Microscope and method for microscopic examination of large samples
A microscope, which is a confocal microscope converted into a light sheet microscope, includes a microscope body and a mechanical receiving apparatus for microscope objectives, through which a microscope beam path extends. An optical module attachable to the receiving apparatus is configured to illuminate a sample volume and collect and transmit light from the sample volume. The optical module comprises: first and second optical arrangements with first and second beam paths that intersect in the sample volume, an optical beam path selector configured to combine the first and/or second beam path with the microscope beam path, and an attachment element arranged between the first or second optical arrangement and the sample volume, wherein the first or second beam path extend at least in sections through the attachment element in order to generate a light sheet. An area sensor is configured to detect light collected from the sample volume.
1 . A microscope, which is a confocal microscope converted into a light sheet microscope, the microscope comprising:
a microscope body;
a mechanical receiving apparatus, through which a microscope beam path extends;
an optical module which is attachable to the mechanical receiving apparatus and which is configured to illuminate a sample volume and collect and transmit light from the sample volume, wherein the optical module comprises:
a first optical arrangement with a first beam path,
a second optical arrangement with a second beam path that intersects the first beam path in the sample volume,
an optical beam path selector which is configured to combine the first beam path and/or the second beam path with the microscope beam path, and
an attachment element arranged between the second optical arrangement and the sample volume, wherein the second beam path extends at least in sections through the attachment element in order to generate a light sheet; and
an area sensor configured to detect light collected from the sample volume,
wherein the microscope is switchable between a first microscopy mode and a light sheet mode,
wherein, in the first microscopy mode, the microscope is configured to illuminate the sample volume along the first beam path; and
wherein, in the light sheet mode, the microscope is configured to illuminate, with the light sheet, the sample volume along the second beam path.
2 . The microscope as claimed in claim 1 , wherein the microscope has at least two operating states of the following list of operating states:
a scanning microscopic mode;
a nonlinear illumination mode;
a confocal mode;
a light field mode; and
the light sheet mode.
3 . The microscope as claimed in claim 2 , further comprising a switchover apparatus having at least one element from the following list of elements:
a wavelength-changeable light source;
a light source with at least two different emission wavelengths that are switchable independently of one another;
a wavelength-changing optical element configured to vary a wavelength of radiated-in light;
an optical retardation element configured to vary a polarization of the radiated-in light; and
a beam path selector changing module configured to alternately or independently introduce at least two beam path selectors into the microscope beam path.
4 . The microscope as claimed in claim 1 , wherein the microscope is switchable between the first microscopy mode and the light sheet mode by a switchover apparatus.
5 . The microscope as claimed in claim 1 , wherein the optical beam path selector is a wavelength-selective optical element.
6 . The microscope as claimed in claim 5 , wherein the wavelength-selective optical element has at least one spectral filter edge which separates a first spectral range from a second spectral range, the wavelength-selective optical element having different transmission and reflection properties for the first spectral range and for the second spectral range, and wherein:
in the light sheet mode, the spectral filter edge lies spectrally between a first wavelength of light radiated into the sample volume and a second wavelength of light collected from the sample volume, and
in the first microscopy mode, the first wavelength and the second wavelength of the light lie together in the first spectral range or in the second spectral range.
7 . The microscope as claimed in claim 6 , further comprising at least one light source which emits light in at least two different wavelength ranges, wherein the light of the at least one light source of the at least two different wavelength ranges is coupleable independently of one another into the microscope beam path, and wherein the at least two different wavelength ranges of the light source lie in different spectral ranges of the wavelength-selective optical element.
8 . The microscope as claimed in claim 6 , wherein at least one of the following is/are provided in the first beam path:
a stop filter for attenuating or blocking a reflected component of the second spectral range which is substantially transmitted to the second optical arrangement by the wavelength-selective optical element, and
a further optical arrangement for displacing a back focal plane of the first optical arrangement to a different position.
9 . The microscope as claimed in claim 8 , wherein at least one element is received in repeatedly interchangeable fashion upon attachment of the module to the mechanical receiving apparatus, the at least one receiving element comprising at least one of:
the first optical arrangement;
the second optical arrangement;
the beam path selector; and
the stop filter.
10 . The microscope as claimed in claim 1 , wherein the second optical arrangement is a light sheet module configured to generate a light sheet that passes through the sample volume.
11 . The microscope as claimed in claim 1 , wherein the microscope beam path is feedable into the optical module in a manner tilted to a normal perpendicular to a back focal plane of the first optical arrangement or the second optical arrangement, and wherein the second optical arrangement or the attachment element is tiltable depending on a tilt of the microscope beam path with respect to the normal of the back focal plane of the second optical arrangement.
12 . The microscope as claimed in claim 1 , further comprising a further imaging beam path which has a further microscope objective and a further detector, wherein the sample volume is able to be imaged on the further detector by the further microscope objective of the further imaging beam path from a side which faces away from the optical module.
13 . The microscope as claimed in claim 12 , wherein the further imaging beam path is movable relative to the optical module, or wherein the first optical arrangement is movable relative to the second optical arrangement.
14 . The microscope as claimed in claim 1 , wherein a further attachment element is arranged between the sample volume and the first optical arrangement.
15 . The microscope as claimed in claim 1 , wherein an optical axis of the microscope beam path and an optical axis of the first optical arrangement, and/or an optical axis of the second optical arrangement and/or an optical axis of a further imaging beam path, intersect a common object field.
16 . The microscope as claimed in claim 1 , wherein the first optical arrangement and/or the second optical arrangement and/or a further imaging beam path have a variable lens or a variably adjustable liquid lens or a zoom optical unit.
17 . The microscope as claimed in claim 1 , further comprising a scanner configured to generate a dynamic light sheet.
18 . The microscope as claimed in claim 1 , further comprising a cylindrical lens configured to generate a static light sheet.
19 . The microscope as claimed in claim 1 , wherein the first optical arrangement or the second optical arrangement comprises a microscope objective with finite optics or infinite optics.
20 . A microscope, which is a confocal microscope converted into a light sheet microscope, the microscope comprising:
a microscope body;
a mechanical receiving apparatus through which a microscope beam path extends;
an optical module which is attachable to the mechanical receiving apparatus and which is configured to illuminate a sample volume and collect and transmit light from the sample volume, wherein the optical module comprises:
a first optical arrangement with a first beam path,
a second optical arrangement with a second beam path that intersects the first beam path in the sample volume,
an input coupling site for illumination light configured to illuminate part of the sample volume by way of the second beam path, and
an attachment element arranged between the second optical arrangement and the sample volume, wherein the second beam path extends at least in sections through the attachment element; and
an area sensor configured to detect light collected from the sample volume,
wherein the microscope is switchable between a first microscopy mode and a light sheet mode,
wherein, in the first microscopy mode, the microscope is configured to illuminate the sample volume along the first beam path; and
wherein, in the light sheet mode, the microscope is configured to illuminate, with a light sheet, the sample volume along the second beam path.
21 . An optical module for illuminating a sample volume and for collecting and transmitting light from the sample volume, which is adaptable to a mechanical receiving apparatus of a microscope with a microscope beam path extending through the mechanical receiving apparatus, the microscope comprising an area sensor for detecting light collected from the sample volume, the optical module comprising:
a first optical arrangement with a first beam path;
a second optical arrangement with a second beam path that intersects the first beam path in the sample volume;
an input coupling site for illumination light or an optical beam path selector, the input coupling site being configured to illuminate part of the sample volume by way of the second beam path, or the optical beam path selector being configured to combine the first beam path and/or the second beam path with the microscope beam path; and
an attachment element arranged between the second optical arrangement and the sample volume, wherein the second beam path extend at least in sections through the attachment element,
wherein the microscope is switchable between a first microscopy mode and a light sheet mode,
wherein, in the first microscopy mode, the microscope is configured to illuminate the sample volume along the first beam path; and
wherein, in the light sheet mode, the microscope is configured to illuminate, with a light sheet of the microscope, the sample volume along the second beam path.
22 . A method for observing a sample volume by a microscope, which is a confocal microscope converted into a light sheet microscope, the method comprising:
transmitting light emitted by the sample volume through an optical module along a first beam path;
guiding a second beam path through an attachment element arranged between a second optical arrangement and the sample volume;
detecting light collected from the sample volume using an area sensor; and
carrying out a switchover between a first microscopy mode and a light sheet mode, wherein:
in the first microscopy mode, the sample volume is illuminated through the optical module along the first beam path, the optical module being attached to a microscope body of the microscope; and
in the light sheet mode, the sample volume is illuminated using a light sheet of the light sheet microscope along a second beam path that intersects the first beam path in the sample volume.
23 . The method as claimed in claim 22 , wherein the switchover comprises at least one method step from the following list of method steps:
exchanging an optical beam path selector which combines the first beam path and/or the second beam path with a microscope beam path;
altering a wavelength of illumination light radiated-in by a wavelength-changeable light source and/or a wavelength-changing optical element; and
varying a polarization of the radiated-in illumination light by an optical retardation element.
24 . The method as claimed in claim 23 , wherein:
a region of interest of the sample volume is identified in the light sheet mode;
upon the switchover into the first microscopy mode, the identified region of interest is examined by a scanning microscope with a first resolution that is higher than the light sheet microscope with a second resolution in the light sheet mode; and
image data which were generated by the area sensor or a point detector and which represent a light distribution emitted by the sample volume are merged and/or represented together.
25 . The method as claimed in claim 23 , wherein, upon illumination of the sample volume, light other than the illumination light is radiated onto or into a sample arranged in the sample volume.