SYSTEMS AND METHODS FOR COUNTING CELLS AND BIOMOLECULES

• US 20100189338A1
• Filed: 03/16/2009
• Published: 07/29/2010
• Est. Priority Date: 04/09/2008
• Status: Abandoned Application

• Current Assignee: Revvity Health Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Nexcelom Bioscience, LLC (Revvity, Inc.)
• Inventors: Lin, Bo, Smith, Timothy, Li, Peter Y., Pirani, Alnoor, Sobolewski, Todd, Qiu, Jean
• Application Number:
  US12/404,803
• Publication Number:
  US 20100189338A1
• Time in Patent Office: Days
• US Class Current: 382/133
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1486 Counting the particles

Abstract

The present invention generally relates to systems and methods for counting biomolecules or cells. In certain embodiments, the invention provides a cell counting or biomolecule counting system including: a covered chamber having a known height and configured to hold a suspension of biomolecules or cells in a sample; at least one fluorescent light source connected to at least one fluorescent light beam narrowing device; a bright-field light source connected to a bright-field light beam narrowing device; a microscope objective; a detection device; a fluorescent filter assembly to allow only excitation light to illuminate the sample and allow only emission light from the sample to be imaged by the detection device; and a movable light shutter to block bright-field light during fluorescent detection.

Claims

1. A system for counting cells or biomolecules comprising:

a covered chamber having a known height and configured to hold a suspension of biomolecules or cells in a sample;

at least one fluorescent light source connected to at least one fluorescent light beam narrowing device;

a bright-field light source connected to a bright-field light beam narrowing device;

a microscope objective;

a detection device;

a fluorescent filter assembly to allow only excitation light to illuminate the sample and allow only emission light from the sample to be imaged by the detection device; and

a movable light shutter to block bright-field light during fluorescent detection.

2. The system according to claim 1, further comprising a computer operably connected to the cell counting system.

3. The system according to claim 2, further comprising analysis software installed on the computer.

4. The system according to claim 1, wherein the fluorescent light beam narrowing device is a collimator.

5. The system according to claim 1, wherein the bright-field light beam narrowing device is a collimator.

6. The system according to claim 1, wherein the detection device is a camera.

7. The system according to claim 6, wherein the camera is a CCD camera.

8. The system according to claim 1, wherein the covered chamber comprises a sample introduction port and an air escape port.

9. The system according to claim 8, wherein the covered chamber further comprises a counting grid.

10. The system according to claim 9, wherein the counting grid is an integral part of a top of the chamber.

11. The system according to claim 9, wherein the counting grid is an integral part of a bottom of the chamber.

12. The system according to claim 9, wherein width of lines of the counting grid range from about 0.1 micrometer to about 1 mm.

13. The system according to claim 9, wherein width of lines of the counting grid range from about 1 micrometer to about 25 micrometer.

14. The system according to claim 9, wherein thickness of lines of the counting grid range from about 0.1 micrometer to about 50 micrometer.

15. The system according to claim 1, wherein each of the fluorescent light source and the bright-field light source is a light emitting diode.

16. The system according to claim 1, wherein the system is configured to capture entirety of the sample with a single image.

17. The system according to claim 1, wherein the system is configured to capture a portion of the sample with a single image.

18. The system according to claim 1, wherein the sample a human tissue or body fluid.

19. The system according to claim 1, wherein the biomolecules are selected from the group consisting of DNA, RNA, and protein.

20. The system according to claim 1, wherein the sample is selected from the group consisting of serum, cell culture supernatant, and cell lysis.

21. The system according to claim 1, wherein the biomolecule or cell is indicative of a disease or disease state.

22. A method for determining a concentration or number count of cells that express a biomarker in a population of cells in a sample comprising:

contacting a sample comprising cells that express a biomarker with a fluorescently labeled agent that specifically binds the biomarker;

loading the sample into a covered chamber having a known height, wherein the population of cells is suspended within the chamber;

acquiring a single static bright-field image of the population of cells in the sample in the chamber;

acquiring a single static fluorescent image of the population of cells in the sample in the chamber; and

comparing cell count from the bright-field image to cell count from the fluorescent image to determine the concentration or number count of the cells that express the biomarker in the population of cells.

23. The method according to claim 22, wherein prior to said contacting step, the method further comprises contacting the population of cells with an agent that makes the population of cells permeable.

24. The method according to claim 22, wherein the biomarker is a cell surface biomarker.

25. The method according to claim 22, wherein the biomarker is an intercellular biomarker.

26. The method according to claim 22, wherein the fluorescently labeled agent is a fluorescently labeled antibody that possesses an epitope for the biomarker.

27. The method according to claim 22, wherein the biomarker is associated with a particular disease or disease state.

28. The method according to claim 22, wherein the sample is a human tissue or body fluid.

29. The method according to claim 22, wherein each of the single static bright-field image and the single static fluorescent image is an image of the entire sample.

30. The method according to claim 22, wherein each of the single static bright-field image and the single static fluorescent image is an image of a portion of the sample.

31. A method for determining a concentration or number count of stem cells in a population of cells in a sample comprising:

contacting a sample comprising stem cells with a fluorescently labeled agent that specifically binds the stem cells in the sample;

loading the sample into a covered chamber having a known height, wherein the population of cells is suspended within the chamber;

acquiring a single static bright-field image of the population of cells in the sample in the chamber;

acquiring a single static fluorescent image of the population of cells in the sample in the chamber; and

comparing cell count from the bright-field image to cell count from the fluorescent image to determine the concentration or number count of stem cells in the population of cells.

32. The method according to claim 31, wherein the fluorescently labeled agent is a fluorescently labeled antibody specific for a stem cell biomarker, or a particle coated with a fluorescently labeled antibody specific for a stem cell biomarker.

33. The method according to claim 31, wherein the biomarker is selected from the group consisting of TRA-1-81, TRA-1-60, Thy-1, SSEA-3, SSEA4, Oct-4, CD9, CD30, and alkaline phosphatase.

34. The method according to claim 31, wherein each of the single static bright-field image and the single static fluorescent image is an image of the entire sample.

35. The method according to claim 31, wherein each of the single static bright-field image and the single static fluorescent image is an image of a portion of the sample.

36. A method for determining infection rates of malaria in a subject comprising:

contacting a sample of red blood cells from a subject having malaria with a fluorescently labeled agent specific for a malaria parasite;

loading the sample into a covered chamber having a known height, wherein the cells are suspended within the chamber;

acquiring a single static bright-field image of the cells in the sample in the chamber;

acquiring a single static fluorescent image of the cells in the sample in the chamber; and

comparing cell count from the bright-field image to cell count from the fluorescent image to determine the infection rate of malaria in the subject.

37. The method according to claim 36, wherein the subject is human.

38. The method according to claim 36, wherein each of the single static bright-field image and the single static fluorescent image is an image of the entire sample.

39. The method according to claim 36, wherein each of the single static bright-field image and the single static fluorescent image is an image of a portion of the sample.

40. A method for identifying or number count adipocytes in sample comprising:

contacting a sample comprising adipocytes with a fluorescently labeled agent that specifically binds the adipocytes;

loading the sample into a covered chamber having a known height, wherein the cells are suspended within the chamber;

acquiring a single static bright-field image in the chamber of the sample;

acquiring a single static fluorescent image in the chamber of the sample; and

comparing the bright-field image to the fluorescent image to identify or number count the adipocytes.

41. The method according to claim 40, wherein the fluorescent label is a DNA fluorescent dye.

42. The method according to claim 40, wherein each of the single static bright-field image and the single static fluorescent image is an image of the entire sample.

43. The method according to claim 40, wherein each of the single static bright-field image and the single static fluorescent image is an image of a portion of the sample.

44. A method for detecting a biomolecule in a sample comprising:

contacting a sample with particles coated with a biotinylated antibody, and streptavidin coupled to a fluorescent indicator;

loading the sample into a covered chamber having a known height, wherein the biomolecules are suspended within the chamber;

acquiring a single static bright-field image of the sample in the chamber;

acquiring a static fluorescent image of the sample in the chamber; and

comparing the bright-field image to the fluorescent image to detect the biomolecule in the sample.

45. The method according to claim 44, wherein prior to acquiring the bright-field image, the method further comprises washing unbound particles.

46. The method according to claim 44, wherein the biomolecule is selected from the group consisting of DNA, RNA, and protein.

47. The method according to claim 44, wherein the biomolecule is associated with a particular disease or disease state.

48. The method according to claim 44, wherein the sample is a human tissue or body fluid.

49. The method according to claim 44, wherein the sample is selected from the group consisting of serum, cell culture supernatant, and cell lysis.

50. The method according to claim 44, wherein each of the single static bright-field image and the single static fluorescent image is an image of the entire sample.

51. The method according to claim 44, wherein each of the single static bright-field image and the single static fluorescent image is an image of a portion of the sample.

52. A method for determining a concentration or number count of viable hepatocytes in a population of hepatocytes in a sample comprising:

contacting a sample comprising hepatocytes with at least one fluorescently labeled agent;

loading the sample into a covered chamber having a known height, wherein the population of hepatocytes is suspended within the chamber;

acquiring two or more static images of the population of hepatocytes, wherein the first and second image are selected from the group consisting of a bright-field image, a fluorescent image of viable hepatocytes, and a fluorescent image of dead hepatocytes, wherein the first image is different from the second image;

comparing cell count from the first image to cell count from the second image to determine the concentration or number count of viable hepatocytes in the population of hepatocytes.

53. The method according to claim 52, wherein the population of hepatocytes is contacted with two different fluorescent agents.

54. The method according to claim 53, wherein a first fluorescent agent specifically binds viable hepatocytes, and a second fluorescent agent specifically binds dead hepatocytes.

55. The method according to claim 54, wherein the first fluorescent agent is acridine orange and the second fluorescent agent is propidium iodine.

56. The method according to claim 54, wherein the first image is the fluorescent image of live hepatocytes, the second image is the fluorescent image of dead hepatocytes, and the concentration of viable hepatocytes in the population of hepatocytes is determined by comparing cell count from the fluorescent image of live hepatocytes to cell count from the fluorescent image of dead hepatocytes.

57. The method according to claim 54, wherein the first image is the bright-field image, the second image is the fluorescent image of dead hepatocytes, and the concentration of viable hepatocytes in the population of hepatocytes is determined by comparing cell count from the bright-field image to cell count from the fluorescent image of dead hepatocytes.

58. The method according to claim 54, wherein the first image is the bright-field image, the second image is the fluorescent image of live hepatocytes, and the concentration of viable hepatocytes in the population of hepatocytes is determined by comparing cell count from the bright-field image to cell count from the fluorescent image of live hepatocytes.

59. The method according to claim 52, wherein the concentration of viable hepatocytes in the population of hepatocytes is correlated with a particular disease or disease state.

60. The method according to claim 52, wherein each of the first and second image is an image of the entire sample.

61. The method according to claim 52, wherein each of the first and second image is an image of a portion of the sample.

62. A system for counting cells or biomolecules comprising:

a closed chamber configured to hold a suspension of biomolecules or cells in a sample, and to allow calculation of a volume of sample that is interrogated;

at least one fluorescent light source connected to at least one fluorescent light beam narrowing device;

a bright-field light source connected to a bright-field light beam narrowing device;

a microscope objective;

a detection device;

a fluorescent filter assembly to allow only excitation light to illuminate the sample and allow only emission light from the sample to be imaged by the detection device; and

a movable light shutter to block bright-field light during fluorescent detection.