Biopolymer array substrate thickness dependent automated focus-distance determination method for biopolymer array scanners

• US 7,361,906 B2
• Filed: 12/23/2004
• Issued: 04/22/2008
• Est. Priority Date: 07/31/2002
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Ghosh, Jayati, Sillman, Debra A., Corson, John F.
• Primary Examiner(s): Hannaher; Constantine
• Application Number:
  US11/022,302
• Publication Number:
  US 20050153349A1
• Time in Patent Office: 1,216 Days
• Field of Search: 250/458.1
• US Class Current: 250/458.1
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/276 with alternation of sample and standard in optical path
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G02B 21/241 Devices for focusing focusing in general G02B7/28
• CPC Class Codes: G02B 21/244 using image analysis techniques

Abstract

A method and system for determining a biopolymer array substrate thickness dependent optimal focus distance for scanning a molecular array by a molecular array scanner are provided. A reference substrate is automatically scanned at successively greater distances of the stage from a light gathering medium, such as an optical fiber, or z-positions, to produce data providing a functional relationship between z-position and measured signal intensities. The data is then processed by an array substrate thickness dependent focus-finding routine that selects an optimal focus-distance for data scans which is optimized for the thickness of the reference substrate. Also provided are methods of determining the thickness of a biopolymer array substrate using a position sensitive device (PSD) component of a biopolymer array scanner. Further methods include determining the thickness of said biopolymeric array and automatically selecting an optimal focus distance using the determined thickness and a calibration function on thickness versus optimal focus distance. The subject invention finds use in a variety of different applications, including both genomic and proteomic applications.

Claims

1. A method comprising:

(a) providing a biopolymeric array optical scanner configured to determine an optimal focus distance based on at least a first and second reference substrate thickness;

(b) calibrating said biopolymer array optical scanner so that the scanner automatically determines an optimal focus distance based on the thickness of an array substate; and

reading a biopolymeric array with said calibrated biopolymer array optical scanner.

2. The method of claim 1, further comprising:

scanning portions of a reference substrate over a range of positions, each position representing a different distance between an illuminated region of the reference substrate and an emitted-light detection component of the scanner, each portion scanned at a different position; and

deriving a function for determining optimal focus-distance or range of optimal focus distances for a given array substrate thickness.

3. The method of claim 2, further comprising deriving the function for each of a plurality of array substrate thicknesses.

4. The method of claim 2, wherein the function is derived from data relating to signal intensity over the range of positions obtained during the scanning.

5. The method according to claim 4, wherein each position is associated with a mean intensity value.

6. The method of claim 2, wherein the method comprises determining an array substrate thickness dependent optimal focus distance or range of focus distances for the biopolymer array scanner for two or more reference substrates of different thickness.

7. The method of claim 2, further comprising recording the function in the memory of the scanner.

8. The method of claim 2, wherein scanning portions of the reference substrate over a range of positions relative to an emitted-light detection component further includes:

for each position,

scanning a set of rows of one or more arrays on the substrate,

filtering the intensity values scanned for each row, and

averaging the intensity values for the set of rows into an average intensity value for the position.

9. The method of claim 1, further comprising identifying a focus distance that provides a signal intensity that varies minimally with variations in focus distance during scanning of the array.

10. The method of claim 1, wherein said first and second reference substrate thicknesses are determined using a sensor for detecting focus distance.

11. The method according to claim 1, wherein the method comprises performing a peak-intensity-focus-distance determination method.

12. The method according to claim 1, wherein the method comprises performing a window-focus-distance determination.

13. The method of claim 1, wherein the method comprises performing an average peak-intensity-focus-distance determination method.

14. The method of claim 13, wherein the average peak-intensity-focus-distance determination method comprises:

determining the position of the peak intensity in the intensity/position function;

identifying a left and right threshold position relative to the determined peak intensity position that have an intensity below a threshold value; and

determining the mean of said left and right threshold positions to determine the array substrate dependent optimal focus distance or range of focus distances.

15. A set of computer instructions for carrying out the method of claim 1 encoded by one of:

storing the computer instructions in a machine readable medium; and

printing the computer instructions in a human readable medium.

16. A biopolymer array scanner comprising:

(a) a probe-molecule excitation system;

(b) a photodetection system

(c) a biopolymer-array-holding stage that holds a biopolymer array for scanning and that can be moved through a range of positions that place an illuminated region of the surface of the biopolymer array at different distances from the photodetection system; and

(d) an automated array substrate thickness dependent focus-distance-determination subsystem.

17. The biopolymer array scanner of claim 16, wherein the automated array substrate thickness dependent focus-distance-determination subsystem comprises a set of computer instructions for performing a method comprising:

(a) scanning portions of a reference substrate over a range of positions, each position representing a different distance between an illuminated region of the reference substrate and a photodetection component of the scanner, each portion scanned at a different position;

(b) deriving a calibration function for determining an optimal focus-distance or range of optimal focus distances for a given array substrate thickness.

18. A method of assaying a sample, said method comprising:

(a) contacting said sample with a biopolymer array of two or more biopolymer ligands immobilized on a surface of a solid support; and

(b) reading the array with a calibrated biopolymer array optical scanner according to claim 16 to obtain a result.

19. The method of claim 18, wherein said reading step (b) includes:

(i) determining the thickness of the biopolymer array; and

(ii) automatically selecting an optimal focus distance using the determined thickness and calibration function.

20. The method of claim 18, wherein the biopolymer array comprises a polypeptide array or a nucleic acid array.

21. The method of claim 18, further comprising transmitting the result from a first location to a second location.

22. The method of claim 21, wherein the second location is a remote location.

23. A method comprising:

(a) providing a biopolymeric array optical scanner configured to determine an optimal focus distance based on at least a first thickness of a first reference substrate and second thickness of a second reference substrate;

(b) calibrating said biopolymer array optical scanner so that the scanner automatically determines an optimal focus distance based on the thickness of an array substrate; and

reading a biopolymeric array with said calibrated biopolymer array optical scanner.