System, method, and product for imaging probe arrays with small feature size

• US 8,520,976 B2
• Filed: 09/23/2011
• Issued: 08/27/2013
• Est. Priority Date: 01/27/2006
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Kaiser, Michael D., Smith, David R.
• Primary Examiner(s): Chu, Randolph I
• Application Number:
  US13/241,970
• Publication Number:
  US 20120051663A1
• Time in Patent Office: 704 Days
• Field of Search: None
• US Class Current: 382/284
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: Y10S 128/922 including image analysis

Abstract

An embodiment of a method for resolving features on a probe array is described that, comprises acquiring a plurality of micro-shifted images of a region of a probe array; reconstructing an image of the probe array using the micro-shifted images; and deriving intensity values for one or more probe features disposed on the probe array from the reconstructed image.

Claims

1. A method for resolving features on a probe array, comprising:

acquiring images of a region of a probe array with an image acquisition device comprising an objective lens and a sensing element;

shifting the images by less than one half a size of a pixel of the sensing element while maintaining a fixed spatial relationship between the probe array and the objective lens to produce micro-shifted images;

reconstructing pixels of the micro-shifted images by unboxing the micro-shifted images, wherein unboxing comprises deconvolving the micro-shifted images using the Richardson-Lucy algorithm, wherein the Richardson-Lucy algorithm is controlled by at least three independent parameters, and wherein the at least three independent parameters comprise over-relaxation or acceleration, momentum, and total variation control; and

deriving intensity values for one or more features from the reconstructed pixels of the micro-shifted images, thereby resolving features on the probe array.

2. The method of claim 1, wherein the reconstructing step further comprises:

tiling a single pixel from a common location within each of the micro-shifted images to preserve spatial orientation of the micro-shifted images.

3. The method of claim 1, wherein the image is shifted in increments of (1/N) multiplied by a pixel size, wherein N is any integer greater than 3, and wherein the pixel size is the size of each of the pixels of the sensing element.

4. The method of claim 1, wherein each of the one or more probe features on the probe array has a feature size from 1 μ

m to 8 μ

m in a dimension, and wherein each of the pixels of the sensing element comprises a size of 1.0 μ

m, 1.5 μ

m, or 2.5 μ

m.

5. The method of claim 1, wherein the unboxing step further comprises deconvolving the micro-shifted images with an optical point spread function value.

6. The method of claim 1, wherein the unboxing step further comprises employing SIR3, algebraic reconstruction technique (ART), or simultaneous algebraic reconstruction technique (SART).

8. The method of claim 1, wherein the shifting step comprises shifting an element of the objective lens relative to the positions of the objective lens and the sensing element.

9. The method of claim 1, wherein the shifting step comprises shifting an element of the sensing element.

7. A method of deriving signal intensity values in a probe array experiment, which comprises:

hybridizing a labeled sample to a probe array;

acquiring images of the labeled sample on the probe array with an image acquisition device comprising an objective lens and a sensing element;

shifting the images by less than one half a size of a pixel while maintaining a fixed spatial relationship between the probe array and the objective lens to create micro-shifted images;

tiling a single pixel from a common location within each of the micro-shifted images to provide a reconstructed image;

unboxing the reconstructed image by applying a set of linear equations to determine subpixel values, thereby providing a final image, wherein unboxing comprises using the Richardson-Lucy algorithm, wherein the Richardson-Lucy algorithm is controlled by at least three independent parameters, wherein the at least three independent parameters comprise over-relaxation or acceleration, momentum, and total variation control; and

deriving intensity values for one of more probe features disposed on the probe array from the final image.

10. The method of claim 7, wherein the shifting step comprises shifting an element of the objective lens relative to the positions of the objective lens and the sensing element.

11. The method of claim 7, wherein the shifting step comprises shifting an element of the sensing element.