Standardizing fluorescence microscopy systems

Patent metadata

Field	Value
Publication number	US-9366628-B2
Application number	US-201414586337-A
Country	US
Kind code	B2
Filing date	Dec 30, 2014
Priority date	Jul 13, 2011
Publication date	Jun 14, 2016
Grant date	Jun 14, 2016

Abstract

Systems and methods for standardizing one or more fluorescence scanning instruments to a reference system by separating the effects of drift and normalization. In an embodiment, a drift image comprising an image of a drift reference slide is captured by a system to be standardized. A drift measurement is calculated using the drift image. A first normalization image comprising an image of a normalization slide is also captured by the system to be standardized. A reference normalization image, also comprising an image of the normalization slide, is captured by a reference system. The first normalization image is compared to the reference normalization image to determine a gamma value and offset value for the system to be standardized.

First claim

The invention claimed is:

1. A method for standardizing a fluorescence microscopy imaging system, the method comprising:
by a first imaging system to be standardized, capturing a first normalization image comprising an image of a normalization slide;
by a reference imaging system, capturing a reference normalization image comprising an image of the normalization slide;
comparing the first normalization image to the reference normalization image; and
determining a gamma value and offset value for the first imaging system based on the comparison.

2. The method of claim 1 , wherein capturing a first normalization image, comparing the first normalization image to the reference normalization image, and determining a gamma value and offset value for the first imaging system are all performed for each of a plurality of normalization slides.

3. The method of claim 1 , wherein the first normalization image and the reference normalization image comprise images of an identical region of the normalization slide.

4. The method of claim 1 , further comprising, for the reference imaging system, setting a gamma value to one and an offset value to zero.

5. The method of claim 1 , further comprising storing the reference normalization image.

6. The method of claim 1 , further comprising by the first imaging system to be standardized,
capturing a drift image comprising an image of a drift reference slide; and
calculating a drift measurement using the drift image.

7. The method of claim 6 , wherein calculating the drift measurement comprises calculating a ratio of average pixel intensity in the drift image to an exposure of the drift image.

8. The method of claim 6 , further comprising:
by the first imaging system, capturing a digital image of a sample, wherein the digital image comprises a plurality of pixels, each pixel associated with a pixel intensity value; and
altering one or more of the pixel intensity values based on the drift measurement, the gamma value, and the offset value.

9. The method of claim 8 , further comprising storing the digital image without alteration of the pixel intensity values.

10. The method of claim 9 , further comprising applying image processing to the digital image, wherein the alteration of one or more pixel intensity values is performed at a time that the application of the image processing is performed.

11. The method of claim 9 , further comprising displaying the digital image, wherein the alteration of one or more pixel intensity values is performed at a time that the digital image is displayed.

12. The method of claim 6 , further comprising by the reference imaging system, capturing a reference drift image comprising an image of a reference drift slide, and calculating a reference drift measurement using the reference drift image.

13. The method of claim 12 , further comprising storing the reference drift measurement.

14. A system for standardizing a fluorescence microscopy imaging system, the system comprising:
at least one hardware processor; and
at least one executable module that, when executed by the at least one hardware processor,
receives a first normalization image comprising an image of a normalization slide captured by the first imaging system,
receives a reference normalization image comprising an image of the normalization slide captured by a reference imaging system,
compares the first normalization image to the reference normalization image, and
determines a gamma value and offset value for the first imaging system based on the comparison.

15. The system of claim 14 , wherein the first normalization image and the reference normalization image comprise images of an identical region of the normalization slide.

16. The system of claim 14 , wherein at least one executable module, when executed by the at least one hardware processor,
receives a drift image comprising an image of a drift reference slide captured by a first imaging system, and
calculates a drift measurement using the drift image.

17. The system of claim 16 , wherein calculating the drift measurement comprises calculating a ratio of average pixel intensity in the drift image to an exposure of the drift image.

18. The system of claim 16 , wherein the at least one executable module further:
receives a digital image of a sample captured by the first imaging system, wherein the digital image comprises a plurality of pixels, each pixel associated with a pixel intensity value; and
alters one or more of the pixel intensity values based on the drift measurement, the gamma value, and the offset value.

19. A non-transitory computer-readable medium having instructions stored thereon, wherein the instructions, when executed by a processor, cause the processor to:
receive a first normalization image comprising an image of a normalization slide captured by a first imaging system;
receive a reference normalization image comprising an image of the normalization slide captured by a reference imaging system;
compare the first normalization image to the reference normalization image; and
determine a gamma value and offset value for the first imaging system based on the comparison.

20. The non-transitory computer-readable medium of claim 19 , wherein the first normalization image and the reference normalization image comprise images of an identical region of the normalization slide.

21. The non-transitory computer-readable medium of claim 19 , wherein the instructions, when executed by the processor, cause the processor to:
receive a drift image comprising an image of a drift reference slide captured by a first imaging system; and
calculate a drift measurement using the drift image.

22. The non-transitory computer-readable medium of claim 21 , wherein calculating the drift measurement comprises calculating a ratio of average pixel intensity in the drift image to an exposure of the drift image.

23. The non-transitory computer-readable medium of claim 21 , wherein the instructions further cause the processor to:
receive a digital image of a sample captured by the first imaging system, wherein the digital image comprises a plurality of pixels, each pixel associated with a pixel intensity value; and
alter one or more of the pixel intensity values based on the drift measurement, the gamma value, and the offset value.

Assignees

• Leica Biosystems Imaging Inc

Inventors

• Crandall Gregory
• Olson Allen

Classifications

• G01N21/278

  Constitution of standards · CPC title

• G01J3/4406

  Fluorescence spectrometry · CPC title

• G01J3/027

  Control of working procedures of a spectrometer; Failure detection; Bandwidth calculation · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G01J3/0235

  using means for replacing an element by another, for replacing a filter or a grating · CPC title