Method and apparatus for detecting fluorescence of a sample

• US 6,970,246 B2
• Filed: 04/11/2001
• Issued: 11/29/2005
• Est. Priority Date: 04/11/2000
• Status: Active Grant

• Current Assignee: ChemoMetec A/S
• Original Assignee: ChemoMetec A/S
• Inventors: Hansen, Frans Ejner Ravn
• Primary Examiner(s): Evans, F. L.
• Application Number:
  US10/257,271
• Publication Number:
  US 20040061070A1
• Time in Patent Office: 1,693 Days
• Field of Search: 356/317, 356/318, 356/417, 250/458.1, 250/459.1, 250/461.1, 250/461.2
• US Class Current: 356/417
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/361 Optical details, e.g. image relay to the camera or image sensor G02B21/364 takes precedence; illumination details G02B21/06 and subgroups

Abstract

The present invention relates to an apparatus and a method for detecting the fluorescence of a sample, wherein the apparatus comprises excitation light source located on the same side of the sample as the detecting means. The apparatus may be constructed as a single-sided as well as a double-sided system, wherein the double-sided system comprises double-sided excitation system and/or double-sided detecting system. The double-sided system may also be a combination of fluorescence system and conventional microscopy. The apparatus may be used for analysis of various types of biological material, liquid as well as solid material, and various other types of material suitable being analysed through fluorescence studies.

Claims

1. An apparatus for detecting the fluorescence of a sample comprising:

a first excitation light system including at least a first light source, the first light source having a main light path;

a sample plane for positioning said sample;

a detection device including at least a first detector for detecting fluorescence signals from the sample, an axis between the detection device and the sample plane being a detection-sample axis;

a processor coupled to receive data from the detector;

a focusing device for focusing the signals onto the detection device, said focusing device having a collection angle, an angle between the main light path of the first light source and the detection-sample axis being between the collection angle/2 and 90°

, and the focusing device providing a magnification in a range of 2/1 to 1/10.

2. The apparatus according to claim 1, wherein at least the first light system is located in a first light plane parallel to the sample plane, said first light plane being between the sample plane and the detection device.

3. The apparatus according to claim 1, wherein the first light source is selected from a light emitting diode, a laser diode, a laser, a thermal light source, and a gas discharge lamp.

4. The apparatus according to claim 1, wherein an excitation light filter is inserted in the main light path from said first light source.

5. The apparatus according to claim 1, wherein said first light system includes a plurality of light sources and substantially identical filters are used for all the light sources.

6. The apparatus according to claim 1, wherein the first excitation light system includes at least two light sources, and excitation light filters for each light source are connected to each other on a supporting material.

7. The apparatus according to claim 6, wherein the supporting material has a shape selected from circular, rectangular, square, semi-circular.

8. The apparatus according to claim 1, wherein the first light source is filtered through a first filter, and a second light source is filtered through a second filter, the first filter and the second filter being different.

9. The apparatus according to claim 1, wherein a second excitation light system is located in a second light plane, said second light plane being parallel with the sample plane and located on the other side of the sample plane relative to the first light plane allowing the sample to receive excitation light from two sides.

10. The apparatus according to claim 9, wherein a filter inserted in a light path from the second light system is different from a filter inserted in the main light path of the first light source.

11. The apparatus according to claim 1, wherein the detection device includes an array of detectors.

12. The apparatus according to claim 1, wherein the detection device includes an array of charged coupled devices.

13. The apparatus according to claim 1, wherein an additional detection device is arranged so that the sample plane is positioned between the detection device and the additional detection device.

14. The apparatus according to claim 13, wherein the detection device is identical with the additional detection device.

15. The apparatus according to claim 1, wherein an emission light filter is inserted in an emission light path to at least the first detector.

16. The apparatus according to claim 15, wherein the emission light filter is selected from interference filters, coloured filters, and polarisation filters.

17. The apparatus according to claim 1, wherein the focusing device is a lens.

18. The apparatus according to claim 1, wherein a detection area of the sample is at least 0.1 mm².

19. The apparatus according to claim 1, wherein an angle between the main light path and the detection-sample is in a range between 35°

and 90°

.

20. The apparatus according to claim 1, wherein at least the first light system is located in a first light plane parallel to the sample plane, said first light plane being positioned at a distance from the sample plane behind the detector.

21. The apparatus according to claim 20, wherein the detector is positioned in a housing having an opening towards the sample.

22. The apparatus according to claim 1, wherein the focusing device provides a magnification in a range of 2/1 to 1/4.

23. The apparatus according to claim 22, wherein the detector is positioned in a housing having an opening towards the sample.

24. A method of assessing a parameter of a sample comprising:

arranging the sample in a sample plane;

exposing a first surface of the sample directly with excitation light from a first light system having at least a first light source;

by use of a focusing device detecting a fluorescence signal from the first surface of the sample onto a detection device including at least a first detector, an axis between the detection device and the sample plane being a detection-sample axis, said focusing device having a collection angle, an angle between an excitation main light path of the first light source and the detection-sample axis being between the collection angle/2 and 90°

, and the focusing device providing a magnification in a range of 2/1 to 1/10;

processing the detected signal obtaining signal data;

correlating the signal data to the parameter to be assessed; and

assessing the parameter.

25. The method according to claim 24, wherein said first light system is located in a first light plane parallel to the sample plane, said first light plane being between the sample plane and the detection device.

26. The method according to claim 24, wherein the first light source is a light emitting diode.

27. The method according to claim 24, wherein an excitation light filter is inserted in an excitation light path from said first light source.

28. The method according to claim 27, wherein the excitation light filter is arranged on a supporting material.

29. The method according to claim 24, wherein said first light system includes a plurality of light sources and substantially identical filters are used for all the light sources.

30. The method according to claim 24, wherein said first light source is filtered through a first filter, and a second light source is filtered through a second filter, the first filter and the second filter being different.

31. The method according to claim 24, further comprising exposing a second surface of the sample directly with excitation light from a second light system having at least one light source.

32. The method according to claim 31, wherein the second excitation light system is located in a second light plane, said second light plane being parallel with the sample plane and located on the other side of the sample plane relative to the first light plane allowing the sample to be exposed on two opposite surfaces.

33. The method according to claim 31, wherein a filter inserted in a light path from the second light system is different from a filter inserted in the main light path of the first light source.

34. The method according to claim 24, wherein the detection device includes an array of detection devices.

35. The method according to claim 34, wherein the detection device includes an array of charged coupled devices.

36. The method according to claim 24, wherein an additional detection device is used that is arranged so that the sample plane is positioned between the detection device and the additional detection device.

37. The method according to claim 36, wherein the detection device is identical with the additional detection device.

38. The method according to claim 24, wherein an emission light filter is inserted in an emission light path to at least the first detector.

39.The method according to claim 24, wherein a collimating lens is arranged in an emission light path to at least the first detector.

39. The method according to claim 24, wherein the sample plane is provided with a detection area.

41. The method according to claim 24, wherein a sample compartment is arranged in the sample plane for housing the sample.

43.The method according to claim 24, wherein the angle between the excitation main light path and the detection-sample axis is in a range between 35°

and 90°

.

42. The method according to claim 24, wherein at least the first light system is located in a first light plane parallel to the sample plane, said first light plane being positioned at a distance from the sample plane behind the detector.

43. The method according to claim 44, wherein the detector is positioned in a housing having an opening allowing emitted signals to reach the detector.

44. The method according to claim 24, wherein the sample is a liquid sample.

48. The method according to claim 49, wherein the number of particles is assessed by the detection device and the morphology of the particles is assessed by an additional detection device.

49. The method according to claim 24, wherein the sample is a part of a solid material capable of emitting a fluorescence signal.

53. The method according to claim 24, wherein the excitation light is from at least two light sources, and excitation light filters for each light source are connected to each other on a supporting material.

40. The method according to claim 40, wherein said detection area is at least 0.1 mm².

46. The method according to claim 46, wherein a number of particles in said sample is assessed.

45. The method according to claim 46, wherein the fluorescence signal is related to a parameter of a particle in the sample.

47. The method according to claim 46, wherein a morphology of a number of particles in said sample is assessed.

51. The method according to claim 51, wherein the sample is a document or a note.

50. The method according to claim 51, wherein the sample is tissue, parts and/or cell aggregates.

52. The method according to claim 50, wherein the sample is a part of metal construction for detecting failure signals from the metal.