Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/06—Means for illuminating specimens
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0068—Optical details of the image generation arrangements using polarisation
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence

Definitions

• the invention relates to a method for imaging a specimen by means of a light-beam microscope, in which the specimen is illuminated from two different illumination directions with two light sheets which have different polarization states and are coplanarly superimposed on one another in a target region of the specimen, and by means of an imaging optics of the light sheet microscope, an image of the illuminated target area is generated. Furthermore, the invention relates to a correspondingly working light-sheet microscope.
• a target is illuminated with a thin light sheet via a lighting optic and the target area illuminated in this way is imaged by means of imaging optics whose optical axis is perpendicular to the optical axis of the illumination optics.
• imaging optics whose optical axis is perpendicular to the optical axis of the illumination optics.
• This type of light sheet illumination is achieved in that an illumination light beam passes through a Wollaston prism, which splits the illumination light beam into two differently linearly polarized sub-beams, which are deflected away from the optical axis of the illumination beam path and thus from different illumination directions in the Lighting level, ie reach the target area of the sample. If shading of the illumination light now occurs in one of the two illumination directions as a result of a scattering center or an absorber, the other illumination direction, which is unimpaired by the scattering center or the absorber, still ensures adequate illumination of the target area.
• overlap adjustment In order to ensure the sharpest possible imaging in a light-beam microscope, there is a precise spatial overlap between the illumination plane, ie the target area defined by the light-sheet thickness, which defines the sample volume excited to emit fluorescence radiation, and the focal plane, ie Depth of field specified focus range of the imaging optics required.
• the fine adjustment required for this purpose referred to hereinafter as overlap adjustment, is used to produce the spatial overlay between the illuminated target area and the focus area of the imaging optics, usually by means of a visual assessment, eg using a Reference preparation containing small fluorescent particles.
• the microscopically recorded sample image itself visually assessed to make the overlap adjustment.
• such a procedure only allows a comparatively rough overlap jus- tage.
• a comparatively complicated adjustment method provides for introducing into the illumination beam path a mirror which deflects the light sheet to a reference position of the detector arranged in the image plane of the imaging beam path, as soon as the adjustment has taken place.
• the object of the invention is to provide a method for imaging a sample by means of a light-sheet microscope and a light-sheet microscope itself, which allow a precise and automated adjustment of the spatial overlap between the illuminated target area of the sample and the focus range of the imaging optics.
• the invention provides a method for imaging a sample by means of a light microscope, in which the sample is illuminated from two different directions of illumination with two leaves that have different polarization states and are coplanarly superimposed on each other in a target area of the sample, and an image of the illuminated target area is generated by means of an imaging optics of the light-sheet microscope.
• an interference pattern is generated in the illuminated target area, whereby the image of the target area is imprinted with an image modulation corresponding to the interference pattern.
• the image modulation is evaluated and the illuminated target area is adjusted as a function of the evaluated image modulation relative to the focus range of the imaging optics.
• the invention provides for an automatic adjustment of the illuminated target area relative to the focus area of the imaging optics, in that the image of the target area captured by the imaging optics is evaluated with regard to image modulation.
• the latter is imprinted on the image by illuminating the reproduced target area with two interfering light sheets.
• an interference pattern illuminating the target area is generated, which is reflected in the image of the illuminated target area in the form of the aforementioned image modulation, which is evaluated, for example, by a control unit provided for this in the light-sheet microscope and used for automatic overlap adjustment.
• the adjustment is completed, for example, when the imprinted image of the illuminated target area image modulation is maximum.
• the modulation present in the picture is strong It depends on how precisely the target area acted upon by the light sheet illumination and the focal range of the imaging optics are spatially superimposed on one another.
• the image modulation is maximal when the illumination plane jointly defined by the two light sheets coincides with the focal plane of the imaging optics.
• the light sheets are to be generated so that they have sufficient time for an interference time and spatial coherence to each other.
• it must be ensured in particular that the two light sheets have polarization states during the adjustment which make it possible at all to interfere with the light sheets in the target area. If, for example, one assumes that the light sheets are differently linearly polarized from the outset, the light sheets in the target area then interfere with one another when their polarization directions are non-orthogonal to one another. If this is ensured, no polarization means is required, which is specially designed to ensure its ability to interfere in the target area by influencing the polarization states of the two light sheets.
• such a polarization means provided specifically for the production of the ability to interfere with the light sheets can advantageously be used to produce a particularly pronounced interference pattern by influencing the polarization states in the target area accordingly.
• the polarizing agent can be used to linearly parallelize the two light sheets in parallel, thereby maximizing the interference of the two light sheets.
• Such a polarization means can be realized, for example, by a birefringent crystal placed in a suitable position in the illumination beam path or a polarizer of a different kind, for example a retardation plate.
• the influencing of the polarization states of the light sheets can be effected by polarization-dependent properties of the illumination optics, for example by using polarization-dependent phases of interference layers or means for stress birefringence.
• electro-optical or liquid crystal-based agent can be used.
• the image modulation is evaluated by determining its amplitude.
• the illuminated target area is then adjusted relative to the focus area of the imaging optics such that the amplitude of the image modulation is maximized.
• the amplitude of the image modulation represents an easily detectable optimization parameter, on the basis of which an automatic overlap adjustment can be carried out in the light-sheet microscope.
• a characteristic of the interference pattern is specified and the image modulation is evaluated on the basis of this predetermined characteristic.
• the aforementioned characteristic is a characteristic of the interference pattern, which can be derived, for example, from the selected geometry of the light sheet illumination and is thus known a priori. This known property of the interference pattern can then be used in a simple manner for evaluating the image modulation.
• f denotes the modulation period
• l the wavelength of the light sheets and a the aforementioned angle which the illumination directions enclose with one another.
• the modulation period f represents a previously known characteristic of the interference pattern, which results from the predetermined wavelength l of the light sheets as well as the likewise predetermined angle a between the two illumination directions of the two light sheets according to the above relationship.
• the alignment of the interference pattern from the geometry of the light sheet illumination, ie the illumination directions of the two light sheets is also known. Due to the knowledge of the modulation period and the orientation of the interference pattern, the amplitude of the image modulation can now be determined simply and reliably, for example by means of a Fourier analysis of the image.
• the narrowly localized modulation period of the interference fringes is convoluted with the spatial spectrum of the sample representing the a priori unknown structure of the sample.
• the captured image of the target area is represented by positive real data
• the sum of this data which is the DC component of the Fourier spectrum, is always positive and real, so that it is the a priori knowledge of the modulation period f of the interference pattern and its alignment allows to determine the amplitude of the image modulation reliably even with a high-frequency spatial spectrum of the sample.
• the interference fringes of the interference pattern for not too large angle a between the illumination directions of the two light sheets on the imaging optics of the light sheet microscope can be detected easily and reliably in the form of a modulation of the recorded image.
• the amplitude of the image modulation imposed on the image by the interference fringes can thus be used as an optimization parameter or quality criterion for the coplanarity between the illumination plane and the focal plane of the imaging optics. Since the amplitude of the image modulation is the only parameter to be optimized, the parameter space for the coplanarity adjustment is one-dimensional. Thus, simple linear search algorithms can be used to maximize the amplitude of the image modulation.
• the two coplanar superimposed light sheets are moved together along the optical axis of the imaging optics.
• This can be done, for example, via a deflection element arranged in the illumination beam path, which is activated as a function of the evaluated image modulation.
• a simultaneous displacement of both the light sheets and the focus range of the imaging optics is possible.
• the two light sheets are converted into interference-capable polarization states relative to the focus range of the imaging optics prior to the adjustment of the illuminated target area.
• the aforementioned polarization states are chosen so that the two light sheets are polarized in the target area linear, non-orthogonal, in particular parallel to each other. This ensures that a pronounced interference pattern is formed in the target area, by means of which a corresponding strong image modulation is generated in the recorded image.
• the two light sheets are brought into non-interference-capable polarization states after the adjustment of the illuminated target area relative to the focus area of the imaging optics. This avoids the actual imaging disturbing modulation of the microscope image.
• This step can be automated using the Fourier analysis of the image.
• a pre-adjustment is provided in which the illuminated target area is adjusted as a function of the brightness of the image relative to the focus area of the imaging optics.
• Such a pre-adjustment can also be carried out in particular in the case of high-frequency local spectrum of the sample as a function of the energy content of the detected total spectrum, which results from the convolution of the location spectrum and the illumination spectrum.
• the light-sheet microscope according to the invention comprises a lighting unit which is designed to illuminate a sample from two different illumination directions with two light sheets which have different polarization states and are coplanarly superimposed on one another in a target area of the sample Imaging optics configured to generate an image of the illuminated target area, and a control unit.
• the control unit is designed to control the lighting unit such that an interference pattern is generated with the two light sheets in the illuminated target area, whereby an image modulation corresponding to the interference pattern is imprinted onto the image of the target area.
• the control unit is further configured to evaluate the image modulation and to control the illumination unit and / or the imaging optics such that the illuminated target area is adjusted in dependence on the evaluated image modulation relative to the focus range of the imaging optics.
• the illumination unit comprises a light source, which is designed to generate an illumination light beam, a first polarization element, which is designed to split the illumination light beam into two differently polarized sub-beams, and an illumination optical system, which is formed from the two sub-beams create two light sheets illuminating the target area.
• the first polarization element is designed, for example, in such a way that it deflects the two sub-beams away from the optical axis of the illumination unit at preferably identical angles.
• Sample generates two sheets of light that propagate at an angle ⁇ ß / 2y to the optical axis of the illumination unit within the sample.
• the first polarization element is formed so that the two sub-beams are linearly polarized, their polarization directions are orthogonal to each other. These orthogonal polarization directions have the advantage in actual imaging that image modulation due to interference between the two light sheets is excluded.
• illumination with these two polarization directions reduces photoselection effects in the excitation of fluorophores.
• the first polarization element is a Wollaston prism.
• a prism consists for example of two rectangular calcite prisms, which are cemented to one another at their base surfaces. The optical axes of the two prisms are orthogonal to each other.
• the illumination unit preferably comprises a deflection element which can be controlled via the control unit, by means of which the two coplanarly superimposed light sheets can be displaced together along the optical axis of the imaging optics.
• the deflection element is, for example, a mirror which is driven via a motor controlled by the control unit in order to move the two coplanar light sheets along the optical axis of the imaging optics.
• the illumination unit comprises a second polarization element which can be controlled via the control unit and which is designed to selectively convert the two light sheets into polarization states capable of interference and polarization states which can not be interfered with.
• this embodiment enables both a precise overlap adjustment and a high-resolution imaging, which in particular is not disturbed by image modulations as a result of interference effects.
• the light-beam microscope according to the invention has two separate, the sample-facing lenses, one of which is assigned to the illumination unit and the other of the imaging optics and their optical axes perpendicular to each other.
• the objective assigned to the illumination unit preferably directs the two light sheets from the same side onto the target area of the sample.
• the light-sheet microscope can also be designed as a so-called oblique plane microscope, which has a single sample-facing objective both for the illumination and the detection.
• the imaging optics of the light-sheet microscope is formed in this embodiment as transport optics, which images the light sheets in the target area of the sample and at the same time generates an image of the illuminated target area.
• the transport optics preferably have a scanning device which is designed to carry out an axial and / or lateral rastering process for volume imaging by moving the light sheets through the sample accordingly.
• Figure 1 shows an embodiment of a light sheet microscope according to the invention in a schematic sectional view
• FIG. 2 shows a further schematic sectional view of the light-pancake microscope according to FIG. 1;
• FIG. 3 is a flow chart which illustrates the method for the overlap adjustment according to the invention on the basis of an exemplary embodiment;
• FIG. 4 shows a diagram which shows the amplitude of an image modulation as a function of the offset between the illuminated target area and the focus area of the imaging optics of the light-leaf microcircuit;
• FIG. 5 shows an interference pattern generated in the target area
• FIG. 6 shows the spatial spectrum of the interference pattern according to FIG. 5 obtained by a Fourier transformation
• FIG. 7 shows an image of the target area which, for the purpose of overlap adjustment, has an image modulation corresponding to the interference pattern
• FIG. 8 shows the spatial spectrum of the image according to FIG. 7 obtained by a Fourier transformation
• FIGS. 1 and 2 show sectional views of a light-sheet microscope 10.
• the light-sheet microscope 10 comprises a lighting unit 12 and a imaging optical system 14.
• the lighting unit 12 and the imaging optical unit 14 are aligned in the present embodiment so that their 0 and 0 'in the region of a sample not explicitly shown in Figures 1 and 2 are perpendicular to each other.
• FIGS. 1 and 2 reference is made in each case to a right-angled xyz coordinate system whose z-axis coincides with the optical axis 0 of the illumination unit 12. Accordingly, the light-sheet microscope 10 is shown in FIG. 1 in an xz-section and in FIG. 2 in a yz-section.
• the illustrations according to FIGS. 1 and 2 are simplified and purely schematic. Thus, only those components are shown which are necessary for the understanding of the invention.
• the illumination unit 12 has a light source 16 and an illumination optics generally designated 18 in FIGS. 1 and 2.
• the illumination optics 18 comprise a first polarization element in the form of a Wollaston prism 20, a motorized second polarization element in the form of a compensator 22, an anamorphic focusing system in the form of a cylindrical lens 24, a motorized adjustment mirror 26, an eyepiece lens 28, a deflection mirror 30 Tube lens 32 and an illumination objective 34 with an objective pupil 36.
• the aforementioned compensator is formed for example of a birefringent crystal, in particular a retardation plate.
• the imaging optics 14 comprises an imaging objective 38 facing the sample to be imaged, a tube lens 40 and a spatially resolving detector in the form of a camera 42.
• the light-sheet microscope 10 further includes a control unit 44 which controls the entire microscope operation. More specifically, in the present embodiment, the control unit 44 serves to control the compensator 22, the motorized adjustment mirror 26, and the camera 42 as well as those detailed below explained image evaluations. Correspondingly, the control unit 44 is connected via control lines 46, 48, 50 to the compensator 22, the input mirror 26 or the camera 42.
• the light source 16 emits a collimated illuminating light beam 52 onto the Wollaston prism 20, which may be formed of, for example, two right-angled prisms, e.g. Calcite prisms cemented together at their base surfaces.
• the Wollaston prism 20 splits the incident illumination beam 52 into two sub-beams 54, 56 having different polarization states, as shown in FIG.
• the plane in which the Wollaston prism 20 splits the illumination light beam 52 into the two sub-beams 54, 56 is parallel to the y-axis, ie. in the section of Figure 2 in the plane and in the section of Figure 1 perpendicular to the plane.
• the two sub-beams 54, 56 pass through the compensator 22, with which the polarization states of the sub-beams 54, 56 can be influenced as required.
• a servo motor (not shown in FIGS. 1 and 2) is provided in the light-beam microscope 10, which under the control of the control unit 44 acts on the compensator 22 in such a way that the polarization states of the two sub-beams 54, 56 are in the desired manner influenced or left unchanged.
• the sub-beams 54, 56 pass through the cylindrical lens 24.
• the latter has the property that it focuses the sub-beams 54, 56 only in a direction parallel to the x-axis direction, while in a direction parallel to the y-axis direction no optical effect the sub-beams 54, 56 has.
• the cylindrical lens 24 generates in the region of its focal plane from the sub-beams 54, 56 respectively a light-sheet-like illumination light distribution, which is focused in the direction of the x-axis and extended in the direction of the x-axis.
• FIGS. 1 and 2 are simplified for the sake of clarity. For example, in FIG.
• the foci of the two partial bundles 54, 56 emerging from the cylindrical lens 24, which correspond to the planes conjugated to the cylindrical lens 24, are arranged on the surface of the motorized adjusting mirror 26. In fact, however, one of these focuses is in the direction of light propagation and the other behind this surface. The same applies to the representation of the foci on the surface of the deflection mirror 30.
• the light deflections made on the adjustment mirror 26 and the deflection mirror 30 are shown in FIGS. 1 and 2 in the same way, although these light deflections are shown in FIG Are given either only in the xz plane or in the yz plane.
• the two sub-beams 54, 56 After reflection on the adjustment mirror 26, the two sub-beams 54, 56 pass through the eyepiece lens 28 and are reflected at the deflection mirror 30. After passing through the tube lens 32, the sub-beams 54, 56 reach the entrance pupil 36 of the illumination objective 34, which directs the sub-beams 54, 56 onto the sample in such a way that the sub-beams 54, 56 illuminate the target area E of the sample from two different illumination directions ,
• the illumination optics 18 of the light-sheet microscope 10 generates, as explained above, two light sheets 58, 60 propagating in different illumination directions, which are superposed coplanarly in the target region E of the sample to be illuminated.
• the two light sheets 58, 60 in the concrete exemplary embodiment are based on the assumption that the compensator 22 initially still has no influence on the polarization states of the sub-beams 54, 56 lets in the target area E polarized linearly orthogonal to each other.
• the light beam associated with the sub-beam 54 is p-polarized
• the light beam associated with the sub-beam 56 is s-polarized.
• the eyepiece lens 28, the tube lens 32 and the illumination objective 34 form intermediate imaging optics which generate the light sheets that the cylinder lens 24 generates by focusing the sub-beams 54, 56 at the location of the deflecting mirror 26 depicts the target area E of the sample.
• the two illumination directions, from which the light sheets 58, 60 are directed into the target region E of the sample enclose an angle a with one another.
• This angle a correlates with an angle .beta.
• step S1 of the flow diagram according to FIG. 3 an automated pre-adjustment of the light sheet illumination to the focus area F of the imaging optical system 14 is carried out.
• the pre-adjustment can be based, for example, on the brightness of the image be taken by the camera 42 recorded image.
• the adjustment mirror 26 is brought under the control of the control unit 44 in a position in which the light sheet illumination ensures maximum image brightness.
• the polarization states of the sub-beams 54, 56 and thus the two light sheets 58, 60 under the control of the control unit 44 by the compensator 22 are set in step S2 so that a maximum interference of the two light sheets 58, 60 in the target area E.
• the compensator 22 for this purpose, the polarization states of the two sub-beams 54, 56 so that they are linearly polarized parallel to each other. In this way, an interference pattern is generated in the target area E by interference between the two light sheets 58, 60, as illustrated purely by way of example in FIG.
• FIG. 5 shows an interference pattern I under the assumption of a completely homogeneous sample.
• the interference pattern I according to FIG. 5 has a plurality of interference fringes which extend in one direction with reference to FIG. 2, which extend in parallel to the angle bisector of the angle a.
• the angle a is enclosed by the two illumination directions in which the two light sheets 58, 60 propagate.
• the aforementioned bisector of the angle a therefore coincides with the optical axis O of the illumination optical system 18.
• the interference fringes extend in the direction of the optical axis O of the illumination optics 18.
• step S3 by the camera 42, an image of the two light sheets 58, 60th illuminated target area E of the sample recorded. Since the two light sheets generate the interference pattern I shown in FIG. 5, the image recorded by the camera 42 is impressed with an image modulation corresponding to the interference pattern. This is illustrated in FIG. 7, which shows an image of the target area recorded by the camera 42, in which the image modulation corresponding to the interference pattern I can be clearly recognized in the form of horizontal stripes.
• step S4 the control unit 44 evaluates the image modulation included in the captured image.
• the control unit 44 uses the a priori knowledge of the characteristic of the interference pattern I as a result of the predetermined light sheet geometry.
• this characteristic is given by the modulation period, ie the distance between adjacent interference fringes, as well as the orientation of the fringe pattern according to FIG.
• the orientation of the interference pattern also results directly from the given light sheet geometry.
• the interference fringes run parallel to the optical axis O of the illumination optical system 18 in the present exemplary embodiment.
• the characteristic of the interference pattern according to FIG. 5 is reflected in its location spectrum generated by a Fourier analysis, which is shown in FIG.
• the horizontal spatial frequencies are indicated along the horizontal axis and the vertical local frequencies are indicated along the vertical axis.
• the units are given by the properties of the discrete Fourier transform, and the signal intensity is represented as gray levels.
• the local spectrum according to FIG. 6 has two signals at spatial frequencies which result from the modulation period of the interference pattern shown in FIG. More precisely, the spatial frequencies in FIG. 6 in accordance with the Fourier transformation respectively represent the inverse of the modulation period of the interference pattern I.
• the signal intensity at zero spatial frequency which represents the DC component of the local spectrometer, is omitted in FIG since this exceeds the signal intensities shown in FIG. 6 by a multiple.
• the DC component signal with the spatial frequency zero lies with respect to the horizontal axis between the two signals shown in FIG. It should be noted that the spatial frequencies in FIG. 6 are given in arbitrary units.
• step S4 therefore, the image represented in FIG. 7, which has the image modulation corresponding to the interference pattern I according to FIG. 5, is subjected to a Fourier transformation and thus generates its spatial spectrum, which is shown in FIG.
• the spatial spectrum according to FIG. 8 shows along the vertical axis two spatial frequency signals which are arranged at equal distances on both sides of a dominant central signal which, at the spatial frequency zero, represents the DC component of the local spectrum.
• the two aforementioned signals reflect the image modulation caused by the interference pattern I.
• the spatial frequency of these signals ie the (positive) distance
• the the signals along the vertical axis of the central DC signal have, determined by the modulation period of the interference pattern.
• the orientation of the two spatial frequency signals representing the image modulation in FIG. 8 corresponds to the direction of the image modula In FIG. 7, it can be seen that the horizontally extending interference strips follow one another in the vertical direction.
• the spatial spectrum according to FIG. 8 is to be evaluated in order to detect the image modulation quantitatively.
• the local spectrum shown in FIG. 8 is to be evaluated precisely at those points at which the signals above or below the central DC component signal are to be found (or only the upper signal with positive spatial frequency).
• This signal represents the amplitude of the image modulation and is therefore used in the following as an optimization parameter for the overlap adjustment.
• step S5 the motorized adjustment mirror 110 is adjusted under the control of the control unit 44 by a predetermined manipulated value, whereby the overlapping of the light sheets 58, 60 together along the optical axis 0 'of the imaging optics 14 are moved. Subsequently, the control flow returns to step S3.
• steps S3 to S5 are performed, for example, using a linear, ie one-dimensional search algorithm (optionally with a Fieranziehung one of an appropriate termination criterion) until the optimization parameter given by the local frequency signal detected in step S4, which represents the amplitude of the image modulation, is maximized.
• FIG. 4 shows purely by way of example how the amplitude of the image modulation changes as a function of an offset which occurs between the illuminated target region E and the sharpening region F of the imaging optical system along its optical axis 0 '. If this offset is equal to zero, the amplitude of the image modulation is maximum and the overlap adjustment is completed.
• step S6 the compensator 22 is actuated by the control unit 44 in such a way that it transfers the sub-beams 54, 56 and thus the light sheets 58, 60 into polarization states in which the light sheets 58, 60 do not interfere with one another.
• the light sheets 58, 60 are polarized in these polarization states linearly orthogonal to each other. This polarization adjustment causes the interference pattern in the target area E to disappear. Accordingly, in the image of the target area E taken by the camera 42, the image modulation is eliminated, as illustrated in FIG.
• the elimination of the image modulation can likewise take place in such a way that the amplitude of the image modulation in a one-dimensional search method corresponding to steps S3 to S5 is used as the optimization parameter, with the difference that the control unit 44 in this case does not use the adjustment mirror 26 but drives the compensator 22 and does not maximize the amplitude of the image modulation, but is to be minimized.
• the image of the target area E freed from image modulation in this way can then be used for the actual imaging.
• the invention is not limited to the embodiment described above. It is possible, for example, to perform the overlap adjustment in a different manner than in the exemplary embodiment described, in which the two light sheets 58, 60 are displaced along the optical axis O 'of the imaging optical unit 14.
• the polarization states of the light sheets 58, 60 can also be influenced in a different way than in the described exemplary embodiment, provided that it is ensured that the two light sheets 58, 60 interfere with one another during the pre-adjustment in the target region E.
• the compensator 22 acts only on one of the two sub-beams 54, 56.
• the invention is not limited to direct the two light sheets 58, 60 as in the above-described embodiment from the same side to the target area E.
• the light microscope can also be embodied as an oblique plane microscope of the type described above, which has a single sample-facing objective for the illumination and the detection.

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• 2018
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