MICROSCOPE AND METHODS FOR THE MICROSCOPIC EXAMINATION OF LARGE SAMPLES

Description

[0001] The invention relates to a microscope and a method for observing a sample volume using a microscope.

[0002] Microscopes of the type mentioned above, i.e., light-sheet microscopes and confocal microscopes, are known from the state of the art, for example, from WO 2018 / 148309. The prior-art solutions are limited in terms of the possible sample size and therefore offer little flexibility and / or accuracy when examining large samples.

[0003] DE 196 29 725 A1 describes a double lens system for the " Theta-Konfokal-Mikroskopie", where the beam paths of illumination and detection intersect at a single point. In JP 2010 072 015 A, the sample is also illuminated pointwise, particularly using a pinhole or a Nipkow disk. In DE 198 51 240 C1, illumination volumes elongated in the direction of illumination intersect with a detection volume, with fluorescence emitted from this illumination volume being detected by a confocally arranged, point-shaped detector.

[0004] Consequently, the object of the invention is to improve the known microscopes of the type mentioned at the beginning so that larger samples can be examined.

[0005] This object is achieved by a microscope according to claim 1.

[0006] Furthermore, the method mentioned at the outset is improved by the present invention in that it comprises the method steps according to claim 15.

[0007] The attachment element according to the invention makes it possible to modify the orientation or distance of the first or second optical arrangement from the sample volume. This allows for a flexible and compact design of the optical module. Furthermore, the attachment element can redirect the first or second beam path.

[0008] The coupling point for the illumination light can be arranged on a housing of the optical module. The coupling point could have a passage for the freely flowing illumination light. Alternatively, a fiber coupler for an optical fiber could be provided on the housing of the optical module, with which the illumination light can be transported via the first beam path and / or the second beam path to illuminate a portion of the sample volume.

[0009] Furthermore, the object mentioned at the outset is achieved with an optical module for illuminating a sample volume and for collecting and transmitting light from the sample volume according to claim 14.

[0010] The microscope according to the invention and the method according to the invention can be further improved by the embodiments described below, each of which is advantageous in its own right. Technical features of the embodiments can be combined with one another or omitted as desired, provided that the technical effect achieved by the omitted feature is not important in the specific embodiment.

[0011] The microscope can be a confocal microscope converted into a light sheet microscope.

[0012] An optical fiber may be provided which is designed to transport the illumination light to the coupling point.

[0013] The optical arrangements can comprise several individual lenses or a lens system and can preferably be designed as a microscope objective. In particular, the design as a microscope objective allows easy access and replacement of the entire optical arrangement. The beam paths are defined by the respective optical arrangement, or the microscope beam path is defined by optical elements of the microscope.

[0014] The attachment element is preferably located on a side of the respective optical arrangement facing the sample and can be understood as a three-dimensional body that is transparent to the light used and through which the light of the illumination or detection is transmitted.

[0015] Preferably, light in the ultraviolet, visible, and near-infrared spectral ranges can be used. For example, the illumination can be provided with ultraviolet light, which preferably has a wavelength corresponding to the excitation wavelength of a fluorescent substance. Thus, fluorescent light emitted by the fluorescent substance can be emitted by the sample in the long-wave visible spectral range or in the near-infrared spectral range compared to the excitation wavelength and can be collected and transmitted by the optical module. When attached to the holding device, the optical module can be mounted on the microscope body so that it can rotate relative to the microscope body.

[0016] In one possible embodiment of the microscope according to the invention, the microscope can have at least two operating states from the following list of operating states: a scanning microscopy mode; a nonlinear illumination mode; a confocal mode; a light field mode; and a light sheet mode.

[0017] Since such a design of the microscope combines a multitude of microscopic methods, it is versatile and a sample that is to be examined using several microscopic methods can remain in a single microscope according to the invention, in particular in the sample volume of the microscope, and does not have to be moved to another microscope for further examination.

[0018] The nonlinear illumination mode can be, for example, and not limited to, a multiphoton mode, a second-harmonic mode, a third-harmonic mode, or a CARS mode. The principles of these microscopy modes are well known in the art and therefore will not be explained in detail here.

[0019] The microscope according to the invention can be further improved in that the microscope has a switching device by means of which the microscope can be reversibly and repeatedly switched at least into a first microscopy mode or a light sheet mode.

[0020] The first microscopy mode can in particular be the scanning microscopy mode or the confocal mode.

[0021] This eliminates the need to move the sample from the sample volume or the microscope to switch between multiple microscopic techniques. Images acquired by the area sensor and / or a point detector can therefore be correlated without complex image data transformations occurring during or after the image acquisition. The sample can thus remain in a fixed location and be examined using different microscopy techniques, ensuring that a sample positioned within the image sections of the different microscopy techniques is always imaged at the same location in the acquired image data.

[0022] A corresponding method according to the invention thus further comprises the method step of switching between a first microscopy mode and a light-sheet mode, wherein in the first microscopy mode, the sample volume is illuminated along the first beam path, and wherein in the light-sheet mode, the sample volume is illuminated along the second beam path. Essentially, switching between the operating states can thus relate to a light path of illumination light. Preferably, the detected light emitted by the sample from the sample volume is always collected and forwarded by the first optical arrangement along the first optical beam path.

[0023] This design of the microscope according to the invention thus allows the microscope's operating modes to be repeatedly changed without the need to readjust the microscope and / or change the position of the sample. Switching between the operating modes thus requires no additional effort and can be seamlessly integrated into existing workflows for the microscopic examination of samples.

[0024] In a further advantageous embodiment of the microscope according to the invention, the optical beam path selector can be a wavelength-selective optical element. A possible wavelength-selective optical element is, for example and not limited to, a dichroic mirror. Such dichroic mirrors are known from the prior art and will not be explained in detail here.

[0025] Furthermore, it is also conceivable that the optical beam path selector according to the invention is based on other optical, wavelength-selective principles, for example diffraction or refraction.

[0026] Preferably, the wavelength-selective optical element can be used at an angle of incidence of approximately 45°. However, angles of incidence deviating from this configuration, ranging from 10° to 80°, are also possible.

[0027] The second optical arrangement is designed as a light sheet module for generating a light sheet traversing the sample volume. This has the advantage that such a light sheet module can be easily exchanged or replaced. Consequently, a light sheet with different properties can be generated in the sample by inserting a light sheet module with different properties into the optical module. A light sheet module can also be designed as a microscope objective. This has the advantage that such a light sheet module can easily replace a microscope objective.

[0028] The light sheet module preferably has a fastening arrangement known from microscopy in the form of a (preferably standardized) bayonet lock, thread or dovetail guide.

[0029] Preferably, the attachment element is arranged on the light sheet module.

[0030] The light sheet generated in the sample volume can be created by an optical element that focuses only in one direction, such as a cylindrical lens. Furthermore, it is possible to rapidly move a focused beam within the sample volume using a deflection unit. If this movement is faster than the detection sampling rate, such a scanning of the sample volume is no longer detectable as movement and is referred to as a virtual light sheet.

[0031] In a further advantageous embodiment of the microscope according to the invention, the wavelength-selective optical element can have at least one spectral filter edge that separates a first spectral range from a second spectral range. The wavelength-selective optical element can have different transmission and reflection properties for the first spectral range and for the second spectral range. In light sheet mode, the spectral filter edge can spectrally lie between a first wavelength of light irradiated into the sample volume and a second wavelength of light collected from the sample volume, whereas in the first microscopy mode, the first wavelength and the second wavelength of the light can both lie in the first spectral range or in the second spectral range.

[0032] Consequently, in the light sheet mode, the incident light of the first wavelength can be transmitted through the wavelength-selective optical element, whereas the light of the second wavelength emitted by the sample is reflected by the wavelength-selective optical element.

[0033] In contrast, in the first microscopy mode, both wavelengths can be transmitted or reflected by the wavelength-selective optical element.

[0034] Furthermore, the wavelength-selective optical element can also have two or more filter edges. For example, the wavelength-selective optical element can comprise a transmissive bandpass filter and a reflective bandstop filter, wherein light from three adjacent spectral ranges is alternately transmitted or reflected by the wavelength-selective optical element. In view of the above-mentioned spectral position of the wavelengths with respect to the filter edge, it is necessary to consider both filter edges in this embodiment. For example, the first and second wavelengths can be arranged on a common side of a first filter edge, but a second filter edge can be arranged between the wavelengths.In such a case, the microscope may be in light sheet mode because the first wavelength is in a spectral region of high reflection and the second wavelength is in a region of high transmission.

[0035] Furthermore, it is possible that the two wavelengths include the transmitting or reflecting band of the bandpass or bandstop filter. In such a case, the microscope can be in the first microscopy mode.

[0036] If alternating reflective and transmissive spectral ranges are assumed, there can be more than two filter edges between the two wavelengths. If the number of filter edges is even, the microscope is in the first microscopy mode. If the number is odd, the two wavelengths are spatially separated by the wavelength-selective optical element, meaning the microscope is in light-sheet mode.

[0037] If one (or an odd number of) filter edges are enclosed by the two wavelengths, then, depending on the transmission or reflection characteristics of the filter, either the shorter wavelength or the longer wavelength can be reflected and, accordingly, the longer wavelength or the shorter wavelength can be transmitted.

[0038] The microscope according to the invention can be further improved in that the switching device selects at least one element from the list of elements comprising a wavelength-variable light source; a light source with at least two independently switchable, differing emission wavelengths; a wavelength-variable optical element for varying the wavelength of the incident light; an optical delay element for varying the polarization of the incident light; and a beam path selector interchangeable module for alternately or independently introducing at least two beam path selectors into the microscope beam path.

[0039] In a corresponding embodiment of the method according to the invention, switching between a first microscopy mode and a light sheet mode can comprise at least one method step from the list of the following method steps: Replacing an optical beam path selector that combines the first beam path and / or the second beam path with a microscope beam path; changing the wavelength of incident illumination light by means of a wavelength-variable light source and / or a wavelength-variable optical element; and varying the polarization of the incident illumination light by means of an optical delay element.

[0040] The light source with independently switchable emission wavelengths can, for example, be a laser with switchable laser lines. Alternatively, this embodiment of the light source can be designed as a module that can comprise multiple lasers. Preferably, one wavelength can be fixed, and two additional wavelengths can be selectively switched on, i.e., alternately, for example. Purely by way of example and not by way of limitation, one laser (for example, at 488 nm) can be switched off and another laser (for example, at 561 nm) switched on. Any other combinations of emission wavelengths of the lasers available in the prior art are conceivable.

[0041] Thus, according to a first general approach, a relative spectral position of the at least two wavelengths of the light radiated into the sample volume or collected therefrom can be changed with respect to the at least one filter edge. In particular, the relative spectral position can be changed such that both wavelengths are on one side of the filter edge or that both wavelengths spectrally enclose the filter edge. The above relations with regard to the spectral position are to be understood with regard to the wavelength of the light or the wavelength at which the filter edge lies. If, for example, a first wavelength and a second wavelength enclose the filter edge, this means that the wavelength at which the filter edge lies is greater than the first wavelength and less than the second wavelength; without loss of generality, the wavelengths can be interchanged.

[0042] With the first approach mentioned above, it is now possible to vary at least one of the two wavelengths while maintaining the spectral position of the filter edge. This can be achieved purely by way of example using a wavelength-variable light source, for example, by spectral selection of the light emitted by a laser, such as a white light laser. Here, the wavelengths of the white light laser or a broadband laser can be selected using suitable devices. For example, and not by way of limitation, such selection can be carried out using acousto-optical devices such as acousto-optical transmission filters (AOTFs) or acousto-optical beam splitters (AOBSs).

[0043] Consequently, if the wavelength of the illuminating light is varied, it can be shifted beyond the spectral filter edge, whereby the transmission or reflection properties of the beam path sector change for the varied wavelength.

[0044] It is also conceivable to use other broadband lasers for illumination, with these lasers allowing the wavelength of the emitted light to be adjusted. The use of at least one diode laser is also conceivable. Diode lasers can be tuned, i.e., varied, within certain limits in terms of the wavelength they emit, for example, by varying the operating current and / or the operating temperature. In an alternative embodiment, a module comprising several switchable lasers, for example, diode lasers, can be used.

[0045] In a second approach, the relative position of the at least two wavelengths with respect to the filter edge is also varied, but here the wavelengths remain constant and the spectral position of the filter edge is varied, i.e., the wavelength at which the filter edge lies is changed. This can be achieved using the beam path selector interchangeable module by replacing one beam path selector with another beam path selector or, alternatively, by inserting the additional beam path selector into the microscope beam path in addition to the beam path selector.

[0046] Alternatively or additionally, a dichroic graduated filter can be used. This is characterized by the fact that the spectral position of the filter edge depends on its position within the graduated filter.

[0047] Two beam path selectors inserted into the microscope beam path can also enable switching of the operating state of the microscope only in combination with their respective transmission and reflection properties, for example reflecting both wavelengths.

[0048] The following is a non-limiting example. Assume two wavelengths, where the second wavelength is greater than the first wavelength, and the beam path selector, as a wavelength-selective element, is equipped with a filter edge located between the two wavelengths. The first wavelength is in a first spectral range of the wavelength-selective element and the second wavelength is in a second spectral range of the wavelength-selective element. In the first spectral range, a (ideally assumed) transmission of 1 can be present, whereas in the second spectral range, a (likewise ideally assumed) reflection of 1 and a transmission of 0 are present.

[0049] With this relative arrangement of the wavelengths and the filter edge, the microscope is in light sheet mode. A second beam selector can now be inserted into the microscope beam path. This second beam selector has a filter edge at a wavelength greater than the second wavelength, with both wavelengths lying in the first spectral range of the additional beam selector and both being reflected. This corresponds to the first microscopy mode. The microscope can provide one or more filter wheels that enable such a change between the beam selectors. A filter wheel is one possible design of a switching device.

[0050] It is also conceivable that, in this example, the additional beam path selector could be inserted into the microscope beam path in addition to the beam path selector already present. In this case, the additional beam path selector could be, for example, a short-pass filter whose filter edge also lies between the two wavelengths. The combination of the beam path selector (long-pass filter) with the additional beam path selector (short-pass filter) also switches the microscope from light-sheet mode to the first microscopy mode.

[0051] In a further advantageous embodiment, the microscope can comprise at least one light source that emits light in at least two different wavelength ranges, for example a laser module with at least two diode lasers, wherein light from the at least one light source of the at least two different wavelength ranges can be coupled into the microscope beam path independently of one another, wherein at least two of the different wavelength ranges of the light source lie in different spectral ranges of the wavelength-selective optical element.

[0052] The two different wavelength ranges can thus be generated by a single light source or by several light sources.

[0053] By selecting the appropriate beam path selector, it is possible to switch very quickly between the two beam paths 1 and 2 without mechanical movement of any part, provided the filter edge of the selector lies between the two wavelengths. This can be used for photomanipulation. The wavelength change can be achieved within microseconds (e.g., by using an AOTF to switch the wavelengths). When using a galvanometer scanner, targeted illumination with a specific wavelength in a specific sample area can be achieved in the millisecond range by simply offsetting the position of the galvo mirror.

[0054] If an acousto-optical adjustment unit is used for beam deflection, positioning can be achieved in microseconds. This is particularly advantageous for fast optogenetically stimulable processes in living organisms, as well as for manipulations that can be triggered by light.

[0055] Preferably, such a light source which emits different wavelength ranges or different wavelengths therein can be combined with the previously described variation of the wavelength.

[0056] If the beam path selector in the optical module is replaced, an optical filter system for changing filters can preferably also be provided in a further plane of the microscope, for example in a detector coupling plane, wherein the filters are selected according to the desired illumination and detection wavelengths and the operating state. The detector coupling plane is the plane to which the detector unit for point and area detection and the at least one light source can be coupled. Filter wheels or filter changing systems known from the prior art can also be used here as a switching device. The filter changing system in this plane can select a filter according to the setting of the beam path selector changing module, particularly preferably automatically and depending on the beam path selector changing module, and insert it into the microscope beam path.In other words, a change of the beam path selector initiates a change of a filter of the filter change system of the detector coupling plane.

[0057] A means for switching the operating mode of the microscope can thus be provided in the microscope itself, for example, in the microscope body. This means can work in close cooperation with the beam path selector interchangeable module.

[0058] In the microscope according to the invention, the area detector is preferably used as the detector for the light sheet mode. If the first microscopy mode is the confocal mode, a point detector can preferably be used for this. However, other combinations of detectors are conceivable, for example, the use of the area detector also for the confocal mode. The detectors can also be used in combination with one another.

[0059] In a further embodiment, the method according to the invention can be improved by in the method in the light sheet mode, a region of interest of the sample volume is identified; after switching to the first microscopy mode, the identified region of interest is examined by means of a scanning microscopy method with a higher resolution than in the light sheet mode; and that image data of the light sheet mode and the first microscopy mode generated by the area sensor or a point detector, which represent a light distribution emitted by the sample volume, are fused and / or displayed together.

[0060] The operating state of the microscope can be switched using one of the process steps described above.

[0061] This embodiment of the method has the advantage that an image can be acquired very quickly in a sample, providing an overview of the sample. Details may not be resolved in these images (intentionally). The region of interest can be identified in the image taken in the microscope's light-sheet mode. This is also called the "region of interest" (ROI for short). The ROI can be selected by the user in a suitable manner on a screen using hand gestures, a mouth switch, or a foot switch. The operating mode of the microscope can then be switched. The preferred scanning method only takes one image of the ROI, i.e., it does not scan / rasterize the entire area acquired in light-sheet mode.

[0062] This method design can be particularly efficient because both operating modes are possible in a single microscope, meaning both measurements / images can be taken without changing the microscope or sample. Furthermore, the ROI selection avoids the need to store large amounts of data that concern areas that may be of little or no interest to the user for the current examination.

[0063] In particular, the image data generated in the light-sheet mode and the first microscopy mode can be fused. This means that the generated image data are correlated with each other, particularly with regard to their spatial position within the sample. For example, if the ROI is enlarged, it can be displayed with higher resolution in this area, whereas this resolution cannot be guaranteed in areas outside the ROI (since the image captured in the light-sheet mode may offer a lower resolution).

[0064] In a further embodiment of the method according to the invention, light which differs from the illumination light can be irradiated onto or into a sample arranged in the sample volume.

[0065] This additional light can be called manipulation light. Depending on the wavelength of the manipulation light, it can either be reflected by the beam selector and redirected along the first beam path or transmitted and passed along the second beam path.

[0066] If the manipulation light is transmitted through the beam path selector, it can irradiate the sample over a large area, i.e. essentially in the form of a light sheet. Due to the different wavelengths, the light sheets formed by the illumination light and the manipulation light can differ slightly in size.

[0067] If, however, the manipulation light is reflected, a point-like irradiation of the sample is possible. The point-like irradiation can then be carried out as a line or area. However, it is conceivable that, with appropriate coupling of the manipulation light (divergent or convergent), a planar irradiation of the sample along the first beam path is also possible.

[0068] The irradiation of the sample can be resonant (at an absorption maximum of the sample) or non-resonant (outside the absorption maximum of the sample), whereby in the first case one can speak of manipulation and in the second case of excitation of the sample.

[0069] In particular, the sample can also be irradiated with near-infrared light to generate multiphoton excitation within the sample. Simultaneously, images can be captured using a light sheet.

[0070] When irradiated with near-infrared light, it can be reflected by the wavelength-selective optical element to the first optical arrangement. Shorter-wavelength fluorescent light (e.g., green) can be generated in the sample and collected by the first optical arrangement. It can also be reflected by the wavelength-selective element from the first optical arrangement toward the microscope body. At the same time, red or blue light (light with wavelengths perceived or defined as red or blue, respectively) can be transmitted through the wavelength-selective optical element toward the second optical arrangement, forming a light sheet (static or virtual light sheet). If the third wavelength-selective optical element is suitably selected, it can reflect the near-infrared, blue, or red light, but transmit the spectral range of fluorescence (green) toward a camera.

[0071] The near-infrared light can be used to generate multiphoton data sets either with a point detector unit or with non-descanning detectors directly above the first optical array (i.e., downstream of the additional wavelength-selective optical element). One advantage of such a setup is that switching is possible without any mechanical movement, allowing for rapid switching between a manipulating method and the light-sheet mode.

[0072] The manipulation light can be coupled into the microscope via a light interface using a suitable additional wavelength-selective element. In the confocal mode of the microscope, such an additional wavelength-selective element can preferably be removed from the beam path or replaced with a (particularly preferably anti-reflective) glass substrate that replicates the beam offset of the additional wavelength-selective element without having any further (spectral) influence on the beam path.

[0073] In the microscope according to the invention, multiphoton light sheet illumination and simultaneous illumination with visible light can be performed to stimulate a biological event. For this purpose, illumination light in the visible spectral range (e.g., less than 500 nm) can be coupled into the microscope beam path via the additional wavelength-selective optical element. It may be possible to adjust an illumination area using a field diaphragm in an axis associated with the additional wavelength-selective optical element.

[0074] The microscope can have an optical interface for connecting a confocal unit. This can be formed by a third wavelength-selective optical element, which can guide light either toward the confocal unit or toward a camera. By appropriately selecting the third wavelength-selective optical element, as well as the wavelength-selective element in the optical module, it is thus possible to confocally scan the sample point by point via the first optical arrangement and either capture the light completely with the camera; capture it completely with a point detector unit; or capture it partially with both the camera and the point detector (with a partial reflector or a chromatic filter in the optical interface). The wavelength-selective optical element of the optical module can be designed as a mirror.

[0075] Possible manipulation methods include laser ablation, optogenetics or photoconversion.

[0076] The microscope according to the invention and the method according to the invention thus allow a sample to be examined using various microscopy techniques. Instead of a single objective lens, as used in the prior art, the module according to the invention can be installed and used on the microscope.

[0077] Simply by using suitable beam path selectors and changing the beam path selector as described in some embodiments, various microscopy techniques can be combined or performed simultaneously. The examinations take place in the same sample area and do not require changing the microscope or moving the sample.

[0078] In addition to the already mentioned confocal scanning microscopy, other possible microscopy techniques are FLIM, FRET, multiphoton microscopy or localization microscopy (PALM / STORM and related techniques).

[0079] In particular, the optical module according to the invention can be used to convert a commercially available, preferably upright, confocal raster scanning microscope into a light sheet-capable microscope.

[0080] Such a conversion saves the space required for an additional microscope and can also be more cost-effective than a complete light-sheet microscope. In particular, the cost of the optical module required for the necessary conversion can be on the order of magnitude of the cost of a new objective.

[0081] By using the optical module according to the invention, an existing confocal microscope can be converted into a versatile microscope with a variety of operating states or operating modes.

[0082] In a further embodiment of the microscope according to the invention, the microscope beam path can be fed into the optical module tilted relative to a normal perpendicular to the rear focal plane of the first and / or second optical arrangement, wherein the second optical arrangement and / or the attachment element can be tilted depending on the tilt of the microscope beam path relative to the normal of the rear focal plane of the second optical arrangement.

[0083] With this configuration of the microscope according to the invention, it can be ensured that light which is essentially transmitted through the beam path selector, i.e. runs along the second optical beam path, does not reach the field of view of the camera (2D detector) via the first optical beam path.

[0084] If wavelength-selective elements in the form of dichroic beam splitters or dichroic mirrors are used as beam path selectors, their coatings can only guarantee 99.0% to 99.9% suppression of unwanted reflections. This means that 0.1% to 1.0% of the incident illumination light intended to propagate along the second beam path can be reflected into the first beam path. This light, which is unintentionally directed toward the first optical arrangement—i.e., a portion of the illumination light—is focused by the first optical arrangement into the sample and can, for example, generate fluorescence in the sample. This fluorescence can overlay the fluorescence excited by the light sheet and produce unwanted artifacts.

[0085] The illumination light is therefore tilted into the optical module so that the portion of the illumination light reflected by the beam path selector is not focused within the sample's image field by the first optical arrangement. In other words, this portion lies outside the acceptance angle of the first optical arrangement and is therefore not imaged by it.

[0086] However, the tilting of the microscope beam path also influences the second beam path, so that preferably the second optical arrangement and / or the attachment element can be tilted in such a way that the first optical axis and the second optical axis in the sample volume again intersect substantially perpendicular to each other.

[0087] In a further embodiment of the microscope according to the invention, a stop filter for attenuating or blocking a reflected portion of the second spectral range (ie the illumination light) which is transmitted from the wavelength-selective optical element essentially to the second optical arrangement and / or a further optical arrangement for displacing the rear focal plane of the first optical arrangement in the first beam path can be provided.

[0088] A stop filter can be a simple alternative to the aforementioned tilting of the microscope beam path. Ideally, the stop filter can completely block the illumination light or at least attenuate it to such an extent that the already only 0.1% to 1.0% of the illumination light cannot generate detectable fluorescence after attenuation.

[0089] To prevent possible vignetting, the additional optical arrangement can be used to shift the rear focal plane of the first optical arrangement. This preferably afocal optical arrangement can shift a plane of a designated tilt axis into the actual rear focal plane of the first optical arrangement.

[0090] This design prevents the beam from tilting around a plane located in the rear focal plane of the optical arrangement. If this were to happen, points at the edge of the image field would not be fully imaged, resulting in vignetting.

[0091] As an alternative to using afocal optics, a state-of-the-art scanner configuration can be used. This configuration comprises two mirrors that can be tilted around parallel axes of rotation, but at different angles. In this case, the position of the tilt plane, i.e., the plane in which the tilt axis lies, can be determined from the distance between the mirrors and the two deflection angles of the mirrors.

[0092] A scanning unit or scanner in a confocal microscope can consist of three scanning devices. Two of these can deflect in the Y direction, one in the X direction. The two mirrors deflecting in the Y direction can direct the tilt axis to the mirror deflecting in the X direction, so that the tilt axes lie in the same plane in both the X and Y directions, as with a gimbal-mounted mirror.

[0093] For the light sheet module, tilting can preferably be achieved with two mirrors in a direction perpendicular to the light sheet plane. The scanning movement to generate the light sheet (virtual light sheet) can be achieved with a single mirror, which can allow for faster scanning. Tilting via the Y scanners and shifting the tilt axis to the rear focal plane of an optical arrangement (e.g., the second optical arrangement) can ensure that the light sheet is not tilted relative to the focal plane of the first optical arrangement. This can prevent the image from becoming blurred in the edge areas.

[0094] The microscope according to the invention can be further improved by using at least one element from the following list of elements, comprising the first optical arrangement; the second optical arrangement; the optical path selector; and the stop filter can be repeatedly and interchangeably mounted when the module is attached to the mounting device.

[0095] Preferably, several of the above-mentioned elements can be exchangeable together so that a corresponding operating state can be selected.

[0096] The microscope can comprise a further imaging beam path which has a further microscope objective and a further detector, wherein the objective of the further imaging beam path can be used to image the sample volume onto the further detector from a side which is facing away from the optical module.

[0097] The microscope's further imaging beam path can be movable relative to the optical module. Alternatively, the first optical arrangement can also be movable relative to the second optical arrangement.

[0098] In addition to the attachment element arranged between the first optical arrangement or the second optical arrangement and the sample volume, a further attachment element can be arranged between the respective other optical arrangement and the sample volume.

[0099] The optical axis of the microscope beam path and the optical axis of the first optical arrangement and / or the optical axis of the second optical arrangement and / or the optical axis of the optionally further imaging beam path can intersect a common object field.

[0100] The first optical arrangement and / or the second optical arrangement and / or optionally the further imaging beam path can have a variable lens or a variably adjustable liquid lens or a vario optic.

[0101] In a further embodiment of the microscope, a dynamic light sheet can be generated using a scanner.

[0102] It is also conceivable that a static light sheet can be generated using a cylindrical lens.

[0103] In a further embodiment of the microscope, the first optical arrangement and / or the second optical arrangement can have a microscope objective with a finite optics or an infinite optics.

[0104] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where an apparatus corresponds to a method step or a function of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding element or property of a corresponding apparatus. Some or all of the method steps may be performed by (or using) a hardware device, such as a processor, a microprocessor, a programmable computer, or an electronic circuit. In some embodiments, one or more of the key method steps may be performed by such an apparatus.

[0105] Depending on specific implementation requirements, embodiments of the invention may be implemented in hardware or software. The implementation may be performed using a non-volatile storage medium, such as a digital storage medium, such as a floppy disk, DVD, Blu-ray, CD, ROM, PROM and EPROM, EEPROM, or FLASH memory, on which electronically readable control signals are stored that interact (or can interact) with a programmable computer system to perform the respective method. Therefore, the digital storage medium may be computer-readable.

[0106] Some embodiments according to the invention comprise a data carrier with electronically readable control signals that can interact with a programmable computer system so that one of the methods described herein is carried out.

[0107] In general, embodiments of the present invention can be implemented as a computer program product with program code, wherein the program code is effective for executing one of the methods when the computer program product is running on a computer. The program code can, for example, be stored on a machine-readable medium.

[0108] Further embodiments include the computer program for carrying out one of the methods described herein, which is stored on a machine-readable carrier.

[0109] In other words, one embodiment of the present invention is therefore a computer program having a program code for carrying out one of the methods described herein when the computer program runs on a computer.

[0110] A further embodiment of the present invention is therefore a storage medium (or a data carrier or a computer-readable medium) comprising a computer program stored thereon for performing one of the methods described herein when executed by a processor. The data carrier, digital storage medium, or recorded medium is typically tangible and / or non-seamless. A further embodiment of the present invention is an apparatus as described herein, comprising a processor and the storage medium.

[0111] A further embodiment of the invention is therefore a data stream or signal sequence representing the computer program for carrying out one of the methods described herein. The data stream or signal sequence can, for example, be configured to be transmitted via a data communication connection, for example, via the Internet.

[0112] A further embodiment comprises a processing means, for example a computer or a programmable logic device, configured or adapted to carry out any of the methods described herein.

[0113] A further embodiment comprises a computer on which the computer program for carrying out one of the methods described herein is installed.

[0114] A further embodiment according to the invention comprises a device or system configured to transmit (e.g., electronically or optically) a computer program for performing one of the methods described herein to a recipient. The recipient may, for example, be a computer, a mobile device, a storage device, or the like. The device or system may, for example, comprise a file server for transmitting the computer program to the recipient.

[0115] In some embodiments, a programmable logic device (e.g., a field-programmable gate array, FPGA) may be used to perform some or all of the functionality of the methods described herein. In some embodiments, a field-programmable gate array may cooperate with a microprocessor to perform any of the methods described herein. In general, the methods are preferably performed by any hardware device.

[0116] The present invention will be explained in more detail below with reference to the accompanying figures. These figures show purely exemplary embodiments of the microscope according to the invention and associated methods according to the invention. The technical features shown in the individual embodiments can be combined with one another as desired and can also be omitted if they are not necessary for a particular embodiment.

[0117] They show:Fig. 1 shows a schematic structure of the microscope according to the invention; Fig. 2 shows a schematic structure of a further embodiment of the microscope according to the invention; Fig. 3 shows a schematic representation of the optical module according to the invention for avoiding vignetting due to a shifted entrance pupil; Fig. 4 shows a schematic representation of a scanning unit with freely adjustable plane in which the tilt axis of the beam lies; Fig. 5 shows a schematic representation of a further embodiment of the optical module according to the invention for avoiding vignetting due to a shifted entrance pupil; Fig. 6a shows a schematic representation of a further embodiment of the optical module according to the invention for avoiding unwanted reflections; Fig. 6b shows a schematic representation of a further embodiment of the optical module according to the invention for avoiding unwanted reflections;Fig. 7 shows a schematic representation of a transmission characteristic of the wavelength-selective optical element; Fig. 8 shows a beam path selector interchangeable module with a plurality of beam path selectors; Figs. 9 and 10 each show a schematic view of a further embodiment of an optical module; Fig. 11 shows a schematic view of a further embodiment of an optical module with a further imaging beam path; and Fig. 12 shows a schematic view of a further embodiment of an optical module.

[0118] In Fig. 1 A microscope 1 is shown, which is a confocal microscope 4 converted into a light sheet microscope 3. The microscope 1 comprises a microscope body 5 and a mechanical mounting device 7 for microscope objectives 37, through which a microscope beam path 9 extends. Attached to the mounting device 7 is an optical module 11 for illuminating a sample volume 13 and for collecting and transmitting light 15 from the sample volume 13. The sample volume 13 is arranged on a sample stage 69.

[0119] The optical module 11 comprises a first optical arrangement 17 with a first beam path 19, a second optical arrangement 21 with a second beam path 23, an optical beam path selector 25 which combines the first beam path 19 and / or the second beam path 23 with the microscope beam path 9.

[0120] The optical beam path selector 25 is designed as a wavelength-selective optical element 39. In particular, this is a dichroic mirror 41.

[0121] The second optical arrangement 21 is in Fig. 1 shown only as a single lens, but like the first optical arrangement 17, it can be a microscope objective 37. In particular, the second optical arrangement 21 can be a light sheet module 43 for generating a light sheet 45 traversing the sample volume 13.

[0122] The microscope 1 further comprises a light source 47, a beam splitter 49, a scanner 51 and a point detector unit 53, all of which are arranged in a confocal module 55.

[0123] In addition to the wavelength-selective optical element 39 of the optical module 11, a further wavelength-selective optical element 57, which is also a beam splitter 49, is provided. Manipulation light 59 can be coupled into the microscope beam path 9 via this. Furthermore, a stop filter 35 is shown, the function of which is described in Fig. 6 described.

[0124] Furthermore, a third wavelength-selective optical element 61 (likewise a beam splitter 49) is provided, which represents an optical interface 63 via which the confocal module 55 (also called a confocal unit or scanning unit in the case of multiphoton excitation) and / or a camera 65 can be connected to the microscope beam path 9. By appropriately selecting the third wavelength-selective optical element 61, both the confocal module 55 and the camera 65 can be simultaneously irradiated with light 15 of the corresponding wavelength, so that multiple measurements of the sample 69 can be performed simultaneously.

[0125] The Fig. 2 shows a further embodiment of the microscope 1 according to the invention, which, in the confocal module 55, has a near-infrared light source 73 in addition to the light source 47, which in this embodiment is a visible light source 71. The near-infrared light source is also coupled into the microscope beam path 9 by means of a beam splitter 49. The scanner 51 thus deflects visible light 73 and near-infrared light 75 equally. This allows, in a confocal mode, scanning mode, or operating state of the microscope 1, multiphoton excitation to be carried out in the sample volume 13 in the same area where the visible light 73 is also radiated for illumination.

[0126] In the optical module 11, in the embodiment shown, the near-infrared light 75 is reflected by the wavelength-selective optical element 39 to the first optical arrangement 17. The visible light 73, for example, blue light, is transmitted through the wavelength-selective optical element 39 and reaches the second optical arrangement 21, which is depicted here as a microscope objective 37. The visible light 73 thus forms the light sheet 45, whereas the near-infrared light 75 is focused through the first optical arrangement 17.

[0127] The near-infrared light 75 can excite fluorescence in the sample volume 13 and emit fluorescent light 77, which is collected by the first optical arrangement 17 and reflected by the wavelength-selective optical element 39.

[0128] The third wavelength-selective optical element 61 is transparent to the fluorescent light 77, allowing it to be detected by the camera 65. The camera is attached to the tube via a tube interface 79 on the microscope 1; alternatively, the tube can be replaced by a tube lens with a camera adapter (not shown).

[0129] The near-infrared light 75 can thus be used to generate multi-photon data sets (not shown) acquired with the point detector unit 53. Alternatively, non-descanning detectors 81 (NDDs for short) can be used, which receive the fluorescent light 77 via the additional wavelength-selective optical element 57. The NDDs 81 are shown only schematically.

[0130] In a further embodiment, the light source 47 can be a broadband light source 83. This is shown in a circle above the microscope 1.

[0131] This broadband light source 83 can transmit emitted light 15 through a wavelength-modifying element 85. The wavelength-modifying element 85 thus represents a switching device 159 and, in the example shown, can be an acousto-optical transmission filter (AOTF) 87. This can be used to select the wavelength of the light 15, so that at the wavelength-selective element 39 in the optical module 11, the light can be directed either to the first optical arrangement 17 or to the second optical arrangement 21 simply by adjusting the wavelength-modifying element 85. A switching frequency of the wavelength-modifying element 85 can determine a possible frame rate for images to be recorded; switching optical components is not necessary.In such an embodiment, the two-wavelength selective optical element 61 can be replaced by a neutral splitter 89, which reflects 50% of the incoming light and transmits 50%. The use of a wavelength-variable light source 84 (this represents a switching device 159) is also possible.

[0132] The illustrated setup allows for several combinations of illumination and stimulation. For example, near-infrared multiphototonal light-sheet illumination and simultaneous area illumination with visible light 73, or light-sheet illumination with visible light 73 in combination with stimulation / excitation of the sample 69 using visible light 73 (a different wavelength) via the first optical arrangement 17.

[0133] In Fig. 3 the optical module 11 is shown, wherein an illumination beam of the light sheet 89, a confocal illumination beam 91 and a confocal illumination beam with shifted tilt plane 93 are shown, furthermore rear focal planes 95 of the first 17 and also of the second optical arrangement 21 are shown.

[0134] It can be seen that with a tilted confocal illumination beam 91, the tilt occurs around a tilt plane 97 and not around the rear focal plane 95 of the first optical arrangement 17. This leads to illumination light 101 being guided in the optics 99 in edge regions 103 of the optics 99. This can lead to shadowing in the edge regions 103, the so-called vignetting.

[0135] This imaging error due to tilted irradiation of the illumination light 101 can be avoided by an additional optic 105, in particular an afocal optic 107. This is in Fig. 5 shown.

[0136] Here, too, the confocal illumination beam 91 is tilted, but is deflected by the additional optics 105 / the afocal optics 107 such that the confocal illumination beam 91 is tilted around the rear focal plane 95 of the first optical arrangement 17. The rear focal plane 95 is thus the tilt plane 97. The original beam path of the confocal illumination beam 91 is shown in dashed lines.

[0137] In contrast to the uncorrected confocal illumination beam 111, the corrected confocal illumination beam 109 is no longer guided through the edge regions 103, but rather in the near-axis region 113. Vignetting is thus attenuated or even completely avoided.

[0138] In the Fig. 4 A scanner 51 is shown in which two mirrors 115 can be tilted about parallel axes of rotation 117. However, a first mirror 115a is tilted by a first angle 119, and a second mirror 115b is tilted by a second angle 121. The first angle 119 and the second angle 121 differ from each other, so that, in combination with a distance 123 between the mirrors 115, the position of the tilt plane 97 can be determined. This method is also known as beam-walking.

[0139] In Fig. 6a The optical module 11 is shown, showing the illumination beam of the light sheet 89 and a tilted illumination beam of the light sheet 125. If illumination light 101 is now to be transmitted along the illumination beam of the light sheet 89 through the wavelength-selective optical element 39, an unwanted reflection can occur at the latter, so that a portion of illumination light 123 is reflected in the direction of the first optical arrangement 17. This portion 123 can amount to 0.1% to 1.0% (depending on the specifications of the filter companies, this portion can also be more than 1%) of the originally incident illumination light 101.

[0140] If the illumination occurs along the illumination beam of the light sheet 89, the portion of illumination light 123, this portion 123 being referred to below as the first portion 123a for differentiation, is focused by the first optical arrangement 17 into an image field 127 of the first optical arrangement 17 and can lead to disturbing artifacts 129 in image data 131 of the sample 69. These artifacts 129 are referred to below as first artifacts 129a for differentiation.

[0141] However, if the illumination light 101 is irradiated along the tilted illumination beam of the light sheet 125, a second reflected portion 133 still occurs. However, after passing through the first optical arrangement 17, this second reflected portion 133 impinges outside the image field 127. This means that this second reflected portion 133 does not reach the sample 69 within the image field 127. These artifacts 129, called second artifacts 129b for differentiation, are therefore not present in the image data 131.

[0142] During the introduced tilting of the illumination beam 89, the position of the first optical arrangement 17 remains unchanged. However, since the first beam path 19 and the second beam path 23 should be perpendicular to each other for a light sheet recording, the attachment element 27 is shifted laterally. This is schematically illustrated by dashed lines.

[0143] The attachment element 27 drawn with a solid line indicates a position 135 for the tilted illumination beam of the light sheet 125. In contrast, an optimal position 137 for the non-tilted illumination beam of the light sheet 89 is indicated by a dashed line.

[0144] To avoid lateral chromatic aberration of the illumination lens when tilting the illumination beam, another alternative is provided with reference to the attached Fig. 6b described: If the second optical arrangement 21 is illuminated at a tilt far outside the center of the image, lateral chromatic aberrations can occur. This becomes apparent when using different illumination wavelengths as a focus offset in the illumination plane (different planes are illuminated). To avoid this, when the illumination beam of the light sheet 125 is tilted, the second optical arrangement 21 together with the attachment element 27 is also tilted by this angular amount (as an example 1°-8°). This, on the one hand, prevents imaging errors in the form of lateral chromatic aberrations and, on the other hand, ensures that the first beam path 19 and the second beam path 23 are perpendicular to one another for the light sheet recording. The detection objective 17 therefore does not need to be tilted accordingly.

[0145] The second optical arrangement 21 and the attachment element 27, drawn with a solid line, mark the position 135 for the tilted illumination beam of the light sheet 125; the optimal position 137 for the non-tilted illumination beam of the light sheet 89 is indicated by a dashed line.

[0146] Also in the Fig. 6b The second artifacts 129b, which arise due to the second reflected portion 133 occurring through the first optical arrangement 17, are shown. However, these second artifacts 129b lie outside the image field 127 and are therefore not present in the image data 131. Also shown are the first artifacts 129a, which occur in the image data 131 for the illumination beam of the light sheet 89 and without the use of a stop filter 35 described below.

[0147] Alternatively, the unwanted reflection described here can be avoided by inserting the above-mentioned stop filter 35 between the wavelength-selective optical element 39 and the first optical arrangement 17. The stop filter 35 is preferably located between a deflection mirror 139 and the first optical arrangement 17. The stop filter 35 blocks the portion of illumination light 123 so that it cannot form disturbing artifacts 129 in the image data 131.

[0148] In Fig. 7 a transmission characteristic of the wavelength-selective optical element 39 is shown. The transmission T is plotted against the wavelength , where the transmission T is ideally zero in a first spectral range 145 and increases to one at a filter edge 143, i.e., in a second spectral range 147. Ideally, the reflection characteristic R (not shown) can be obtained from the transmission characteristic 141 using the formula T+R=1.

[0149] In a light sheet mode 149, which is schematically represented by horizontal hatching, a first wavelength 151 lies in the first spectral range 145 and a second wavelength 153 lies in the second spectral range 147, so that light 15 of the first wavelength 151 is reflected at the wavelength-selective optical element 39 (transmission T is zero), whereas light 15 of the second wavelength 153 is transmitted. The light 15 of the second wavelength 153 can thus represent illumination light 101 for the light sheet 45.

[0150] In a first microscopy mode 155 - this is also represented by a hatched area - both the first wavelength 151 and the second wavelength 153 are in the first spectral range 145.

[0151] Switching between the light sheet mode 149 and the first microscopy mode 155 can thus be effected by (a) varying the position of the filter edge 143 or (b) shifting at least one wavelength beyond the filter edge 143 into the same spectral range of the further wavelength.

[0152] A procedure according to (a) can be realized by replacing the wavelength-selective optical element 39. This can be done, for example, by means of a Fig. 8 shown switching device 159 designed as beam path selector exchange module 157, which in the case shown can alternately introduce different beam path selectors 25 into the microscope beam path 9.

[0153] In the case of a procedure according to (b), the Fig. 2 The wavelength-changing element 85 shown can be used, for example, to shift the second wavelength 153 into the first spectral range 145.

[0154] Fig. 9 shows a further embodiment of an optical module 11, which is mounted on a mechanical receiving device 7 of a Fig. 9 not shown microscope or microscope body with a microscope beam path 9. This optical module 11 has a housing 160 in which the first optical arrangement 17 with the first beam path 19 and the second optical arrangement 21 with the second beam path 23 intersecting the first beam path 19 in the sample volume 13 are arranged. An attachment element 27 is arranged between the second optical arrangement 21 and the sample volume 13, with the second beam path 23 passing through the attachment element 27. With the aid of the cylindrical lens 196, a static light sheet can be generated with the second optical arrangement 21.

[0155] On the housing 160 of the optical module 11, a coupling point 162 is provided, via which illumination light for illuminating a part of the sample volume 13 is passed via the second beam path 23. Thus, the illumination light could, on the one hand, be collimated and coupled as a freely running light beam into the second beam path 23, wherein in this case the coupling point is designed, for example, in the form of a passage in the housing 160 of the optical module 11 (in Fig. 9 not shown). Specifically, a housing part 164 is arranged at the coupling point 162, on which a fiber coupler 166 of an optical fiber 168 is arranged. Illumination light from a source not shown in Fig. 9 shown light source is coupled into the second beam path 23. For this purpose, a collimation optic is provided with which the illumination light emerging divergently from the fiber coupler is collimated.

[0156] Fig. 10 shows a further embodiment in which the housing part 164 is arranged from above or on another side of the housing 160 of the optical module 11, whereby a further deflection mirror 139 arranged downstream of the collimation optics 170 is required, but where other requirements for the external installation space can be met than in the embodiment of the Fig. 9 is the case.

[0157] Fig. 11 shows a further embodiment in which a further imaging beam path 172 is provided. The further imaging beam path 172 has a microscope objective 174 and a further, in Fig. 11 not shown detector, which could be arranged, for example, below the one in the form of a suitably designed dichroic steel splitter 139. With the objective 174 of the further imaging beam path 172, the sample volume 13 can be imaged onto the further detector from a side facing away from the optical module 11. In other words, the same object or sample volume 13 can be imaged with the further imaging beam path 172 from the lower side of the Fig. 11 The sample volume 13 can be imaged with the optical module 11 from above relative to the sample container 176. This can be done simultaneously or sequentially. It is also possible to manipulate the object in the sample volume in a targeted manner via the additional imaging beam path 172, for example, using pulsed or continuous light of a suitable wavelength, intensity, and / or beam profile. In this respect, it can also be provided that the additional imaging beam path 172 serves solely for object manipulation.

[0158] In a preferred embodiment, the further imaging beam path 172 is arranged to be movable relative to the optical module 11. Alternatively or additionally, the first optical arrangement 17 can be arranged to be movable relative to the second optical arrangement 21, for example in a substantially horizontal direction of the Fig. 9 .

[0159] Preferably, in addition to the attachment element 27 arranged between the first optical arrangement 17 or the second optical arrangement 21 and the sample volume 13, a further attachment element (not shown in the figures) could be arranged between the respective other optical arrangement 21, 17 and the sample volume 13. Thus, the angle α between the optical axis 180 of the microscope objective 37 of the first beam path 19 of the first optical arrangement 17 and the optical axis 182 of the second beam path 23 (after the attachment element 27) of the second optical arrangement 21 can also be changed and, in particular, reduced or even set to an angle of substantially zero degrees if, for example, the further attachment element is designed as a deflection element, which deflects the optical axis of the first beam path 19 of the first optical arrangement 17 accordingly.

[0160] Most preferably, the optical axis 178 of the microscope beam path 9 and the optical axis 180 of the first optical arrangement 17 and / or the optical axis 182 of the second optical arrangement 21 and / or the optical axis 184 of the optionally further imaging beam path 172 intersect a common object field or a common area in the sample volume 13, which e.g. in Fig. 11 is indicated. In this context, the common object field is to be understood in particular as the area of the microscope objective 37 of the first optical arrangement 17 that lies in the focal plane of the microscope objective 37 and can be imaged therefrom onto a detector. By designing the beam paths of the microscope body, the first optical arrangement 17, the second optical arrangement 21 and / or the optionally further imaging beam path 172, the same object can be imaged from different sides simultaneously or with a time delay in a particularly advantageous manner, namely, for example, with the microscope objective 37 of the first optical arrangement 17 or with the further microscope objective 174 of the further imaging beam path 172.

[0161] The first optical arrangement 17 and / or the second optical arrangement 21 and / or optionally the further imaging beam path 172 could each comprise a variable lens 186 (see Fig. 9 ) or a variably adjustable liquid lens—based, for example, on electrowetting technology—or a varifocal lens. This allows, for example, the focus position and / or the effective (illumination) aperture of the second optical arrangement 21 to be varied relative to the first beam path 19 or the microscope objective 37.

[0162] An influence on the sample illumination generated by the second optical arrangement 21 can alternatively or additionally also be achieved with the Fig. 12 shown further embodiment (which has a similar structure to the embodiment of the Fig. 10 has) can be realized. For this purpose, a polarization beam splitter 194 is suitably arranged downstream of the collimation optics 170, which in this embodiment deflects the linearly polarized illumination light coming from the collimation optics 170 in the direction of the optics 188. The optics 188 are configured to focus the illumination light. The illumination light then propagates to the movably arranged mirror 190. The mirror 190 can be Fig. 12 be moved horizontally to the left or right and / or tilted relative to the optical axis of the optics 188 (see the two double arrows). After the illumination light has passed through the delay plate 192 twice, the polarization direction of the illumination light is rotated relative to the polarization direction of the light coming from the collimation optics 170 such that it can pass through the polarization beam splitter 194 essentially in a straight line and thus illuminate the sample volume 13 via the second beam path 23 of the second optical arrangement 21. By moving the movable mirror 190, the illumination situation in the sample volume 13 can be changed accordingly. Thus, the focus position of the generated light sheet can be varied in the propagation direction of the illumination light if the mirror 190 is moved horizontally to the left or right.Tilting the light sheet is possible by tilting the mirror 190 accordingly relative to the optical axis of the optics 188. The retardation plate 192 can be arranged either between the optics 188 and the mirror 190 or between the optics 188 and the polarization beam splitter 194. In . Fig. 12 the two positions for the delay plate 192 are shown, but only one delay plate 192 is required in the beam path.

[0163] Now a light sheet in the sense of a virtual light sheet could be connected, for example, with a Fig. 11 The image can be generated dynamically using the scanner of the confocal microscope (not shown). In this case, no cylindrical lens is arranged in the second beam path 23. Alternatively, the Fig. 11 A static light sheet can be generated by the scanner of the confocal microscope (not shown) if, for example, the scanning mirrors of the scanner of the confocal microscope are "parked" in a suitable position, wherein then in the second beam path 23 a suitable cylindrical lens is arranged at a suitable position (see, for example, the cylindrical lens 196 in Fig. 9 ).

[0164] Now, the first optical arrangement 17 and / or the second optical arrangement 21 of the optical module 11 can have a microscope objective 37 with a finite optic (e.g., calculated at 160 mm and then without a tube lens) or an infinite optic. If a microscope objective 37 with an infinite optic is used, a tube lens is provided, which can be arranged in a suitable position in the optical module 11.

[0165] The term "and / or" includes all combinations of one or more of the related listed elements and may be abbreviated as " / ".

[0166] Although some aspects have been described in the context of a device, it is clear that these aspects also represent a description of the corresponding method, with a block or device corresponding to a method step or a function of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block, element, or property of a corresponding device. Bezugszeichen

[0167] 1 Microscope 3 Light sheet microscope 4 Confocal microscope 5 Microscope body 7 Mechanical holding device 9 Microscope beam path 11 Optical module 13 Sample volume 15 Light 17 First optical arrangement 19 First beam path 21 Second optical arrangement 23 Second beam path 25 Optical beam path selector 27 Attachment element 29 Rotation axis 31 Section 33 Aperture 35 Stop filter 37 Microscope objective 39 Wavelength-selective optical element 41 Dichroic mirror 43 Light sheet module 45 Light sheet 47 Light source 49 Beam splitter 51 Scanner 53 Point detector unit 55 Confocal module 57 Further wavelength-selective optical element 59 Manipulation light 61 Third wavelength-selective optical element 63 Optical interface 65 Camera 67 Sample stage 69Probe 71Visible light source 73Visible light 75Near-infrared light 77Fluorescent light 79Interface for tubes 81Non-descanning detectors 83Broadband light source 84Wavelength-variable light source 85Wavelength-variable element87Acousto-optical transmission filter (AOTF) 89Illumination beam of the light sheet 91Confocal illumination beam 93Confocal illumination beam with shifted tilt plane 95Rear focal plane 97Tilt planes 99Optics 101Illumination light 103Peripheral region 105Further optics 107Afocal optics 109Corrected confocal illumination beam 111Uncorrected confocal illumination beam 113Near-axis region 115Mirror 115aFirst mirror 115bSecond mirror 117Axis of rotation 119First angle 121Second angle 123Component of illumination light 123aFirst component 125Tilted illumination beam of the light sheet 127Image field 129Artifact 129aFirst artifacts 129bSecond artifacts 131Image data 133Second reflected portion 135Position for the tilted illumination beam of the light sheet 137Optimal position for the non-tilted illuminator beam of the light sheet 139Deflection mirror 141Transmission characteristic 143Filter edge 145First spectral range 147Second spectral range 149Light sheet mode 151FirstWavelength 153 Second wavelength 155 First microscopy mode 157 Beam path selector - interchangeable module 159 Switching device 160 Housing of (11) 162 Coupling point 164 Housing part of (11) 166 Fiber coupler 168 Optical fiber 170 Collimation optics 172 Further imaging beam path 174 Further microscope objective of (172) 176 Sample holder 178 Optical axis of (9) 180 Optical axis of (17 or 19) 182 Optical axis of (21 or 23) 184 Optical axis of (172) 186 Variable lens 188 Optics 190 Movably arranged mirror 192 Retardation plate 194 Polarization beam splitter 196 Cylindrical lens

Claims

1. Microscope (1), comprising a microscope body (5), a mechanical holding device (7) for microscope objectives, through which a microscope beam path (9) extends, and an optical module (11) attachable to the holding device (7) for illuminating a sample volume (13) and for collecting and transmitting light (15) from the sample volume (13), wherein the optical module (11) has: - a first optical arrangement (17) with a first beam path (19); - a second optical arrangement (21) with a second beam path (23) intersecting the first beam path (19) in the sample volume (13), wherein the second optical arrangement (21) is configured as a light sheet module (43) for generating a light sheet (45) traversing the sample volume (13); - a coupling point (162) for illuminating light for illuminating a part of the sample volume (13) via the first beam path (19) and / or the second beam path (23); and - an attachment element (27) between the first optical arrangement (17) and / or the second optical arrangement (21) and the sample volume (13), wherein the first beam path (19) or the second beam path (23) extends at least partially through the attachment element (27), wherein the microscope (1) comprises an area sensor for detecting light (15) collected from the sample volume (13).

2. The microscope (1) according to claim 1, wherein the microscope (1) is a confocal microscope (4) converted into a light sheet microscope (3).

3. The microscope (1) according to claim 1 or 2, wherein an optical fiber (168) is provided which is configured to transport the illumination light to the coupling point.

4. The microscope (1) according to any one of claims 1 to 3, wherein the microscope (1) has a switching device (159) by means of which the microscope (1) can be reversibly and repeatedly switched to at least a first microscopy mode (155) from the following list of operating states: - a scanning microscopic mode; - a non-linear lighting mode; - a confocal mode; and - a light field mode; or a light sheet mode (149).

5. The microscope (1) according to any one of claims 1 to 4, wherein the microscope has a beam path selector configured as a wavelength-selective optical element (39).

6. The microscope (1) according to claim 5, wherein the wavelength-selective optical element (39) has at least one spectral filter edge (143) which separates a first spectral range (145) from a second spectral range (147), wherein the wavelength-selective optical element (39) has different transmission and reflection properties (T, R) for the first spectral range (145) and for the second spectral range (147), wherein - in the light sheet mode (149), the spectral filter edge (149) lies spectrally between a first wavelength (151) of light (15) radiated into the sample volume (13) and a second wavelength (153) of light (15) collected from the sample volume (13) and wherein - in the first microscopy mode (155), the first wavelength (151) and the second wavelength (151) of the light (15) are together in the first spectral range (145) or in the second spectral range (147).

7. The microscope (1) according to any one of claims 4 to 6, wherein the switching device (159) comprises at least one element from the list of elements, comprising - a wavelength-variable light source (84); - a light source (84) having at least two independently switchable, differing emission wavelengths; - a wavelength-changing optical element (85) for varying the wavelength of the incident light (15); - an optical delay element for varying the polarization of the incident light; and - a beam path selector exchange module (157) for alternately or independently introducing at least two beam path selectors (25) into the microscope beam path (9).

8. The microscope (1) according to claim 7 or 8, wherein the microscope (1) comprises at least one light source (47) which emits light (15) in at least two different wavelength ranges, wherein the light of the at least one light source (45) of the at least two different wavelength ranges (151, 153) can be coupled independently of one another into the microscope beam path (9), and wherein at least two of the different wavelength ranges (151, 153) of the light source (47) lie in different spectral ranges (145, 147) of the wavelength-selective optical element (39).

9. The microscope (1) according to any one of claims 1 to 8, wherein the microscope beam path can be fed into the optical module (11) tilted relative to a normal perpendicular to the rear focal plane (95) of the first (17) and / or second optical arrangement (21), and that the second optical arrangement (21) and / or the attachment element (27) can be tilted depending on the tilt of the microscope beam path (9) to the normal of the rear focal plane (95) of the second optical arrangement (21).

10. The microscope (1) according to any one of claims 7 to 9, wherein - a stop filter (35) for attenuating or blocking a reflected portion (123) of the second spectral range (153) which is transmitted from the wavelength-selective optical element (39) essentially to the second optical arrangement (21) and / or - a further optical arrangement (105) for displacing the rear focal plane (95) of the first optical arrangement (17) is provided in the first beam path (19).

11. The microscope (1) according to any one of claims 1 to 10, with a further imaging beam path (172) which has a further microscope objective (174) and a further detector, wherein with the objective (174) of the further imaging beam path (172) the sample volume (13) can be imaged onto the further detector from a side which is facing away from the optical module (11).

12. The microscope (1) according to claim 11, wherein the further imaging beam path (172) is movable relative to the optical module (11) or wherein the first optical arrangement (17) is movable relative to the second optical arrangement (21).

13. The microscope (1) according to any one of claims 1 to 12, wherein the optical axis (178) of the microscope beam path (9) and the optical axis (180) of the first optical arrangement (17) and / or the optical axis (182) of the second optical arrangement (21) and / or the optical axis (184) of the optionally further imaging beam path (172) intersect a common object field.

14. Optical module for illuminating a sample volume (13) and for collecting and transmitting light (15) from the sample volume (13), which is adaptable to a mechanical holding device (7) of a microscope (1) for microscope objectives, wherein a microscope beam path (9) extends through the holding device (7), wherein the optical module (11) has: - a first optical arrangement (17) with a first beam path (19); - a second optical arrangement (21) with a second beam path (23) intersecting the first beam path (19) in the sample volume (13), wherein the second optical arrangement (21) is configured as a light sheet module (43) for generating a light sheet (45) traversing the sample volume (13); - a coupling point (162) for illumination light, preferably transported by means of an optical fiber (168), for illuminating a part of the sample volume (13) via the first beam path (19) and / or the second beam path (23); and - an attachment element (27) between the first optical arrangement (17) and / or the second optical arrangement (21) and the sample volume (13), wherein the first beam path (19) or the second beam path (23) extends at least partially through the attachment element (27), wherein the microscope (1) comprises an area sensor for detecting light (15) collected from the sample volume (13).

15. Method for observing a sample volume (13) by means of a microscope (1) - preferably configured according to any one of claims 1 to 14 - in particular a confocal microscope (4) converted into a light sheet microscope (3), comprising the following method steps: - illuminating the sample volume (13) through an optical module (11) attached to a microscope body (5) along a first beam path (19) or along a second beam path (23) intersecting the first beam path, wherein the second optical arrangement (21) is configured as a light sheet module (43) for generating a light sheet (45) traversing the sample volume (13); - collecting and transmitting light (15) emitted by the sample volume (13) through the optical module (11) along the first beam path (19); - passing the first (19) or second beam path (1) through an attachment element (27) arranged between a first (17) and / or second optical arrangement (21) and the sample volume (13); and - detecting light (15) collected from the sample volume (13) with an area sensor.

16. The method according to claim 15, further comprising switching between a first microscopy mode (155) and a light sheet mode (149), wherein - in the first microscopy mode (155) the sample volume (13) is illuminated along the first beam path (19) and - in the light sheet mode (149) the sample volume (13) is illuminated along the second beam path (23).

17. The method according to claim 16, wherein the switching comprises at least one method step from the list of the following method steps: - replacing an optical beam path selector (120) which combines the first beam path (19) and / or the second beam path (23) with a microscope beam path (9); - changing the wavelength of incident illumination light by means of a wavelength-variable light source and / or a wavelength-variable optical element; and - varying the polarization of the incident illumination light (101) by means of an optical delay element.

18. The method according to claim 17, wherein - in the method in light sheet mode (149) a region of interest of the sample volume (13) is identified; - after switching to the first microscopy mode (150), the identified region of interest is examined by means of a scanning microscopic method with a higher resolution than the light sheet mode (41); and wherein - image data generated by the area sensor or a point detector, which represent a light distribution emitted by the sample volume (13), are fused and / or displayed together.

19. The method according to any one of claims 15 to 18, wherein at the same time as illuminating the sample volume (13), light (15) which differs from the illuminating light (101) is irradiated onto or into a sample (69) arranged in the sample volume (13).