Compensator for multiple surface imaging
A system and method for imaging biological samples on multiple surfaces of a support structure are disclosed. The support structure may be a flow cell through which a reagent fluid is allowed to flow and interact with the biological samples. Excitation radiation from at least one radiation source may be used to excite the biological samples on multiple surfaces. In this manner, fluorescent emission radiation may be generated from the biological samples and subsequently captured and detected by detection optics and at least one detector. The detected fluorescent emission radiation may then be used to generate image data. This imaging of multiple surfaces may be accomplished either sequentially or simultaneously. In addition, the techniques of the present invention may be used with any type of imaging system. For instance, both epifluorescent and total internal reflection methods may benefit from the techniques of the present invention.
1. A method for detecting features of an array, comprising:
(a) providing a flow cell comprising a first interior surface and a second interior surface that are in a generally mutually facing relation, wherein the first interior surface and the second interior surface are separated by a fluid passage, wherein an array of nucleic acid sites is present on the first interior surface and on the second interior surface, wherein the nucleic acid sites are present at a density of at least 1,000 sites per square millimeter; and
(b) performing a sequencing by ligation reaction, comprising:
(i) introducing fluorescent oligonucleotides to the flow cell to add fluorescent tags to the nucleic acid sites;
(ii) detecting the fluorescent tags at the nucleic acid sites of the array on the first interior surface at a resolution to distinguish the nucleic acid sites on the first interior surface; and
(iii) repeating steps (b)(i) and (b)(ii).
2. The method of claim 1 , wherein (a) further comprises amplifying different template nucleic acids on the first interior surface and on the second interior surface to provide the array of nucleic acid sites that is present on the first interior surface of the array.
3. The method of claim 1 , wherein each of the nucleic acid sites constitutes a population of nucleic acids having identical sequences.
4. The method of claim 1 , wherein the detecting further comprises translating the flow cell, whereby the detecting occurs at successive regions of the first interior surface.
5. The method of claim 1 , wherein detecting further comprises detecting the fluorescent tags at the nucleic acid sites on the second interior surface at a resolution to distinguish the nucleic acid sites on the second interior surface.
6. The method of claim 5 , wherein the first interior surface and the second interior surface are detected from the same side of the flow cell.
7. The method of claim 6 , wherein the first interior surface is disposed between an excitation source and the second interior surface during the detecting.
8. The method of claim 6 , wherein the first interior surface is disposed between a detector and the second interior surface during the detecting.
9. The method of claim 6 , further comprising inserting or removing corrective optics between a detector and the flow cell after the detecting of the fluorescent tags at the nucleic acid sites on the first interior surface.
10. The method of claim 1 , wherein the detecting produces one or more images of the first interior surface.
11. The method of claim 10 , wherein the one or more images have a resolution of 10 microns or less.
12. A method for detecting features of an array, comprising:
(a) providing a flow cell comprising a first interior surface and a second interior surface that are in a generally mutually facing relation, wherein the first interior surface and the second interior surface are separated by a fluid passage, wherein an array of nucleic acid sites is present on the first interior surface and on the second interior surface; and
(b) performing a sequencing by ligation reaction, comprising:
(i) introducing fluorescent oligonucleotides to the flow cell to add fluorescent tags to the nucleic acid sites;
(ii) detecting the fluorescent tags at the nucleic acid sites of the array on the first interior surface at a resolution between 0.1 and 50 microns to distinguish the nucleic acid sites on the first interior surface; and
(iii) repeating steps (b)(i) and (b)(ii).
13. The method of claim 12 , wherein (a) further comprises amplifying different template nucleic acids on the first interior surface and on the second interior surface to provide the array of nucleic acid sites that is present on the first interior surface of the array.
14. The method of claim 13 , wherein each of the nucleic acid sites constitutes a population of nucleic acids having identical sequences.
15. The method of claim 12 , wherein the detecting further comprises translating the flow cell, whereby the detecting occurs at successive regions of the first interior surface.
16. The method of claim 12 , wherein detecting further comprises detecting the fluorescent tags at the nucleic acid sites on the second interior surface at a resolution to distinguish the nucleic acid sites on the second interior surface.
17. The method of claim 16 , wherein the first interior surface and the second interior surface are detected from the same side of the flow cell.
18. The method of claim 17 , wherein the first interior surface is disposed between an excitation source and the second interior surface during the detecting.
19. The method of claim 17 , wherein the first interior surface is disposed between a detector and the second interior surface during the detecting.
20. The method of claim 17 , further comprising inserting or removing corrective optics between a detector and the flow cell after the detecting of the fluorescent tags at the nucleic acid sites on the first interior surface.
21. The method of claim 12 , wherein the detecting produces one or more images of the first interior surface.
22. The method of claim 12 , wherein the nucleic acid sites are present at a density of at least 1,000 sites per square millimeter.