Luminescence-scanning microscopy process and a luminescence scanning microscope utilizing picosecond or greater pulse lasers

• US 5,777,732 A
• Filed: 04/25/1996
• Issued: 07/07/1998
• Est. Priority Date: 04/28/1994
• Status: Expired due to Term

• Current Assignee: Hell, Stefan, Pekka Hanninen
• Original Assignee: Hell, Stefan, Pekka Hanninen
• Inventors: Hell, Stefan, Hanninen, Pekka
• Primary Examiner(s): McGraw, Vincent P.
• Application Number:
  US08/571,839
• Time in Patent Office: 803 Days
• Field of Search: 356/318, 250/459.1
• US Class Current: 356/318
• CPC Class Codes: G02B 21/002 Scanning microscopes scanning near field optical microscopes G01Q60/18
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence

Abstract

The description relates to a process for luminescence scanning microscopy with two-photon excitation, especially for examining biological objects (5). A laser pulse excites luminescent, especially fluorescing molecules and the luminescence emitted by the object (5) is measured and evaluated. In the process, the luminescent molecules in the object (5) are excited by laser pulses of over 10^-12 second duration. A luminescence scanning microscope for implementing the process has a detector (2), a filter (7, 8) for separating the light emitted by the sample from the laser light (4) and a laser light source which is a laser (1) emitting pulsed or continuous radiation.

Claims

1. Process for luminescence-scanning microscopy with two-photon excitation of an object in which a laser pulse excites luminescing and/or fluorescing molecules of the object, and the luminescence light emitted by the object is measured and evaluated, wherein the excitation of the luminescing molecules present in the object results from laser pulses whose duration is greater than 10^-12 seconds.

2. Process according to claim 1, wherein the evaluation of the light of luminescence emitted by the object (5) is accomplished in the process of photon counting.

3. Process according to claim 1, wherein several scanning points of the object (5) are excited simultaneously and the light of luminescence emitted by these scanning points evaluated simultaneously.

4. Process according to claim 1, wherein said measurement and evaluation step is implemented with a luminescence-scanning microscope comprising at least one laser-light source, at least one detector and at least one filter for the separation of the light emitted by the sample from the laser light, and wherein said at least one laser-light source is a laser (1) with pulsed radiation.

5. Luminescence-scanning microscope according to claim 4, wherein the laser is a semiconductor laser.

6. Luminescence-scanning microscope according to claim 4, wherein the laser is a gas laser.

7. Luminescence-scanning microscope according to claim 6, wherein the laser is an HeNe laser or a krypton-ion laser.

8. Luminescence-scanning microscope according to claim 4, wherein said at least one detector (2) is a high, signal-noise ratio detector.

9. Luminescence-scanning microscope according to claim 4, wherein said microscope further comprises a beam-scanning unit (6) for the controlled scanning of the object (5).

10. Luminescence-scanning microscope according to claim 4, wherein said at least one laser-light source comprises an array of lasers (1) and wherein said at least one detector comprises an array of detectors (2) arranged in an optically conjugated plane for the focusing of the object (5).

11. Luminescence-scanning microscope according to claim 4, wherein said at least one detector is an avalanche photodiode.

12. Process for luminescence-scanning microscopy with two-photon excitation of an object in which a laser pulse excites luminescing and/or fluorescing molecules of the object, and the luminescence light emitted by the object is measured and evaluated, wherein the excitation of the luminescing molecules present in the object results from laser pulses whose duration is 10^-12 seconds.

13. Process according to claim 12, wherein the evaluation of the light of luminescence emitted by the object (5) is accomplished in the process of photon counting.

14. Process according to claim 12, wherein several scanning points of the object (5) are excited simultaneously and the light of luminescence emitted by these scanning points evaluated simultaneously.

15. Process according to claim 12, wherein said measurement and evaluation step is implemented with a luminescence-scanning microscope comprising at least one laser-light source, at least one detector and at least one filter for the separation of the light emitted by the sample from the laser light, and wherein said at least one laser light source is a laser (1) with pulsed radiation.

16. Luminescence-scanning microscope according to claim 15, wherein the laser is a semiconductor laser.

17. Luminescence-scanning microscope according to claim 15, wherein the laser is a gas laser.

18. Luminescence-scanning microscope according to claim 17, wherein the laser is an HeNe laser or a krypton-ion laser.

19. Luminescence-scanning microscope according to claim 15, wherein said at least one detector is a high signal-noise ratio detector.

20. Luminescence-scanning microscope according to claim 15, wherein said microscope further comprises a beam-scanning unit (6) for the controlled scanning of the object (5).

21. Luminescence-scanning microscope according to claim 15, wherein said at least one laser-light source comprises an array of lasers (1) and wherein said at least one detector comprises an array of detectors (2) arranged in an optically conjugated plane for the focusing of the object (5).

22. Luminescence-scanning microscope according to claim 15, wherein said at least one detector is an avalanche photodiode.

23. Process for luminescence-scanning microscopy with two-photon excitation for an object, in which a laser light excites luminescing and/or fluorescing molecules of the object, and the luminescence light emitted by the object is measured and evaluated, wherein the excitation of the luminescing molecules present in the object results from continuous laser light.

24. Process according to claim 23, wherein said measurement and evaluation step is implemented with a luminescence-scanning microscope comprising at least one laser-light source, at least one detector and at least one filter for the separation of the light emitted by the sample from the laser light, and wherein said at least one laser-light source is a laser (1) with continuous radiation.

25. Process according to claim 23 wherein the evaluation of the light of luminescence emitted by the object (5) is accomplished in the process of photon counting.

26. Process according to claim 23 wherein several scanning points of the object (5) are excited simultaneously and the light of luminescence emitted by these scanning points evaluated simultaneously.

27. Luminescence-scanning microscope according to claim 24, wherein the laser is a semiconductor laser.

28. Luminescence-scanning microscope according to claim 24, wherein the laser is a gas laser.

29. Luminescence-scanning microscope according to claim 28, wherein the laser is an HeNe laser or a krypton-ion laser.

30. Luminescence-scanning microscope according to claim 24, wherein said at least one detector is a high signal-noise ratio detector.

31. Luminescence-scanning microscope according to claim 24, wherein said microscope further comprises a beam-scanning unit (6) for the controlling scanning of the object (5).

32. Luminescence-scanning microscope according to claim 24, wherein said at least one laser-light source comprises an array of lasers (1) and wherein said at least one detector comprises an array of detectors (2) arranged in an optically conjugated plane for the focusing of the object (5).

33. Luminescence-scanning microscope according to claim 24, wherein said at least one detector is an avalanche photodiode.