METHOD AND MICROSCOPE FOR HIGH SPATIAL RESOLUTION EXAMINATION OF SAMPLES

Abstract

A method and a microscope, in particular a laser scanning fluorescence microscope, for high spatial resolution examination of samples, the sample (1) to be examined comprising a substance that can be repeatedly converted from a first state (Z1, A) into a second state (Z2, B), the first and the second states (Z1, A; Z2, B) differing from one another in at least one optical property, comprising the steps that the substance in a sample region (P) to be recorded is firstly brought into the first state (Z1, A), and that the second state (Z2, B) is induced by means of an optical signal (4), spatially delimited subregions being specifically excluded within the sample region (P) to be recorded, are defined with regard to increasing resolution in any desired direction and with regard to an increased imaging rate by the fact that the optical signal (4) is simultaneously concentrated at a number of focal points, and the focal points are focused into various sites of the sample (1).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred refinements and developments of the teaching are also explained in general in conjunction with explanations of the preferred exemplary embodiments and with the aid of the drawing, in which

FIG. 1 shows a schematic of a cyclic illumination scheme of a method for high spatial resolution examination of samples, and

FIG. 2 shows a schematic of an exemplary embodiment of a microscope according to the invention.

Claims

1. A method for high spatial resolution examination of samples, the sample (1) to be examined comprising a substance that can be repeatedly converted from a first state (Z1, A) into a second state (Z2, B), the first and the second states (Z1, A; Z2, B) differing from one another in at least one optical property, comprising the steps that the substance in a sample region (P) to be recorded is firstly brought into the first state (Z1, A), and that the second state (Z2, B) is induced by means of an optical signal (4), spatially delimited subregions being specifically excluded within the sample region (P) to be recorded, wherein the optical signal (4) is simultaneously concentrated at a number of focal points, and the focal points are focused into various sites of the sample (1).

2. The method as claimed in claim 1, wherein the pupil functions of the individual focal points are modulated.

3. The method as claimed in claim 2, wherein the modulation is carried out in such a way that at least one intensity zero point (5) is produced at each focal point.

4. The method as claimed in claim 2, wherein the modulation is carried out by means of a phase filter (20).

5. The method as claimed in claim 4, wherein the phase filter (20) is arranged in a plane that is conjugate with the pupil of an objective (31) through which the sample (1) is illuminated, and that is situated between the objective (31) and the plane of the focal points produced.

6. The method as claimed in claim 4, wherein a phase filter array is used as phase filter (20).

7. The method as claimed in claim 1, wherein the focal points are produced by means of a lens arrangement (12) with a number of microlenses (13).

8. The method as claimed in claim 1, wherein the focal points are produced by means of a number of sequentially arranged beam splitters.

9. The method as claimed in claim 1, wherein the focal points are produced by means of a rotating diaphragm, by means of an array composed of optical fibers and/or by means of holographic elements.

10. The method as claimed in claim 7, wherein the devices (11) producing the focal points are arranged such that the focal points are produced in the image, in the intermediate image or in a plane conjugate therewith.

11. The method as claimed in claim 4, wherein a switching signal (2) for converting the substance into the first state (Z1, A), a test signal (7) for reading out the first state (Z1, A), and a measuring signal (8) emanating from the sample (1) are not influenced by the phase filter (20).

12. The method as claimed in claim 11, wherein the switching signal (2) and/or the test signal (7) and/or the measuring signal (8) are spatially separated from the optical signal (4) upstream of the phase filter (20).

13. The method as claimed in claim 12, wherein the spatial separation is carried out by means of one or more dichroic filters (21) and/or polarization filters.

14. The method as claimed in claim 13, wherein the switching signal (2) and/or the measuring signal (8) are/is coupled into or out of the beam path at a location between the phase filter (20) and the objective (31).

15. The method as claimed in claim 7, wherein the sample (1) is scanned by means of suitable movement of the devices (11) producing the focal points, in particular by lateral movement or rotation of the microlenses (13).

16. The method as claimed in claim 1, wherein the sample (1) is scanned by means of suitable movement of a scanning mirror (40) arranged in the beam path.

17. The method as claimed in claim 15, wherein the sample (1) is scanned synchronously with a cyclic irradiation of the switching signal (2), of the optical signal (4) and of the test signal (7), and with the reading out of the measuring signal (8).

18. The method as claimed in claim 11, wherein the measuring signal (8) emanating from the sample (1) is detected by means of a CCD camera (39) or of an EMCCD camera.

19. The method as claimed in claim 11, wherein the measuring signal (8) emanating from the sample (1) is detected by means of a detector array, preferably an APD array.

20. The method as claimed in claim 18, wherein the focal points produced are respectively assigned defined detector areas, preferably individual camera pixels and/or camera pixel areas.

21. The method as claimed in claim 18, wherein the detector areas are assigned pinholes.

22. The method as claimed in claim 18, wherein the recorded images are processed by means of electronic image processing.

23. A microscope, in particular a laser scanning fluorescence microscope, for high spatial resolution examination of samples and, in particular, for carrying out a method as claimed in claim 1, the sample (1) to be examined comprising a substance that can be repeatedly converted from a first state (Z1, A) into a second state (Z2, B), the first and the second states (Z1, A; Z2, B) differing from one another in at least one optical property, comprising the steps that the substance in a sample region (P) to be recorded is firstly brought into the first state (Z1, A), and that the second state (Z2, B) is induced by means of an optical signal (4), spatially delimited subregions being specifically excluded within the sample region (P) to be recorded, defined by a device (11) for simultaneously concentrating the optical signal (49) at a number of focal points, it being possible to focus the focal points into various sites of the sample (1).

24. The microscope as claimed in claim 23, defined by a phase filter (20) for modulating the pupil functions of the individual focal points.

25. The microscope as claimed in claim 24, wherein the phase filter (20) is arranged in a plane that is conjugate with the pupil of an objective (31) through which the sample (1) is illuminated, and that is situated between the objective (31) and the plane of the focal points produced.

26. The method as claimed in claim 24, wherein the phase filter (20) is designed as a phase filter array.

27. The microscope as claimed in claim 24, wherein the phase filter (20) is designed as a vapor-deposited structure on a substrate, as an achromatic phase filter or as an LCD.

28. The microscope as claimed in claim 24, wherein the phase filter (20) is designed in the form of a semicircle.

29. The microscope as claimed in claim 24, wherein the phase filter (20) is designed in the form of a circle.

30. The microscope as claimed in claim 24, wherein the phase filter (20) is designed as a phase clock.

31. The microscope as claimed in claim 23, defined by a lens arrangement (12) with a number of microlenses (13) for producing the focal points.

32. The microscope as claimed in claim 31, wherein the phase filter (20) is arranged in the plane of the lens arrangement (12) or in a plane conjugate therewith.

33. The microscope as claimed in claim 31, wherein the phase filter (20) is designed as a structure vapor-deposited onto the microlenses (13).

34. The microscope as claimed in claim 23, defined by a number of sequentially arranged beam splitters for producing the focal points.

35. The microscope as claimed in claim 23, defined by a rotating diaphragm, an array composed of optical fibers, and/or holographic elements for producing the focal points.

36. The microscope as claimed in claim 23, wherein the devices (11) producing the focal points are arranged such that the focal points are produced in the image, in the intermediate image or in a plane conjugate therewith.

37. The microscope as claimed in claim 23, defined by a modular design.

38. The microscope as claimed in claim 23, wherein individual components and/or subassemblies and/or the overall structure are mounted in a housing.

39. The microscope as claimed in claim 23, defined by preferably electronically controlled adjusting means.

40. The microscope as claimed in claim 23, defined by sensors, arranged in the beam path, for detecting the beam position and/or focal position.

41. The microscope as claimed in claim 23, defined by a control for automatic readjustment.