Confocal imaging methods and apparatus

• US 7,589,315 B2
• Filed: 10/11/2007
• Issued: 09/15/2009
• Est. Priority Date: 11/23/2005
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Triener, Alexander, Wang, Mark, Che, Diping, Feng, Wenyi, Kain, Robert, Kotseroglou, Theofilos
• Primary Examiner(s): Lee; John R
• Application Number:
  US11/871,085
• Publication Number:
  US 20080290263A1
• Time in Patent Office: 705 Days
• Field of Search: 250/234, 250/235, 250/236, 250/208.1, 250/208.2, 250/458.1, 250/459.1, 250/586
• US Class Current: 250/234
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• 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 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0032 Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06
• CPC Class Codes: G02B 21/004 fixed arrays, e.g. switchable aperture arrays

Abstract

The invention provides imaging apparatus and methods useful for obtaining a high resolution image of a sample at rapid scan rates. A rectangular detector array having a horizontal dimension that is longer than the vertical dimension can be used along with imaging optics positioned to direct a rectangular image of a portion of a sample to the rectangular detector array. A scanning device can be configured to scan the sample in a scan-axis dimension, wherein the vertical dimension for the rectangular detector array and the shorter of the two rectangular dimensions for the image are in the scan-axis dimension, and wherein the vertical dimension for the rectangular detector array is short enough to achieve confocality in a single axis.

Claims

1. An imaging apparatus comprising:

(a) a radiation source positioned to send excitation radiation to at least a portion of a sample region;

(b) a rectangular detector array comprising a plurality of pixel elements in a horizontal axis and a plurality of pixel elements in a vertical axis, wherein said horizontal axis is longer than said vertical axis;

(c) imaging optics positioned to direct a rectangular image of said portion to said rectangular detector array, wherein the shorter of the two rectangular dimensions for said image correlates with the vertical axis of said rectangular array, thereby achieving confocality in a single axis of said detector, wherein said single axis is said vertical axis; and

(d) a scanning device configured to scan said sample region in a scan-axis dimension, wherein the vertical axis for said rectangular detector array and said shorter of the two rectangular dimensions for said image are in said scan-axis dimension.

2. The apparatus of claim 1, wherein said rectangular detector array is configured to collect electrical responses from pixel elements in the vertical axis in a common bin.

3. The apparatus of claim 1, further comprising a line generator positioned to receive excitation radiation from said radiation source and to send a radiation line to said sample region.

4. The apparatus of claim 3, wherein the ratio of the shorter of two rectangular dimensions for said radiation line to the Rayleigh resolution of the imaging optics is in the range of 0.1 to 10.

5. The apparatus of claim 1, wherein the ratio of said vertical axis and the product of the Rayleigh resolution of the imaging optics is in the range of 0.1 to 10.

6. The apparatus of claim 1, wherein said apparatus is configured to obtain an image of said sample comprising a Rayleigh resolution between 0.2 and 10 micrometers.

7. A method of obtaining an image of a sample in an imaging apparatus, comprising

(a) contacting at least a first portion of a sample with excitation radiation under conditions wherein radiation is emanated from said first portion;

(b) directing said radiation emanated from said first portion to form a rectangular image of said first portion at a rectangular detector array, wherein said rectangular detector array comprises a plurality of pixel elements in a horizontal axis and a plurality of pixel elements in a vertical axis, wherein said horizontal axis is longer than said vertical axis, wherein the shorter of the two rectangular dimensions for said image correlates with the vertical axis of said rectangular array, thereby achieving confocality in a single axis of said detector, wherein said single axis is said vertical axis; and

(c) scanning said sample region in a scan-axis dimension, thereby repeating steps (a) and (b) to form a rectangular image of a second portion of said sample at said rectangular detector array.

8. The method of claim 7, further comprising collecting electrical responses from a plurality of pixel elements of the vertical axis in a common bin, whereby said image is obtained by reading the collected charge from said common bin as a single value.

9. The method of claim 7, wherein step (a) comprises contacting at least a first portion of a sample with a radiation line under conditions wherein radiation is emanated from said first portion.

10. The method of claim 9, wherein the ratio of the shorter of two rectangular dimensions for said radiation line to the Rayleigh resolution of the imaging apparatus is in the range of 0.1 to 10.

11. The method of claim 7, wherein the ratio of said vertical axis and the product of the Rayleigh resolution of the imaging apparatus is in the range of 0.1 to 10.

12. The method of claim 7, wherein said image is obtained at a Rayleigh resolution between 0.2 and 10 micrometers.

13. An imaging apparatus comprising:

(a) a radiation source;

(b) a line generator positioned to receive excitation radiation from said radiation source and to send a radiation line to at least a portion of a sample region;

(c) a rectangular detector array;

(d) imaging optics positioned to direct a rectangular image of said portion to said rectangular detector array; and

(e) a scanning device configured to scan said sample region in a scan-axis dimension,

wherein the ratio of the shorter of two rectangular dimensions for said radiation line to the Rayleigh resolution of the imaging optics is in the range of 0.1 to 10.

14. The apparatus of claim 13, wherein said rectangular detector array comprises a plurality of pixel elements in a horizontal axis and a plurality of pixel elements in a vertical axis, wherein said horizontal axis is longer than said vertical axis.

15. The apparatus of claim 14, wherein said rectangular detector array is configured to collect electrical responses from pixel elements in the vertical axis in a common bin.

16. The apparatus of claim 14, wherein the ratio of said vertical axis and the product of the Rayleigh resolution of the imaging optics is in the range of 0.1 to 10.

17. The apparatus of claim 13, wherein said apparatus is configured to obtain an image of said sample comprising a Rayleigh resolution between 0,2 and 10 micrometers.

18. The apparatus of claim 13, wherein the shorter of the two rectangular dimensions for said image correlates with the vertical axis of said rectangular array, thereby achieving confocality in a single axis of said detector, wherein said single axis is said vertical axis.

19. A method of obtaining an image of a sample in an imaging apparatus, comprising

(a) contacting at least a first portion of a sample with a radiation line under conditions wherein radiation is emanated from said first portion;

(b) directing said radiation emanated from said first portion to form a rectangular image of said first portion at a rectangular detector array, wherein the ratio of the shorter of two rectangular dimensions for said radiation line to the Rayleigh resolution of said imaging apparatus is in the range of 0.1 to 10; and

(c) scanning said sample region in a scan-axis dimension, thereby repeating steps (a) and (b) to form a rectangular image of a second portion of said sample at said rectangular detector array.

20. The method of claim 19, wherein said rectangular detector array comprises a plurality of pixel elements in a horizontal axis and a plurality of pixel elements in a vertical axis, wherein said horizontal axis is longer than said vertical axis.

21. The method of claim 20, further comprising collecting electrical responses from a plurality of pixel elements of the vertical axis in a common bin, whereby said image is obtained by reading the collected charge from said common bin as a single value.

22. The method of claim 20, wherein the ratio of said vertical axis and the product of the Rayleigh resolution of the imaging apparatus is in the range of 0.1 to 10.

23. The method of claim 19, wherein said image is obtained at a Rayleigh resolution between 0.2 and 10 micrometers.

24. The method of claim 19, wherein the shorter of the two rectangular dimensions for said image correlates with the vertical axis of said rectangular array, thereby achieving confocality in a single axis of said detector, wherein said single axis is said vertical axis.