Methods and systems for determining a baseline during image processing

• US 8,050,516 B2
• Filed: 09/13/2007
• Issued: 11/01/2011
• Est. Priority Date: 09/13/2006
• Status: Expired due to Fees

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Sun, Gang, Dube, Simant, Zhao, Lian-She
• Primary Examiner(s): Vanore; David A
• Application Number:
  US11/900,926
• Publication Number:
  US 20080075322A1
• Time in Patent Office: 1,510 Days
• Field of Search: 382/128, 382/133, 382/168, 382/170, 382/181, 382/190, 382/195, 382/199, 422/50, 422/504, 422/509, 435/5, 435/6, 435/86
• US Class Current: 382/276
• CPC Class Codes: G06V 20/693 Acquisition

Abstract

A method of determining a plurality of positions associated with a plurality of reaction chambers of a microfluidic device includes a) providing a baseline image; b) providing a template image of a reaction chamber; and c) selecting a region of the baseline image. The method also includes d) performing a matching process including matching the template image to one or more portions of the region of the baseline image; e) determining a position of a first chamber; and f) predicting a position of a second chamber. The method further includes g) repeating steps c) through f) for subsequent chambers.

Claims

1. A method of determining a plurality of positions associated with a plurality of reaction chambers of a microfluidic device, the method comprising:

a) providing a baseline image;

b) providing a template image of a reaction chamber;

c) selecting a region of the baseline image;

d) performing a matching process including matching the template image to one or more portions of the region of the baseline image;

e) determining a position of a first chamber;

f) predicting a position of a second chamber; and

g) repeating steps c) through f) for subsequent chambers.

2. The method of claim 1 further comprising analyzing a fit between predicted positions of the chambers to determine if a desired accuracy is obtained.

3. The method of claim 2 further comprising repeating steps a) through g) if the desired accuracy is not obtained.

4. The method of claim 1 wherein the template image is characterized by a shape substantially similar to a shape of the reaction chambers.

5. The method of claim 1 wherein predicting a position of a second chamber comprises utilizing data related to a design of the microfluidic device.

6. The method of claim 1 wherein the microfluidic device comprises an array of reaction chambers.

7. The method of claim 6 wherein the array of reaction chambers comprises a 48×

48 array.

8. The method of claim 7 wherein the array of reaction chambers comprises an array larger than 48×

48 reaction chambers.

9. The method of claim 6 wherein each of the chambers in the 48×

48 array is in fluidic isolation from any of the other chambers in the 48×

48 array.

10. A method of determining positions of a plurality of chambers of a microfluidic device, the method comprising:

providing a passive reference image of the microfluidic device;

determining a plurality of first chamber positions based on the passive reference image;

providing an additional passive reference image of the microfluidic device;

determining a plurality of second chamber positions based on the additional passive reference image;

providing a reporter dye image of the microfluidic device;

determining a plurality of third chamber positions based on the reporter dye image; and

determining one or more statistical parameters based on the reporter dye image.

11. The method of claim 10 wherein the passive reference image is acquired at a first cycle.

12. The method of claim 10 wherein the additional passive reference image is acquired at a subsequent cycle.

13. The method of claim 10 further comprising updating the plurality of first chamber positions.

14. The method of claim 10 wherein the reporter dye image is acquired at a subsequent cycle.

15. The method of claim 10 wherein the one or more statistical parameters includes an average value of an intensity associated with chamber pixels.

16. The method of claim 15 wherein the one or more statistical parameters includes an average value of an intensity associated with chamber pixels and a pixel boundary with a predetermined width greater than a width associated with the chamber pixels.

17. The method of claim 16 wherein the predetermined width comprises a two-pixel width.

18. A method of removing a baseline from PCR ratio data, the method comprising:

obtaining a plurality of images, each of the plurality of images associated with a cycle of a PCR process;

determining a ratio of reporter dye to passive reference dye for each of the plurality of images, thereby providing a ratio data curve;

determining a corner point of the ratio data curve;

determining a baseline value associated with the ratio data curve; and

subtracting the baseline value from each value in the ratio data curve, thereby providing a baseline removed ratio data curve.

19. The method of claim 18 wherein determining a baseline value associated with the ratio data curve comprises computing a statistical parameter associated with ratio data curve points for cycles less than the corner point.

20. The method of claim 19 wherein the statistical parameter comprises an average value.

21. A method of determining a change in threshold value for a plurality of ratio data curves, the method comprising:

determining a first quality factor for a first ratio data curve of the plurality of ratio data curves;

determining that the first quality factor is greater than a predetermined value;

determining a second quality factor for a second ratio data curve of the plurality of ratio data curves;

determining that the second quality factor is greater than the predetermined value; and

determining the change in threshold value based on the first ratio data curve and the second ratio data curve.

22. The method of claim 21 further comprising:

determining a third quality factor for a third ratio data curve of the plurality of ratio data curves;

determining that the third quality factor is less than the predetermined value;

determining that the third ration data curve should not be used in determining the change in threshold value.

23. The method of claim 21 wherein the first quality factor and the second quality factor range from zero to one.

24. The method of claim 23 wherein the first quality factor and the second quality factor range from 0.5 to 0.7.

25. The method of claim 21 wherein the predetermined value is 0.65.