Integrated illumination of optical analytical devices
Optical analytical devices and their methods of use are provided. The devices are useful in the analysis of highly multiplexed optical reactions in large numbers at high densities, including biochemical reactions, such as nucleic acid sequencing reactions. The devices include integrated illumination elements and optical waveguides for illumination of the optical reactions. The devices further provide for the efficient coupling of optical excitation energy from the waveguides to the optical reactions. Optical signals emitted from the reactions can thus be measured with high sensitivity and discrimination using features such as spectra, amplitude, and time resolution, or combinations thereof. The devices of the invention are well suited for miniaturization and high throughput.
1. A method of nucleic acid sequencing comprising:
providing an analytical device comprising;
a substrate comprising;
a plurality of illumination volumes, each illumination volume comprising a nucleic acid synthesis complex comprising at least one polymerase enzyme, at least one template nucleic acid, at least one primer sequence that is complementary to a portion of the at least one template sequence, and at least one labeled nucleotide;
a plurality of discrete light sources, at least one discrete light source disposed below at least one illumination volume and optically coupled to the at least one illumination volume; and
a plurality of detector elements, at least one detector element disposed below the at least one illumination volume and optically coupled to the at least one illumination volume;
illuminating the at least one illumination volume with the at least one discrete light source; and
detecting light emitted from the at least one illumination volume by the at least one detector element, wherein light detected by the at least one detector element indicates incorporation of the at least one labeled nucleotide into the at least one primer sequence.
2. The method of claim 1 wherein each illumination volume comprises a plurality of different labeled nucleotides.
3. The method of claim 1 wherein the at least one labeled nucleotide comprises a fluorescent label.
4. The method of claim 1 , wherein the discrete light sources are semiconductor laser diodes, light-emitting diodes, solid-state lasers, or vertical cavity surface-emitting lasers.
5. The method of claim 1 , wherein the discrete light sources are diode light sources.
6. The method of claim 5 , wherein the diode light sources comprise an electrically pumped p-n junction.
7. The method of claim 1 , wherein the illumination volumes are illuminated by light emitted from a top surface of the discrete light sources.
8. The method of claim 7 , wherein the light is emitted in a cone shape from the top surface of the discrete light sources.
9. The method of claim 1 , wherein the illumination volumes are illuminated by an evanescent field emanating from a top surface of the discrete light sources.
10. The method of claim 1 , wherein the discrete light sources are arranged in a strip structure.
11. The method of claim 1 , wherein the analytical device comprises at least 1,000 illumination volumes.
12. The method of claim 1 , wherein the analytical device comprises at least 10,000 illumination volumes.
13. The method of claim 1 , wherein the analytical device comprises at least 1 million illumination volumes.
14. The method of claim 1 , wherein each illumination volume comprises four different nucleotides, and wherein each nucleotide is labeled with a different spectrally distinguishable fluorescent label.
15. The method of claim 1 , wherein each illumination volume comprises four different nucleotides, each nucleotide labeled with a spectrally distinguishable fluorescent label the one or more labeled nucleotides comprise four labeled nucleotides, each distinguishable by signal intensity, excitation spectrum, or both.
16. The method of claim 1 , wherein each illumination volume is contained in a nanowell.
17. The method of claim 16 , wherein the nanowell is a zero mode waveguide.