MICROSCOPE AND METHOD FOR SPIM MICROSCOPY

• US 20150168732A1
• Filed: 06/26/2013
• Published: 06/18/2015
• Est. Priority Date: 07/02/2012
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Inventors: Singer, Wolfgang, Anhut, Tiemo, Wolleschensky, Ralf, Siebenmorgen, Jorg
• Granted Patent: US 10,698,225 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G02B 21/0032 Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/06 Means for illuminating specimens
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/361 Optical details, e.g. image relay to the camera or image sensor G02B21/364 takes precedence; illumination details G02B21/06 and subgroups
• 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
• CPC Class Codes: G02B 27/0927 Systems for changing the beam intensity distribution, e.g. Gaussian to top-hat
• CPC Class Codes: G02B 27/0988 Diaphragms, spatial filters, masks for removing or filtering a part of the beam

Abstract

A microscope, in particular according to any of the preceding claims, consisting of an illuminating device, comprising an illumination light source and an illumination beam path for illuminating the specimen with a light sheet, a detection device for detecting light emitted by the specimen, and an imaging optical unit, which images the specimen via an imaging objective in an imaging beam path at least partly onto the detection device, wherein the light sheet is essentially planar at the focus of the imaging objective or in a defined plane in proximity of the geometrical focus of the imaging objective, and wherein the imaging objective has an optical axis, which intersects the plane of the light sheet at an angle that is different from zero, preferably perpendicularly, wherein an amplitude and/or phase filter is provided in the illumination beam path, said filter acting as a sine spatial filter in that it limits the illumination light in at least one spatial direction by filtering the spatial frequencies that occur with a sine function and/or in that the illumination light is limited with regard to the phase and amplitude thereof in at least one spatial direction by a sine filter function, and/or a combined amplitude and phase filter is provided in the illumination beam path, said filter shaping the light sheet by influencing the transmission profile of the illumination light distribution with a sine filter function.

Claims

1. A microscope, comprising

an imaging optical unit, which absorbs light emitted by a specimen into a first aperture angle via an imaging objective in a first direction,

a detection device for detecting the fight emitted by the specimen into the first aperture angle, said light being absorbed by means of the imaging optical unit, and

an illuminating device, having an illumination light source with a wavelength λ

and an illumination beam path for illuminating the specimen with an illumination light distribution in a second direction, wherein the second direction is perpendicular to the first direction,

said illuminating device having means such that said illumination light distribution is constant within a section in the second direction,

the power of the illumination light being less than 20% within a cone put up by the first aperture angle outside an area of an expansion in the first direction, wherein the following shall apply;

LY<

2λ

,

LZ>

8λ

.

2. A microscope according to claim 1, wherein said means comprises a complex filter, which limits the light distribution in an aperture diaphragm of the illuminating device to a circular area.

3. A microscope according to claim 2, wherein the filter generates an illumination light distribution that consists of a superimposition of at least two sine functions in the aperture diaphragm of the illuminating device.

4. A microscope according to claim 3, wherein one of the two sine functions corresponds to a sinc(z) function preferably projected into the X-Y plane according to the Ewald summation.

5. A microscope according to claim 1, wherein the filter generates an illumination distribution, which corresponds to the spectrum of a Mathieu beam in the aperture diaphragm of the illuminating device.

10. A microscope according to claim 1, further comprising

a scanning Mathieu beam for the generation of a light sheet with SPIM.

6. A microscope comprising

an illuminating device, having an illumination light source and an illumination beam path for illuminating the specimen with a light sheet,

a detection device for detecting light emitted by the specimen,

an imaging optical unit, which images the specimen via an imaging objective in an imaging beam path at least partly onto the detection device,

said light sheet being planar at the focus of the imaging objective or in a defined plane in proximity of the geometrical focus of the imaging objective, and said imaging objective having an optical axis, which intersects the plane of the light sheet at an angle that is different from zero,

wherein an amplitude and/or phase filter provided in the illumination beam path, said filter acting as a sine spatial filter in that it limits the illumination light in at least one spatial direction by filtering the spatial frequencies that occur with a sine function and/or in that the illumination light is limited with regard to the phase and amplitude thereof in at least one spatial direction by a sine filter function and/or a combined amplitude and phase filter is provided in the illumination beam path, said filter shaping the light sheet by influencing the transmission profile of the illumination light distribution with a sine filter function.

7. A microscope according to claim 6, wherein said amplitude and/or phase filter is arranged at least partly in or in proximity of a plane of the aperture diaphragm of the illumination optical unit and/or the filter is realized with a hologram (CGH) and/or a diffractive optical unit and/or the filter is formed at least partly by a spatial tight modulator (SLM) and/or the light sheet is displaced perpendicular to the direction of the light with the SLM and/or the focal plane of the imaging objective is displaced when the light sheet is displaced and/or that an asymmetrical sine function/off-axis distribution is generated in the plane of the aperture diaphragm and/or that a sine filter acting extra-axially to the optical axis is provided and/or a superimposition of a plurality of sine filters is provided for structuring the light sheet and/or a sine function resealed with the Ewald summation is provided as filter function and/or the filter is combined with an anamorphic optical unit in the illumination beam path for forming the beam.

14. The microscope according to claim 6, wherein said angle that is different than zero being ninety degrees.

8. (canceled)

9. A microscope, comprising

an illuminating device, comprising an illumination light source and an illumination beam path for illuminating the specimen with a light sheet,

a detection device for detecting light emitted by the specimen,

an imaging optical unit, which images the specimen via an imaging objective in an imaging beam path at least partly onto the detection device,

said light sheet being planar at the focus of the imaging objective or in a defined plane in proximity of the geometrical focus of the imaging objective, said imaging objective having an optical axis, which intersects the plane of the light sheet at an angle that is different from zero, and

a Mathieu beam used for the generation of a light sheet with SPIM.

15. The microscope according to claim 9, wherein said angle that is different than zero being ninety degrees.

11. A microscope comprising

an illuminating device, comprising an illumination light source and an illumination beam path for illuminating the specimen with a light sheet,

a detection device for detecting light emitted by the specimen,

an imaging optical unit, which images the specimen via an imaging objective in an imaging beam path at least partly onto the detection device,

said light sheet being planar at the focus of the imaging objective or in a defined plane in proximity of the geometrical focus of the imaging objective, and wherein the imaging objective has an optical axis, which intersects the plane of the light sheet at an angle that is different from zero, and means for generating Mathieu beam with an intensity and/or phase modulator, by means of at least one of the following optical elements:

1) partial diaphragm ring

2) with a spatial light modulator (SLM)

3) axicon

4) hologram

5) diffractive element.

12. A microscope according to claim 11, further comprising means for

the generation of an elliptical Gaussian distribution in front of the diaphragm ring and/or the generation of a Gaussian distribution in front of the diaphragm ring, wherein an upper and lower part of the diaphragm ring is not involved and/or a beam formation via an anamorphic optical unit and/or an intensity modulator such as an SLM and/or a generation of an elliptical Gaussian distribution on the axicon and/or a generation of a Gaussian distribution on the axicon, wherein an upper and lower part of the axicon is not involved and/or the use of an intensity modulator for optimizing the light distribution and/or a combination of an anamorphic optical unit and intensity modulator for optimizing the light distribution and/or the suppression of frequency ratios of the Mathieu beam for the prevention of the periodic intensity modulation of the beam and/or the generation of a structured illumination and/or the row-by-row detection on a surface detector, in particular a CCD receiver or EMCCD receiver or CMOS or sCMOS camera and/or the surface detection on a surface detector, whereby the surface of the detection is smaller than the total detection area of the surface detector.

16. The microscope according to claim 11, wherein said angle that is different than zero being ninety degrees.

17. The microscope according to claim 11, wherein said partial diaphragm ring has a diaphragm consisting of two opposing semi-circles.

13. (canceled)