METHOD AND DEVICE FOR ILLUMINATING A SAMPLE PLANE

Abstract

An illumination apparatus for illuminating a sample plane and a sample that is optionally arranged therein along an illumination beam path includes: a first light source (for outputting light of at least one first wavelength (λ.sub.photo), a second light source for outputting light of at least one second wavelength (λ.sub.exc), and a diffraction grating in the illumination beam path between the first and second light sources and the sample plane. Light of the first wavelength (λ.sub.photo) is not diffracted by the diffraction grating, and light of the second wavelength (λ.sub.exc) is diffracted due to the effect of the diffraction grating. The illumination apparatus can be used in a microscope.

Claims

1-8. (canceled)

9. An illumination apparatus for illuminating a sample plane and a sample that is optionally arranged therein along an illumination beam path, the appratus comprising: a first light source configured for outputting light of at least one first wavelength (λ.sub.photo); a second light source configured for outputting light of at least one second wavelength (λ.sub.exc); and a diffraction grating disposed in the illumination beam path between the first and second light sources and the sample plane, wherein light of the first wavelength (λ.sub.photo) is not diffracted by the diffraction grating and light of the second wavelength (λ.sub.exc) is diffracted due to the effect of the diffraction grating.

10. The illumination apparatus according to claim 9, wherein the diffraction grating is configured to generate a predetermined illumination pattern of the light of the second wavelength (λ.sub.exc) in the sample plane.

11. The illumination apparatus according to claim 10, wherein the light of the first wavelength (λ.sub.photo) is selected for changing a first property of an emitter in the sample, and wherein light of the second wavelength (λ.sub.exc) is selected for changing a second property of the emitter.

12. The illumination apparatus according to claim 11, wherein the light of the first wavelength (λ.sub.photo) is selected to place the emitter into an activated state and the light of the second wavelength (λ.sub.exc) is selected to place the emitter into an excited state.

13. The illumination apparatus according to claim 12, wherein the emitter fluoresces in the excited state.

14. The illumination apparatus according to claim 11, wherein the light of the first wavelength (λ.sub.photo) is selected to place the emitter into a deexcited or bleached state.

15. A microscope comprising: a first light source configured for outputting light of at least one first wavelength (λ.sub.photo) to illuminate a sample plane and a sample that is optionally arranged therein along an illumination beam path; a second light source configured for outputting light of at least one second wavelength (λ.sub.exc) to illuminate the sample plane; a diffraction grating disposed in the illumination beam path between the first and second light sources and a sample plane, wherein light of the first wavelength (λ.sub.photo) is not diffracted by the diffraction grating and light of the second wavelength (λ.sub.exc) is diffracted due to the effect of the diffraction grating; and a detector configured for detection light emitted from the sample in repsonse to the light of the at least one second wavelength.

16. The microscope of claim 15, wherein the diffraction grating is configured to generate a predetermined illumination pattern of the light of the second wavelength (λ.sub.exc) in the sample plane.

17. A method comprising: illuminating a sample plane and a sample that is optionally arranged therein along an illumination beam path with light of a first wavelength (λ.sub.photo); illuminating the sample plane and the sample that is optionally arranged therein along the illumination beam path with light of a second wavelength (λ.sub.exc), diffracting, with a diffraction grating in the illumination beam path, light of the second wavelength (λ.sub.exc) but not difracting the light of the first wavelength (λ.sub.photo) with the diffraction grating.

18. The method of claim 17, wherein diffracting, with the diffraction grating in the illumination beam path, light of the second wavelength (λ.sub.exc) generates a predetermined illumination pattern of the light of the second wavelength (λ.sub.exc) in the sample plane.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The invention will be explained in more detail below on the basis of figures and exemplary embodiments. In the drawings:

[0032] FIG. 1 shows an exemplary embodiment of a microscope having an illumination apparatus;

[0033] FIG. 2 shows examples of point spread functions (PSF) that are not according to the invention for objectives with objective pupil diameters of 7.2 mm, 7.6 mm, and 11.5 mm of a photostimulation light λ.sub.photo=488 nm after interaction with a 27.5 μm 2D phase diffraction grating in the object plane of the objective; and

[0034] FIG. 3 shows examples of point spread functions (PSF) according to the invention for objectives with objective pupil diameters of 7.2 mm, 7.6 mm, and 11.5 mm of a photostimulation light λ.sub.photo=488 nm after interaction with a 27.5 μm 2D phase diffraction grating in the object plane of the objective.

DETAILED DESCRIPTION

[0035] FIG. 1 shows a microscope 1 having an illumination apparatus as an exemplary embodiment. The microscope 1 has a photostimulation unit SU for providing excitation light of at least one first wavelength λ.sub.photo and a unit ZEXC for providing excitation light of at least one second wavelength λ.sub.exc.

[0036] A second light source 2.2 for providing light of at least one second wavelength λ.sub.exc (symbolized by the reference signs carrying indices λ.sub.exc1, λ.sub.exc2) is present in the unit EXC. The light of the second wavelength kn λ.sub.exc is coupled into a light-guiding fiber 3 and guided in a beam path 4. A collimator 5 is arranged downstream of the fiber 3 to focus the light beam that diverges at the end of the fiber 3.

[0037] Following this, a pupil plane P and a further collimator 5 for producing a collimated beam are arranged in the beam path 4. In the region of said collimated beam, a beam combiner 7 is arranged, as a result of whose effect light of the photostimulation unit SU is coupled or can be coupled into the beam path 4.

[0038] The light of the first wavelength λ.sub.photo travels from the first light source 2.1, again via a light-guiding fiber 3, to a collimator 5 and is steered onto a spatial light modulator (SLM) 13 or alternatively onto a scanner 12 by means of a mirror 21. The spatial light modulator 13 can be in the form of a LCoS-SLM (Liquid Crystal on Silicon) and the scanner in the form of a galvo scanner. From the spatial light modulator 13 or the scanner 12, the light of the first wavelength λ.sub.photo travels via an optical lens system to the beam combiner 7.

[0039] From the beam combiner 7, light of the first and the second wavelength λ.sub.photo, λ.sub.exc, propagates along a common beam path 4. In each case, a wavelength-selective grating 8 is able to be pushed into the beam path 4. In the exemplary embodiment, a plurality of wavelength-selective gratings 8 are arranged as a grating group 9 on a slide whose movement can be controlled by means of a drive 10. The drive 10 is connected to a control unit 11 and is actuatable by means of the latter. Using the control unit 11, it is also possible to control, or to be able to control, for example, the first and the second light sources 2.1 and 2.2, the spatial light modulator 13 and/or the scanner 12, and movements of any wobble plates/phase shifters that are present and/or displaceable tube lenses (20) (see below).

[0040] A phase shifter (wobble plate) and a tube lens 14 are arranged downstream of the grating group 9. The illumination light of the first wavelength λ.sub.photo or of the second to n-th wavelength λ.sub.exc passes via a dichroic beam splitter 15 to an objective 16 and is focused by its effect into a sample plane 17 in which a sample 18 is arranged. An optional provision of light of different first wavelengths is symbolized by the reference signs carrying indices λ.sub.photo1, λ.sub.photo2.

[0041] The sample 18 is provided with at least one emitter that is activated by the effect of the light of the first wavelength λ.sub.photo and is excited by the effect of the light of the second wavelength λ.sub.exc and emits fluorescent light.

[0042] Fluorescent light brought about in this way is captured by means of the objective 16 and passes into a detection beam path via the dichroic beam splitter 15, which is transmissive to the wavelength of the fluorescent light. A filter 19 and optionally selectable tube lenses 20 are present in said detection beam path. The tube lenses can be moved into the detection beam path according to the captured wavelengths of the fluorescent light. The fluorescent light (i.e., detection radiation) passes via a mirror 21 to a downstream system of beam splitters 15, fixed and/or displaceable optical lenses, mirrors 21, and optional filters 19. Said system steers the corresponding components of the detection radiation onto a first detector 22.1 or onto a second detector 22.2.

[0043] The exemplary embodiment shown in simplified form in FIG. 1 relates to the use of the spatial phase structure element (e.g., wavelength-selective grating 8) in a combination device including an SIM microscope 1 and a photostimulation unit SU. The SIM microscope 1 has a spatially structuring phase element (e.g., wavelength-selective grating 8) with a 1D or 2D grating structure that does not affect the photostimulation wavelengths λ.sub.photo.

[0044] FIG. 2 shows the case that the 2D grating (structuring spatial phase element) of a microscope Elyra7 (Carl Zeiss Microscopy GmbH) interacts with the photostimulation light λ.sub.photo=488 nm, for example, of a photostimulation module SU based on a galvo scanner. It can be seen from FIG. 2 that the respectively shown point spread function (PSF) is broken up. In order to prevent such a destruction of the PSF, a phase grating or the entire grating group in an illumination apparatus according to the prior art would have to be moved out of the beam path to perform a photostimulation event. However, this causes the overall experiment consisting of image and photostimulation event sequences to be extremely slow.

[0045] However, as shown in FIG. 3, when using the techniques described herein, the point spread function is not destroyed (FIG. 3) and the wavelength-selective grating 8 or the grating group 9 does not need to be removed from the beam path 4 during the photostimulation event sequences, thus leading to a greater speed and better time resolution during image recording.

REFERENCE SIGNS

[0046] 1 Microscope [0047] 2.1 First light source [0048] 2.2 Second light source [0049] 3 Fiber [0050] 4 Beam path [0051] 5 Collimator [0052] 6 Pinhole stop [0053] 7 Beam combiner [0054] 8 Wavelength-selective grating [0055] 9 Grating group [0056] 10 Drive [0057] 11 Control unit [0058] 12 Scanner [0059] 13 SLM [0060] 14 Tube lens [0061] 15 Beam splitter [0062] 16 Objective [0063] 17 Sample plane [0064] 18 Sample [0065] 19 Filter [0066] 20 Tube lenses displaceable [0067] 21 Mirror [0068] 22.1 First detector [0069] 22.2 Second detector [0070] P Pupil plane [0071] SU Photostimulation unit [0072] EXC Unit for providing excitation light