Methods and systems for image processing of microfluidic devices

• US 8,055,034 B2
• Filed: 09/13/2007
• Issued: 11/08/2011
• Est. Priority Date: 09/13/2006
• Status: Expired due to Fees

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Dube, Simant, Zhao, Lian-She, Sun, Gang
• Primary Examiner(s): Vanore; David A
• Application Number:
  US11/900,927
• Publication Number:
  US 20080075380A1
• Time in Patent Office: 1,517 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, 435/86
• US Class Current: 382/128
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G06T 2207/10064 Fluorescence image
• CPC Class Codes: G06T 2207/30004 Biomedical image processing
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/74 involving reference images or patches

Abstract

A method of processing data associated with fluorescent emissions from a microfluidic device. The method includes performing an auto-focus process associated with a first image of the microfluidic device and performing an auto-exposure process associated with the first image of the microfluidic device. The method also includes capturing a plurality of images of the microfluidic device. The plurality of images are associated with a plurality of thermal cycles. The method further includes performing image analysis of the plurality of captured images to determine a series of optical intensities and performing data analysis of the series of optical intensities to provide a series of change in threshold values.

Claims

1. A method of processing data associated with fluorescent emissions from a microfluidic device, the method comprising:

performing an auto-focus process associated with a first image of the microfluidic device;

performing an auto-exposure process associated with the first image of the microfluidic device;

capturing a plurality of images of the microfluidic device, wherein the plurality of images are associated with a plurality of thermal cycles;

performing image analysis of the plurality of captured images to determine a series of optical intensities; and

performing data analysis of the series of optical intensities to provide a series of changes in threshold values.

2. The method of claim 1 wherein the first image comprises a passive reference image.

3. The method of claim 1 wherein performing an auto-focus process comprises computing contrast scores based on chamber pixels.

4. The method of claim 1 wherein performing an auto-exposure process comprises determining an exposure time based on chamber pixels.

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

6. The method of claim 5 wherein the array of reaction chambers comprises an array larger than or equal to 48×

48 reaction chambers.

7. The method of claim 5 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.

8. The method of claim 1 further comprising performing an auto-exposure process associated with a second image of the microfluidic device.

9. The method of claim 8 wherein the second image comprises a reference dye image.

10. A method of performing an auto-focus process for a microfluidic device including a plurality of chambers, the method comprising:

acquiring an image stack comprising a plurality of images;

determining a focal plane characterized by a predetermined contrast score;

determining a plurality of positions associated with the plurality of chambers;

computing a contrast score for each of the plurality of images in the image stack;

determining an updated focal plane characterized by a second predetermined contrast score; and

storing the updated focal plane in one or more memories.

11. The method of claim 10 wherein computing a contrast score for each of the plurality of images in the image stack is performed utilizing chamber pixels.

12. The method of claim 11 wherein computing a contrast score for each of the plurality of images in the image stack is performed utilizing only chamber pixels.

13. The method of claim 10 wherein the plurality of images in the image stack are associated with a series of increasing focal distances.

14. The method of claim 10 wherein the contrast scores are computed using an edge detection algorithm.

15. The method of claim 10 wherein the plurality of chambers are adapted to receive a reagent and a sample.

16. The method of claim 15 wherein the reagent and the sample are adapted to perform an PCR process.

17. The method of claim 10 wherein the second predetermined contrast score comprises a contrast score greater than a predetermined threshold.

18. The method of claim 17 wherein the second predetermined contrast score comprises a maximum contrast score.

19. The method of claim 10 further comprising:

acquiring a second stack of images, each image in the second stack of images being associated with a second predetermined focal distance;

determining, for each image in the second stack of images, a contrast score associated with each image;

selecting a second updated image associated with a contrast score selected from the contrast scores associated with each image in the second stack of images; and

storing a second focal distance associated with the second updated image.

20. The method of claim 19 wherein at least one of the second predetermined focal distances is within a range defined by the predetermined focal distance.

21. A method of determining a baseline value associated with fluorescent emissions from a microfluidic device, the method comprising:

computing an initial baseline value;

providing a first baseline value having a different value than the initial baseline value;

providing a series of emission data points as a function of PCR cycle number;

subtracting the initial baseline value from the series of emission data points to provide an initial baseline removed curve;

computing an initial quality factor associated with initial baseline removed curve;

subtracting the first baseline value from the series of emission data points to provide a first baseline removed curve;

computing a first quality factor associated with the first baseline removed curve;

determining that the first quality factor is greater than the second quality factor; and

determining that the baseline value is the first baseline value.

22. The method of claim 21 wherein computing an initial baseline value comprises:

determining a cycle number; and

computing a statistical parameter associated with emission data points for cycles less than the cycle number.

23. The method of claim 22 wherein the statistical parameter comprises an average value.

24. The method of claim 21 wherein the first baseline value is less than the initial baseline value.

25. The method of claim 21 wherein the first baseline value is greater than the initial baseline value.