Automated system for combining bright field and fluorescent microscopy

• US 7,272,252 B2
• Filed: 06/12/2003
• Issued: 09/18/2007
• Est. Priority Date: 06/12/2002
• Status: Expired due to Fees

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Clariant GmbH (Clariant AG)
• Inventors: De La Torre-Bueno, Jose, McBride, James
• Primary Examiner(s): Johns; Andrew W.
• Application Number:
  US10/461,786
• Publication Number:
  US 20030231791A1
• Time in Patent Office: 1,559 Days
• Field of Search: 382/128, 382/133, 382/134, 382/318, 382/319, 356/39, 128/922
• US Class Current: 382/133
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 5/50 using two or more images, e.g. averaging or subtraction
• CPC Class Codes: G06T 7/0012 Biomedical image inspection

Abstract

A method and apparatus for automated analysis of transmitted and fluorescently labeled biological samples, wherein the apparatus automatically scans at a low magnification to acquire images which are analyzed to determine candidate cell objects of interest. Once candidate objects of interest are identified, further analysis is conducted automatically to process and collect data from samples having different staining agents.

Claims

1. A method for processing a biological sample, comprising

(a) contacting a biological sample with an agent or combination of agents that stains the biological sample for objects of interest and at least one fluorescent agent that interacts with an at least one molecule in the biological sample, wherein the at least one molecule is associated with the object of interest, and wherein the at least one fluorescents agent comprises a fluorescent indicator;

(b) acquiring a plurality of images of the biological sample at a plurality of locations/coordinates in transmitted light;

(c) processing the plurality of images to identify a stained object of interest;

(d) determining a coordinate for each identified stained object of interest;

(e) storing each of the determined coordinates corresponding to each identified stained object of interest;

(f) applying an excitation light to the biological sample thereby causing the fluorescent indicator of the at least one fluorescent agent to fluoresce;

(g) acquiring a fluorescent images at each of the identified coordinates; and

(h) processing the fluorescent images to identify a fluorescently labeled at least one molecule.

2. The method of claim 1, wherein the biological sample is a tissue sample.

3. The method of claim 2, wherein the tissue sample is suspected of comprising cells having a cell proliferative disorder.

4. The method of claim 3, wherein the cell proliferative disorder is a neoplasm.

5. The method of claim 4, wherein the cell proliferative disorder is breast cancer.

6. The method of claim 1, wherein the agent is a stain selected from the group consisting of DAB, New Fuchsin, AEC, and hematoxalin.

7. The method of claim 6, wherein the object of interest is a cell.

8. The method of claim 6, wherein the object of interest is a nucleus of a cell.

9. The method of claim 1, wherein the agent is an antibody.

10. The method of claim 9, wherein the antibody specifically interacts with a cancer marker comprising a protein or polypeptide.

11. The method of claim 9, wherein the antibody is selected from the group consisting of an anti-HER2/neu antibody, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, and anti-cyclin D1 antibody.

12. The method of claim 9, wherein the antibody is enzymatically labeled.

13. The method of claim 1, wherein the plurality of images are acquired at a low or a high magnification.

14. The method of claim 1, wherein the at least one fluorescent agent is selected from the group consisting of an antibody, a protein, a polypeptide, a peptidomimetic, a polynucleotide, an oligonucleotide, a small molecule, and any combination thereof.

15. The method of claim 1, wherein the fluorescent image is acquired at a low or a high magnification.

16. The method of claim 1, wherein the method is automated.

17. A method for processing a biological sample, comprising

(a) dividing a biological sample into a plurality of subsamples with respect to a z-axis;

(b) contacting a first subsample with an agent or combination of agents that stains the first subsample for objects of interest;

(c) acquiring a plurality of images of the first subsample at a plurality of locations/coordinates in transmitted light;

(d) processing the plurality of images to identify a stained object of interest;

(e) determining a coordinate for each identified stained object of interest;

(f) storing each of the determined coordinates corresponding to each identified stained object of interest;

(g) contacting a second subsample with an at least one fluorescent agent that interacts with an at least one molecule in the biological sample, wherein the at least one molecule is associated with the object of interest, and wherein the at least one fluorescent agent comprises a fluorescent indicator;

(h) applying an excitation light to the second subsample thereby causing the fluorescent indicator of the at least one fluorescent agent to fluoresce;

(i) acquiring a fluorescent images of the second subsample at each of the identified coordinates; and

(j) processing the fluorescent images to identify a fluorescently labeled molecule.

18. The method of claim 17, wherein the biological sample is a tissue sample.

19. The method of claim 18, wherein the tissue sample is suspected of comprising cells having a cell proliferative disorder.

20. The method of claim 19, wherein the cell proliferative disorder is a neoplasm.

21. The method of claim 19, wherein the cell proliferative disorder is breast cancer.

22. The method of claim 19, wherein the fluorescent agent further characterizes the cells having a cell proliferative disorder.

23. The method of claim 17, wherein the agent is a stain selected from the group consisting of DAB, New Fuchsin, AEC, and hematoxalin.

24. The method of claim 17, wherein the object of interest is a cell.

25. The method of claim 17, wherein the object of interest is a nucleus of a cell.

26. The method of claim 17, wherein the agent is an antibody.

27. The method of claim 26, wherein the antibody is enzymatically labeled.

28. The method of claim 26, wherein the antibody specifically interacts with a cancer marker comprising a protein or polypeptide.

29. The method of claim 26, wherein the antibody is selected from the group consisting of an anti-HER2/neu antibody, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, and anti-cyclin D1 antibody.

30. The method of claim 17, wherein the plurality of images are acquired at a low or a high magnification.

31. The method of claim 17, wherein the at least one fluorescent agent is selected from the group consisting of an antibody, a protein, a polypeptide, a peptidomimetic, a polynucleotide, an oligonucleotide, and a small molecule.

32. The method of claim 17, wherein the fluorescent image is acquired at a low or a high magnification.

33. The method of claim 17, wherein the method is automated.

34. A method for identifying a therapeutic treatment for a subject having a cell proliferative disorder, comprising:

(a) dividing a biological sample from the subject into a plurality of subsamples with respect to a z-axis, wherein adjacent subsamples in the z-axis will comprise substantially identical x- and y-coordinates with respect to an object of interest found in the adjacent subsamples;

(b) contacting a first subsample with an agent or combination of agents that stains the first subsample for cells suspected of having a cell proliferative disorder;

(c) acquiring a plurality of images of the first subsample at a plurality of locations/coordinates in transmitted light;

(d) processing the plurality of images to identify a stained cell;

(e) determining a coordinate for each identified stained cell;

(f) storing each of the determined coordinates corresponding to each identified stained cell;

(g) contacting a second subsample with an at least one fluorescent agent that interacts with an at least one molecule in the biological sample, wherein the at least one molecule is associated with a biological pathway in cells that causes cell proliferative disorders, and wherein the at least one fluorescent agent comprises a fluorescent indicator;

(h) applying an excitation light to the second subsample thereby causing the fluorescent indicator of the at least one fluorescent agent to fluoresce;

(i) acquiring a fluorescent images of the second subsample at each of the identified coordinates; and

(j) processing the fluorescent images to identify a fluorescently labeled molecule, wherein the presence of a fluorescent labeled molecule is indicative of the biological pathway in the cell that causes the cell proliferative disorder thereby allowing the identification of a therapeutic treatment.

35. A system for processing of a biological sample, comprising:

an image acquisition system, comprising a computer;

a monitor in operable communication with the computer;

an input device in communication with the computer;

a storage device for storing data; and

an optical system in operable communication with the computer, the optical system comprising;

a movable stage;

a visible wavelength light source adjacent to the movable stage such that light from the visible wavelength light source illuminates a sample on the movable stage;

a fluorescent excitation light source adjacent to the movable stage such that light from the fluorescent excitation light source illuminates a sample on the movable stage;

an optical sensing array in optical communication with the movable stage and configured to acquire an image at a plurality of locations in a scan area; and

an image processor in electrical communication with the sensing array and operable to process the image to detect an object of interest;

a computer program on computer readable medium with instructions to cause the image acquisition system to carry out a method comprising;

processing a first subsample that has been stained with an agent that stains an object of interest;

acquiring a plurality of images of the first subsample at a plurality of locations/coordinates in transmitted visible light;

processing the plurality of images to identify a stained object of interest;

determining a coordinate for each identified stained object of interest;

storing each of the determined coordinates corresponding to each identified stained object of interest;

processing a second subsample that has been contacted with at least one fluorescent agent that interacts with an at least one molecule in the biological sample, wherein the at least one fluorescent agent comprises a fluorescent indicator;

applying a fluorescent excitation light to the second subsample thereby causing the fluorescent indicator of the at least one fluorescent agent to fluoresce;

acquiring a fluorescent images of the second subsample at each of the identified coordinates; and

processing the fluorescent images to identify a fluorescently labeled molecule.

36. The system of claim 35, further comprising:

a filter wheel comprising a plurality of fluorescent filters interposed between the fluorescent excitation light source and the stage; and

a filter wheel control device for changing fluorescent filters during operation, wherein the filter wheel control device is in electrical communication with the computer.

37. The system of claim 35, further comprising an identification device for identifying and determining a processing parameter for a slide.

38. The system of claim 35, further comprising an automated loading and unloading member for loading and unloading of the slide.

39. The system of claim 37, wherein the processing parameter is an indication as to the visible wavelength light source or the fluorescent excitation light source and the fluorescent filter.

40. The system of claim 35, wherein the biological sample is a tissue sample.

41. The system of claim 40, wherein the tissue sample is suspected of comprising cells having a cell proliferative disorder.

42. The system of claim 41, wherein the cell proliferative disorder is a neoplasm.

43. The system of claim 41, wherein the cell proliferative disorder is breast cancer.

44. The system of claim 35, wherein the agent is a stain selected from the group consisting of DAB, New Fuchsin, AEC, and hematoxalin.

45. The system of claim 35, wherein the object of interest is a cell.

46. The system of claim 35, wherein the object of interest is a nucleus of a cell.

47. The system of claim 35, wherein the agent is an antibody.

48. The system of claim 47, wherein the antibody specifically interacts with a cancer marker comprising a protein or polypeptide.

49. The system of claim 47, wherein the antibody is selected from the group consisting of an anti-HER2/neu antibody, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, and anti-cyclin D1 antibody.

50. The system of claim 47, wherein the antibody is enzymatically labeled.

51. The system of claim 35, wherein the plurality of images are acquired at a low or a high magnification.

52. The system of claim 35, wherein the at least one fluorescent agent is selected from the group consisting of an antibody, a protein, a polypeptide, a peptidomimetic, a polynucleotide, an oligonucleotide, and a small molecule.

53. The system of claim 35, wherein the fluorescent image is acquired at a low or a high magnification.

54. The system of claim 52, wherein the fluorescent agent further characterizes the cells having a cell proliferative disorder.