Method and Arrangement for Examining a Specimen by Means of Light Sheet Microscopy

• US 20180081159A1
• Filed: 03/11/2016
• Published: 03/22/2018
• Est. Priority Date: 03/16/2015
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Inventors: LIPPERT, Helmut, KAUFHOLD, Tobias, KALKBRENNER, Thomas, SIEBENMORGEN, Joerg
• Granted Patent: US 10,642,015 B2
• Time in Patent Office: Days
• CPC Class Codes: G02B 21/10 affording dark-field illumination G02B21/14 and G02B21/125 take precedence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Abstract

The invention relates to a light sheet microscope which comprises an illumination apparatus, which generates coherent illumination light for several illumination wavelengths, a beam-shaping module (8) for generating a light sheet from illumination light, an illumination objective (10) for the illumination of a specimen (1) with the light sheet and a detection objective (12) for imaging light which is emitted by the specimen onto a laminar detector, wherein the optical axis of the detection objective (12) forms an angle different from 0° and 180° with the optical axis of the illumination objective (10).

In such a light sheet microscope, the beam-shaping module (8) comprises a phase-selective element (19) on which several selection areas (20) separated from each other spatially are arranged, wherein in each case one selection area (20) is assigned to one specific illumination wavelength, and wherein a phase distribution predefined for the respective illumination wavelength is impressed on each selection area (20). In addition, the beam-shaping module (8) comprises means for the sequential or simultaneous selection of the selection areas (20) in dependence on the respective illumination wavelength.

Claims

1. A light microscope, comprising

an illumination apparatus that generates coherent illumination light having several illumination wavelengths,

a beam-shaping module that generates a light sheet from the illumination light,

an illumination objective for the illumination of a specimen with the light sheet,

a detection objective for imaging light emitted by the specimen onto a laminar detector, wherein the optical axis of the detection objective forms an angle different from 0°

and 180°

with the optical axis of the illumination objective,

wherein the beam-shaping module comprises a phase-selective element arranged in or near an intermediate plane and having a plurality of selection areas that are separated from each other spatially,

wherein one selection area is assigned to one specific illumination wavelength, and

wherein a phase distribution predefined for the respective illumination wavelength is impressed on each selection area, and

wherein the beam-shaping module comprises selecting means for sequentially or simultaneously selecting the selection areas in dependence on the respective illumination wavelength, and

wherein the selecting means is formed to direct light of the respective illumination wavelength onto an assigned selection area.

2. The light sheet microscope according to claim 1,

wherein the phase-selective element comprises a plurality of individual elements, and

wherein each individual element is assigned to one illumination wavelength.

3. The light sheet microscope according to claim 1,

wherein the selecting comprises:

an optical path selection element for deflecting the light onto the respective selection area in dependence on the illumination wavelength arranged between a light source and the phase-selective element, and

a segmented mirror comprising a plurality of segments arranged downstream of the phase-selective element, the segmented mirror reflecting the incident light onto the selection area of the phase-selective element which is assigned to the illumination wavelength,

wherein one segment is assigned to one illumination wavelength, and a λ

/4 plate is arranged between the segmented mirror and the phase-selective element, and

wherein the phase-selective element is designed not to modify the phase of the light in the case of a first polarization direction and is designed to modify the phase in the case of a second polarization direction perpendicular to the first.

4. The light sheet microscope according to claim 1,

wherein the selecting means comprises at least one selective deflecting element arranged between a light source and the phase-selective element,

wherein each of the deflecting elements is assigned to one illumination wavelength and is arranged to direct light onto the respective selection area, a plane mirror arranged downstream of the phase-selective element and a λ

/4 plate arranged between plane mirror and phase-selective element, and

wherein the phase-selective element is designed not to modify the phase of the light in the case of a first polarization direction and is designed to modify the phase in the case of a second polarization direction perpendicular to the first.

5. The light sheet microscope according to claim 4, wherein impressed on each selection area is a blazed grating which is designed to modify the angle of reflection of the light in the case of the second polarization direction.

7. The light sheet microscope according to claim 1,

wherein the selecting means comprises at least one selective input deflecting element arranged between a light source and the phase-selective element,

wherein each of the input deflecting elements is assigned to one illumination wavelength and is arranged to direct light onto the respective selection area, and,

wherein the selecting means, for beam combining, further comprises at least one output deflecting element arranged downstream of the phase-selective element, and

wherein each of the output deflecting elements is assigned in each case to one illumination wavelength and deflects light out of the respective selection area.

8. The light sheet microscope according to claim 7, wherein, between the phase-selective element and the output deflecting elements, compensating elements are arranged for compensating for differences in path length between paths of the light of the individual illumination wavelengths.

9. The light sheet microscope according to claim 1, wherein the selecting means comprises at least one dichroic beam-splitting element arranged between a light source and the phase-selective element for separating the incident light into at least one reflected partial beam and one transmitted partial beam.

10. The light sheet microscope according to claim 9,

wherein the selecting means further comprises a first deflecting mirror, which deflects the at least one reflected partial beam onto the phase-selective element, and a second deflecting mirror, which deflects the transmitted partial beam onto the phase-selective element, and

wherein the phase-selective element is formed reflective and is positioned in the beam path between the two deflecting mirrors.

11. The light sheet microscope according to claim 9

wherein the dichroic beam-splitting element is formed as a plane-parallel plate with two parallel large areas,

wherein a first colour-splitting layer is applied to a first large area and a second colour-splitting layer is applied to a second large area, and

wherein the two colour-splitting layers act reflectively for different illumination wavelengths or ranges for illumination wavelengths.

12. The light sheet microscope according to claim 1, wherein the selecting means further comprises a dispersive or diffractive optical element arranged between a light source and the phase-selective element, the dispersive or diffractive optical element being capable of deflecting light of each of the illumination wavelengths at a different angle.

13. The light sheet microscope according to claim 12, further comprising:

an optical imaging element arranged between the dispersive or diffractive optical element and the phase-selective element,

wherein the dispersive or diffractive optical element and the phase-selective element are each arranged in or near to a focal plane of the optical imaging element, and

wherein the phase-selective element is designed to reflect the light into the optical imaging element with phase modification.

14. The light sheet microscope according to claim 13, wherein the phase-selective element is arranged in the beam path tilted with respect to the optical path of the incident light, with the result that, in the dispersive or diffractive optical element, a beam displacement is generated between entering and exiting beam for each of the illumination wavelengths.

25. The light sheet microscope according to claim 12, wherein the dispersive or diffractive optical element comprises a glass plate, a prism, or a grating.

26. The light sheet microscope according to claim 13, wherein the optical imaging element comprises a concave mirror.

6. (canceled)

15. A method for examining a specimen by light sheet microscopy, comprising:

selecting several illumination wavelengths for composing illumination light for the specimen,

impressing a predefined phase distribution on a phase-selective element for each of the illumination wavelengths, wherein each of the predefined phase distributions is impressed on the phase-selective element in its own selection area, which is assigned to the respective illumination wavelength and does not overlap with the other selection areas,

illuminating the phase-selective element with illumination light in or near to an intermediate image plane in an illumination beam path, wherein only light of the respective illumination wavelength is directed onto each selection area and structured by the phase-selective element before the light of the different illumination wavelengths is combined to form a common beam,

shaping, in a focal plane of the downstream illumination objective, a structured light sheet of the respective illumination wavelength with a light sheet plane which is perpendicular to the focal plane of the illumination objective, and

illuminating the specimen with the structured light sheets of the respective illumination wavelengths in the light sheet plane, and

detecting light emitted by the specimen in a detection direction which forms an angle different from zero with the light sheet plane.

16. The method according to claim 15, wherein the electric field of the light sheet is determined by coherently superimposing at least two sinc³ beams in the focal plane of the illumination objective to shape the light sheet, and calculating the electric field resulting in the focal plane.

17. The method according to claim 15, further comprising:

determining the electric field of the light sheet by using Bessel beams of predefined shape to shape the light sheet,

determining the electric field of such a Bessel beam in the focal plane, and

arithmetically determining a superimposition of this electric field and identical electric fields spaced apart from each other in each case by a predefined amount Δ

in the focal plane.

18. The method according to claim 15, wherein a diffractive optical element is used as phase-selective element.

19. The method according to claim 15, wherein the phase-selective element, as an assembly, is composed of several individual elements, and each individual element comprises one selection area assigned to an illumination wavelength.

20. The method according to claim 15, wherein, for each selection area, the modulation depth is fixed at π

in dependence on the illumination wavelength.

21. The method according to claim 15, wherein the specimen is illuminated in the light sheet plane simultaneously or sequentially with the structured light sheets of the respective illumination wavelengths.

22. The method according to claim 15, wherein the structured illumination light is imaged into an aperture plane which is arranged downstream of the phase-selective element and in which a frequency spectrum of the illumination light of the respective illumination wavelength is generated, and the aperture structure is adapted such that the zero orders of the structured illumination light of the respective illumination wavelength are substantially removed in the aperture plane.

23. The method according to claim 22,

wherein a predefined phase distribution is impressed on a phase-selective element for each of the illumination wavelengths, in that in the focal plane of an illumination objective for a light sheet of predefined shape, its electric field is determined with light of the respective illumination wavelength, and therefrom the phase distribution to be predefined in each case is calculated,

wherein, in the aperture plane, a middle range based on the position of the light sheet is removed, with the result that the aperture structure removes the zero order of the structured light of the respective illumination wavelength.

24. The method according to claim 18, wherein the diffractive optical element comprises a spatial light modulator.