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, A), 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 in that the optical signal (4) is provided in the form of a focal line (10) with a cross-sectional profile having at least one intensity zero point (5) with laterally neighboring intensity maxima (9).

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,

FIG. 2 shows a schematic of the production of a focal line with a cross-sectional profile having a single intensity maximum,

FIG. 3 shows a schematic of the inventive production of a focal line with a cross-sectional profile having at least one intensity zero point with laterally neighboring intensity maxima,

FIG. 4 shows a schematic of the design of a first exemplary embodiment of an inventive microscope, and

FIG. 5 shows a schematic of the design of a second exemplary embodiment of an inventive microscope.

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, A), 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 provided in the form of a focal line (10) with a cross-sectional profile having at least one intensity zero point (5) with laterally neighboring intensity maxima (9).

2. The method as claimed in claim 1, the sample (1) being illuminated by an objective (3, 13), wherein the focal line (10) is produced by means of a linear illumination in a pupil plane (P3) conjugate with the pupil (P1) of the objective (3, 13)—pupil line (14)—and by suitable phase modulation along the pupil line (14).

3. The method as claimed in claim 2, wherein the phase modulation is undertaken in such a way that one or more phase jumps are introduced along the pupil line (14).

4. The method as claimed in claim 3, wherein a phase jump is introduced at the pupil midpoint.

5. The method as claimed in claim 4, wherein the introduced phase jump corresponds to the length of half a wave train.

6. The method as claimed in claim 2, wherein a number of phase jumps by in each case half a wave train are introduced along the pupil line (14).

7. The method as claimed in claim 2, wherein the phase modulation along the pupil line (14) is implemented by means of a spatial liquid-based phase modulator in an intermediate image of the pupil (P1).

8. The method as claimed in claim 2, wherein the phase modulation along the pupil line (14) is implemented by means of an optical component (19, 27) having a phase-retarding optical coating (PV) and arranged in an intermediate image of the pupil (P1).

9. The method as claimed in claim 2, wherein the phase modulation along the pupil line (14) is implemented by means of an optical component arranged in an intermediate image of the pupil (P1), the component firstly generating a phase jump by total reflection, and subsequently generating a reflection at a metal layer.

10. The method as claimed in claim 2, characterized in that the phase modulation along the pupil line (14) is implemented by means of an optical element arranged in an intermediate image of the pupil (P1), the element having one or more deformable and/or movable mirrors and/or mirror elements.

11. The method as claimed in claim 2, wherein the pupil line (14) is produced by focusing an illuminating light beam of an illuminating light source with a cylindrical lens (17) or a Powell lens.

12. The method as claimed in claim 2, wherein the pupil line (14) is produced by imaging a split diaphragm into the pupil plane.

13. The method as claimed in claim 2, wherein the pupil line (14) is produced by means of holographic elements.

14. The method as claimed in claim 2, wherein the pupil line (14) is coupled into the beam path by means of an Achrogate filter.

15. The method as claimed in claim 14, wherein the phase modulation along the pupil line (14) is implemented by means of additional phase-retarding layers on the Achrogate filter.

16. The method as claimed in claim 1, wherein a laser is used as an illuminating light source for providing the optical signal (4).

17. The method as claimed in claim 2, wherein the light of the optical signal (4) is polarized perpendicular to the pupil line (14).

18. The method as claimed in claim 1, wherein the focal line (10) is produced sequentially in different spatial directions.

19. The method as claimed in claim 18, wherein the sample region (P) to be recorded is multiply scanned in each case in accordance with the respective spatial direction of the focal line (10).

20. The method as claimed in claim 19, wherein the individual images are assembled mathematically to form an overall image.

21. The method as claimed in claim 1, wherein the transition from the first state (Z1, A) into the second state (Z2, B) is implemented by means of multiphoton absorption.

22. The method as claimed in claim 1, wherein the measuring signal (8) emanating from the sample (1) is read out by means of multiphoton excitation.

23. The method as claimed in claim 1, wherein the optical signal (4) and a test signal (7) are generated in order to read out the first state (Z1, A) by means of pulsed light sources.

24. The method as claimed in claim 23, wherein the pulsed light sources are synchronized.

25. 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, wherein the optical signal (4) can be provided in the form of a focal line (10) with a cross-sectional profile having at least one intensity zero point (5) with laterally neighboring intensity maxima (9).

26. The microscope as claimed in claim 25, defined by an optical component (11) for producing an illumination line in a pupil plane (P3) conjugate with the pupil (P1) of the microscope objective (3, 13).

27. The microscope as claimed in claim 26, defined by a phase modulating element (19, 27) for producing the cross-sectional profile of the focal line (10) by phase modulation of the pupil line (14).

28. The microscope as claimed in claim 27, wherein the optical component (11) for producing the pupil line (14), and the phase modulating element (19, 27) are designed as a structural unit.

29. The microscope as claimed in claim 28, wherein the optical component (11) for producing the pupil line (14), and the phase modulating element (19, 27) are respectively arranged as separate units in the illuminating beam path.

30. The microscope as claimed in claim 27, wherein the phase modulating element (19, 27) is designed as a spatial liquid-based phase modulator arranged in an intermediate image of the pupil (P1), or as an optical component having a phase retarding dielectric coating (PV).

31. The microscope as claimed in claim 27, wherein the optical component (11) for producing the pupil line (14) is designed as a cylindrical lens (17) and/or as a Powell lens and/or as a holographic element.

32. The microscope as claimed in claim 25, wherein the illuminating light source for generating the optical signal (4) is designed as a laser.

33. The microscope as claimed in claim 25, wherein the illuminating light source for generating the optical signal (4) is combined with at least one further light source in order to read out the measuring signal (8) emanating from the sample (1), or to induce the first state (Z1, A).

34. The microscope as claimed in claim 33, wherein the further light source illuminates the sample (1) entirely or partially, preferably linearly.

35. The microscope as claimed in claim 25, wherein the focal line (10) of the optical signal (4) is spatially superposed by a further line.

36. The microscope as claimed in claim 35, wherein the intensity zero point (5) of the focal line (10) of the optical signal (4) is spatially superposed by the maximum of a line (15) in order to read out the measuring signal (8) emanating from the sample (1).

37. The microscope as claimed in claim 35, wherein each line (10, 15) is assigned a scanning device (21), preferably in the form of a scanning mirror.

38. The microscope as claimed in claim 37, wherein the scanning direction corresponds to the running direction of the pupil line (14).

39. The microscope as claimed in claim 25, defined by a row detector (26) for the line detection of the measuring signal (8) emanating from the sample (1).

40. The microscope as claimed in claim 39, wherein the row detector (26) is designed as a CCD row, as an EMCCD or as an APD.

41. The microscope as claimed in claim 39, wherein the row detector (26) is arranged confocally with the focal line (10).

42. The microscope as claimed in claim 39, wherein the row detector (26) is arranged in the detection direction downstream of the scanning device(s) (21) or upstream of the scanning device(s) (21).

43. The use of a microscope as claimed in claim 25 for the optically induced transition of dye molecules between various molecular states that differ from one another in at least one optical property.