EP3465316A1 - Light sheet microscope and microscopic method using a light sheet microscope - Google Patents

Abstract

In view of the realization of highly resolved images of a specimen by constructively simple means, a light sheet microscope having a specimen-side objective (1), having an illumination device for providing a first illumination beam (3), which is focused for forming a first light sheet for illuminating a sample from a first direction, said light sheet being inclined obliquely in relation to an optical axis (2) of the objective (1) and being guided through the objective (1), and having a detection device for detection light passing through the objective (1), is configured and developed in such a way that the illumination device is embodied, moreover, for providing at least one second illumination beam (4), which is focused for forming at least one second light sheet for illuminating the sample from at least one second direction that differs from the first direction, said at least one second light sheet being inclined obliquely in relation to the optical axis (2) of the objective (1) and being guided through the objective (1). Moreover, a microscopic method using a light sheet microscope, in particular using a light sheet microscope as described above, is specified.

Description

 Light sheet microscope and microscopic method with a

 Light sheet microscope

The invention relates to a light sheet microscope with a sample-side lens, with a lighting device for providing a first illumination beam, which is focused to form a obliquely inclined to an optical axis of the lens and guided through the lens first light sheet for illuminating a sample from a first direction, and with a detection device for passing through the lens detection light.

The invention further relates to a microscopic method with a light-beam microscope, wherein a first illumination beam is provided by means of an illumination device which is used to form a first light sheet inclined obliquely to an optical axis of a sample-side objective and guided through the objective for illuminating a sample from a first direction is focused, and wherein passing through the lens detection light is detected with a detection device.

Light sheet microscopes and microscopic methods with such light sheet microscopes are known in practice and exist in different embodiments. For example, light-sheet microscopes for illuminating a sample with an obliquely tilted against an optical axis light sheet from US 8 619 237 B2, WO 2015/109323 A2 and US 8 582 203 B2 are known. According to this prior art, for example, in the OPM / SCAPE (Oblique Plane Microscopy / Swept Confocal Aligned Planar Excitation) microscopy, a sample is always illuminated from a fixed side or from one direction and detected approximately perpendicular thereto. The ability to merge and unfold images taken from multiple directions is therefore not possible without rotating the object or sample. This eliminates a simple, proven and established way to generate image data with isotropic resolution. It would be complicated, expensive and expensive to integrate a possibility into the system which allows the illumination of the sample from several directions, for example by the rotation of the optical path. It could be required in a particularly complex manner, with an erection unit including camera and possibly other components to rotate with. Illumination and viewing of a sample from two or more directions was further achieved by SPIV (Single Plane Illumination Microscopy) microscopy by rotation of the sample.

In the light-sheet microscope according to WO 2015/109323 A2, for example, fluorophores are excited by an illumination beam, which is guided over a sample-side objective and tilted with respect to the optical axis of the objective. The light emanating from the light sheet is picked up by the same sample-side lens. Via a telecentric lens and a second lens, an image is generated by this light, which is tilted in the same way with respect to the optical axis of the second lens. This so-called "aerial image" can be imaged by a further optics, typically a third objective and a tube lens, onto a sensor, wherein the optical axis or the optical path of the optics and the sensor is tilted so that the optics is directed perpendicular to the aerial image and the aerial image lies in the focal plane of the optics, eg the third objective.

Based on this prior art, the present invention has the object, a light sheet microscope and a microscopic method with a light sheet microscope of the type mentioned in such a way and further, that high-resolution images of a sample with structurally simple means can be realized.

According to the invention the above object is achieved by a light sheet microscope having the features of claim 1 and by microscopic method having the features of claims 1 1 and 12.

Thereafter, according to claim 1, the light-sheet microscope is configured and further developed such that the illumination device is further configured to provide at least a second illumination beam which is used to form an obliquely inclined to the optical axis of the lens and guided through the lens at least a second sheet of light Illumination of the sample is focused from a different from the first direction at least one second direction. Furthermore, the method according to claim 11 is then designed and refined in such a way that at least one second illumination beam is provided by means of the illumination device, which is used to form an at least one second light sheet inclined at an angle to the optical axis of the objective and guided through the objective Illumination of the sample is focused from a different from the first direction at least one second direction.

In accordance with the invention, it has first been recognized that the above object is achieved in a surprisingly simple manner by skilfully implementing the beam guidance in the light-beam microscope and by skilfully designing the illumination device. In a further inventive manner, it has been specifically recognized for this purpose that the additional design of the illumination device for providing at least one second illumination beam can enable reliable additional illumination of the sample from at least one further direction. In this case, the at least one second illumination beam is focussed in a further concrete manner to form a at least one second light sheet inclined obliquely to the optical axis of the objective lens and guided through the objective for illuminating the sample from a at least one second direction which differs from the first direction. As a result, the sample is illuminated from two directions, with the illumination of the sample in each case with a light sheet, wherein the sample does not have to be moved, for example, to receive three-dimensional image stacks from the at least two directions. The images can be offset in a post-processing step to obtain an image stack with approximately isotropic resolution.

Consequently, the light-sheet microscope according to the invention and the method according to the invention disclose a light-sheet microscope and a method according to which high-resolution images of a sample can be realized with structurally simple means, in particular without the need for movement of a sample.

In a specific embodiment of a light-beam microscope and a microscopic method, for example, a second illumination and detection beam path can be integrated into an existing optical system such that the illumination and the detection direction are respectively interchanged. In other words, the equivalent Illumination beam path of the first illumination beam to the detection beam path of the second illumination beam and vice versa.

The sample-side objective of the light-sheet microscope is an objective which serves both to illuminate the sample with at least two illumination beams and to detect the detection light generated by these illumination beams, for example excited fluorescent light.

In a specific embodiment of the light-beam microscope, for example in the case of two provided illumination beams, the first direction and the second direction of at least two illumination beams may be perpendicular to one another or approximately perpendicular to one another. In principle, the illumination beams may be directed into the periphery of the pupil or the back focal plane of the sample-side objective to ensure the required oblique illumination of the sample from the side. If the aperture of the sample-side objective is insufficient to radiate the illumination beams into the sample volume at an angle of 90 °, the first direction and the second direction may not be perpendicular to one another but at a smaller angle to one another.

With regard to a particularly reliable detection of a detection light, the detection device may comprise a first sensor for the first detection beam caused by the first illumination beam and along a first detection beam path in the lens first detection light and at least one second sensor for caused by the at least one second illumination beam and along at least one second detection beam path have at least second detection light extending in the lens. To that extent, a separate sensor can be provided for each detection light caused by a respective illumination beam, in order to then precisely detect this detection light which runs along its individual detection beam path. In this case, the beam paths can be suitable and / or set up to erect oblique image planes to an optical axis. By realizing such multiple sensors for a plurality of detection lights, a particularly individual operation is still possible, as required, since it is possible to select which illumination beams are generated and to be processed. This ensures a particularly high flexibility in the use of the light sheet microscope.

In a specific exemplary embodiment, the illumination device can be designed to simultaneously provide the first illumination beam and the at least one second illumination beam or to provide the first illumination beam and the at least one second illumination beam at different times, preferably alternately or in a predeterminable sequence.

Alternatively or additionally, the detection device or the sensors of the detection device for simultaneous detection of the first detection light and the at least one second detection light or for detecting the first detection light and the at least one second detection light at different times, preferably alternately or in a predeterminable sequence be trained. Not only by a predefinable chronological sequence of the provision of the illumination beams but also by a predeterminable sequence of the detection function of the detection device or the sensors, a particularly high flexibility and adaptation to different requirements and examination methods can be achieved with the light-sheet microscope according to the invention. In a simple situation, a recording of images can be realized such that the illumination beam is switched so that the first beam path is used for illumination at a first time and a first sensor takes an image and then - at a second time - a second illumination beam path activated and with a second sensor an image from, for example, approximately orthogonal direction is recorded.

In a further advantageous embodiment of the light-sheet microscope, an emission filter, bandpass filter or filter for the first and / or the at least one second detection light can be arranged in the beam path in front of the detection device or in front of at least one sensor. Such emission filters can be implemented in various forms, for example, a prism or a grid can be used. In this case, an emission filter, bandpass filter or filter can be positioned in a beam path that is not assigned to at least one of the sensors. In other words, the light in the Beam path, in which the filter is located, mapped to at least one sensor and not shown on at least one other sensor. Such filters are usually arranged in an infinity beam path, ie in a beam path in which the detection light is neither focused nor divergent, but is collimated.

With regard to a structurally particularly simple embodiment of the light-beam microscope, the illumination device may comprise a dichroic, a galvanometer, plane-parallel glass blocks, optical fibers or a polarization-rotating element with a polarization-dependent beam splitter for coupling the illumination beams into an illumination beam path. Depending on the application, a particularly suitable coupling technique for this application can be used. In a further advantageous embodiment, the detection device can comprise means for merging first detection light caused by the first illumination beam and along a first detection beam path in the objective and at least a second detection light caused by the at least one second illumination beam and along at least one second detection beam path in the objective have on a sensor. In this case, detection light from different detection beam paths is guided on one and the same sensor. In this embodiment, it is therefore not necessary to assign a separate sensor to each detection beam path. In principle, the at least one second detection light can be deflected by suitable optical means to a sensor or the first sensor, so that no further sensor is required. This Umlenkaufwand may be cheaper than the realization of one or more other sensors, on the one hand in terms of system costs and on the other hand in terms of a required Justage- and calibration effort with respect to the possible other sensors or cameras. It should be noted in particular that it is usually quite complicated to match several sensors or cameras to each other, also because they usually differ in their performance from each other. The guiding or deflecting of the at least one second detection light onto one or the first sensor lends itself, for example, when a detection of the detection light differs Illumination beams should be done alternately. Detection light from different detection beam paths can also be guided side by side on one and the same sensor. With regard to a particularly reliable generation of high-resolution images of a sample, the illumination device may comprise means for displacing the first light sheet and / or the at least one second light sheet for generating image stacks from preferably mutually parallel image planes, and the detection device may comprise an evaluation device for processing the images. be assigned to the stack.

In a further concrete embodiment, the first illumination beam and the at least one second illumination beam can be scanned by a single common optical element along a direction perpendicular to the optical axis of the lens through the sample, preferably this optical element for Entscannen returned via this optical element detection light can be used. Such an optical element may, for example, have a tilting mirror or a galvanometer. Upon descent, the variable position of the illumination plane in the sample can be imaged onto a stationary sensor.

Further, a microscopic method is provided with a light sheet microscope, characterized by the steps of: taking images of a sample, wherein the angle between the image planes and the optical axis is ideally equal and the angle between the image planes is ideally 90 °, producing image stacks of Images of ideally mutually parallel planes and fusion of the image stack, for example, by a multiview deployment.

There are now various possibilities for designing and developing the teaching of the present invention in an advantageous manner. For this purpose, on the one hand to refer to the subordinate claims and on the other hand to the following explanation of preferred embodiments of the invention with reference to the drawings. In conjunction with the explanation of the preferred embodiments of the invention with reference to the drawing, generally preferred embodiments and further developments of the teaching are explained. In the drawing show 1 is a schematic representation of a beam path in a light-sheet microscope according to an embodiment of the invention, wherein the beam path is shown on the one hand in the yz plane and the other in the xz plane,

Fig. 1 a in a schematic representation of an embodiment with three

 FIG. 2 is a schematic representation of two possibilities of coupling two illumination beams, FIG.

3a, b, c in schematic representations a beam path in a light-sheet microscope according to further embodiments,

4 shows a schematic representation of a beam path in a light-sheet microscope according to a further exemplary embodiment,

Fig. 5 is a schematic representation of an image taken to the same

 Timing of emissions at different wavelengths,

6 to 8 in schematic representations of beam paths, illumination angles and apertures for illumination objectives with NA = 1, 2, 1, 1 and 1, 0.

Fig. 1 shows a schematic representation of a beam path in a light-sheet microscope according to a first embodiment of the invention. The beam path is shown in the upper half of Fig. 1 in a yz plane and in the lower half of Fig. 1 in an xz plane. The exemplary embodiment comprises a sample-side first objective 1 with an optical axis 2, wherein a first illumination beam 3 is generated at a time 1 which is used to form a first light sheet inclined at an angle to the optical axis 2 of the objective 1 and guided through the objective 1 first image plane 28 is focused to illuminate a sample from a first direction. Furthermore, the illumination device is for providing a second illumination beam 4 at a time 2 formed, wherein the second illumination beam 4 is focused to form a obliquely to the optical axis 2 of the lens 1 and guided by the lens 1 second light sheet in a second image plane 29 for illuminating the sample from a second direction different from the first direction.

The detection device has a first sensor 5 for first detection beam 21 caused by the first illumination beam 3 and along a first detection beam path in objective 1 and a second sensor 6 for second detection light 22 caused by second illumination beam 4 and along a second detection beam path in objective 1 on. A shift of the light sheets along the y-direction by means of a galvanometer 7 to produce a stack of images consisting of parallel image planes. The coupling of the illumination beams 3, 4 via a dichroic eighth

Through a second lens 9, an aerial image of the first and second detection light 21 and 22 is generated. As described above, the detection light 21 caused by the first illumination beam 3 generates an aerial image in an image plane which is imaged onto the first sensor 5 by a first detection optic (comprising a third objective 10 and a tube lens 24) perpendicular to this image plane. In addition, the second detection light 22 also generates a second aerial image in a second image plane, which is mirrored on the optical axis 23 of the objective 9. This aerial image is imaged onto a second sensor 6 by means of a second detection optics (comprising a fourth objective 11 and a tube lens 25) perpendicular to this second image plane. In summary, the aerial images are captured by detection systems and imaged aberration-free on sensors 6 and 5, wherein the detection systems for the first and the second aerial image are "mirrored" to each other on the optical axis of the lens 9. It would be alternatively possible (when not using a second detection optics ), the system consisting of the third lens 10, a tube lens 24 and the sensor 5 to rotate about the optical axis 23 of the second lens 9, however, this is very complicated and due to the required precision in the dimensions and mass of the components to be moved The advantage of constructing a second detection system with a second sensor 6 for detecting the images from the second direction, as shown in FIG is also due to the fact that images from the first and the second direction can be recorded simultaneously or within a very short period of time, which would be inconceivable with a mechanical rotation of the entire detection system. The coupling of illumination light can also take place via one or more dichroic mirrors between the third objectives 10, 11 and / or the tube lenses 24, 25. In this case, no dichroic mirror between galvanometer 7 and second lens 9 is necessary.

With the embodiment shown in FIG. 1, a simple recording of image stacks is possible, in which case a tilting of a galvanometer 7 located in a rear focal plane of the sample-side detection objective 1 takes place. Alternatively, the displacement of the aerial image-generating objective 9 along its optical axis 23, for example by means of a piezo-actuator (SPIM according to Dunsby), enable the generation and recording of image stacks. Further alternatively, the object may simply be displaced relative to the lens 1 for generating and picking up image stacks, preferably along the Y axis. The advantages of the first two variants are that the object does not have to be moved. The last variant is visually simpler and cheaper to implement.

FIG. 1 further shows a control unit 12, which may be formed for example by a computing device, a computer or the like, wherein the control unit 12 may be configured to take over a plurality of tasks. This can be, for example, the control of the actuators for the relative offset of the illumination / detection plane relative to the object, the three particularly advantageous methods being described above. Furthermore, the control unit 12 may perform, alternatively or additionally, for example, the reading out and processing of the images of the sensors 5, 6 or of the sensor elements, see FIG. 3 c. Alternatively or additionally, the control unit 12, the billing of the images to a 3D image stack, for example, a stack of images according to FIG. 5, for example by means of multi-view deconvolution perform.

In principle, a light-beam microscope according to the invention is also conceivable with more than two such erection units 26 and 27, which here according to FIG Elements 5, 24 and 10 or 6, 25 and 1 1 have. With three units, these could then be arranged so that the optical axes are each at angles of 120 ° to each other, wherein respective focal planes can be parallel to the surfaces of a tetrahedron, see Fig. 1 a. In this case, a galvanometer or galvanometer mirror could be designed as a preferably 2-axis gimbal galvanometer in order to be able to operate the lenses oriented in different directions. Another alternative is four pairs of opposing lenses. Their advantage is that each two opposite directions can be operated simultaneously. However, with more than two opposing lenses, the resolution of the system can be only slightly increased, since the main advantage of merging or merging images from multiple directions is to charge two preferably vertically oriented image stacks, so that instead of - worse - axial Resolution in one image stack the better lateral resolution of the other image stack can be used. The main advantage of illumination and detection from more than two directions is the possibility of reducing shadowing effects that may result from scattering and / or absorbing parts in the sample. A method for capturing images may be configured such that a first illumination beam 3 is used at a first time to illuminate the sample, and a sensor 5 captures / picks up a corresponding image. Thereafter, a second illumination beam path is activated with a second illumination beam 4, wherein a second sensor 6 captures an image from an approximately orthogonal direction at a second time.

Such images can also be taken at the same time or in the same period of time when different dyes are excited in the sample with light from illumination beams 3 and 4 having different wavelengths. It is advantageous if the emission spectra of the dyes or fluorophores overlap each other as little as possible and with the excitation wavelengths of the illumination beams 3 and 4. In a further advantageous manner, emission filters, bandpass filters or filters can be positioned in front of the sensors 5 and 6, which respectively cut out regions from the regions of the spectra of the fluorophores or dyes where neither the other fluorophore radiates light nor the light used to excite it. In these cases, one can measure from two directions in time. Essentially, there are two possibilities for accommodating 3D volumes. For imaging a dye, images of mutually parallel planes from one direction can be recorded successively by tilting the galvanometer 7. Subsequently, the lighting is switched and taken with the second sensor 6, a stack of images from the other direction. This can happen either on the "return path" of the galvanometer, sending a zigzag control signal to the galvanometer, or in the same direction, sending a sawtooth signal to the galvanometer Alternatively, the illumination can be switched so fast that images of the sample are taken from two directions for each angular position of the galvanometer 7. The movement of the galvanometer 7 can take place stepwise, advantageously however, continuously at a speed which avoids image blurring due to the relative movement of the imaged plane relative to the sample during the exposure time, ie, the motion is significantly less than the effective depth of field of detection. Whether the first or second mode is preferred lies in FIG In essence, how fast the circuit bez ie the switching of the lighting can be made. If fast and wear-free switches are used, the second mode can be advantageously used. Otherwise, the first mode represents a simple alternative.

According to the embodiment of Fig. 1, the illumination beams 3 and 4 are coupled via a dichroic 8 between the galvanometer 7 and the second lens 9. In contrast to the realization and representation according to the embodiment in Fig. 1, however, it is particularly advantageous to reflect the illumination beams 3 and 4 via the dichroic 8 in the beam path and not to transmit. In a corresponding manner, the image or detection light would then be transmitted and not reflected-as shown in FIG. For switching the illumination, a further actuator may be necessary, which can generate the necessary parallel beam offset of the illumination beams 3 and 4. Unfortunately, it is necessary that an illumination beam can be provided alternately at two positions. Therefore, an embodiment with a continuously operating component such as a galvanometer or a wobble plate does not appear to be necessary, but would be able to directly take into account a flexible adjustment of the position, in particular tilt, of the light sheet as a function of the refractive index of the sample. According to FIG. 2, a further exemplary embodiment of a device for providing two illumination beams 3, 4 at two different positions is shown at the upper position a). In this case, one or more plane-parallel glass blocks 34 can be introduced into the beam path 33 of the light source, which deflect the beam respectively into one or the other beam path in order to generate the two illumination beams 3, 4. Only by turning a glass block 34, the switching operation can be realized.

Alternatively, FIG. 2 shows at b) a combination of a fast polarization-rotating element 35 such as an EOM (electro-optical modulator) and a polarization-dependent beam splitter 36 in the beam path 33 of the light source. Depending on the polarization of the light beam, reflection takes place through the polarization-dependent beam splitter at one or the other position. In order to align the two illumination beams 3, 4 thus generated in parallel for better further use, a downstream mirror 37 can optionally be used, for example.

Alternatively, two optical fibers could be placed in a suitable position, the light between the fibers being switchable with a fast switch.

Alternatively, for example, if no beam splitter can or should be introduced between the second objective 9 and the galvanometer 7, dichroics can also be used in the erection units 26 and 27. 3a, b and c show further embodiments of a beam path in a light sheet microscope, wherein two different detection beam paths are combined in order to be able to image them on a single sensor 5 can. For elements of the figures, which are designated by the same reference numeral as corresponding elements in Fig. 1, reference is made to the description of the figures to Fig. 1. The tube lens 43 can also be configured in duplicate, in each case in the two beam paths. Instead of the mirrors 40, 41, 42 shown here, it is also possible, for example, to use dichroic cubes with long-pass or short-pass filters if detection is simultaneous with two colors.

For changing the guidance of the detection beam paths to the sensor 5, a tilting mirror 39 and further mirrors 40, 41 and a tube lens 43 are used in the exemplary embodiment in FIG. The tilting mirror can assume two orientations (mirror positions), which are defined by the fact that in the first mirror position (at a time 1) the first detection light 21 via a first detection beam path and in the case of the second mirror position (at a time 2) the second detection light 22 passes to the sensor 5 via a second detection beam path. The times must be selected so that they correspond to the times of illumination by means of the illumination beams 3, 4 suitably, so are defined so that detection light generated by the illumination beam 3 at one of the two mirror positions of the tilting mirror 39 reaches the sensor 5 and generates detection light through the illumination beam 4 at the other mirror position. By contrast, in the exemplary embodiment shown in FIG. 3b, a fixed mirror system (formed by the mirrors 40, 41, 42) is used in a simplified manner. Compared with the exemplary embodiment from FIG. 3 a, this has the disadvantage that it can potentially lead to disturbing artifacts in the detection or to a lower image quality, partly because of the non-ideal image, since both detection beam paths are always open.

The image (via the mirrors 39, 40, 41, 42 and the lens 43) takes place in the case of the embodiments shown in FIGS. 3a and 3b at two detection times, for example alternately, on the same area of the sensor 5. The beam paths for the two But views can also be designed so that the images are offset next to each other on the sensor 5. A corresponding embodiment is shown in Fig. 3c.

The illustration of the detection beam path between sample-side lens 1 and objective 9 is shown in simplified form in FIGS. 3 a, 3 b, 3 c without galvanometer 7 and lens 31. The scan actuator (galvanometer mirror) and / or any number of telecentric optical imaging systems may be inserted at designated location 45. For imaging volumes, an optical system as shown in Figure 1 may be used or the objective 9 may be mechanically slidably mounted along its optical axis.

4 shows a further exemplary embodiment of a beam path in a light-beam microscope, this embodiment being designed to avoid a steric collision in the case of a realization with third lenses 10, 11 with a high numerical aperture. The illustration is realized analogously to the figures 3a, 3b, 3c simplified without galvanometer. Similar to FIG. 3a, a switchable mirror is used here, but this is used to split the detection path even before an objective producing the aerial image (objective 9 in the other exemplary embodiments). The mirrors 50, 51 serve in this embodiment, only the compactness of the structure. The lenses 52 and 53 take over the function of the objective 9 from the previous embodiments. As in the previous examples, the scan actuator (galvanometer mirror) and / or any number of telecentric optical imaging systems may be inserted at the designated location 45, for example. Also in this embodiment, elements designated by the same reference numerals as in FIG. 1 correspond to the elements shown in FIG.

5 shows a schematic illustration of a scanning movement in which different dyes 1 and 2 having different wavelengths are excited to emit detection light with likewise different wavelengths. In this case, both illumination beams 3, 4 are always directed simultaneously to the sample at successive points in time, whereby the different dyes 1 and 2 contained in the sample are excited at the same time to emit detection light 21, 22. Such (for example with a Structure as shown in Fig. 1) generated and collected detection light 21, 22 of both dyes can be gradually processed from time 1 to time n to an image stack, as shown in the lower part of Fig. 5. Again, elements of the figure designated by the same reference numerals as in FIG. 1 correspond to the elements shown in FIG.

FIGS. 6 to 8 show the beam paths, illumination angles and apertures for illumination objectives with a numerical aperture NAw = 1, 2 (FIG. 6), 1, 1 (FIG. 7) and 1, 0 (FIG. 8) in water (FIG. Refractive index n = 1, 33), wherein in each case two exemplary variants for the beam path, once in detection perpendicular to the light sheet, and once in non-perpendicular detection, are located. In the drawings, the angles relevant to the following considerations are plotted, with variable names including the character "a" referring to half the opening angle, while variable names having the character "β" denote angles to the optical axis 2. The indices are marked with "S" angles referring to vertical detection, and "D" angles appearing in non-perpendicular detection.

Typically, the light generated by the illumination beam 3 light sheet is tilted against the optical axis 2 by an angle ßi_s. The angle ai_s corresponds to half the aperture angle of the illumination beam 3 at a numerical aperture NA = n sin (ai_s). As described above, two beam paths 60, 61 for the vertical and 62, 63 and the non-perpendicular detection are drawn in each case, wherein the main beams 61 and 63 are shown in dashed lines and in each case an edge beam 60, 62 is drawn. 60, 61 a beam path is shown, which guarantees a detection perpendicular to the illumination and in which the edge rays 60 are arranged symmetrically about the main beam 61. The main beam 61 is then tilted by the angle βs = 90 ° - ßi_s against the optical axis. The symmetrical (sagittal) aperture for the vertical case then results from NA _S = n sin (as) with as = aobj-βs, where aobj is the half-aperture angle of the sample-side objective. Table 1 :

As can be seen in FIG. 6, with a high aperture of the objective of NAw = 1.2, a high numerical aperture of up to NAd.max = 0.75 can be detected. However, in this case, no two lenses 10, 1 1 can be mounted on the opposite side, since the two lenses would collide. There are no collision problems in FIGS. 7 and 8, ie in the case of lenses with NAw = 1, 1 and NAw = 1, 0, however, the possible aperture is given away by the symmetrical use of the objective and the vertical alignment on the light sheet. FIG. 8 shows that a practical detection aperture of 0.07 remains.

It is therefore shown in an alternative beam path 62, 63, which on the one hand makes maximum use of the detection aperture and, on the other hand, additionally keeps 5 ° solid angle to the optical axis in order to mount two objectives (ie to avoid the steric collision discussed above). From this follows ßD = CID - 5 °. The values are summarized in Table 2. The maximum numerical aperture that can be used to avoid collisions of the two third and fourth lenses 10, 11 used to detect the aerial images may be larger for NAw = 1, 0 and NAw = 1, 1 than would be necessary for the transmission of optical information. In other words, the sub-aperture used for detection by the sample-side lens 1 may be smaller than that of the lenses 10, 11, without collision / overlap of the lenses. It should be noted in this case that the asymmetrical use of the pupil leads to an asymmetrical numerical aperture or aperture utilization, and therefore the resolution is also asymmetric. The third and fourth objectives 10, 11 are tilted by the angle y = β _D + β _LS against the plane illuminated by the light sheet. While the third and fourth lens 10, 1 1, the solid angle 2 a 3 holds ready (in the figures, this corresponds to the angle between the edge beam 62 and the dashed line 65), effectively the angular range 2 o used. Here, half the aperture angle 0: 3 of these lenses is given by the tilt angle between the optical axis 2 and the main beam 61 less the above-mentioned angle to avoid the steric collision, ie in this example 5 °. While for a NA of the sample-side lens 1 of 1, 2 so the third and fourth lens 10, 1 1 limit the effective detection aperture, this is not the case for smaller values of the aperture from the sample-side lens 1 of 1, 1 or 1, 0.

Table 2:

According to FIG. 6, the beam path 60, 61 ensures the detection perpendicular to the excitation. The alternative beam path 62, 63 is dualview-compatible, ie it is possible to use two objectives with a half opening angle CID tilted by ß _D against the optical axis, in order to tap off tilted aerial images.

With regard to further advantageous embodiments of the light-beam microscope according to the invention and the microscopic method according to the invention with a light-sheet microscope, reference is made to avoid repetition to the general part of the specification and to the appended claims.

Finally, it should be expressly understood that the embodiments described above are only for the purpose of discussion of the claimed teaching, but not limit these to the embodiments.

Claims

Expectations 1. Light sheet microscope with a sample-side lens (1), with an illumination device for providing a first illumination beam (3), which is used to Formation of a first light sheet which is inclined obliquely to an optical axis (2) of the objective (1) and guided through the objective (1) for illuminating a sample from a first direction, and with a detection device for detection light passing through the objective (1). , d a d u r c h g e k e n n n e t that the illumination device is further designed to provide at least one second illumination beam (4), which is used to form at least one second light sheet which is inclined obliquely to the optical axis (2) of the lens (1) and guided through the lens (1). Illumination of the sample is focused from an at least one second direction that differs from the first direction. 2. Light sheet microscope according to claim 1, characterized in that the first direction and the second direction of at least two illumination beams (3, 4) are perpendicular to one another or approximately perpendicular to one another or the illumination beams (3, 4) are in the edge region of the pupil or the rear focal plane of the sample-side lens (1). 3. Light sheet microscope according to claim 1 or 2, characterized in that the detection device has a first sensor (5) for first detection light caused by the first illumination beam (3) and running along a first detection beam path in the objective (1) and at least one second sensor (6) for at least second detection light caused by the at least one second illumination beam (4) and running along at least one second detection beam path in the objective (1). 4. Light sheet microscope according to one of claims 1 to 3, characterized in that the illumination device for simultaneously providing the first illumination beam (3) and the at least one second illumination beam (4) or for providing the first illumination beam (3) and the at least one second illumination beam (4) is formed at different times, preferably alternately or in a predeterminable sequence. 5. Light sheet microscope according to one of claims 1 to 4, characterized in that the detection device or the sensors (5, 6) of the detection device for simultaneous detection of the first detection light and the at least one second detection light or for detection of the first detection light and of the at least one second detection light is or are formed at different times, preferably alternately or in a predeterminable sequence. 6. Light sheet microscope according to one of claims 1 to 5, characterized in that an emission filter, bandpass filter or filter for the first and / or the at least one second detection light is arranged in the beam path in front of the detection device or in front of at least one sensor (5, 6). 7. Light sheet microscope according to one of claims 1 to 6, characterized in that the illumination device has a dichroic (8), a galvanometer, plane-parallel glass blocks, optical fibers or a polarization-rotating element with a polarization-dependent beam splitter for coupling the illumination beams (3, 4) into an illumination beam path having. 8. Light sheet microscope according to one of claims 1 to 7, characterized in that the detection device has means for combining the first detection light caused by the first illumination beam (3) and running along a first detection beam path in the objective (1) and through the at least one second illumination beam (4) and at least second detection light which runs along at least one second detection beam path in the objective (1) onto a sensor (5). 9. Light sheet microscope according to one of claims 1 to 8, characterized in that the lighting device has means for displacing the first light sheet and / or the at least one second light sheet for generating of image stacks from preferably parallel image planes and that the detection device is assigned an evaluation device for processing the image stacks. 10. Light sheet microscope according to one of claims 1 to 9, characterized in that the first illumination beam (3) and at least one second illumination beam (4) pass through a single common optical element along a perpendicular to the optical axis (2) of the objective (1). Direction through the sample can be scanned, with this optical element preferably being used for descanning detection light returned via this optical element. 1 1. Microscopic method with a light sheet microscope, in particular with a light sheet microscope according to one of claims 1 to 10, wherein a first illumination beam (3) is provided by means of an illumination device, which is used to form an oblique to an optical axis (2) of a sample side Lens (1) inclined and guided through the lens (1) first light sheet for illuminating a sample is focused from a first direction, and detection light passing through the lens (1) is detected with a detection device, so that at least a second Illumination beam (4) is provided by means of the illumination device, which is used to form at least one second light sheet, which is inclined obliquely to the optical axis (2) of the objective (1) and guided through the objective (1), for illuminating the sample from one different from the first Direction distinguishing at least a second direction is focused. 12. Microscopic method with a light sheet microscope, in particular with a light sheet microscope according to one of claims 1 to 10, characterized by the steps: - taking images of a sample, where the angle between the image planes and the optical axis is ideally the same and the angle between the image planes is ideally 90°, - Creating image stacks of images that are ideally parallel to each other and - Fusion of the image stacks, for example through a multiview unfolding.