Methods, software, and apparatus for focusing an optical system using computer image analysis

• US 20060001955A1
• Filed: 06/30/2004
• Published: 01/05/2006
• Est. Priority Date: 06/30/2004
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Kinney, Patrick D., King, Howard G., Naley, Mark, Pallas, Michael C.
• Granted Patent: US 7,394,943 B2
• Time in Patent Office: Days
• US Class Current: 359/391
• CPC Class Codes: G02B 21/244 using image analysis techniques
• CPC Class Codes: G02B 7/38 measured at different points on the optical axis , e.g. focussing on two or more planes and comparing image data

Abstract

Methods, software, and apparatus for focusing an image in biological instrument are disclosed. Focusing elements are moved to various focus positions within a focus element travel range, and sample images are captured at the various focus positions. The sample images are resolved into subregions and an optimal focus position is determined based on the image intensity statistical dispersions within the identified subregions.

Claims

1. A method for focusing an image in a biological instrument, the method comprising:

moving a focusing element to a plurality of focus positions within a focusing element movement range;

capturing a sample image of a target sample at the plurality of focus positions;

resolving the sample image into a plurality of subregions;

calculating image intensity statistical dispersion values within the plurality of subregions;

identifying subregion focus positions for the plurality of subregions based on the calculated image intensity statistical dispersion values; and

determining an optimal focus position based on the identified subregion focus positions,

wherein the target sample is a high-density microarray.

2. The method of claim 1, wherein the focusing element comprises a focus stage.

3. The method of claim 1, wherein the focusing element comprises at least one lens associated with the imaging instrument.

4. The method of claim 1, wherein the focusing element comprises an image detector associated with the imaging instrument.

5. The method of claim 1, wherein the plurality of focus positions comprises positions above and positions below a predicted optimal focal position.

6. The method of claim 1, wherein the high-density microarray comprises densities of 4 binding sites per square millimeter to 10⁴ binding sites per square millimeter.

13. The method of claim 1, wherein the plurality of subregions numbers between four and one-hundred.

14. The method of claim 1, wherein the sample image is substantially rectangular in configuration and wherein the plurality of subregions comprises substantially rectangular images, which are formed by separating the sample image into rows and columns of subimages.

15. The method of claim 1, wherein the sample image is captured by an image detector comprising a plurality of pixels and calculating the image intensity statistical dispersion values comprises calculating standard deviations of pixel intensity values associated with the plurality of pixels.

16. The method of claim 1, wherein determining the optimal focus position further comprises determining whether a predetermined percentage of the subregion focus positions fit within a focal window associated with the imaging instrument.

17. The method of claim 1 further comprising:

identifying extreme foci within the subregion focus positions; and

determining the optimal focus position based on the extreme foci.

18. The method of claim 17, wherein the optimal focus position is determined by calculating a midpoint between the extreme foci.

19. The method of claim 17, wherein the optimal focus position is determined by using an average of the subregion focus positions within a focal window associated with the imaging instrument.

20. The method of claim 17, further comprising:

ignoring outlier positions in the subregion focus positions.

21. The method of claim 1, wherein determining the optimal focus position comprises determining the optimal focus position for received electromagnetic energy of a particular wavelength.

22. The method of claim 1, wherein determining the optimal focus position comprises determining the optimal focus position for received electromagnetic energy of different wavelengths.

7. A method for focusing an image in a biological instrument, the method comprising:

moving a focusing element to a plurality of focus positions within a focusing element movement range;

capturing a sample image of a target sample at the plurality of focus positions;

resolving the sample image into a plurality of subregions;

calculating image intensity statistical dispersion values within the plurality of subregions;

identifying subregion focus positions for the plurality of subregions based on the calculated image intensity statistical dispersion values; and

determining an optimal focus position based on the identified subregion focus positions,

wherein the target sample is a high-density biological sample container.

8. The method of claim 7, wherein the container is a reaction plate comprising between 96 and 1536 wells.

9. The method of claim 7, wherein the container is a custom multi-well reaction plate.

10. The method of claim 7, wherein the container is a plurality of capillaries in an array.

11. The method of claim 7, wherein the container is a plurality of individual sample tubes arranged in an array configuration.

12. The method of claim 7, wherein the container is a plurality of individual sample locations in a microfluidic card.

23. A method for focusing an image in an imaging instrument, the method comprising:

moving a focusing element to a plurality of focus positions within a focusing element movement range;

scattering electromagnetic radiation off a surface of a target sample;

capturing a sample image of the target sample at the plurality of focus positions based on the scattered electromagnetic radiation;

resolving the sample image into a plurality of subregions;

calculating image intensity statistical dispersion values within the plurality of subregions;

identifying subregion focus positions for the plurality of subregions based on the calculated image intensity statistical dispersion values; and

determining an optimal focus position based on the identified subregion focus positions,

wherein the target sample is a high-density microarray.

24. The method of claim 23, wherein the target sample comprises a microarray.

25. The method of claim 24, wherein the scattered light from fiducials associated with the microarray is used to determine the optimal focus position.

26. Software for focusing an image in an imaging instrument, the software comprising:

instructions to cause an electromechanical movement to move a focusing element to a plurality of focus positions within a focusing element movement range;

instructions to cause a digital camera to capture a sample image of a target sample at the plurality of focus positions;

instructions to cause an image processor to resolve the sample image into a plurality of subregions; and

instructions to calculate image intensity statistical dispersion values within the plurality of subregions and to identify subregion focus positions for the plurality of subregions based on the calculated image intensity statistical dispersion values and to determine an optimal focus position based on the identified subregion focus positions,

wherein the target sample is a high-density microarray or high-density biological sample container.

27. An instrument for analyzing biological samples, the instrument comprising:

an electromechanical movement coupled to and operable to move a focusing element to a plurality of focus positions within a focusing element movement range;

a digital camera coupled to the imaging instrument operable to capture a sample image of a target sample at the plurality of focus positions;

an image processor operable to resolve the sample image into a plurality of subregions; and

a digital processor operable to execute instructions to calculate image intensity statistical dispersion values within the plurality of subregions and to identify subregion focus positions for the plurality of subregions based on the calculated image intensity statistical dispersion values and to determine an optimal focus position based on the identified subregion focus positions,

wherein the target sample is a high-density microarray or high-density biological sample container.