Fluorescent intensity measuring method and apparatus

• US 7,224,826 B2
• Filed: 06/26/2003
• Issued: 05/29/2007
• Est. Priority Date: 12/26/2000
• Status: Expired due to Term

• Current Assignee: Olympus Corporation
• Original Assignee: Olympus Corporation
• Inventors: Shibazaki, Takami, Okawa, Kaneyasu
• Primary Examiner(s): Bella; Matthew C.
• Assistant Examiner(s): GORADIA, SHEFALI DINESH
• Application Number:
  US10/606,518
• Publication Number:
  US 20040001196A1
• Time in Patent Office: 1,433 Days
• Field of Search: 382/128, 382141-152, 382/254, 382/274, 382/275, 356/317, 356/318, 356/417, 356/517, 250/458.1, 250/459.1, 250/461.1, 250/461.2, 422/82.07, 422/82.08, 436/172
• US Class Current: 382/128
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging

Abstract

A fluorescent image is obtained by emitting the light with a wavelength which excites a labelling fluorescent substance, a mask is created by a foreign matter area image extracted from an image of foreign matter adhering to the measurement object obtained by emitting light with a wavelength which does not excite the fluorescent substance, and logical multiplication of the mask and the fluorescent image is executed, thereby obtaining a fluorescent image that a foreign matter area is eliminated from the fluorescent image.

Claims

1. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imaging step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask;

an expansion step of expanding the foreign matter area of the binarized image by a determined quantity;

a normalization step of normalizing the measured intensity of the minute point by using a reference area of the minute point; and

a reliability judgment step of obtaining an area of each minute point after the foreign matter elimination step and judging the reliability of the measurement value by using a ratio of the obtained area and the reference area of the minute point.

2. A fluorescent intensity measuring method according to claim 1, further comprising a correction step of correcting the second image by using a reference image.

3. A fluorescent intensity measuring method according to claim 1, wherein the extraction step obtains the binarized image by using a differential image acquired from the second image.

4. A fluorescent intensity measuring method according to claim 3, wherein the extraction step determines a binarization level of the binarized image by using a frequency distribution of the differential signal corresponding to each pixel.

5. A fluorescent intensity measuring method according to claim 4, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

6. A fluorescent intensity measuring method according to claim 3, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

7. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imaging step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask;

an expansion step of expanding the foreign matter area of the binarized image by a determined quantity;

a normalization step of normalizing the measured intensity of the minute point by using a reference area of the minute point; and

a correction step of correcting the second image by using a reference image.

8. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imagine step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask;

an expansion step of expanding the foreign matter area of the binarized image by a determined quantity; and

a normalization step of normalizing the measured intensity of the minute point by using a reference area of the minute point;

wherein the extraction step obtains the binarized image by using a differential image acquired from the second image.

9. A fluorescent intensity measuring method according to claim 8, wherein the extraction step determines a binarization level of the binarized image by using a frequency distribution of the differential signal corresponding to each pixel.

10. A fluorescent intensity measuring method according to claim 9, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

11. A fluorescent intensity measuring method according to claim 8, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

12. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imaging step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask;

an expansion step of expanding the foreign matter area of the binarized image by a determined quantity; and

a reliability judgment step of obtaining an area of each minute point after the foreign matter elimination step and judging the reliability of the measurement value by using a ratio of the obtained area and the reference area of the minute point.

13. A fluorescent intensity measuring method according to claim 12, further comprising a correction step of correcting the second image by using a reference image.

14. A fluorescent intensity measuring method according to claim 12, wherein the extraction step obtains the binarized image by using a differential image acquired from the second image.

15. A fluorescent intensity measuring method according to claim 14, wherein the extraction step determines a binarization level of the binarized image by using a frequency distribution of the differential signal corresponding to each pixel.

16. A fluorescent intensity measuring method according to claim 15, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

17. A fluorescent intensity measuring method according to claim 14, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

18. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imaging step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask; and

an expansion step of expanding the foreign matter area of the binarized image by a determined quantity;

wherein the extraction step obtains the binarized image by using a differential image acquired from the second image.

19. A fluorescent intensity measuring method according to claim 18, wherein the extraction step determines a binarization level of the binarized image by using a frequency distribution of the differential signal corresponding to each pixel.

20. A fluorescent intensity measuring method according to claim 19, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

21. A fluorescent intensity measuring method according to claim 18, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

22. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imaging step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask;

a normalization step of normalizing the measured intensity of the minute point by using a reference area of the minute point; and

a reliability judgment step of obtaining an area of each minute point after the foreign matter elimination step and judging the reliability of the measurement value by using a ratio of the obtained area and the reference area of the minute point.

23. A fluorescent intensity measuring method according to claim 22, further comprising a correction step of correcting the second image by using a reference image.

24. A fluorescent intensity measuring method according to claim 22, wherein the extraction step obtains the binarized image by using a differential image acquired from the second image.

25. A fluorescent intensity measuring method according to claim 24, wherein the extraction step determines a binarization level of the binarized image by using a frequency distribution of the differential signal corresponding to each pixel.

26. A fluorescent intensity measuring method according to claim 25, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

27. A fluorescent intensity measuring method according to claim 24, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

28. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imaging step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask;

a normalization step of normalizing the measured intensity of the minute point by using a reference area of the minute point; and

a correction step of correcting the second image by using a reference image.

29. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imagine step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask; and

a normalization step of normalizing the measured intensity of the minute point by using a reference area of the minute point;

wherein the extraction step obtains the binarized image by using a differential image acquired from the second image.

30. A fluorescent intensity measuring method according to claim 29, wherein the extraction step determines a binarization level of the binarized image by using a frequency distribution of the differential signal corresponding to each pixel.

31. A fluorescent intensity measuring method according to claim 30, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

32. A fluorescent intensity measuring method according to claim 29, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

33. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imaging step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image;

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask; and

a reliability judgment step of obtaining an area of each minute point after the foreign matter elimination step and judging the reliability of the measurement value by using a ratio of the obtained area and the reference area of the minute point.

34. A fluorescent intensity measuring method according to claim 33, further comprising a correction step of correcting the second image by using a reference image.

35. A fluorescent intensity measuring method according to claim 33, wherein the extraction step obtains the binarized image by using a differential image acquired from the second image.

36. A fluorescent intensity measuring method according to claim 35, wherein the extraction step determines a binarization level of the binarized image by using a frequency distribution of the differential signal corresponding to each pixel.

37. A fluorescent intensity measuring method according to claim 36, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

38. A fluorescent intensity measuring method according to claim 35, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

39. A fluorescent intensity measuring method which measures the intensity of minute points which are arranged on a substrate having a substantially flat surface and include a fluorescent substance, comprising:

a first imaging step of emitting light with a wavelength with which the fluorescent substance can be excited and obtaining an image of each minute point including the fluorescent substance as a first image;

a second imaging step of obtaining an image of foreign matter adhering on the substrate as a second image by light with a wavelength which does not excite the fluorescent substance;

an extraction step of obtaining a binarized image by extracting a foreign matter area from the second image; and

a foreign matter elimination step of disabling an image at a part overlapping the foreign matter area in the first image with the binarized image being used as a mask;

wherein the extraction step obtains the binarized image by using a differential image acquired from the second image.

40. A fluorescent intensity measuring method according to claim 39, wherein the extraction step determines a binarization level of the binarized image by using a frequency distribution of the differential signal corresponding to each pixel.

41. A fluorescent intensity measuring method according to claim 40, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.

42. A fluorescent intensity measuring method according to claim 39, wherein the differential signal is standardized with an intensity in a minute area corresponding to the differential signal.