Devices and compositions for use in luminescent imaging of multiple sites within a pixel, and methods of using the same
A device for luminescent imaging includes an array of imaging pixels, a photonic structure over the array of imaging pixels, and an array of features over the photonic structure. A first feature of the array of features is over a first pixel of the array of imaging pixels, and a second feature of the array of features is over the first pixel and spatially displaced from the first feature. A first luminophore is within or over the first feature, and a second luminophore is within or over the second feature. The device includes a radiation source to generate first photons having a first characteristic at a first time, and generate second photons having a second characteristic at a second time. The first pixel selectively receives luminescence emitted by the first and second luminophores responsive to the first photons at the first time and second photons at the second time, respectively.
1 . A device for use in luminescent imaging, the device comprising:
an imaging pixel;
a first nanowell corresponding to the imaging pixel, and
a second nanowell corresponding to the imaging pixel, the second nanowell being spatially displaced from the first nanowell;
a first luminophore disposed within or over the first nanowell;
a second luminophore disposed within or over the second nanowell;
at least one radiation source configured to generate first photons having a first characteristic, and configured to generate second photons having a second characteristic, the second characteristic being different than the first characteristic; and
a controller coupled to the at least one radiation source and configured to cause the at least one radiation source to illuminate the first and second features nanowells with the first photons at a first time and to illuminate the first and second features nanowells with the second photons at a second time which is different from the first time,
wherein the first photons interfere with one another in such a manner as to selectively illuminate the first nanowell with a first interference pattern, the first interference pattern having a field strength that is more intense at the first nanowell than at the second nanowell and thus selectively excites the first luminophore to a greater extent than the second luminophore at the first time, and
wherein the second photons interfere with one another in such a manner as to selectively illuminate the second nanowell with a second interference pattern, the second interference pattern having a field strength that is more intense at the second nanowell than at the first nanowell and thus excites the second luminophore to a greater extent than the first luminophore at the second time, the second interference pattern being different than the first interference pattern; and
wherein the imaging pixel is configured to receive luminescence emitted by the first luminophore of the first nanowell responsive to the selective illumination of the first nanowell by the first interference pattern at the first time, and is configured to receive luminescence emitted by the second luminophore of the second nanowell responsive to selective illumination of the second nanowell by the second interference pattern at the second time.
2 . The device of claim 1 , wherein the first and second characteristics are selected independently from the group consisting of polarization, wavelength, and angle.
3 . The device of claim 1 , wherein the at least one radiation source is configured to flood illuminate the first and second nanowells at the first time, and is configured to flood illuminate the first and second nanowells at the second time.
4 . The device of claim 1 , wherein the at least one radiation source comprises a first laser.
5 . The device of claim 4 , wherein the at least one radiation source further comprises a second laser, wherein the first laser generates a first laser beam and the second laser generates a second laser beam, wherein the first laser beam and the second laser beam generate the first and second interference patterns using multi-laser interference illumination.
6 . The device of claim 1 , the device further comprising a photonic structure, wherein:
the first photons interfere with one another via the photonic structure to generate the first interference pattern, and
the second photons interfere with one another via the photonic structure to generate the second interference pattern.
7 . The device of claim 6 , wherein the photonic structure is selected from the group consisting of a photonic crystal, a photonic superlattice, a microcavity array, and an array of plasmonic nanoantennae.
8 . The device of claim 1 , wherein the first luminophore is coupled to a first nucleic acid, wherein the second luminophore is coupled to a second nucleic acid, wherein the first nucleic acid is coupled to a first polynucleotide to be sequenced, and wherein the second nucleic acid is coupled to a second polynucleotide to be sequenced.
9 . The device of claim 8 , further comprising:
a third polynucleotide, the third polynucleotide being complementary to and coupled to the first polynucleotide;
a fourth polynucleotide, the fourth polynucleotide being complementary to and coupled to the second polynucleotide;
a first polymerase, the first polymerase being configured to add a first nucleic acid to the third polynucleotide, the first polynucleotide being coupled to the first nanowell;
a second polymerase, the second polymerase being configured to add the second nucleic acid to the fourth polynucleotide, the second polynucleotide being coupled to the second nanowell;
a liquid, the liquid comprising the first and second nucleic acids and the first and second polymerases; and
a channel, the channel being configured to allow the liquid to flow through and into or over the first and second nanowells.
10 . A method for use in luminescent imaging, the method comprising:
at a first time, illuminating first and second nanowells with first photons having a first characteristic, wherein the first photons interfere with one another in such a manner as to selectively illuminate the first nanowell with a first interference pattern having a field strength that is more intense at the first nanowell than at the second nanowell of the plurality of nanowells;
at a second time different than the first time, illuminating the first and second nanowells with second photons having a second characteristic that is different than the first characteristic, wherein the second photons interfere with one another in such a manner as to selectively illuminate the second nanowell with a second interference pattern being different than the first interference pattern and having a field strength that is more intense at the second nanowell than at the first nanowell;
receiving, by an imaging pixel, luminescence emitted by a first luminophore disposed within or over the first nanowell responsive to the selective illumination of the first nanowell by the first interference pattern at the first time; and
receiving, by the imaging pixel, luminescence emitted by a second luminophore disposed within or over the second nanowell responsive to the selective illumination of the second nanowell by the second interference pattern at the second time.
11 . The method of claim 10 , wherein the first and second characteristics are selected independently from the group consisting of polarization, wavelength, and angle.
12 . The method of claim 10 , wherein the first and second nanowells are flood-illuminated at the first and second times.
13 . The method of claim 10 , wherein the first and second nanowells are illuminated by a first laser beam.
14 . The method of claim 13 , wherein the first and second nanowells are further illuminated by a second laser beam, wherein the first laser beam and the second laser beam generate the first and second interference patterns using multi-laser interference illumination.
15 . The method of claim 10 , wherein the first photons interfere with one another via a photonic structure to generate the first interference pattern, and
wherein the second photons interfere with one another via a photonic structure to generate the second interference pattern.
16 . The method of claim 15 , wherein the photonic structure is selected from the group consisting of a photonic crystal, a photonic superlattice, a microcavity array, and an array of plasmonic nanoantennae.
17 . The method of claim 10 , further comprising flowing one or more analytes to the first and second nanowells.
18 . The method of claim 10 , wherein the first luminophore is coupled to a first nucleic acid, wherein the second luminophore is coupled to a second nucleic acid, wherein the first nucleic acid is coupled to a first polynucleotide to be sequenced, and wherein the second nucleic acid is coupled to a second polynucleotide to be sequenced.
19 . The method of claim 18 , further comprising:
adding, by a first polymerase, the 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, and the first polynucleotide being coupled to the first nanowell;
adding, by a second polymerase, the 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, and the second polynucleotide being coupled to the second nanowell; and
flowing, by a channel, a liquid including the first and second nucleic acids and the first and second polymerases into or over the first and second nanowells.