Methods and systems for image processing of microfluidic devices

• US 8,600,168 B2
• Filed: 10/05/2011
• Issued: 12/03/2013
• Est. Priority Date: 09/13/2006
• Status: Expired due to Fees

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Dube, Simant, Sun, Gang, Zhao, Lian-She
• Primary Examiner(s): Vanore, David A
• Application Number:
  US13/253,857
• Publication Number:
  US 20120245888A1
• Time in Patent Office: 790 Days
• Field of Search: 382/128, 382/133, 382/168, 382/170, 382/181, 382/190, 382/195, 382/199, 382/206, 422/50, 422/504, 422/509, 435/5, 435/6, 435/86
• US Class Current: 382/190
• 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 for an imaging system, the method comprising:

providing a histogram of intensities of pixels of an image;

computing an average intensity for a range of pixels; and

computing an exposure time;

computing a number of saturated pixels;

determining that the exposure time is less than a predetermined time;

determining that the number of saturated pixels is less than a predetermined value; and

utilizing the exposure time during an image acquisition process, wherein the image acquisition process images both chamber pixels and background pixels.

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

3. The method of claim 2 wherein the pixel intensities are associated with chamber pixels.

4. The method of claim 1 wherein the range of pixels runs from a first intensity value to a second intensity value.

5. A method of acquiring reporter images during a polymerase chain reaction (PCR) process, the method comprising:

acquiring a passive reference image;

determining an exposure time for the passive reference image;

determining a focal plane for the passive reference image; and

acquiring a plurality of reporter images at the determined focal plane using the determined exposure time.

6. The method of claim 5 wherein the passive reference image includes one or more chambers containing a passive reference dye.

7. The method of claim 5 wherein determining an exposure time comprises:

providing a histogram of pixel intensities;

computing an average intensity for a range of pixels; and

computing an exposure time;

computing a number of saturated pixels;

determining that the exposure time is less than a predetermined time; and

determining that the number of saturated pixels is less than a predetermined value.

8. The method of claim 5 wherein the plurality of reporter images include one or more chambers containing a reporter dye.

9. The method of claim 8 wherein the plurality of reporter images are characterized by unsaturated emission from the reporter dye.

10. The method of claim 5 wherein determining a focal plane comprises:

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; and

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

11. A method of determining a corner point for a polymerase chain reaction (PCR) ratio data curve, the method comprising:

a) computing an estimate of an amplification factor for a predetermined cycle number;

b) determining a fitting window for the PCR ratio data curve, wherein the fitting window is associated with the predetermined cycle number;

c) fitting a first function to the PCR ratio data curve over the fitting window;

d) determining a fitting error for the first function;

e) fitting a second function to the PCR ratio data curve over the fitting window

f) determining a fitting error for the second function;

g) computing a difference between the fitting error for the first function and the fitting error for the second function,

h) repeating steps a) through g) for subsequent cycle numbers; and

i) identifying a maximum of the computed difference as the corner point.

12. The method of claim 11 wherein the first function comprises a second order polynomial.

13. The method of claim 11 wherein the second function comprises an exponential curve.

14. The method of claim 13 wherein the exponential curve is a function of the amplification factor.

15. The method of claim 11 wherein the fitting error comprises a sum of the squared differences between the fitted values and the actual values at a predetermined cycle number.