Integrated optoelectronic read head and fluidic cartridge useful for nucleic acid sequencing

• US 9,193,996 B2
• Filed: 02/13/2013
• Issued: 11/24/2015
• Est. Priority Date: 04/03/2012
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Buermann, Dale, Moon, John A., Crane, Bryan, Wang, Mark, Hong, Stanley S., Harris, Jason, Hage, Matthew, Nibbe, Mark J.
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US13/766,413
• Publication Number:
  US 20130260372A1
• Time in Patent Office: 1,014 Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/06 Valves, specific forms thereof
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/527 for a plurality of reagents
• 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 2021/0307 Insert part in cell
• CPC Class Codes: G01N 2021/0325 Cells for testing reactions, e.g. containing reagents
• CPC Class Codes: G01N 2021/058 Flat flow cell
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

Abstract

A detection apparatus having a read head including a plurality of microfluorometers positioned to simultaneously acquire a plurality of the wide-field images in a common plane; and (b) a translation stage configured to move the read head along a substrate that is in the common plane. The substrate can be a flow cell that is included in a cartridge, the cartridge also including a housing for (i) a sample reservoir; (ii) a fluidic line between the sample reservoir and the flow cell; (iii) several reagent reservoirs in fluid communication with the flow cell, (iv) at least one valve configured to mediate fluid communication between the reservoirs and the flow cell; and (v) at least one pressure source configured to move liquids from the reservoirs to the flow cell. The detection apparatus and cartridge can be used together or independent of each other.

Claims

1. A detection apparatus, comprising

(a) a carriage comprising a plurality of microfluorometers,

wherein the plurality of microfluorometers form a read head,

wherein the microfluorometers are permanently fixed in the read head such that the microfluorometers are not independently moveable with respect to each other in a direction parallel to the common plane,

wherein the read head comprises a co-molded assembly of the plurality of microfluorometers,

wherein each of the microfluorometers comprises an objective having a field diameter of at least 1 mm,

wherein the plurality of microfluorometers is positioned to simultaneously acquire a plurality of wide-field images in a common plane, and

wherein each of the wide-field images is from a different area of the common plane;

(b) a translation stage configured to move the carriage in at least one direction parallel to the common plane; and

(c) a sample stage configured to hold a substrate in the common plane.

2. The detection apparatus of claim 1, wherein each of the microfluorometers further comprises a dedicated autofocus module.

3. The detection apparatus of claim 2, wherein the autofocus module for a first microfluorometer of the apparatus is configured to integrate data from an autofocus module for a second microfluorometer of the apparatus, whereby the autofocus module for a first microfluorometer alters the focus of the first microfluorometer based on the focus position of the first microfluorometer and the focus position of the second microfluorometer.

4. The detection apparatus of claim 1, wherein each of the microfluorometers further comprises a beam splitter and a detector, wherein the beam splitter is positioned to direct excitation radiation from an excitation radiation source to the objective and to direct emission radiation from the objective to the detector.

5. The detection apparatus of claim 4, wherein each of the microfluorometers further comprises the excitation radiation source.

6. The detection apparatus of claim 5, wherein the excitation radiation source directs the excitation radiation to the objective of an individual microfluorometer in the plurality of microfluorometers, whereby each microfluorometer comprises a separate excitation radiation source.

7. The detection apparatus of claim 5, wherein the carriage further comprises a heat sink in thermal contact with the radiation source.

8. The detection apparatus of claim 4, wherein the excitation radiation source directs the excitation radiation to the objectives of two or more microfluorometers in the plurality of microfluorometers, whereby two or more microfluorometers share an excitation radiation source.

9. The detection apparatus of claim 4, wherein each of the microfluorometers further comprises at least two excitation radiation sources.

10. The detection apparatus of claim 1, wherein the objective of each of the microfluorometers has a numerical aperture between 0.2 and 0.5.

11. The detection apparatus of claim 1, wherein each of the microfluorometers is configured to detect at a resolution sufficient to distinguish features that are less than 50 microns apart.

12. The detection apparatus of claim 11, wherein the objective of each of the microfluorometers has a numerical aperture between 0.2 and 0.5.

13. The detection apparatus of claim 1, wherein the carriage prevents lateral movement between the microfluorometers.

14. A method of imaging a substrate, comprising the steps of:

(a) providing a substrate to the sample stage of the detection apparatus of claim 1, the substrate comprising fluorescent features on a surface;

(b) acquiring a plurality of wide-field images of a first portion of the surface using the plurality of microfluorometers,

wherein each of the microfluorometers acquires a wide-field image from a different location of the surface,

wherein the plurality of microfluorometers are affixed to the carriage; and

(c) translating the carriage in a direction parallel to the surface and repeating step (b) for a second portion of the surface.

15. The method of claim 14, further comprising individually focusing each microfluorometer in the plurality of microfluorometers using an autofocus technique.

16. The method of claim 15, wherein the autofocus technique uses at least one dedicated detector that is not used to acquire the wide-field image.

17. The method of claim 15, wherein the autofocus technique uses at least one detector that is also used to acquire the wide-field image.

18. The method of claim 15, wherein the autofocus technique used for a first microfluorometer of the plurality of microfluorometers integrates data from an autofocus technique used for a second microfluorometer of the plurality of microfluorometers, whereby the focus of the first microfluorometer is adjusted based on the focus position of the first microfluorometer and the focus position of the second microfluorometer.

19. The method of claim 14, wherein each of the microfluorometers acquires at least two wide-field images of the surface in step (b), each of the at least two wide-field images detecting fluorescence at a different excitation or emission wavelength.

20. The method of claim 14, wherein the fluorescent features have an average center-to-center spacing for nearest neighbors that is less than 50 microns apart and the fluorescent features are individually resolved in the wide-field images.

21. The method of claim 14, wherein the method further comprises a step:

(d) returning the carriage to a position to acquire a second plurality of wide-field images of the first portion of the surface.

22. The method of claim 21, further comprising modifying the fluorescent features on the surface after step (c) and before step (d), wherein the second plurality of wide-field images are different from the first plurality of wide-field images.

23. The method of claim 22, wherein the fluorescent features comprise nucleic acids and the modifying comprises altering nucleic acids in a sequencing-by-synthesis technique.

24. The method of claim 22, further comprising repeating step (c) a plurality of times prior to step (d), thereby imaging at least a third portion and fourth portion of the surface.

25. The detection apparatus of claim 1, wherein the plurality of microfluorometers that form the read head comprises more than two microfluorometers.

26. The detection apparatus of claim 25, wherein each of the microfluorometers in the read head has the same optical components as the other microfluorometers in the read head.

27. The detection apparatus of claim 1, wherein the plurality of microfluorometers is positioned such that each of the wide-field images in the common plane is contiguous with another of the wide field images in the common plane.

28. The detection apparatus of claim 1, wherein the plurality of microfluorometers is further positioned such that each of the wide-field images in the common plane is contiguous and non-overlapping with another of the wide field images in the common plane.

29. The detection apparatus of claim 1, wherein the plurality of microfluorometers is further positioned such that each of the wide-field images in the common plane is partially overlapping with another of the wide field images in the common plane.

30. The detection apparatus of claim 1, wherein the substrate comprises a flow cell, and wherein the flow cell is present at the sample stage.

31. The detection apparatus of claim 30, wherein an inner surface of the flow cell comprises nucleic acid features that are placed in the common plane.