the features can be filled with one or more additional materials, which can each have a refractive index different from that of the material and different from that of each other. 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 on 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 No. 62/312,704, filed on March 24, 2016, 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.

As used herein, a "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 one another by varying the properties of an excitation source (e.g., the wavelength, polarization, or angle of the excitation source). For additional details on 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 two-dimensional coupled photonic crystal microcavity arrays," Opt. Lett. 30:1422-1428 (2011), the entire contents of which are incorporated herein by reference.

As used herein, a "plasmonic nanoantenna array" means a periodic, two-dimensional arrangement of plasmonic nanostructures that supports multiple (e.g., at least two, at least three, or at least four) resonances that can be excited independently of one another by varying the properties of an excitation source, such as the wavelength, polarization, or angle of a polarization source. For additional details regarding 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), the entire contents of which are incorporated herein by reference.

The 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 glass (e.g., inorganic glass such as silica or modified or functionalized glass) and polymers (e.g., acrylic resins, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TEFLON ^™ , cycloolefins, polyimides, or nylon). Examples of semisolid dielectric materials include gels, such as hydrogels. Additionally or alternatively, the one or more materials of the photonic structure may be or include an optically transparent solid semiconductor material.

As used herein, the term "gel" is intended to mean a semisolid or semirigid material that is permeable to liquids and gases. Typically, a gel material can expand when liquid is drawn in and can shrink when the liquid is removed by drying. Exemplary gels include, but are not limited to, those having a colloidal structure, such as agarose or a hydrogel; a polymer network structure, such as gelatin; or a cross-linked polymer structure, 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 feature in which it is present.

As used herein, the term "well" means a discrete concave feature in a material having a surface opening (pore) that is 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., the spatial location of the well within the 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 does not necessarily need to be, uniform along the length of the well.

As used herein, the term "pillar" means a discrete convex feature protruding from the surface of a material and completely surrounded by the interpulsal region of the surface. Pillars 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 having concentric pillars separated by dielectric material), and distribution (e.g., the spatial location of the pillars 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.

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

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

As used herein, the term "luminescent" means emitting cold-body radiation, and the term "luminophoric" means an article that emits light. The term "luminescent" is intended to be distinguished 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 atom's electron 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, which emit cold-body radiation relatively quickly (e.g., less than 1 millisecond) after the excitation radiation, and "phosphorescent" articles, which emit cold-body radiation relatively slowly (e.g., greater than or equal to 1 millisecond) after the excitation radiation. Photoluminescence can be perceived as the emission of radiation by an article at one wavelength 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."

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.

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. The set 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 characteristics, such as size (such as volume, diameter, and depth), shape (such as circular, elliptical, triangular, square, polygonal, star-shaped (with any suitable number of vertices), irregular or with concentric features separated by dielectric materials) and distribution (such as, the spatial position of features in a dielectric material, 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.

As used herein, the term "site" refers to the position of a particular species of molecule or cell (or other analyte) in an array. A site may comprise only a single molecule (or cell or other analyte) or it may comprise 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 specific analyte. In other embodiments, the site is created by attaching molecules or cells (or other analytes) 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. A feature may function as a component of a lattice, an array, or both.

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

As used herein, the term "spacing" when used with reference to features of a lattice (e.g., a photonic structure) or array is intended to refer to the center-to-center spacing of adjacent features of the lattice or array. The pattern of features can be characterized in terms of an average spacing. The pattern can be ordered, such 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 on the order 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 (approximately 380-700 nm), the UV spectrum (less than about 380 nm to about 10 nm), and the IR spectrum (greater than about 700 nm to about 1 mm). In a photonic structure, features can have different spacings from one another in different directions. For example, in a photonic superlattice, different types of features can have different spacings and patterns from one another. 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).

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, have variable spacing between each other. Alternatively, the spacing between features of the same type or of different types may be ordered, e.g., forming a regular pattern, such as a rectilinear grid or a hexagonal grid.

As used in this article, the term "nucleotide" or "nucleic acid" is intended to mean a molecule comprising a sugar and at least one phosphate group, and optionally also comprises 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 [0014] In some embodiments, the present invention relates to a reversible nucleoside triphosphate (rbNTP), adenosine triphosphate (adenosine triphosphate (adenosine triphosphate), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), reversibly blocked adenosine triphosphate (rbATP), reversibly blocked thymidine triphosphate (rbTTP), reversibly blocked cytidine triphosphate (rbCTP), and reversibly blocked guanosine triphosphate (rbGTP). For more details on reversibly blocked nucleoside triphosphates (rbNTPs), see U.S. Patent Publication No. 2013/0079322, the entire contents of which are incorporated herein by reference.

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 a polynucleotide, 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, 2-thiothymine, 2-thiocytosine, 15-halouracil ... In some embodiments, the polynucleotides may be nucleotides containing 5'- thiouracil, 5'- thiouracil, 5'- thiouracil, 6 ...

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, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag), genomic DNA, a genomic DNA fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer, or an amplified copy of any of the foregoing.

As used herein, "chemical coupling" is intended to mean the attachment between the first component and the second component. In some embodiments, this attachment is generally irreversible under the conditions in which the attached component 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., an analysis step for detecting the subunits 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 suitably used to couple the first component to the second component. 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, attachment can be formed using a physical or biological interaction, such as an interaction between a first protein and a second protein that 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.

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

Provided herein are compositions and devices including photonic structures, for example, for enhancing the monochromatic or polychromatic luminescent signal from an analyte (e.g., a DNA cluster) in a plurality of excitation and/or cold light emission bands, optionally 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, for example, miniaturizing a DNA sequencer. The throughput of a sequencing device based on CMOS may be limited by the size of imaging pixels. For example, a relatively large pixel size may be useful for providing enough signal collection from a single DNA molecule or a cluster of identical molecules. Although pixels can 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.

An alternative approach to increasing 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 particular embodiments, the compositions, devices, and methods of the present invention can image multiple sites using imaging pixels by selectively exciting different sites at different times from one another using an excitation source and obtaining a corresponding image at each such time, each site may include a corresponding analyte. Illustratively, an array of imaging pixels can be provided, and multiple sites can be arranged on each such imaging pixel. The multi-site per pixel configuration of the present invention can significantly increase the number of sites that can be imaged using a given pixel array relative to a configuration in which only one site is arranged on each given pixel. 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 that the pixel generates in response to receiving such luminescence.

Optical techniques, such as those provided herein, can be used to selectively excite only a single site among a plurality of sites arranged on a given imaging pixel at a given time to obtain an electrical signal from the pixel in response to luminescence from only that site at that time, and subsequently excite a second site among a plurality of sites on the imaging pixel at a second time to obtain a second electrical signal from the pixel in response to 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, apparatus, and methods of the present invention can provide luminescence 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.

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

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 light and imaged under 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 appreciated that the photonic structure-based devices, compositions and methods of the present invention can be suitably used in any type of luminescence imaging or any other suitable application, and are not limited to use in sequencing of polynucleotides such as DNA.

Previously, the patterning of dielectric substrates has been successfully utilized 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. 2014/0243224 A1, which is incorporated herein by reference. However, the reduction in cluster size causes a significant reduction in the quantity of collected multicolor fluorescent signals. For example, when the number of labeled nucleotides in the DNA cluster is reduced (for example, reduced to the single molecule level or the resolution limit of the imaging system), the detection of the faint multicolor fluorescent signals from a large sampling area may become increasingly difficult. Therefore, significant fluorescence signal enhancement can contribute to promoting nucleotide identification and increasing the throughput of next-generation SBS systems.

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 Ta ₂ O ₅ 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 slabs with ultra-low background emission toward highly sensitive fluorescence spectroscopy” (Opt. Express 19:1422-1428 (2011)) to Pokhriyal et al. enhanced fluorescence using a quartz substrate to reduce limits of detection” (Opt. Express 18:24793-24808(2010)).

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 angles of incidence).

Figure 1A schematically illustrates a perspective view of an exemplary photonic structure-based device for use in luminescent imaging of sites within a pixel. The device shown in Figure 1A includes an imaging pixel, such as a complementary metal oxide semiconductor (CMOS)-based image sensor; a photonic structure, such as a PhC layer, disposed on the imaging pixel; and nanowells defined within a third material disposed on the PhC layer. The PhC layer may include a first material having a refractive index of _n1 (shown in black) and a regular pattern of wells of uniform shape and size defined within the first material and filled with a second material having a refractive index of _n2 (shown in white), where _n1 and _n2 differ from each other. Sites comprising one or more luminophores, such as one or more analytes, such as one or more nucleotides, respectively coupled to the luminophores, may be disposed within the nanowells. The luminophores may be disposed in the near field of the PhC layer and transiently excited by an excitation wavelength, such as a photon with appropriate characteristics (shown as a large downward-pointing arrow in Figure 1A). The imaging pixels may be suitably electrically coupled to detection circuitry (not specifically shown) that may be configured to receive and analyze electrical signals generated by the imaging pixels in response to luminescence generated by the luminophores. Although the imaging pixels are shown in FIG1A as having dimensions of 1.75 μm on each side, it will be appreciated that any suitable size of imaging pixels may be used.

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

For example, FIG2A schematically illustrates a perspective view of an exemplary excitation of the array of sites shown in FIG1B. Illustratively, the array of sites can be illuminated with uniform (top-hat) illumination from a single light source (e.g., a laser). Such illumination can appropriately excite one or more resonant modes in a PhC beneath such sites (e.g., shown in FIG1A and FIG1C). For example, FIG2B schematically illustrates simulated exemplary field strengths within an array of devices, such as those shown in FIG1A and FIG1C, in response to excitation such as that shown in FIG2A. The characteristics of the PhC can be adjusted to provide a relatively high field strength immediately below nanowell 200, thereby selectively exciting the luminophores at the sites disposed within the nanowell.

As provided herein, by selectively exciting different ones of such sites at different times from one another, the number of sites can be increased to an integer multiple n>1 of the number of imaging pixels. For example, FIG3A schematically illustrates a perspective view of an exemplary array of sites, for example, provided herein, wherein a plurality of sites corresponds to one pixel. In the non-limiting example shown in FIG3A , four sites (represented as circles having different fills from one another) are provided per pixel (represented as rectangles), although it will be appreciated that any suitable number of sites per pixel may be provided, for example, 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. Such sites may be provided using any suitable features. For example, FIG3B schematically illustrates a cross-sectional view of an apparatus, for example, provided herein, wherein a plurality of sites corresponds to the pixels, for example, shown in FIG3A . In the exemplary embodiment of the apparatus shown in FIG3A-3B , an optional photonic structure may be arranged on, for example, any suitable imaging pixel known in the art. The optional photonic crystal may include a first material, such as silicon nitride (SiN), disposed on the imaging pixel; a second material, such as silicon dioxide ( _SiO2 ), disposed 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), may be disposed on the PhC layer. A plurality of features, such as a plurality of nanowells, may be defined in a sixth material, such as _SiO2 , and a seventh material, such as TaO, may be disposed on the plurality of nanowells. As shown in Figures 3A-3B, a plurality of features, such as a plurality of nanowells, may be disposed on each imaging pixel. Thus, each imaging pixel may receive luminescence from a luminophore disposed within or above each such feature, such as within each such nanowell above the pixel, at different times and generate appropriate electrical signals in response to receiving such luminescence at such different times. The imaging pixel, the optional photonic structure, and the features may optionally be monolithically integrated with one another.

It should be appreciated that the optional photonic structures shown in Figure 3B are intended to be exemplary, rather than limiting. For example, the photonic structure may include a photonic crystal or a photonic structure or a microcavity array or a plasmonic nanoantenna array.

For example, with reference to Figures 3A-3B, any suitable technique can be used to selectively excite sites such as those provided herein. For example, the photonic structure can optionally be omitted, and sites on a given pixel can be selectively excited by directing photons to such sites. Illustratively, a focused laser beam can be scanned over different sites at different times relative to one another so as to selectively excite sites in different sites at such times, with the pixels generating electrical signals at such times in response to luminescence from the specific sites being excited. For example, Figure 4A schematically illustrates a perspective view of an exemplary excitation of selected sites of the array of sites shown in Figure 3A using, for example, scanning focused beam illumination provided herein. 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)), the entire contents of both of which are incorporated herein by reference.

As another example, a site 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 luminescence from the corresponding sites. For example, FIG4B schematically illustrates a perspective view of an exemplary excitation of selected sites of the array of sites shown in FIG3A using, for example, multi-laser interference illumination as provided herein. These sites can be selectively illuminated using multi-laser interferometry 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)), the entire contents of each of which are incorporated herein by reference.

As 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 in response to illumination with photons having a first characteristic at a first time, and to selectively excite another of the sites on the pixel in response to illumination with photons having a second characteristic at a second time. The pixel can generate corresponding electrical signals at the first and second times in response to luminescence from the corresponding site. For example, Figure 5 schematically illustrates an exemplary photonic structure that can be included in, for example, a device provided herein and shown in Figures 3A, 3B. In the specific embodiment shown in Figure 5, the photonic structure can include a photonic crystal (PhC), but it will be appreciated that the photonic structure can include a photonic superlattice or a microcavity array or a plasmonic nanoantenna array. The exemplary PhC shown in Figure 5 includes a hexagonal array of features defined within the material, wherein the spacing ΛPhC between the features is on the order of the wavelength of the excitation wavelength _λexcitation .

Photonic structures, such as PhCs, can be tuned (e.g., features of the PhC can be selected) so that photons with distinct properties can selectively excite different resonances within the PhC. Photonic structure design parameters can be computationally tuned to adjust the resonances to the desired locations and/or excitation or emission peaks of the 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, within a desired physical and/or spectral region. For example, the refractive index of the material the photonic structure will comprise, the desired spatial location of high field intensity, 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 program can be used to adjust other parameters of the structure, such as the size, shape, and distribution of features within the structure, in order to explore the design parameter space of the structure and identify a combination of parameters that aligns the structure's spectral and spatial features with the desired emitter location and/or excitation or emission wavelength.

For example, Figures 6A-6D schematically illustrate exemplary simulated field intensities within a photonic crystal, such as that shown in Figure 5, for a radiation source that generates photons having different characteristics at different times. The simulated photonic crystal includes a hexagonal array of pores in a Ta ₂ O ₅ film located on top of a SiO ₂ substrate. More specifically, Figure 6A shows the simulated field intensities within the exemplary photonic crystal of Figure 5 for a photon having a first polarization (e.g., X polarization) at a first time; Figure 6B shows the simulated field intensities within the photonic crystal for a photon having a second polarization (e.g., Y polarization) at a second time; Figure 6C shows the simulated field intensities within the photonic crystal for a photon having a third polarization (e.g., RX polarization) at a third time; and Figure 6D shows the simulated field intensities within the photonic crystal for a photon having a fourth polarization (e.g., RY polarization) at a fourth time. Based on Figures 6A-6D, it can be understood that by changing the polarization of the photons, different patterns of field intensities within the photonic crystal can be excited. It will be appreciated that by varying other properties of the photons, such as their wavelength or angle, different patterns of field intensity within a photonic crystal can be similarly excited according to different patterns of field intensity. Similar variations in the pattern of field intensity can be achieved for any suitable type of photonic structure, such as a photonic crystal, a photonic superlattice, a microcavity array, or a plasmonic nanoantenna array, by appropriately adjusting the characteristics of the photonic structure and appropriately varying the properties of the photons that illuminate the structure at different times relative to one another.

In some embodiments, the devices, compositions and methods of the present invention can provide a plurality of light emitters comprising sites that spatially overlap with different patterns of field intensities excited at different times relative to one another. For example, Figure 7A schematically illustrates a plan view of an exemplary photonic structure-based device, such as provided herein and illustrated in Figures 3A-3B, comprising first and second sites (e.g., clusters) per pixel. The device can 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 can, for example, include a photonic crystal, a photonic superlattice, a microcavity array, or an array of plasmonic nanoantennas. The array of imaging pixels, the photonic structure, and the array of features can optionally be monolithically integrated with one another. In one non-limiting example, the photonic structure can include a hexagonal lattice, and the imaging pixels can be rectangular.

A first feature of the feature array can be arranged over a first pixel of the imaging pixel array, and a second feature of the feature array can be arranged over the first pixel and spatially displaced from the first feature. For example, in the non-limiting example shown in FIG7A , a first feature (referred to as "Cluster 1") and a second feature (referred to as "Cluster 2") are arranged over the same pixel. The second feature can be laterally displaced from the first feature, such as in the manner shown in FIG7A . In one example, the first and second features are located at the lower right and upper left corners, respectively, of a metal light shield above the pixel. A first light emitter can be arranged within or above the first feature, and a second light emitter can be arranged within or above the second feature. For example, in some embodiments, the feature array can include a plurality of wells; the first feature can include a first well in which the first light emitter is arranged, and the second feature can include a second well in which the second light emitter is arranged, such as in a manner similar to that shown in FIG3B . In other embodiments, the feature array can include a plurality of pillars; the first feature can include a first pillar on which the first light emitter is arranged, and the second feature can include a second pillar on which the second light emitter is arranged. Illustratively, the first and second features (e.g., wells or pillars) can each have a substantially circular cross-section.

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 and the second time being different from the first time. In contrast to the embodiments described herein with reference to Figures 4A-4B, the radiation source need not necessarily be configured to selectively direct radiation to different locations at different times. Instead, 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 the characteristics of the photonic structure may selectively direct radiation to different locations. In addition 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.

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

In some embodiments, a first pixel can selectively receive luminescence emitted by a first light emitter in response to a first photon at a first time, and can selectively receive luminescence 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 can generate a first resonant pattern within a photonic structure at a first time, the first resonant pattern selectively exciting the first light emitter relative to the second light emitter. Illustratively, FIG7B schematically illustrates exemplary simulated field intensities for a radiation source within an array of devices, such as those provided herein and shown in FIG7A and 3A-3B, that generates photons having a first characteristic that selectively excites a first light source at a first time. As can be seen, 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 can selectively excite the first light emitter relative to the second light emitter at the first time. Thus, the imaging pixel can generate an electrical signal at a first time that substantially corresponds to the selective excitation of the first light emitter disposed within or on the first feature.

Additionally, 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 luminophore relative to the first luminophore. Illustratively, FIG7C schematically illustrates exemplary simulated field intensities within the array of devices, such as those provided herein and shown in FIG7A and FIG3A-3B, for a radiation source generating photons having a second characteristic that selectively excites a second site at a second time. As can be seen, 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 thus can selectively excite the second luminophore relative to the first luminophore at the second time. Consequently, the imaging pixel can generate an electrical signal at the second time that substantially corresponds to the selective excitation of the second luminophore disposed within or on the second feature. Thus, two or more luminophores within a detection region of a particular pixel can be distinguished from one another using spatial patterns of excitation light applied to the luminophores at different times. This combination of spatial and temporal separation of excitation events can allow a pixel to distinguish between two or more luminophores within its detection region.

Note that although a first luminophore may be selectively excited relative to a second luminophore at a first time, such as shown in FIG7B , the second luminophore may still be excited to a lesser extent than the first luminophore at the first time. Similarly, although a second luminophore may be selectively excited relative to a first luminophore at a second time, such as shown in FIG7C , the first luminophore may still be excited to a lesser extent than the second luminophore at the second time. This excitation of the second luminophore at the first time and this excitation of the first luminophore at the second time may be referred to as “crosstalk.” FIG7D schematically illustrates an exemplary crosstalk term resulting from the selective excitation of first and second sites, such as provided herein and shown in FIG7B and FIG7C , respectively. The photonic structure and/or each of the characteristics of the first and second photons may be adjusted to reduce the crosstalk to a level at which the respective luminescence from the first and second luminophores can be properly distinguished from one another.

In embodiments such as those shown in Figures 7B and 7C, the first and second characteristics of the first and second photons can be independently selected from the group consisting of wavelength, polarization, and angle. Illustratively, the first characteristic can include a first linear polarization, and the second characteristic can include a second linear polarization different from the first linear polarization. As an example, the first linear polarization can be substantially orthogonal to the second linear polarization. Illustratively, photons having a first linear polarization (e.g., X polarization) are used to produce the pattern of field intensities shown in Figure 7B, which selectively excites a first light emitter within or above a first feature; and photons having a second linear polarization, such as Y polarization, which is substantially orthogonal to the first linear polarization, are used to produce the pattern of field intensities shown in Figure 7C, which selectively excites a second light emitter within or above a second feature. However, it should be recognized that the photons at the first time and the second time can have any suitable polarization. For example, the first linear polarization can be rotated by 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, as described herein with reference to Figures 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 each have any other suitable characteristics. For example, the first characteristic of the photon at the first time can include a first wavelength, and the second characteristic of the photon at the second time can include a second wavelength different from the first wavelength.

The properties 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 suitably configured to control the optical component so as to impose the first property on the first photon and to impose the second property on the second photon. For example, in embodiments where the corresponding photon properties 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 embodiments where the corresponding photon properties include wavelength, the optical component may include an electronically tunable filter arranged in the path of the photons that can be adjusted to control the wavelength of the photons 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 photons generated by the radiation source at a given time by the controller in response to the control signal. In embodiments where the corresponding photon characteristic includes an angle, the optical component may include reflective or transmissive optics, such as lenses and/or mirrors, that can be adjusted by the controller in response to a control signal to control the angle of the photons reaching the photonic structure. It should be appreciated that more than one photon characteristic can be varied at a time. For example, any suitable combination of two or more of the wavelength, angle, and polarization of the photons can be adjusted to selectively excite a given light emitter disposed at a pixel relative to another light emitter disposed at the pixel.

In some embodiments, the first and second photons can strike the photonic structure at any suitable angle. For example, the first and second photons can each strike the photonic structure at substantially the same angle as one another, 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.

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

It will also be appreciated that any suitable number of sites per pixel may be provided. Illustratively, the device, for example, shown in Figures 3A-3B and 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 illuminant arranged within or above 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, the third time being different from the first and second times. The first pixel may selectively receive cold light emitted by the third illuminant in response to the third photon at the third time. For example, Figure 8A schematically illustrates a plan view of an exemplary photonic structure-based device, for example, provided herein and shown in Figures 3A-3B, comprising first, second, and third sites (e.g., clusters) per pixel (represented as circles). In a manner similar to that described above with reference to Figures 6A-6D and 7A-7C, the photonic structure can be illuminated with photons having first, second, and third characteristics at the first, second, and third times, respectively, so as to excite the first, second, and third light emitters at the first, second, and third locations, respectively, at such times.

For example, FIG8B schematically illustrates exemplary simulated field intensities within a device array, such as those provided herein and illustrated in FIG8A and 3A-3B, for a radiation source generating photons having a first characteristic that selectively excites a first site at a first time. FIG8C schematically illustrates exemplary simulated field intensities within such a device array for a radiation source generating photons having a second characteristic that selectively excites a second site at a second time. FIG8D schematically illustrates exemplary simulated field intensities within such a device array for a radiation source generating photons having a third characteristic that selectively excites a third site at a third time. As an example, the first characteristic can include a first linear polarization, such as a Y polarization, the second characteristic can include a second linear polarization, such as a R Y polarization, and the third characteristic can include a third linear polarization, such as a R X polarization. Note that one or more of these polarizations can be orthogonal to each other, but need not necessarily be. For example, the R X polarization and the R Y polarization can be orthogonal to each other and each form an angle between approximately 15 degrees and approximately 75 degrees, such as 45 degrees, with the Y polarization. For example, the first linear polarization can be rotated by an angle between about 15 degrees and about 75 degrees relative to the second linear polarization, for example, to produce other patterns of field intensity, as described herein with reference to Figures 6A-6D. As another example, the polarization axis can be rotated 30 degrees clockwise, and rotated X-polarized and Y-polarized beams (RX-polarized and RY-polarized beams) can be used. Also note that, in a manner similar to that described above with reference to Figure 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. Figure 8E schematically illustrates exemplary crosstalk terms generated by selective excitation of the first, second, and third sites, for example, as provided herein and shown in Figures 8B-8D, respectively, according to some embodiments. 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 light from the first and second luminophores can be appropriately distinguished from each other.

The device of the present invention may also include a greater number of sites arranged at each pixel. For example, the device described above with reference to Figures 3A-3B and 8A-8E may optionally further include a fourth feature of the feature array, which is arranged at the first pixel and spatially displaced from each of the first, second, and third features. The device may also include a fourth luminescent body arranged within or above 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 luminescence emitted by the fourth luminescent body in response to the fourth photon at the fourth time. Illustratively, Figures 9A-9D each schematically illustrate perspective views of exemplary selective excitation of the first, second, third, and fourth sites within the device array, such as provided herein and shown in Figures 3A-3B, using a radiation source that generates photons having different characteristics at different times. For example, in the manner shown in Figure 9A, the photonic structure may be illuminated at a first time with photons having a first characteristic (e.g., a first polarization, such as X polarization) to selectively excite the first site arranged at each pixel. Subsequently, in a manner such as shown in FIG9B , the photonic structure may be irradiated with photons 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 a manner such as shown in FIG9C , the photonic structure may be irradiated with photons 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 a manner such as shown in FIG9D , the photonic structure may be irradiated with photons having a fourth characteristic (e.g., a fourth polarization, such as Y polarization) at a fourth time. The pixel may generate electrical signals at a first time, a second time, a third time, and a fourth time, respectively, based on which the first, second, third, and fourth sites arranged on such a pixel may be distinguished from each other.

The compositions, apparatus and methods of the present invention can be suitably used to produce luminescent images in SBS sequencing fluorescence signal enhancement under normal incidence illumination. For example, the device can further include 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. Additionally or alternatively, the compositions, apparatus and methods 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 at four resonant wavelengths (λ ₁ , λ ₂ , λ ₃ and λ ₄ ) in a 4-channel SBS chemistry scheme, or the excitation efficiency at two excitation wavelengths (λ ₁ and λ ₂ ) in a 2-channel SBS chemistry scheme, or the excitation efficiency at one excitation wavelength (λ ₁ ) in a 1-channel SBS chemistry scheme can be enhanced. Exemplary 4-channel, 3-channel, 2-channel or 1-channel SBS scheme is for example described in U.S. Patent No. 2013/0079232A1 (incorporated herein by reference), and can be modified to use for the equipment and method of setting forth herein.For example, again with reference to the embodiment described for example with reference to Figure 7A-7D, wherein 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, with reference to the optional embodiment described with reference to Figure 7A-7D, wherein 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 another example, again with reference to the illustrative embodiment described with reference to Figure 9A, 9D, 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 luminescence imaging, the first luminophore may be coupled to A, the second luminophore may be coupled to G, the third luminophore may be coupled to C, and the fourth luminophore may be coupled to T. As another example, in a composition for use in sequencing RNA using luminescence imaging, the first luminophore may be coupled to A, the second luminophore may be coupled to G, the third luminophore may be coupled to C, and the fourth luminophore may be coupled to U.

In devices such as those provided herein, such as those described with reference to Figures 3A-3B, 7A-7D, 8A-8E, or 9A-9D, a first luminophore can be coupled to a first polynucleotide to be sequenced, and a second luminophore can be coupled to a second polynucleotide to be sequenced. For example, the first polynucleotide can be coupled to a first feature, and the second polynucleotide can be coupled to a second feature. The device may further include a first polymerase that adds a first nucleic acid to a third polynucleotide that is complementary to and coupled to the first polynucleotide, the first nucleic acid being coupled to the first luminophore. The device may further include a second polymerase that adds a second nucleic acid to a fourth polynucleotide that is complementary to and coupled to the second polynucleotide, the second nucleic acid being coupled to the second luminophore. The device may further include a channel for flowing a first liquid comprising the first and second nucleic acids and the first and second polymerases into or over the first and second features. For example, the first and second polynucleotides can be coupled to first and second features disposed on a first pixel and sequenced using a suitable SBS protocol. The first and second luminophores can be coupled to first and second nucleic acids, respectively, that are incorporated into the first and second polynucleotides, respectively, using, for example, first and second polymerases. Following 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 one another, e.g., 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).

It will be appreciated that any suitable method may be used to image a luminophore at multiple locations using a given pixel. For example, FIG10 shows an exemplary flow of steps in a method provided herein for use in luminescence imaging. The method 1000 shown in FIG10 may include providing an imaging pixel array (1001). For example, the imaging pixel array is commercially available. The method 1000 shown in FIG10 may also include providing a photonic structure (1002) arranged on the imaging pixel array. For example, a photonic crystal, a photonic superlattice, a microcavity array, or a plasmonic nanoantenna array may be arranged on the imaging pixel array using any suitable combination of material fabrication and patterning techniques, such as those known in the art.

The method 1000 shown in FIG10 may also include providing an array of features arranged on the photonic structure (1003). For example, an array of wells or pillars may be arranged on the photonic structure using any suitable combination of material fabrication and patterning techniques known in the art. The array of features may be aligned with the photonic crystal and the pixel array such that an integer number n>2 of features is arranged on each pixel. For example, a first feature of the array of features may be arranged on a first pixel of an imaging pixel array, and a second feature of the array of features 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 one non-limiting example, the photonic structure includes a hexagonal lattice and the imaging pixels are rectangular. The first and second features, such as first and second pillars or wells, may optionally each 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, and may be fabricated as a single structure, for example, using a series of CMOS processing steps.

The method 1000 shown in Figure 10 can 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 can include a plurality of wells. The first feature can include a first well in which the first luminophore is disposed, and the second feature can include a second well in which the second luminophore is disposed. As another example, the feature array can include a plurality of columns. The first feature can include a first column on which the first luminophore is disposed, and the second feature can include a second column on which the second luminophore is disposed. Optionally, the first and second luminophores can be coupled directly or indirectly to the first and second features, respectively. As a non-limiting example, the first and second luminophores can be coupled to the first and second nucleic acids, respectively, and/or can be coupled to the first and second polynucleotides that are sequenced, for example, in a manner described elsewhere herein.

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

A second photon having a second characteristic can produce 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. Exemplary radiation sources, photon characteristics, and resonant patterns are described elsewhere herein. Illustratively, the first and second photon characteristics can be independently selected from the 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 relative to the second linear polarization by an angle between about 15 degrees and about 75 degrees. 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.

Optionally, the first and second photons each strike the photonic structure at substantially the same angle as each other. For example, the first and second photons may each strike the photonic structure at an angle approximately perpendicular to a major surface of the photonic structure. Or, for example, the first and second photons may each strike the photonic structure at an angle approximately parallel to a major surface of the photonic structure. In some embodiments, the radiation source may include an optical component, and method 1000 may further include controlling the optical component so as to impose a first characteristic on the first photon and being configured to impose a second characteristic on the second photon. Illustratively, the optical component may 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 the controller in response to a second control signal. Additionally or alternatively, the optical component may control the wavelength or angle of the first and second photons by the controller in response to the control signal.

The method 1000 shown in FIG10 may further include selectively receiving, by the first pixel, luminescent light emitted by the first light emitter in response to the first photon at a 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 a second time (1009). The first pixel may generate corresponding electrical signals at the first and second times, and the first and second light emitters may be distinguished from each other based on the electrical signals, for example, in a manner as described elsewhere herein.

Any suitable number of features, such as three or four features, can be arranged on the first pixel in a manner, such as described herein with reference to Figures 3A-3B, 7A-7D, 8A-8E, or 9A-9D. For example, a third feature of the array of features can optionally be arranged on the first pixel and spatially displaced from each of the first and second features, and method 1000 can further include providing a third luminophore arranged within 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, the third time being different from the first and second times; and selectively receiving, by the first pixel, luminescent light emitted by the third luminophore at the third time in response to the third photon. Alternatively, a fourth feature of the feature array can be disposed on the first pixel and spatially displaced from each of the first, second, and third features, and method 1000 can further include providing a fourth luminophore disposed within 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, 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 luminophore at the fourth time in response to the fourth photon. In one non-limiting example, 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.

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

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 flowing one or more analytes to first and second features via the 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, the first luminophore may be coupled to a first polynucleotide to be sequenced, and the second luminophore may be coupled to a second polynucleotide to be sequenced. The first polynucleotide may be coupled to the first feature, and the second polynucleotide may be coupled to the 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 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 coupled to the second luminophore. Method 1000 may also include flowing a first liquid comprising the first and second nucleotides and the first and second polymerases through a channel into or over the first and second features.

As mentioned elsewhere in this article, any suitable combination of material processing and patterning techniques can be used to prepare the apparatus and method of the present invention. For example, Figure 11 shows an exemplary order of steps that can be used to prepare an apparatus or composition such as provided herein. Illustratively, two nanoimprint lithography steps, followed by a conformal dielectric deposition step, can be used to prepare an apparatus or composition such as described herein with reference to Figures 3A-3B. For example, in step (a) of Figure 11, a first optically transparent material such as a dielectric or semiconductor such as a polymer (such as a resin) can be arranged on a substrate such as a glass substrate. In step (b) of Figure 11, nanoimprint lithography can be used to pattern the first material, such as to define multiple features, such as wells or pillars. In step (c) of Figure 11, a third optically transparent material such as a dielectric or semiconductor material with a higher refractive index than the first material can be arranged (such as conformally coated) on the features defined in the first material. In step (d) of Figure 11, a fourth optically transparent material such as a dielectric or semiconductor such as a polymer (such as a resin) can be arranged on the third material. In step (e) of Figure 11 , nanoimprint lithography can be used to pattern the fourth material, for example, to define a plurality of wells or nanowells. The fourth material optionally fills a space within the photonic structure, for example, as shown in step (e) of Figure 11 . A second material (not specifically shown) comprising one or more luminophores can be arranged within the well or nanowell, for example, on the photonic structure. Although Figure 11 shows the preparation of a single well per pixel, it will be understood that multiple wells per pixel can be easily prepared, for example, by varying the distribution and size of the features formed in the second nanoimprint step.

As another example, a combination of two photolithography steps, two RIE steps, dielectric deposition, and CMP can be used to prepare devices or compositions such as those described herein with reference to Figures 3A-3B. For example, Figure 12 shows another exemplary sequence of steps that can be used to prepare devices or compositions such as those provided herein. In step (a) of Figure 12, a first optically transparent material, such as a dielectric or semiconductor, such as a polymer (e.g., a resin), can be arranged on a substrate, such as a glass substrate. In step (b) of Figure 12, photolithography, followed by reactive ion etching (RIE), can be used to pattern the first material, such as to define multiple features, such as wells or pillars. In step (c) of Figure 12, a third optically transparent material, such as a dielectric or semiconductor material having a higher refractive index than the first material, can be arranged (e.g., conformally coated) on the features defined within the first material. In step (d) of Figure 12, a fourth optically transparent material, such as a dielectric or semiconductor, such as a polymer (e.g., a resin), can be arranged on the third material. In step (e) of Figure 12, chemical mechanical polishing (CMP) can be used, for example, to flatten the fourth material. In step (f) of Figure 12 , a fourth material can be patterned using photolithography followed by RIE, for example, to define a plurality of wells or nanowells. The fourth material optionally fills a space within the photonic structure, for example, as shown in step (f) of Figure 12 . A second material (not specifically shown) comprising one or more luminophores can be arranged within the wells or nanowells, for example, on the photonic structure. Although Figure 12 illustrates the preparation of a single well per pixel, it will be appreciated that multiple wells per pixel can be readily prepared, for example, by varying the distribution and size of the features formed in the second photolithography and RIE steps.

It should be understood that the devices of the present invention may be suitably used in any of a variety of applications, such as for luminescent imaging. For example, Figure 13 shows an exemplary device for use in luminescent imaging, such as provided herein. Figure 13 shows an exemplary device including a photonic structure 1310, an optical component 1330, an imaging pixel 1350, and a detection circuit 1340. 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. The features (e.g., wells) in the first plurality of features 1313 may differ from the 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 the exemplary photonic structure 1310 shown in FIG13 , the features (e.g., wells) in the first plurality of features 1313 differ in size (e.g., width) and distribution (e.g., spacing) compared to the features in the second plurality of features 1314. In the non-limiting example shown in FIG13 , a second material can be disposed within or between the first and second pluralities 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). In other embodiments, such as those discussed above with reference to FIG3A-3B , the second material can be disposed on both the first and second pluralities of features. Illustratively, the first material can include a polymer, glass, or other suitable material, or the second material can include a fluid, gel, or other suitable material. Optionally, the photonic structure 1310 further comprises a third material having a third refractive index different from the first and second refractive indices, the third material being disposed on at least one of the first and second plurality of features, and the second material being disposed on the third material, in a manner such as described herein with reference to Figures 3A-3B. 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.

The photonic structure 1310 can selectively support a first resonant pattern in response to illumination with photons having a first characteristic at a first time, and the first light emitter 1321 can emit a first wavelength λ ₁ in response to the illumination. The photonic structure 1310 can selectively support a second resonant pattern in response to illumination with photons having a second characteristic at a second time, and the second light emitter 1322 can emit a second wavelength λ ₂ in response to the illumination. The first and second wavelengths can optionally be different from each other, for example, and 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 disposed 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 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 with the second photons at a second time. In the exemplary apparatus shown in FIG. 13 , the photonic structure 1310 is illuminated with first and second photons at angles 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.

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 λ ₁ and second wavelength λ ₂ 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, can be arranged to be in contact with the second major 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, all of which are incorporated herein by reference in their entirety.

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.

Note that the apparatus of the present invention, such as the apparatus 1300 shown in FIG13 , may optionally include one or more microfluidic features, such as those described elsewhere herein. For example, the apparatus 1300 may optionally include at least one microfluidic feature that contacts the photonic structure and is configured to provide for the flow of one or more analytes within, between, or over the first and second plurality of features disposed above the pixels. Such analytes may optionally include one or more reagents for nucleic acid sequencing, such as nucleotides, nucleic acids, or polymerases.

Thus, provided herein are devices, compositions, and methods comprising photonic structures that can provide monochromatic or multicolor luminescence signal enhancement at a greater number of sites than the number of pixels used in luminescence imaging, for example, the devices, compositions, and methods are compatible with previously known fluorescence microscope scanning systems, such as sequencing platforms commercially available 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 the wavelength, angle, and/or polarization state of the excitation beam. Placing a luminophore (e.g., a biomolecule coupled to such a luminophore) near such a photonic structure can enhance the luminescence signal, but resonantly enhances luminophore excitation, luminescence collection, or both. Thus, photonic structures are attractive platforms for achieving luminescence signal enhancement from multiple imaging sites on a single pixel, for example, using uniform illumination, where selective imaging site excitation can be achieved by controlling the characteristics of the excitation beam at different times. The photonic structure can be adjusted to reduce crosstalk terms such as described herein with reference to Figures 7D and 8E. Alternatively, the photonic structure can be omitted and the excitation beam can be directed to selected ones of the imaging sites using, for example, free space optics or multi-laser interferometry.

Other optional embodiments

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 will be understood that the compositions, systems, and methods herein may be suitably adapted for luminescent imaging associated with any suitable object. The appended claims are intended to encompass all such changes and modifications that fall within the true spirit and scope of the present invention.

Claims (146)

1. An apparatus for use in luminescent imaging, the apparatus comprising: An array of imaging pixels; A photonic structure arranged on the array of imaging pixels; A trap array, which is arranged on the photonic structure, The first well of the well array is disposed on the first pixel of the array of imaging pixels. The second well of the well array is arranged on the first pixel and spatially shifted from the first well; A first light-emitting element is arranged inside the first trap; A second light emitter, which is arranged within the second trap; and A radiation source is configured to generate a first photon having a first characteristic at a first time, and 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, the first pixel selectively receiving cold light emitted by the first light emitter in response to the first photon at the first time, and selectively receiving cold light emitted by the second light emitter in response to the second photon at the second time. 2. The device of claim 1, wherein the first photon having the first characteristic generates a first resonant pattern within the photon structure at the first time, the first resonant pattern selectively exciting the first light emitter relative to the second light emitter; and The second photon having the second characteristic generates a second resonant pattern within the photon structure at the second time, and the second resonant pattern selectively excites the second light emitter relative to the first light emitter. 3. The device according to claim 1 or claim 2, wherein the array of imaging pixels, the photonic structure, and the trap array are monolithically integrated with each other. 4. The device according to claim 1 or claim 2, wherein the photonic structure comprises a photonic crystal. 5. The device according to claim 1 or claim 2, wherein the photonic structure comprises a photonic superlattice. 6. The device according to claim 1 or claim 2, wherein the photonic structure comprises a microcavity array. 7. The device according to claim 1 or claim 2, wherein the photonic structure comprises a plasma nanoantenna array. 8. The device according to claim 1 or claim 2, wherein the first characteristic and the second characteristic are independently selected from the group consisting of wavelength, polarization and angle. 9. The device according to claim 1 or claim 2, wherein the first characteristic includes a first linear polarization, and wherein the second characteristic includes a second linear polarization different from the first linear polarization. 10. The device of claim 9, wherein the first linear polarization is substantially orthogonal to the second linear polarization. 11. The device of claim 9, wherein the first linear polarization is rotated relative to the second linear polarization by an angle between 15 degrees and 75 degrees. 12. The device according to any one of claims 1, 2, 10 and 11, wherein the first characteristic includes a first wavelength, and wherein the second characteristic includes a second wavelength different from the first wavelength. 13. The device according to any one of claims 1, 2, 10, and 11, wherein the radiation source includes optical components. The device further includes a controller coupled to the optical component and configured to control the optical component to apply the first characteristic to the first photon and to apply the second characteristic to the second photon. 14. The device of claim 13, wherein the optical component comprises 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. 15. The device according to any one of claims 1, 2, 10, 11 and 14, wherein the first photon and the second photon each illuminate the photon structure at substantially the same angle to each other. 16. The device according to any one of claims 1, 2, 10, 11 and 14, wherein each of the first photon and the second photon illuminates the photon structure at an angle perpendicular to the main surface of the photon structure. 17. The device according to any one of claims 1, 2, 10, 11 and 14, wherein each of the first photon and the second photon illuminates the photon structure at an angle parallel to the main surface of the photon structure. 18. The device according to any one of claims 1, 2, 10, 11 and 14, wherein the second well is laterally displaced from the first well. 19. The device according to any one of claims 1, 2, 10, 11 and 14, wherein: The third well of the well array is arranged on the first pixel and is spatially shifted from each of the first well and the second well; The device also includes a third light-emitting element disposed within the third well; The radiation source is configured to generate a third photon with 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 The first pixel selectively receives cold light emitted by the third light emitter in response to the third photon at the third time. 20. The apparatus according to claim 19, wherein: The fourth well of the well array is arranged on the first pixel and is spatially shifted from each of the first well, the second well, and the third well; The device also includes a fourth light-emitting element disposed within the fourth well; The radiation source is configured to generate a fourth photon with a fourth characteristic at a fourth time, the fourth characteristic being different from the first characteristic, the second characteristic, and the third characteristic, and the fourth time being different from the first time, the second time, and the third time; and The first pixel selectively receives cold light emitted by the fourth light emitter in response to the fourth photon at the fourth time. 21. The device of claim 20, wherein the first light emitter is coupled to a first nucleic acid, the second light emitter is coupled to a second nucleic acid, the third light emitter is coupled to a third nucleic acid, and the fourth light emitter is coupled to a fourth nucleic acid. 22. The device according to any one of claims 1, 2, 10, 11 and 14, wherein: The third well of the well array is arranged on the second pixel of the array of imaging pixels; The fourth well of the well array is arranged on the second pixel and spatially shifted from the third well; The device also includes a third light-emitting element disposed within the third well; The device also includes a fourth light-emitting element disposed within the fourth well; The second pixel selectively receives cold light emitted by the third light emitter in response to the first photon at the first time or in response to the second photon at the second time; and The second pixel selectively receives cold light emitted by the fourth light emitter in response to the first photon at the first time or in response to the second photon at the second time. 23. The device of claim 22, wherein the first light emitter is coupled to a first nucleic acid, the second light emitter is coupled to a second nucleic acid, the third light emitter is coupled to a third nucleic acid, and the fourth light emitter is coupled to a fourth nucleic acid. 24. The device according to any one of claims 1, 2, 10, 11, 14, 20, 21 and 23, wherein each of the first well and the second well has a substantially circular cross-section. 25. The device according to any one of claims 1, 2, 10, 11, 14, 20, 21 and 23, wherein the photonic structure comprises a hexagonal lattice and wherein the imaging pixel is rectangular. 26. The device according to any one of claims 1, 2, 10, 11, 14, 20, 21 and 23, wherein the radiation source is configured to illuminate the photonic structure with the first photon and the second photon floodlight. 27. The device according to any one of claims 1, 2, 10, 11, 14, 20, 21 and 23, wherein the radiation source comprises a laser. 28. The device according to any one of claims 1, 2, 10, 11, 14, 20, 21 and 23, wherein the first photon and the second photon independently have wavelengths between 300 nm and 800 nm. 29. The device according to any one of claims 1, 2, 10, 11, 14, 20, 21 and 23, wherein the first light emitter is coupled to a first nucleic acid, and wherein the second light emitter is coupled to a second nucleic acid. 30. The device according to any one of claims 1, 2, 10, 11, 14, 20, 21 and 23, further comprising at least one microfluidic feature that contacts the trap array and is configured to provide a flow of one or more analytes to the first trap and the second trap. 31. The device according to any one of claims 1, 2, 10, 11, 14, 20, 21 and 23, wherein the first luminescent element is coupled to a first polynucleotide to be sequenced, and the second luminescent element is coupled to a second polynucleotide to be sequenced. 32. The device of claim 31, wherein the first polynucleotide is coupled to the first well, and wherein the second polynucleotide is coupled to the second well. 33. The device according to claim 32, further comprising: A first polymerase adds a first nucleic acid to a third polynucleotide that is complementary to and coupled to the first polynucleotide, the first nucleic acid being coupled to the first luminescent organism; and A second polymerase adds a second nucleic acid to a fourth polynucleotide that is complementary to and coupled to the second polynucleotide, the second nucleic acid being coupled to the second luminescent organism. 34. The apparatus of claim 33 further includes channels for flowing a first liquid comprising the first nucleic acid and the second nucleic acid, as well as the first polymerase and the second polymerase, into the first trap and the second trap. 35. An apparatus for use in luminescent imaging, the apparatus comprising: An array of imaging pixels; A photonic structure arranged on the array of imaging pixels; A columnar array, arranged on the photonic structure. The first pillar of the pillar array is arranged on the first pixel of the imaging pixel array. The second column of the column array is arranged on the first pixel and spatially shifted from the first column; A first light-emitting body is arranged on the first pillar; A second light-emitting element, which is arranged on the second pillar; and A radiation source is configured to generate a first photon having a first characteristic at a first time, and 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, the first pixel selectively receiving cold light emitted by the first light emitter in response to the first photon at the first time, and selectively receiving cold light emitted by the second light emitter in response to the second photon at the second time. 36. The device of claim 35, wherein the first photon having the first characteristic generates a first resonant pattern within the photon structure at the first time, the first resonant pattern selectively exciting the first light emitter relative to the second light emitter; and The second photon having the second characteristic generates a second resonant pattern within the photon structure at the second time, and the second resonant pattern selectively excites the second light emitter relative to the first light emitter. 37. The device of claim 35 or claim 36, wherein the array of imaging pixels, the photonic structure, and the column array are monolithically integrated with each other. 38. The device according to claim 35 or claim 36, wherein the photonic structure comprises a photonic crystal. 39. The device according to claim 35 or claim 36, wherein the photonic structure comprises a photonic superlattice. 40. The device of claim 35 or claim 36, wherein the photonic structure comprises a microcavity array. 41. The device of claim 35 or claim 36, wherein the photonic structure comprises a plasma nanoantenna array. 42. The device according to claim 35 or claim 36, wherein the first characteristic and the second characteristic are independently selected from the group consisting of wavelength, polarization and angle. 43. The device of claim 35 or claim 36, wherein the first characteristic includes a first linear polarization, and wherein the second characteristic includes a second linear polarization different from the first linear polarization. 44. The device of claim 43, wherein the first linear polarization is substantially orthogonal to the second linear polarization. 45. The device of claim 43, wherein the first linear polarization is rotated relative to the second linear polarization by an angle between 15 degrees and 75 degrees. 46. The device according to any one of claims 35, 36, 44 and 45, wherein the first characteristic includes a first wavelength, and wherein the second characteristic includes a second wavelength different from the first wavelength. 47. The device according to any one of claims 35, 36, 44, and 45, wherein the radiation source includes optical components. The device further includes a controller coupled to the optical component and configured to control the optical component to apply the first characteristic to the first photon and to apply the second characteristic to the second photon. 48. The device of claim 47, wherein the optical component comprises a birefringent material configured to rotate the first photon to a first linear polarization in response to a first control signal by the controller, and configured to rotate the second photon to a second linear polarization in response to a second control signal by the controller. 49. The device according to any one of claims 35, 36, 44, 45 and 48, wherein the first photon and the second photon each illuminate the photon structure at substantially the same angle to each other. 50. The device according to any one of claims 35, 36, 44, 45 and 48, wherein each of the first photon and the second photon illuminates the photon structure at an angle perpendicular to the main surface of the photon structure. 51. The device according to any one of claims 35, 36, 44, 45 and 48, wherein each of the first photon and the second photon illuminates the photon structure at an angle parallel to the main surface of the photon structure. 52. The device according to any one of claims 35, 36, 44, 45 and 48, wherein the second post is laterally displaced from the first post. 53. The device according to any one of claims 35, 36, 44, 45 and 48, wherein: The third column of the column array is arranged on the first pixel and is spatially shifted from each of the first and second columns; The device also includes a third light-emitting element arranged on the third column; The radiation source is configured to generate a third photon with 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 The first pixel selectively receives cold light emitted by the third light emitter in response to the third photon at the third time. 54. The device according to claim 53, wherein: The fourth column of the column array is arranged on the first pixel and is spatially shifted from each of the first, second, and third columns; The device also includes a fourth light-emitting element arranged on the fourth column; The radiation source is configured to generate a fourth photon with a fourth characteristic at a fourth time, the fourth characteristic being different from the first characteristic, the second characteristic, and the third characteristic, and the fourth time being different from the first time, the second time, and the third time; and The first pixel selectively receives cold light emitted by the fourth light emitter in response to the fourth photon at the fourth time. 55. The device of claim 54, wherein the first light emitter is coupled to a first nucleic acid, the second light emitter is coupled to a second nucleic acid, the third light emitter is coupled to a third nucleic acid, and the fourth light emitter is coupled to a fourth nucleic acid. 56. The device according to any one of claims 35, 36, 44, 45 and 48, wherein: The third column of the column array is arranged on the second pixel of the array of imaging pixels; The fourth column of the column array is arranged on the second pixel and spatially shifted from the third column; The device also includes a third light-emitting element arranged on the third column; The device also includes a fourth light-emitting element arranged on the fourth column; The second pixel selectively receives cold light emitted by the third light emitter in response to the first photon at the first time or in response to the second photon at the second time; and The second pixel selectively receives cold light emitted by the fourth light emitter in response to the first photon at the first time or in response to the second photon at the second time. 57. The device of claim 56, wherein the first light emitter is coupled to a first nucleic acid, the second light emitter is coupled to a second nucleic acid, the third light emitter is coupled to a third nucleic acid, and the fourth light emitter is coupled to a fourth nucleic acid. 58. The device according to any one of claims 35, 36, 44, 45, 48, 54, 55 and 57, wherein each of the first column and the second column has a substantially circular cross-section. 59. The device according to any one of claims 35, 36, 44, 45, 48, 54, 55 and 57, wherein the photonic structure comprises a hexagonal lattice and wherein the imaging pixel is rectangular. 60. The device according to any one of claims 35, 36, 44, 45, 48, 54, 55 and 57, wherein the radiation source is configured to illuminate the photonic structure with the first photon and the second photon floodlight. 61. The device according to any one of claims 35, 36, 44, 45, 48, 54, 55 and 57, wherein the radiation source comprises a laser. 62. The device according to any one of claims 35, 36, 44, 45, 48, 54, 55 and 57, wherein the first photon and the second photon independently have wavelengths between 300 nm and 800 nm. 63. The device according to any one of claims 35, 36, 44, 45, 48, 54, 55 and 57, wherein the first light emitter is coupled to a first nucleic acid, and wherein the second light emitter is coupled to a second nucleic acid. 64. The device according to any one of claims 35, 36, 44, 45, 48, 54, 55 and 57, further comprising at least one microfluidic feature that contacts the column array and is configured to provide a flow of one or more analytes to the first column and the second column. 65. The device according to any one of claims 35, 36, 44, 45, 48, 54, 55 and 57, wherein the first luminescent element is coupled to a first polynucleotide to be sequenced, and the second luminescent element is coupled to a second polynucleotide to be sequenced. 66. The device of claim 65, wherein the first polynucleotide is coupled to the first column, and wherein the second polynucleotide is coupled to the second column. 67. The apparatus of claim 66, further comprising: A first polymerase adds a first nucleic acid to a third polynucleotide that is complementary to and coupled to the first polynucleotide, the first nucleic acid being coupled to the first luminescent organism; and A second polymerase adds a second nucleic acid to a fourth polynucleotide that is complementary to and coupled to the second polynucleotide, the second nucleic acid being coupled to the second luminescent organism. 68. The apparatus of claim 67, further comprising a channel for flowing a first liquid comprising the first nucleic acid and the second nucleic acid, as well as the first polymerase and the second polymerase, over the first column and the second column. 69. A method for use in luminescent imaging, the method comprising: Provides an array of imaging pixels; Provide a photonic structure arranged on the array of the imaging pixels; Provide a trap array arranged on the photonic structure, The first well of the well array is disposed on the first pixel of the array of imaging pixels. The second well of the well array is arranged on the first pixel and spatially shifted from the first well; Provide a first light emitter disposed within the first trap; A second light emitter is provided, disposed within the second trap; The radiation source generates a first photon with the first characteristics at the first instant. The radiation source generates a second photon with a second characteristic at a second time, the second characteristic being different from the first characteristic, and the second time being different from the first time. The first pixel selectively receives cold light emitted by the first light emitter in response to the first photon at the first time; and The first pixel selectively receives cold light emitted by the second light emitter in response to the second photon at the second time. 70. The method of claim 69, wherein the first photon having the first characteristic generates a first resonant pattern within the photonic structure at the first time, the first resonant pattern selectively exciting the first light emitter relative to the second light emitter; and The second photon having the second characteristic generates a second resonant pattern within the photon structure at the second time, and the second resonant pattern selectively excites the second light emitter relative to the first light emitter. 71. The method of claim 69 or claim 70, wherein the array of imaging pixels, the photonic structure, and the trap array are monolithically integrated with each other. 72. The method of claim 69 or claim 70, wherein the photonic structure comprises a photonic crystal. 73. The method of claim 69 or claim 70, wherein the photonic structure comprises a photonic superlattice. 74. The method of claim 69 or claim 70, wherein the photonic structure comprises a microcavity array. 75. The method of claim 69 or claim 70, wherein the photonic structure comprises a plasma nanoantenna array. 76. The method according to claim 69 or claim 70, wherein the first characteristic and the second characteristic are independently selected from the group consisting of wavelength, polarization and angle. 77. The method of claim 69 or claim 70, wherein the first characteristic includes a first linear polarization, and wherein the second characteristic includes a second linear polarization different from the first linear polarization. 78. The method of claim 77, wherein the first linear polarization is substantially orthogonal to the second linear polarization. 79. The method of claim 77, wherein the first linear polarization is rotated relative to the second linear polarization by an angle between 15 degrees and 75 degrees. 80. The method according to any one of claims 69, 70, 78 and 79, wherein the first characteristic comprises a first wavelength, and wherein the second characteristic comprises a second wavelength different from the first wavelength. 81. The method according to any one of claims 69, 70, 78, and 79, wherein the radiation source comprises optical components. The method further includes controlling the optical components to apply the first characteristic to the first photon and to apply the second characteristic to the second photon. 82. The method of claim 81, wherein the optical component comprises a birefringent material, the birefringent material rotating the first photon to a first linear polarization by means of a controller in response to a first control signal, and rotating the second photon to a second linear polarization by means of the controller in response to a second control signal. 83. The method according to any one of claims 69, 70, 78, 79 and 82, wherein the first photon and the second photon each illuminate the photon structure at substantially the same angle to each other. 84. The method according to any one of claims 69, 70, 78, 79 and 82, wherein each of the first photon and the second photon illuminates the photon structure at an angle perpendicular to the main surface of the photon structure. 85. The method according to any one of claims 69, 70, 78, 79 and 82, wherein each of the first photon and the second photon illuminates the photon structure at an angle parallel to the main surface of the photon structure. 86. The method according to any one of claims 69, 70, 78, 79 and 82, wherein the second well is laterally displaced from the first well. 87. The method according to any one of claims 69, 70, 78, 79, and 82, wherein the third well of the well array is arranged on the first pixel and spatially shifted from each of the first well and the second well, and the method further comprises: A third light emitter is provided, which is arranged within the third well; A third photon with a third characteristic is generated 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 The first pixel selectively receives cold light emitted by the third light emitter in response to the third photon at the third time. 88. The method of claim 87, wherein the fourth well of the well array is disposed on the first pixel and spatially shifted from each of the first well, the second well, and the third well, and the method further comprises: A fourth light emitter is provided, which is arranged within the fourth well; A fourth photon with a fourth characteristic is generated at a fourth time, the fourth characteristic being different from the first characteristic, the second characteristic, and the third characteristic, and the fourth time being different from the first time, the second time, and the third time; and The first pixel selectively receives cold light emitted by the fourth light emitter in response to the fourth photon at the fourth time. 89. The method of claim 88, wherein the first luminescent body is coupled to a first nucleic acid, the second luminescent body is coupled to a second nucleic acid, the third luminescent body is coupled to a third nucleic acid, and the fourth luminescent body is coupled to a fourth nucleic acid. 90. The method according to any one of claims 69, 70, 78, 79 and 82, wherein: The third well of the well array is arranged on the second pixel of the array of imaging pixels; The fourth well of the well array is arranged on the second pixel and spatially shifted from the third well; and The method further includes: A third light emitter is provided, which is arranged within the third well; A fourth light emitter is provided, which is arranged within the fourth well; The second pixel selectively receives cold light emitted by the third light emitter in response to the first photon at the first time or in response to the second photon at the second time; and The second pixel selectively receives cold light emitted by the fourth light emitter in response to the first photon at the first time or in response to the second photon at the second time. 91. The method of claim 90, wherein the first luminescent body is coupled to a first nucleic acid, the second luminescent body is coupled to a second nucleic acid, the third luminescent body is coupled to a third nucleic acid, and the fourth luminescent body is coupled to a fourth nucleic acid. 92. The method according to any one of claims 69, 70, 78, 79, 82, 88, 89 and 91, wherein each of the first well and the second well has a substantially circular cross-section. 93. The method according to any one of claims 69, 70, 78, 79, 82, 88, 89 and 91, wherein the photonic structure comprises a hexagonal lattice and wherein the imaging pixel is rectangular. 94. The method according to any one of claims 69, 70, 78, 79, 82, 88, 89 and 91, comprising illuminating the photonic structure with the first photon and the second photon floodlight. 95. The method according to any one of claims 69, 70, 78, 79, 82, 88, 89 and 91, comprising generating the first photon and the second photon with a laser. 96. The method according to any one of claims 69, 70, 78, 79, 82, 88, 89 and 91, wherein the first photon and the second photon independently have wavelengths between 300 nm and 800 nm. 97. The method according to any one of claims 69, 70, 78, 79, 82, 88, 89 and 91, wherein the first luminescent body is coupled to a first nucleic acid, and wherein the second luminescent body is coupled to a second nucleic acid. 98. The method according to any one of claims 69, 70, 78, 79, 82, 88, 89 and 91, further comprising providing at least one microfluidic feature in contact with the trap array and causing one or more analytical streams to flow into the first trap and the second trap through the at least one microfluidic feature. 99. The method according to any one of claims 69, 70, 78, 79, 82, 88, 89 and 91, wherein the first luminescent body is coupled to a first polynucleotide to be sequenced, and the second luminescent body is coupled to a second polynucleotide to be sequenced. 100. The method of claim 99, wherein the first polynucleotide is coupled to the first trap, and wherein the second polynucleotide is coupled to the second trap. 101. The method of claim 100, further comprising: A first nucleic acid is added by a first polymerase to a third polynucleotide that is complementary to and coupled to the first polynucleotide, and the first nucleic acid is coupled to the first luminescent body; and A second nucleic acid is added by a second polymerase to a fourth polynucleotide that is complementary to and coupled to the second polynucleotide, and the second nucleic acid is coupled to the second luminescent body. 102. The method of claim 101, further comprising allowing a first liquid comprising the first nucleic acid and the second nucleic acid, as well as the first polymerase and the second polymerase, to flow through channels into the first trap and the second trap. 103. A method for use in luminescent imaging, the method comprising: Provides an array of imaging pixels; Provide a photonic structure arranged on the array of the imaging pixels; Provides a columnar array arranged on the photonic structure. The first pillar of the pillar array is arranged on the first pixel of the imaging pixel array. The second column of the column array is arranged on the first pixel and spatially shifted from the first column; A first light-emitting element is provided, which is arranged on the first column; A second light-emitting element is provided, which is arranged on the second column; The radiation source generates a first photon with the first characteristics at the first instant. The radiation source generates a second photon with a second characteristic at a second time, the second characteristic being different from the first characteristic, and the second time being different from the first time. The first pixel selectively receives cold light emitted by the first light emitter in response to the first photon at the first time; and The first pixel selectively receives cold light emitted by the second light emitter in response to the second photon at the second time. 104. The method of claim 103, wherein the first photon having the first characteristic generates a first resonant pattern within the photon structure at the first time, the first resonant pattern selectively exciting the first light emitter relative to the second light emitter; and The second photon having the second characteristic generates a second resonant pattern within the photon structure at the second time, and the second resonant pattern selectively excites the second light emitter relative to the first light emitter. 105. The method of claim 103 or claim 104, wherein the array of imaging pixels, the photonic structure, and the pillar array are monolithically integrated with each other. 106. The method of claim 103 or claim 104, wherein the photonic structure comprises a photonic crystal. 107. The method of claim 103 or claim 104, wherein the photonic structure comprises a photonic superlattice. 108. The method of claim 103 or claim 104, wherein the photonic structure comprises a microcavity array. 109. The method of claim 103 or claim 104, wherein the photonic structure comprises a plasma nanoantenna array. 110. The method according to claim 103 or claim 104, wherein the first characteristic and the second characteristic are independently selected from the group consisting of wavelength, polarization and angle. 111. The method of claim 103 or claim 104, wherein the first characteristic includes a first linear polarization, and wherein the second characteristic includes a second linear polarization different from the first linear polarization. 112. The method of claim 111, wherein the first linear polarization is substantially orthogonal to the second linear polarization. 113. The method of claim 111, wherein the first linear polarization is rotated relative to the second linear polarization by an angle between 15 degrees and 75 degrees. 114. The method according to any one of claims 103, 104, 112 and 113, wherein the first characteristic includes a first wavelength, and wherein the second characteristic includes a second wavelength different from the first wavelength. 115. The method according to any one of claims 103, 104, 112, and 113, wherein the radiation source comprises optical components. The method further includes controlling the optical components to apply the first characteristic to the first photon and to apply the second characteristic to the second photon. 116. The method of claim 115, wherein the optical component comprises a birefringent material, the birefringent material rotating the first photon to a first linear polarization by a controller in response to a first control signal, and rotating the second photon to a second linear polarization by the controller in response to a second control signal. 117. The method according to any one of claims 103, 104, 112, 113 and 116, wherein the first photon and the second photon each illuminate the photon structure at substantially the same angle to each other. 118. The method according to any one of claims 103, 104, 112, 113 and 116, wherein each of the first photon and the second photon illuminates the photon structure at an angle perpendicular to the main surface of the photon structure. 119. The method according to any one of claims 103, 104, 112, 113 and 116, wherein each of the first photon and the second photon illuminates the photon structure at an angle parallel to the main surface of the photon structure. 120. The method according to any one of claims 103, 104, 112, 113 and 116, wherein the second post is laterally displaced from the first post. 121. The method according to any one of claims 103, 104, 112, 113, and 116, wherein the third pillar of the pillar array is arranged on the first pixel and spatially shifted from each of the first and second pillars, and the method further comprises: Provide a third light-emitting element arranged on the third pillar; A third photon with a third characteristic is generated 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 The first pixel selectively receives cold light emitted by the third light emitter in response to the third photon at the third time. 122. The method of claim 121, wherein the fourth pillar of the pillar array is arranged on the first pixel and spatially shifted from each of the first pillar, the second pillar, and the third pillar, and the method further comprises: Provide a fourth light-emitting element arranged on the fourth pillar; A fourth photon with a fourth characteristic is generated at a fourth time, the fourth characteristic being different from the first characteristic, the second characteristic, and the third characteristic, and the fourth time being different from the first time, the second time, and the third time; and The first pixel selectively receives cold light emitted by the fourth light emitter in response to the fourth photon at the fourth time. 123. The method of claim 122, wherein the first luminescent body is coupled to a first nucleic acid, the second luminescent body is coupled to a second nucleic acid, the third luminescent body is coupled to a third nucleic acid, and the fourth luminescent body is coupled to a fourth nucleic acid. 124. The method according to any one of claims 103, 104, 112, 113, and 116, wherein: The third column of the column array is arranged on the second pixel of the array of imaging pixels; The fourth pillar of the pillar array is arranged on the second pixel and spatially shifted from the third pillar; and The method further includes: Provide a third light-emitting element arranged on the third pillar; Provide a fourth light-emitting element arranged on the fourth pillar; The second pixel selectively receives cold light emitted by the third light emitter in response to the first photon at the first time or in response to the second photon at the second time; and The second pixel selectively receives cold light emitted by the fourth light emitter in response to the first photon at the first time or in response to the second photon at the second time. 125. The method of claim 124, wherein the first luminescent body is coupled to a first nucleic acid, the second luminescent body is coupled to a second nucleic acid, the third luminescent body is coupled to a third nucleic acid, and the fourth luminescent body is coupled to a fourth nucleic acid. 126. The method according to any one of claims 103, 104, 112, 113, 116, 122, 123 and 125, wherein each of the first column and the second column has a substantially circular cross-section. 127. The method according to any one of claims 103, 104, 112, 113, 116, 122, 123 and 125, wherein the photonic structure comprises a hexagonal lattice and wherein the imaging pixel is rectangular. 128. The method according to any one of claims 103, 104, 112, 113, 116, 122, 123 and 125, comprising illuminating the photonic structure with the first photon and the second photon floodlight. 129. The method according to any one of claims 103, 104, 112, 113, 116, 122, 123 and 125, comprising generating the first photon and the second photon with a laser. 130. The method according to any one of claims 103, 104, 112, 113, 116, 122, 123 and 125, wherein the first photon and the second photon independently have wavelengths between 300 nm and 800 nm. 131. The method according to any one of claims 103, 104, 112, 113, 116, 122, 123 and 125, wherein the first luminescent body is coupled to a first nucleic acid, and wherein the second luminescent body is coupled to a second nucleic acid. 132. The method according to any one of claims 103, 104, 112, 113, 116, 122, 123 and 125, further comprising providing at least one microfluidic feature in contact with the column array and flowing one or more analytical streams into the first column and the second column through the at least one microfluidic feature. 133. The method according to any one of claims 103, 104, 112, 113, 116, 122, 123 and 125, wherein the first luminescent body is coupled to a first polynucleotide to be sequenced, and the second luminescent body is coupled to a second polynucleotide to be sequenced. 134. The method of claim 133, wherein the first polynucleotide is coupled to the first column, and wherein the second polynucleotide is coupled to the second column. 135. The method of claim 134, further comprising: A first nucleic acid is added by a first polymerase to a third polynucleotide that is complementary to and coupled to the first polynucleotide, and the first nucleic acid is coupled to the first luminescent body; and A second nucleic acid is added by a second polymerase to a fourth polynucleotide that is complementary to and coupled to the second polynucleotide, and the second nucleic acid is coupled to the second luminescent body. 136. The method of claim 135, further comprising allowing a first liquid comprising the first nucleic acid and the second nucleic acid, and the first polymerase and the second polymerase, to flow through channels on the first column and the second column. 137. An apparatus for use in luminescent imaging, the apparatus comprising: An array of imaging pixels; A photonic structure arranged on the array of imaging pixels; A trap array, which is arranged on the photonic structure, The first well of the well array is disposed on the first pixel of the array of imaging pixels. The second well of the well array is arranged on the first pixel and spatially shifted from the first well. The photonic structure is adjusted to selectively illuminate the first well with first-polarized light in contrast to light with a second polarization. The photonic structure is adjusted to selectively illuminate the second trap with light of the second polarization, in contrast to light of the first polarization. 138. The apparatus of claim 137, further comprising a radiation source configured to generate a first photon having the first polarization at a first time, and configured to generate a second photon having the second polarization at a second time. 139. The device according to claim 137 or claim 138, further comprising a first light emitter disposed in the first well and a second light emitter disposed in the second well. 140. The apparatus according to any one of claims 137-138, further comprising a first target analyte disposed in the first well and a second target analyte disposed in the second well, wherein the first target analyte is different from the second target analyte. 141. The apparatus of claim 140, wherein the first target analyte and the second target analyte comprise nucleic acids having different sequences. 142. An apparatus for use in luminescent imaging, the apparatus comprising: An array of imaging pixels; A photonic structure arranged on the array of imaging pixels; A columnar array, arranged on the photonic structure. The first pillar of the pillar array is arranged on the first pixel of the imaging pixel array. The second pillar of the pillar array is arranged on the first pixel and spatially shifted from the first pillar. The photonic structure is adjusted to selectively illuminate the first pillar with first polarized light in contrast to light with a second polarization. The photonic structure is adjusted to selectively illuminate the second column with light of the second polarization, in contrast to light of the first polarization. 143. The apparatus of claim 142 further includes a radiation source configured to generate a first photon having the first polarization at a first time, and configured to generate a second photon having the second polarization at a second time. 144. The device according to claim 142 or claim 143 further includes a first light-emitting element disposed on the first column and a second light-emitting element disposed on the second column. 145. The apparatus according to any one of claims 142-143, further comprising a first target analyte disposed on the first column and a second target analyte disposed on the second column, wherein the first target analyte is different from the second target analyte. 146. The apparatus of claim 145, wherein the first target analyte and the second target analyte comprise nucleic acids having different sequences.