Methods and apparatus for detection of fluorescently labeled materials

• US 8,233,735 B2
• Filed: 09/17/2008
• Issued: 07/31/2012
• Est. Priority Date: 02/10/1994
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Stern, David, Fiekowsky, Peter
• Primary Examiner(s): Lu, Tom Y
• Assistant Examiner(s): CONWAY, THOMAS A
• Application Number:
  US12/212,320
• Publication Number:
  US 20090010524A1
• Time in Patent Office: 1,413 Days
• Field of Search: None
• US Class Current: 382/255
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: G01N 2021/174 either absorption-reflection or emission-fluorescence
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

Claims

1. A scanning system, comprising:

a probe array;

one or more optical elements constructed and arranged to direct an excitation beam at the probe array;

one or more detectors constructed and arranged to receive reflected intensity data responsive to the excitation beam, wherein the reflected intensity data is responsive, at least in part, to a focusing distance between an optical element and the probe array;

a transport frame constructed and arranged to adjust the focusing distance in a first direction with respect to the probe array;

an auto-focuser constructed and arranged to determine a best plane of focus based, at least in part, upon one or more characteristics of the reflected intensity data as received at two or more focusing distances;

and wherein;

the one or more detectors are further constructed and arranged to receive a plurality of pixel intensity values based, at least in part, upon detected emissions from a plurality of probe features disposed on the probe array at the best plane of focus; and

the system further comprises an image generator constructed and arranged to associate each of the pixel intensity values with one or more image pixel positions of a probe array based, at least in part, on comparison to a synthesis file.

2. The scanning system of claim 1, wherein:

the reflected intensity data correspond to reflected light from one or more reflection spots.

3. The scanning system of claim 2, wherein:

the best plane of focus is based, at least in part, upon associating the one or more reflection spots with one or more characteristics of a beam diameter.

4. A method, comprising the acts of:

directing an excitation beam at a probe array;

receiving reflected intensity data responsive to the excitation beam, wherein the intensity data is responsive, at least in part, to a focusing distance between an optical element and the probe array;

adjusting the focusing distance in a first direction with respect to the probe array;

repeating the steps of receiving and adjusting for a number of iterations;

determining a best plane of focus based, at least in part, upon one or more characteristics of the reflected intensity data at the adjusted focusing distances;

dividing the reflected intensity values into one or more pixels to create pixel intensity values; and

generating an image of the probe array by associating each of the one or more pixel intensity values with one or more image pixel positions of the probe array based, at least in part, on comparison to a synthesis file.

5. The method of claim 4, wherein:

the first direction is away from or toward the probe array.

6. The method of claim 4, wherein:

the reflected intensity data are responsive to reflection of the excitation beam from one or more focus features.

7. The method of claim 6, wherein:

the reflected intensity data correspond to one or more reflection spots.

8. The method of claim 7, wherein:

the best plane of focus is based, at least in part, upon associating the one or more spots with one or more characteristics of a beam diameter.

9. The method of claim 7, wherein:

the one or more focus features are positioned at one or more corners of the probe array.

10. A scanning system, comprising:

a probe array;

one or more optical elements constructed and arranged to direct an excitation beam at the probe array;

one or more detectors constructed and arranged to receive reflected intensity data responsive to the excitation beam, wherein the intensity data is determined, at least in part, by a focusing distance between an optical element and the probe array, and wherein the one or more detectors are further constructed and arranged to receive a plurality of pixel intensity values based, at least in part, upon detected emissions from a plurality of probe features disposed on the probe array at the best plane of focus;

an auto-focuser constructed and arranged to determine a best plane of focus based, at least in part, upon one or more characteristics of the reflected intensity data as received at two or more focusing distances, and

an image generator constructed and arranged to associate each of the pixel intensity values with one or more image pixel positions of a probe array based, at least in part, on comparison to a synthesis file.

11. The scanning system of claim 10, wherein:

the reflected intensity data are responsive to reflection of the excitation beam from one or more focus features.

12. The scanning system of claim 11, wherein:

the one or more focus features are positioned at one or more corners of the probe array.

13. The scanning system of claim 11, wherein:

the reflected intensity data correspond to one or more reflection spots.

14. The scanning system of claim 13, wherein:

the best plane of focus is based, at least in part, upon associating the one or more spots with one or more characteristics of a beam diameter.