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

• US 8,055,098 B2
• Filed: 01/26/2007
• Issued: 11/08/2011
• 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: Smith, David R., Kaiser, Michael D.
• Primary Examiner(s): CHU, RANDOLPH I
• Assistant Examiner(s): CHU, RANDOLPH I
• Application Number:
  US11/627,876
• Publication Number:
  US 20070197911A1
• Time in Patent Office: 1,747 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 obtaining an image of a plurality of identifiable signals from a plurality of features on a probe array that are captured by a plurality of pixels of a detector to form the image of the probe array, comprising;

providing a probe array in an image acquisition device comprising an objective lens, a shift element, a detector having pixels, and a lens outside of the detector;

acquiring an image of the probe array;

moving the lens outside of the detector by micro-shifting the shift element by less than one half the size of a pixel of the detector while maintaining a fixed spatial relationship between the probe array and the objective lens of the image acquisition device to produce a set of micro-shifted images;

repeating as necessary, in both X and Y dimensions;

reconstructing a single image of the probe array by taking at least one pixel from a common location within each of the set of the micro-shifted images and tiling the pixels in the single image according to a spatial orientation corresponding to a shift direction and a magnitude used during the acquisition of the micro-shifted images; and

creating a deconvolved image using a Richardson-Lucy algorithm with a Total Variation Regularization algorithm controlled by three independent parameters,

wherein the three independent parameters are alpha, including over-relaxation or acceleration, and beta, including momentum, on a per-iteration basis, and lambda including total variation control.

2. A method in accordance with claim 1 further comprising;

creating an unboxed image from the single image, wherein the individual pixels of the single image are made up of a plurality of subpixels, the individual pixels in proximity to each other within a single image share common subpixels; and

solving a plurality of values for the identifiable signals from the plurality of subpixels using a series of linear equations to create a new image at a higher subpixel resolution image than a pixel resolution.

3. A method in accordance with claim 2 further comprising;

creating a deconvolved image by using the single image as a starting image, the unboxed image as an initial guess, and a plurality of physical properties such as a point spread function of an optical system.

4. A method for obtaining an image of a plurality of intensity values from a plurality of features on a nucleic acid probe array that are captured by a plurality of individual pixels of a CCD camera to form the image of the probe array, comprising;

acquiring an image of the nucleic acid probe array using an image acquisition device comprising the CCD camera, an objective lens, and a tube lens;

translating the tube lens relative to the CCD by less than one half a pixel size of the CCD to produce a set of shifted images;

repeating as necessary;

creating a single image from the set of shifted images using one pixel from a common location within each of the shifted images and tiling the individual pixels in the single image according to a spatial orientation corresponding to a shift direction and a magnitude used during the acquisition of the image;

creating an unboxed image from the single image, wherein it is recognized that the individual pixels of the single image are made up of a plurality of subpixels, the individual pixels in proximity to each other within an single image share common subpixels;

creating a deconvolved image by using the single image as a starting image, the unboxed image as a initial guess, and a plurality of a point spread function of an optical system, the deconvolved image is created using a Richardson-Lucy with a Total Variation Regularization algorithm controlled by three independent parameters,

wherein the three independent parameters are alpha, including over-relaxation or acceleration, and beta, including momentum, on a per-iteration basis, and lambda including total variation control; and

deriving a plurality of intensity values for one or more probe features disposed on the probe array from the deconvolved image.

5. A method for obtaining an image of a plurality of intensity values from a plurality of features on a probe array that are captured by a plurality of individual pixels of a sensing element to form the image of the probe array, comprising;

providing a probe array in an image acquisition device comprising an objective lens, a shift element, and a sensing element having a plurality of pixels;

acquiring an image of the probe array;

moving the shift element by less than one half the size of a pixel of the sensing element while maintaining a fixed spatial relationship between the probe array and the objective lens of the image acquisition device to produce a set of shifted images;

repeating as necessary, in both X and Y dimensions; and

creating a single image from a set of shifted images by taking one pixel from a common location within each of the shifted images and tiling the pixels in the single image according to a spatial orientation corresponding to a shift direction and a magnitude used during the acquiring of the image;

creating an unboxed image from the single image, wherein the individual pixels of the single image are made up of a plurality of subpixels, the individual pixels in proximity to each other within a single image share common subpixels; and

, solving a plurality of values for the plurality of subpixels using a series of linear equations to create a new image at a higher subpixel resolution image than a pixel resolution; and

creating a deconvolved image by using the single image as a starting image, the unboxed image as an initial guess, and a point spread function of an optical system, wherein the deconvolved image is created using a Richardson-Lucy with a Total Variation Regularization algorithm controlled by three independent parameters, wherein the three independent parameters are alpha, including over-relaxation or acceleration, and beta, including momentum, on a per-iteration basis, and lambda including total variation control.