System, Method, and Product for Imaging Probe Arrays with Small Feature Sizes

• US 20070197911A1
• Filed: 01/26/2007
• Published: 08/23/2007
• 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.
• Granted Patent: US 8,055,098 B2
• Time in Patent Office: Days
• US Class Current: 600/437
• 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 a plurality of micro-shifted images of a region of a probe array using an image acquisition device comprising an objective lens and a sensing element having pixels;

shifting the images by less than one half a size of the pixel of a sensing element while maintaining a fixed spatial relationship between the probe array and an objective lens of the image acquisition device;

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.

2. A method in accordance with claim 1, wherein the method further comprises 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 the spatial orientation corresponding to the shift direction and magnitude used during image acquisition.

3. A method in accordance with claim 2, wherein the method further comprises creating an Unboxed image from the single image, wherein the individual pixels of the single image are made up of subpixels, the pixels in proximity of each other within an single image share common subpixels;

and,

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

4. A method in accordance with claim 3 wherein the image is shifted in increments of (1/N)*PixelSize, where N is any integer greater than 3.

5. A method for obtaining subpixel resolution, comprising:

creating a set of uniformly micro-shifted images using an image acquisition device comprising an objective lens and a sensing element having pixels;

shifting the images by less than one half the size of the pixel of a sensing element while maintaining a fixed spatial relationship between the probe array and an objective lens of the image acquisition device;

applying a set of linear equations to the images, the equations describing the relationship between subpixel intensities and the pixel intensities in the micro-shifted images caused by the subpixel intensities; and

applying techniques of linear algebra to solve for those subpixel intensities.

6. A method in accordance with claim 5, wherein the method further comprises 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 the spatial orientation corresponding to the shift direction and magnitude used during image acquisition.

7. A method in accordance with claim 6, wherein the method further comprises creating an Unboxed image from the single image, wherein the individual pixels of the single image are made up of subpixels, the pixels in proximity to each other within an single image share common subpixels;

and,

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

8. A method in accordance with claim 7 wherein the image is shifted in increments of (1/N)*PixelSize, where N is any integer greater than 3.

9. A method for obtaining an image, comprising;

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

acquiring a second image of the substrate;

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

processing the images using a set of linear equations.

10. A method in accordance with claim 9, wherein the processing step involves 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 the spatial orientation corresponding to the shift direction and magnitude used during image acquisition.

11. A method in accordance with claim 9 further comprising;

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

,

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

12. A method in accordance with claim 11 further comprising;

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

13. A method in accordance with claim 12 wherein the deconvolved image is created using the Richardson-Lucy algorithm.

14. A method in accordance with claim 13, further comprising using the Richardson-Lucy with Total Variation Regularization algorithm which has been modified to use, on a per iteration basis, parameters alpha, which includes overrelaxation or acceleration, and beta, which includes momentum.

15. A method for obtaining an image, comprising;

acquiring an image of a nucleic acid probe array using an image acquisition device comprising a 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;

acquiring a second image of the probe array;

repeating as necessary;

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

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

creating a deconvolved image by using the single image as the starting image, the Unboxed image as the initial guess, and physical properties such as the point spread function of the optical system, the deconvolved image is created using the Richardson-Lucy with Total Variation Regularization algorithm which has been modified to use, on a per iteration basis, parameters alpha, which includes overrelaxation or acceleration, and beta, which includes momentum; and

deriving intensity values for one or more probe features disposed on the probe array from the reconstructed image.