Light scanning microscope and use

• US 7,561,326 B2
• Filed: 10/19/2004
• Issued: 07/14/2009
• Est. Priority Date: 07/16/2004
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: CARL Zeiss Microimaging GmbH (Carl-Zeiss-Stiftung)
• Inventors: Wolleschensky, Ralf, Steinert, Joerg, Funk, Joerg-Michael
• Primary Examiner(s): Amari; Alessandro
• Application Number:
  US10/967,638
• Publication Number:
  US 20060012871A1
• Time in Patent Office: 1,729 Days
• Field of Search: 359/368, 359370-377, 359/385, 359/389
• US Class Current: 359/368
• CPC Class Codes: G01J 2003/1217 Indexed discrete filters or choppers
• CPC Class Codes: G01J 3/02 Details
• CPC Class Codes: G01J 3/0202 Mechanical elements; Supports for optical elements
• CPC Class Codes: G01J 3/021 using plane or convex mirrors, parallel phase plates, or particular reflectors
• CPC Class Codes: G01J 3/0227 using notch filters
• CPC Class Codes: G01J 3/0237 Adjustable, e.g. focussing
• CPC Class Codes: G01J 3/06 Scanning arrangements arrangements for order-selection
• CPC Class Codes: G01J 3/10 Arrangements of light sources specially adapted for spectrometry or colorimetry
• CPC Class Codes: G01J 3/44 Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
• CPC Class Codes: G02B 21/0044 moving apertures, e.g. Nipkow disks, rotating lens arrays
• CPC Class Codes: G02B 21/0064 multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/008 Details of detection or image processing, including general computer control

Abstract

In a confocal laser scanning microscope with an illuminating configuration (2), which provides an illuminating beam for illuminating a specimen region (23), with a scanning configuration (3, 4), which guides the illuminating beam over the specimen while scanning, and with a detector configuration (5), which via the scanning configuration (3, 4) images the illuminated specimen region (23) by means of a confocal aperture (26) on to at least one detector unit (28), it is provided that the illuminating configuration (2) of the scanning configuration (3, 4) provides a line-shaped illuminating beam, that the scanning configuration (3, 4) guides the line-shaped illuminating beam over the specimen f while scanning and that the confocal aperture is designed as a slit aperture (26) or as a slit-shaped region (28, 48) of the detector unit (28) acting as a confocal aperture.

Claims

1. Confocal laser scanning microscope for the detection of at least one specimen region, comprising:

illuminating means for providing a line-shaped illuminating beam for illuminating a specimen in several points or regions in parallel,

an illuminating beam path traveled by the illuminating beam,

scanning means for guiding the illuminating beam over the specimen while scanning and for descanning radiation emitted by the specimen,

detector means for simultaneously detecting several illuminated specimen points lying on a line, the detector means including;

(a) at least one detector unit, and

(b) confocal image producing means for producing an at least partially confocal image of the specimen from the descanned radiation and imaging it onto the at least one detector unit, the confocal image producing means including a slit aperture unit comprising a slider and a plurality of fixed-width slits on the slider, each of the slits being stationary in its position on the slider and having a slit width different from the slit widths of the other slits, wherein the slit width establishes the depth of focus with which the descanned radiation can be detected, and wherein the confocal image is imaged through a single one of the slits, and the slider is movable to change the slit through which the confocal image is imaged.

2. Laser scanning microscope in accordance with claim 1, further comprising:

an objective positioned in the illuminating beam path for detecting the specimen,

an optical zoom system for producing an intermediate image therein, the optical zoom system being positioned in front of the objective in the illuminating beam path and having an exit pupil, the optical zoom system further forming an entrance pupil of the illuminating beam path and having at least one of variable magnification and variable imaging length into the exit pupil.

3. Laser scanning microscope in accordance with claim 2, further comprising aperture means arranged in the exit pupil for affecting the size of the exit pupil independently from the setting on the optical zoom system, wherein the size of the exit pupil is preferably smaller than the size of the entrance pupil (OP[EP]/EnP) of the objective.

4. Laser scanning microscope in accordance with claim 2, further comprising control means for providing variable control of the optical zoom system in a first mode of operation with variable magnification and constant imaging length and a second mode of operation with variable imaging length with constant magnification.

5. Laser scanning microscope in accordance with claim 2, wherein the scanning means includes a cylindrical telescope which magnifies the line length of the illuminating beam and which, for the optical zoom system, is switched to a zoom factor of smaller than one in the beam path.

6. Laser scanning microscope in accordance with claim 1, further comprising converting means for generating a line-shaped illuminating beam from a cross-sectionally inhomogeneous source beam, specifically from a Gaussian source beam, wherein the converting means includes an aspherical, convex mirror which is more pronouncedly curved in the region of the point of incidence of the source beam than in regions remote from the point of incidence.

7. Laser scanning microscope in accordance with claim 6, wherein the mirror is wedge-shaped with a rounded off vertex.

8. Laser scanning microscope in accordance with claim 7, wherein the mirror has a reflecting surface that satisfies the function y²/[c+(c²−

(1−

Q)y²)^1/2] relative to the Cartesian (x, y, z)-coordinates, wherein “

c”

is a radius of curvature of the vertex of the cone and “

Q”

is the conic constant.

9. Laser scanning microscope in accordance with claim 7, wherein the mirror has a reflecting surface which is additionally curved along the longitudinal axis of the dome.

10. Laser scanning microscope in accordance with claim 9, wherein the aspheric mirror satisfies the function f (x, y)=[(a(y)−

r_x)²−

x²]^1/2, wherein r_x is the radius of curvature along the longitudinal axis of the vertex and a(y)=y²/[c+(c²−

(1−

Q)y²)^1/2].

11. Laser scanning microscope in accordance with one of the claims 6, wherein the mirror has a symmetrical axis that lies at a 4°

to 20°

angle to the incident axis of the source beam.

12. Laser scanning microscope in accordance with one of the claims 6, further comprising a concentrating reflecting mirror arranged after the aspherical mirror.

13. Laser scanning microscope in accordance with claim 12, wherein the concentrating reflecting mirror is one of cylindrical and toric.

14. Laser scanning microscope in accordance with claim 13, wherein the concentrating reflecting mirror exhibits a radius of curvature equal to (r_x+2d), wherein d is the distance between the aspheric mirror and the concentrating reflecting mirror.

15. Laser scanning microscope in accordance with claim 1, wherein the scanning means includes two independently controllable and operable scanning units, one of the scanning units effecting a decentralized zoom function.

16. Laser scanning microscope in accordance with claim 1, wherein the detector unit includes a two-dimensional radiation sensor, and a spectrometer which spectrally splits up the line-shaped beam crosswise to the beam and guides it to the two-dimensional radiation sensor.

17. Laser scanning microscope in accordance with claim 16, wherein the spectrometer includes an entrance slit which serves as a confocal aperture.

18. Laser scanning microscope in accordance with claim 1, wherein the detector unit includes a two-dimensional radiation sensor and a streak camera, which temporally splits up the line-shaped beam crosswise to the beam and guides it to the two-dimensional radiation sensor.

19. Laser scanning microscope in accordance with claim 1, wherein the illuminating means includes at least one polarizer and wherein the detector means includes at least one polarization analyzer in the detector assembly.

20. Laser scanning microscope in accordance with claim 1, wherein the detector means includes several spectral channels, and one detector unit for each respective spectral channel.

21. Laser scanning microscope in accordance with claim 20, further comprising a common slit aperture preceding all of the spectral channels.

22. Laser scanning microscope in accordance with claim 21, wherein the slit aperture has an adjustable aperture width.

23. Laser scanning microscope in accordance with claim 1, wherein the slit aperture unit precedes all of the spectral channels.

24. Laser scanning microscope in accordance with claim 23, wherein the at least one plane parallel transparent plate comprises at least one of a pivoting plate and at least one biaxial tilting plate.

25. Laser scanning microscope in accordance with claim 23, wherein the at least one plane parallel transparent plate comprises at least two different uniaxial tilting plates.

26. Laser scanning microscope in accordance with claim 23, wherein the at least one plane parallel transparent plate comprises at least two different uniaxial pivoting plates.

27. Laser scanning microscope in accordance with claim 26, further comprising interchangeable elements in the path of the beam, and wherein the setting unit comprises a configuration of the interchangeable elements as operating parameters.

28. Laser scanning microscope in accordance with claim 26, further comprising positionally adjustable elements in the path of the beam and wherein the setting unit comprises a configuration of the adjustable elements as operating parameters.

29. Laser scanning microscope in accordance with claim 26, wherein the detector means performs the function of analyzing radiation of different wave lengths and the setting unit interprets the wave lengths in the beam path as operating parameters.

30. Laser scanning microscope in accordance with claim 23, further comprising a setting unit for setting the tilt position of the plate dependent on the value of an operating parameter of the optical configuration.

31. Laser scanning microscope in accordance with claim 30, wherein the control loop means performs at least one of the further functions of maximizing the radiation intensity in the detector means and minimizing the image shift/misalignment.

32. Laser scanning microscope in accordance with claim 23, further comprising control loop means for balancing the effects of at least one temperature errors and long term drift errors using the tilt position of the plate as a correcting variable.

33. Laser scanning microscope in accordance with claim 23, wherein the at least one plane parallel transparent plate comprises two independently drivable plates composed of materials with different dispersions for the purpose of setting a chromatically independent or targeted, chromatically dependent parallel offset/shift.

34. Laser scanning microscope in accordance with claim 23, wherein the at least one plane parallel transparent plate is constructed of two graduated plates with materials of different dispersions in order to compensate for chromatic cross aberrations in the path of the beam.

35. Laser scanning microscope in accordance with claim 23, wherein the at least one plane parallel transparent plate is arranged in front of the detector means in the beam path in order to center the image of the specimen region on the detector means.

36. Laser scanning microscope in accordance with claim 23, wherein the at least one plane parallel transparent plate is arranged in front of the detector means in the beam path in order to center an image of the slotted aperture (26) on the detector unit (28).

37. Laser scanning microscope in accordance with claim 23, further comprising at least one of a biaxial tilting plate and a pivotable plate and pivotable plates provided between the specimen region and the confocal aperture as well as between the aperture and the detector unit.

38. Laser scanning microscope in accordance with claim 23, further comprising at least one of two different uniaxial tilting plates and pivotable plates provided between the specimen region and the confocal aperture as well as between the aperture and the detector unit.

39. Process in accordance with claim 38, wherein the analyzing step comprises analyzing the specimen using one of coherent Stoke'"'"'s spectroscopy and anti-Stoke'"'"'s spectroscopy and hyper-Raman spectroscopy and stimulated Raman spectroscopy and Raman processes of the higher order.

40. Laser scanning microscope in accordance with claim 1, further comprising a correcting unit, provided in at least one of the illuminating means and the detector means, wherein the correcting unit includes a holding fixture in the beam path and at least one plane parallel transparent plate held in the holding fixture, and which can be driven by the holding fixture into at least one of a tilting movement and a pivoting movement around at least one axis so as to set a specific parallel offset/shift (dx, dy) of the beams in the beam path by changing the tilt position of the plate.

41. Process for laser scanning microscopy, using the laser scanning microscope according to claim 1, comprising the step of:

analyzing the specimen using Raman spectroscopy.

42. Process for analyzing developmental processes, comprising the step of:

analyzing dynamic processes ranging from tenths of seconds to hours, at the level of cell groups and entire organisms, using the laser scanning microscope according to claim 1.

43. Process for the analysis of intracellular transport processes, comprising the step of:

representing small motile structures, e.g. proteins with high speeds (usually in the range of hundredths of seconds), in particular, for applications such as FRAP with ROI bleaching, using the laser scanning microscope according to claim 1.

44. Process for the representation of molecular and other subcellular interactions, comprising the step of:

representing very small structures with high speeds for the resolution of submolecular structures, using the laser scanning microscope according to claim 1.

45. Process for analyzing rapid signal transmission processes, comprising the step of:

analyzing neurophysiological processes with high temporal resolution, for analyses in the muscle system and in the nervous system, using the laser scanning microscope according to claim 1.

46. Laser scanning microscope in accordance with claim 1, wherein the detector unit further includes a spectrally resolving element, and wherein the confocal imaging means images the descanned radiation onto the spectrally resolving element of the detector unit.