Compensator for multiple surface imaging

• US 8,586,947 B1
• Filed: 08/23/2013
• Issued: 11/19/2013
• Est. Priority Date: 05/05/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Feng, Wenyi, Bryant, Jason, Buermann, Dale
• Primary Examiner(s): Kim, Kiho
• Application Number:
  US13/974,976
• Time in Patent Office: 88 Days
• Field of Search: 250/459.1
• US Class Current: 250/459.1
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0024 Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
• CPC Class Codes: G02B 2207/113 Fluorescence

Abstract

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.

Claims

1. A method for detecting nucleic acids, comprising:

(a) providing a support structure comprising a first manufactured surface and a second manufactured surface that are separated by a fluid passage, wherein the first manufactured surface and the second manufactured surface are in contact with a fluid in the fluid passage, and wherein an array of nucleic acid sites is distributed on the first manufactured surface and on the second manufactured surface, wherein the nucleic acid sites are present at a density of at least 1,000 sites per square millimeter;

(b) detecting the array using an optical system having a numerical aperture of at least 0.6 and at a resolution to distinguish the nucleic acid sites on the first manufactured surface; and

(c) detecting the array using the optical system having a numerical aperture of at least 0.6 at a resolution to distinguish the nucleic acid sites on the second manufactured surface.

2. The method of claim 1, wherein (a) further comprises randomly disposing nucleic acids on the first manufactured surface and on the second manufactured surface, thereby forming the array of nucleic acid sites that is distributed on the first manufactured surface and on the second manufactured surface.

3. The method of claim 2, wherein (a) further comprises amplifying the nucleic acids on the first manufactured surface and on the second manufactured surface to form the array of nucleic acid sites that is distributed on the first manufactured surface and on the second manufactured surface.

4. The method of claim 1, wherein the detecting in (b) is done at a resolution between 0.1 and 50 microns on the first manufactured surface.

5. The method of claim 4, wherein the detecting in (c) is done at a resolution between 0.1 and 50 microns on the second manufactured surface.

6. The method of claim 1, wherein the nucleic acid sites comprise fluorescent labels, and wherein the detecting in (b) and (c) comprises detecting the fluorescent labels.

7. The method of claim 6, wherein the first manufactured surface and the second manufactured surface are detected from the same side of the support structure in (b) and (c).

8. The method of claim 7, wherein the first manufactured surface is disposed between an excitation source and the second manufactured surface during the detecting of (c).

9. The method of claim 8, wherein the first manufactured surface and the second manufactured surface are excited by total internal reflection in (b) and (c).

10. The method of claim 8, wherein the first manufactured surface and the second manufactured surface are excited and detected by epifluorescence in (b) and (c).

11. The method of claim 7, wherein the first manufactured surface is disposed between a detector and the second manufactured surface during the detecting of (c).

12. The method of claim 11, further comprising inserting corrective optics between the detector and the support structure after (b) and before (c).

13. The method of claim 11, further comprising removing corrective optics between the detector and the support structure after (b) and before (c).

14. The method of claim 1, wherein the detecting in (b) and (c) produce images of the first manufactured surface and the second manufactured surface.

15. The method of claim 14, wherein the images have a resolution of 10 microns or less.

16. The method of claim 1, further comprising:

(d) repeating (b) and (c) in a process of sequencing the nucleic acids at the nucleic acid sites.

17. The method of claim 1, further comprising:

(d) introducing a fluorescent reagent to the support structure to add fluorescent tags to the nucleic acid sites.

18. The method of claim 17, further comprising:

(e) repeating (b), (c) and (d) in a process of sequencing the nucleic acids at the nucleic acid sites.

19. The method of claim 18, wherein the process of sequencing comprises sequencing by synthesis and the fluorescent reagent comprises a nucleotide having a reversible termination property.

20. The method of claim 18, wherein the process of sequencing comprises sequencing by ligation or pyrosequencing.

21. The method of claim 1, wherein the first manufactured surface and the second manufactured surface are detected from opposite sides of the support structure in (b) and (c).