LIGHT SHEET MICROSCOPE

Description

Field of the invention

[0001] The invention relates to a light sheet microscope comprising illumination optics for illuminating a sample. The sample is located on a sample carrier in a medium, for example a liquid or in a gel. The sample carrier is aligned parallel to a flat reference surface. This reference surface can be, for example, the plane of a sample stage on which the sample carrier is located, for example a plane-parallel glass plate of a predetermined thickness or a corresponding vessel with walls whose transparent bottom is aligned parallel to this reference surface. The vessel can be open, but it can also be closed with a transparent cover glass. The illumination optics serve to illuminate the sample via an illumination beam path. The illumination is achieved with a light sheet whose light sheet plane encloses an illumination angle other than zero with the normal to the reference surface.The light sheet microscope also includes a detection optics with a detection beam path with an optical axis that encloses a non-zero detection angle with the normal to the reference surface in the area of ​​the sample carrier. The angle between the detection and illumination angles is often 90°, but this is not a mandatory requirement.

[0002] The light sheet microscope also includes an interface system with at least one layer made of a specified material with a specified thickness, which separates the medium in which the sample is located from the illumination and detection optics. The interface system, with a base surface aligned parallel to the reference surface, is in contact with the medium at least in the area accessible for illumination and detection. In the case of an inverted light sheet microscope, in which illumination and detection are arranged below the sample, the transparent base of the sample vessel or the sample carrier plate, together with the air layer or an immersion medium, which are located between the base and the optics, form the interface system. When observing from above, for example, a transparent lid of the sample vessel, together with the air layer or an immersion medium, forms the interface system.the immersion medium between the lid and the illumination and detection optics, the separation layer system.

[0003] Since the illumination and detection optics each enclose an angle other than zero with the normal of the reference surface, at least one correction element is arranged in the illumination and / or detection beam path to reduce aberrations that arise due to the oblique passage of light to be detected or of light for illuminating the sample through interfaces of the interface system. State of the art

[0004] Light sheet microscopes are used primarily for the examination of biological samples, where the sample is illuminated with a light sheet whose plane intersects the optical axis of the detection at an angle other than zero. Typically, the light sheet forms a right angle with the detection direction, which can, for example, correspond to the optical axis of a detection objective used solely for detection. This technique, also known as SPIM (Selective Plane Illumination Microscopy), allows three-dimensional images of even thicker samples to be created in a relatively short time. Based on optical sectioning combined with relative movement between the sample and the light sheet, i.e. in a direction perpendicular to the sectioning plane, a spatially extended visual representation of the sample is possible.

[0005] The SPIM technique is primarily used in fluorescence microscopy, where it is referred to as LSFM (Light Sheet Fluorescence Microscopy). Compared to other established techniques such as confocal laser scanning microscopy or two-photon microscopy, LSFM techniques and setups offer several advantages: Since detection can be performed in the wide field, larger sample areas can be captured. Although the resolution is somewhat lower than with confocal laser scanning microscopy, thicker samples can be analyzed using LSFM due to the greater penetration depth. Furthermore, the light exposure of the samples is lowest with this technique, which, among other things, reduces the risk of unwanted bleaching of the sample, since it is only illuminated by a thin light sheet at a non-zero angle to the detection direction.

[0006] Both a static light sheet, generated using cylindrical lenses, for example, and a quasi-static light sheet can be used. The latter can be generated by quickly scanning the sample with a light beam. Light sheet-like illumination is created by subjecting the light beam to very rapid relative movement to the observed sample and thereby repeatedly sequentially arranging it. The integration time of the camera, on whose sensor the sample is ultimately imaged, is selected so that scanning is completed within the integration time. Instead of a camera with a two-dimensional sensor field, a line sensor can be used in combination with a rescan in the detection optics. Detection can also be performed confocally.

[0007] The SPIM technique is now established and has been described many times in the literature, for example in DE 102 57 423 A1 and the subsequent WO 2004 / 053558 A1 or in the review article "Selective plane illumination microscopy techniques in developmental biology" by J. Huisken et al., published in 2009 in the journal Development, Vol. 136, p. 1963.

[0008] Light sheet microscopes that operate according to the SPIM method have so far largely only been used for the observation of samples. However, for more in-depth examination of samples it is often desirable to carry out optical manipulation on them. In the context of the present invention, the term optical manipulation is understood to mean photostimulation, i.e. the deposition of light intensity at predetermined locations and times in the sample in laterally and axially restricted areas whose shape, size and, for simultaneous manipulation, number can be predetermined, whereby the sample must not be completely penetrated by the manipulation light and whereby the deposition of the light intensity causes an optically driven reaction at the predetermined locations. Such reactions can consist, for example, of bleaching the sample, photoconversion, uncaging, etc.The optical manipulation in samples in which the manipulation light interacts with the sample along the manipulation direction only in areas that are to be manipulated will be referred to below as local optical manipulation.

[0009] Various methods are known in the prior art. For example, any position in a plane, for example in the light sheet plane, can be quickly targeted using a point scanner, i.e. a laser scanning microscope where detection is not confoccal. High intensities can be generated at the focus. The disadvantage of this is that the individual positions have to be targeted one after the other. Positioning in the axial direction must be achieved via focusing, for example internal focusing in the objective, or via a corresponding movement of the sample. A further disadvantage is that areas above and below the target position are completely penetrated in the axial direction, whereby, depending on the intensity, interactions with the sample can also occur outside the actually focused area.Using appropriate optics, a multi-point scanner can also be implemented, in which several points are controlled simultaneously.

[0010] To generate a two-dimensional intensity pattern, a digital micromirror array (DMD array) can be used, for example, which is arranged in an intermediate image plane in the illumination optics. The individual pixels can be switched on or off and directly define the illuminated regions in the sample. This is described, for example, in the article "Probing the function of neuronal populations: combining micromirror-based optogenetic photostimulation with voltage-sensitive dye imaging" by S. Tsuda et al., published in "Neuroscience Research" 75 (1), p. 76 in 2013. Instead of a DMD array, an LCD array in transmission can also be used, but this results in high losses in the incident light power. LED arrays can also be used instead of DMDs; these are then used directly for illumination, and, as with the DMD, the amplitude of the illumination is modulated on a pixel-by-pixel basis.The advantage over DMD arrays lies in the higher modulation frequency in the MHz range, whereas DMD arrays have a modulation frequency in the kHz range. The use of an LED array to structure the illumination light is described, for example, in the article "Multi-site optical excitation using ChR2 and micro-LED array" by N. Grossman et al., published in the "Journal of Neural Engineering" 7 (1), p. 016004 in 2010.

[0011] The use of spatial light modulators (SLMs) allows photostimulation in spatially extended areas, i.e., not only in a plane perpendicular to the detection or illumination direction, for example, but also axially in this direction. The spatial light modulator is positioned in a pupil plane of the manipulation beam path, and a phase pattern is imprinted on it, thus changing the phase of the light used for manipulation. The imaging then results in the desired intensity distribution. This approach is described, for example, in the article "Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning" by F. Anselmi et al., published in "Proceedings of the National Academy of Sciences" 108 (49), pp. 19504 ff. in 2011.An intensity distribution is specified, which can be determined, for example, based on the sample setup from a previously acquired overview image or from a spatial overview scan. From this specified intensity distribution, the required phase pattern, which must be imposed on the SLM, is iteratively calculated using algorithms based on inverse Fourier transforms. This procedure is described, for example, in the article "Experimental demonstration of holographic three-dimensional light shaping using a Gerchberg-Saxton algorithm" by G. Whyte et al., published in "New Journal of Physics" 7, p. 117 in 2005. However, due to the complexity of this procedure and the lower frame rate of spatial light modulators, which are usually based on liquid crystal elements, the patterns can only be changed in a very slow temporal sequence, approximately at a frequency of a few tens of Hz.Compared to two-dimensional structuring, these methods are about a hundred times slower.

[0012] Particularly good local confinement of optical manipulation to an essentially point-shaped area in space can also be achieved with the concept of multiphoton excitation. A laser pulse is imaged into the area, exhibiting extremely high intensity only at the focus. Such a pulse can be generated, for example, with a femtosecond laser. The wavelength of this laser is chosen so that any dyes present in the sample are not excited to fluorescence. These are generally near-infrared wavelengths. However, the intensity of the light at the focus is so high that the probability that two or more photons are absorbed simultaneously and trigger a corresponding fluorescence-exciting transition is particularly high. This enables targeted excitation only in the focal plane and not above or below the focal plane as with single-photon excitation, since the intensity there is too low.The penetration depth during imaging can also be increased using scanning systems with multiphoton excitation. Furthermore, since wavelengths in the near-infrared range are used, this approach is particularly advantageous for living biological samples. The multiphoton excitation method can be combined with point scanning, but also with multipoint illumination, in which the sample is illuminated at many points simultaneously. This can be achieved, for example, by a spatial light modulator in the manipulation beam path, to which an iteratively calculated phase pattern is applied. This approach using two-photon excitation is described, for example, in the article "SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators" by V. Nikolenko et al., published in the journal "Frontiers in Neural Circuits," Vol. 2 (5), p. 1, December 19, 2008.

[0013] While depth discrimination can be easily achieved with point scanners using multiphoton excitation, particularly two-photon excitation, due to the quadratic intensity dependence of the excitation in combination with strong in-plane focusing, this is not possible in wide-field imaging without additional tools. However, using a diffractive optical element, such as a grating, short laser pulses can be spectrally split. This diffractive optical element is then imaged onto the sample via the microscope objective. As a result, the different spectral elements of the illuminating light pulses take different optical paths and only reconverge in the focal plane, where they then form the original, very short laser pulse.In this way, the peak power of the pulse is maximum only in the focal plane, which, in conjunction with the quadratic intensity dependence of the two-photon excitation mentioned above, leads to the desired depth discrimination even in wide-field illumination. This approach, also referred to as "temporal focusing," is described, for example, in the article "Scanningless depth-resolved microscopy" by D. Oron et al., published in the journal "Optics Express" 13 (5), p. 14687 in 2005.

[0014] All of the previously described methods for gentle photostimulation have largely not yet been used for light sheet microscopy. However, US Pat. No. 8,547,634 B2 proposes various arrangements for photomanipulation of a sample in light sheet microscopy. All of these arrangements always include manipulation via the illumination beam path, in which the manipulation light is also formed into a light sheet. The light source used to generate the light sheet or a separate manipulation light source can be used. Axially locally limited photostimulation is not possible in this way. US Pat. No. 8,547,634 B2 also describes the possibility of using a second manipulation optics in addition to the first manipulation illumination using a light sheet, in which the manipulation light is directed onto the sample via the detection optics.It is proposed to use a laser scanning microscope as the second manipulation optics. With this second manipulation optics, manipulation is in principle possible exclusively in the light sheet plane, although the manipulation light from the second light source generally penetrates or penetrates the sample over a larger area along the normal direction of the light sheet. Even in the light sheet plane, manipulation or illumination with manipulation light takes place axially in the illumination light along the entire extent of the light sheet.

[0015] Further examples of light sheet microscopes with manipulation illumination are known from WO 2015 / 109323 A2 and US 2012 / 206798 A1. Description of the invention

[0016] The object of the invention is therefore to further develop a light sheet microscope of the type described above in such a way that a gentle observation of samples is possible.

[0017] This object is achieved in a light sheet microscope of the type described above, which is designed in particular for the inverse observation of a sample, in that the light sheet microscope comprises means, which are independent of the light sheet generation, for exposing the sample to light intensity in essentially point-shaped areas in the light sheet plane or in a predetermined volume which at least temporarily encompasses the light sheet plane via at least one manipulation beam path.

[0018] By restricting the manipulation to essentially point-shaped areas, i.e., particularly laterally restricted areas, for example—but not exclusively—for manipulation in one plane, but preferably also axially restricted areas relative to the entire volume of the sample, the size of which naturally depends on various factors such as lateral and axial focusing, the excitation mechanisms used, the manipulation tools themselves, etc., a particularly sample-friendly manipulation can be achieved. The essentially point-shaped areas can, in turn, define a larger area in the sense of a "region of interest" (ROI) in both two- and three-dimensional space, from which, in turn, several independent and non-contiguous ROIs can be defined within a volume to be manipulated.The term "point" should not be understood here in a mathematical sense, but rather to make it clear that the areas to be selected and manipulated should be as small as possible.

[0019] Manipulation and observation / illumination are fundamentally independent of each other. For example, the light sheet can be used to observe one area or ROI of the volume while manipulation is taking place in another area. This other area is only subsequently illuminated by the light sheet. Therefore, the volume being manipulated, or the essentially point-shaped areas, do not need to be in the plane of the light sheet while intensity is being deposited. Only then are they recorded by the light sheet, possibly after a predefined time.

[0020] The illumination optics of the light sheet microscope usually expediently comprise an illumination objective, and the detection optics a detection objective. Illumination and detection can also be performed by the same objective, meaning the illumination objective can be identical to the detection objective. Illumination and detection can also share individual optical elements in the beam path, for example, correction elements. According to the invention, the means for manipulation comprise a separate manipulation objective in the manipulation beam path. In an embodiment not corresponding to the claimed invention, the manipulation can also be performed by the illumination objective or by the detection objective; in these cases, the manipulation objective is identical to the illumination objective or the detection objective, respectively.These two variants have the advantage that no additional lens is required, but the configuration is somewhat less flexible compared to using a dedicated manipulation lens. Among these variants not according to the invention, manipulation is particularly preferably performed through the detection lens, as this is often arranged perpendicular to the light sheet, which facilitates planar manipulation in the light sheet plane or in a volume surrounding the light sheet plane.

[0021] In principle, the manipulation can be performed using any of the three lenses mentioned, and the elements explained in detail below, which supplement the manipulation means, can generally be used with any of the three lenses. However, according to the invention, the light used for manipulation is coupled into a dedicated manipulation beam path. When the illumination beam path is used in a manner not consistent with the invention, the manipulation and illumination share optical elements, but they do so independently and without influencing each other. In particular, the light used for manipulation is not formed into a light sheet.

[0022] In a simple embodiment, the manipulation means comprise a point scanner for sequentially exposing individual, essentially point-shaped regions of the sample to light intensity, or a multipoint scanner for sequentially exposing a plurality of essentially point-shaped regions of the sample, a number of which can be exposed to light intensity simultaneously. The multipoint scanner is constructed analogously to the point scanner; for example, a microlens array can be used to generate multipoints.

[0023] Using a first spatial light modulator, the sample can also be exposed to a predetermined intensity distribution, which then occurs essentially at a constant amplitude. To impose a predetermined amplitude distribution on the predetermined intensity distribution, the manipulation means can comprise a second spatial light modulator.

[0024] In a further embodiment, the means for manipulation comprise a digital micromirror array (DMD) for generating a predetermined intensity distribution in a plane that is preferably parallel to the light sheet plane, particularly preferably in the light sheet plane. When positioned parallel to the light sheet plane, the manipulation is expediently performed by the detection lens or a separate manipulation lens.

[0025] In a particularly preferred embodiment, the aforementioned manipulation means can be combined to increase the manipulation speed, since the DMDs can be switched more quickly than spatial light modulators. In a preferred embodiment, the light sheet microscope therefore also comprises switching means, preferably a switching mirror, for switching between the first spatial light modulator, the digital micromirror array, or a combination of both. In addition to the first spatial light modulator, the second spatial light modulator can also be used. In this way, for example, the spatial light modulator can be used to define several spatial regions with a corresponding phase pattern, which can then be quickly individually switched on and off by the DMD.

[0026] If the manipulation lens is identical to the detection lens, the first or second spatial light modulator can be used, in a particularly preferred embodiment, both for manipulating the sample and for correcting aberrations. For this purpose, a first region of one of the spatial light modulators is designed to apply a predetermined intensity or amplitude distribution to the sample and to serve as a correction element, while a second region of the spatial light modulator is designed exclusively as a correction element in the detection beam path. This can be achieved via appropriate control of the SLM. Deflecting means are provided that deflect the light to be detected to the second region and the light used for manipulation to the first region.This can also be realized equivalently with the illumination lens if the manipulation is carried out through the illumination lens, i.e. this is identical to the manipulation lens, or - if necessary - also with the manipulation lens.

[0027] In this way, costs can be reduced because no separate correction element needs to be provided for the detection beam path or the illumination beam path. Of course, correction elements known from the prior art can also be used - in addition or as an alternative, for example so-called Alvarez plates, which consist of two essentially plate-shaped elements that can be displaced relative to one another, onto which a corresponding surface structure that corrects the aberrations is imprinted, and which can be displaced laterally relative to one another. The surface structures can be freeform surfaces, and the plates function as wavefront modulators. One such correction element is described, for example, in DE 10 2014 104 977 A1.The correction element can also be configured, additionally or alternatively, as an optical transmission system, a so-called virtual relay, as described, for example, in DE 10 2013 112 600 A1. This correction element can be dimensioned so that it can be used for both the illumination and detection lenses, and it is placed between these lenses and the separation layer system. Further correction elements, which can also be used individually or in combination with those already mentioned, include deformable mirrors or, as already mentioned, spatial light modulators.

[0028] The deflection means preferably comprise a dichroic element whose parallel, active optical surfaces are preferably arranged perpendicular to the active optical surface of the spatial light modulator. The beam path is guided such that the light used for manipulation and the light to be detected are each deflected twice onto the dichroic element, and the deflection to the respective area is achieved by means of the thickness of the dichroic element. The dichroic element does not necessarily have to be arranged vertically as described, but may need to be made even thicker if tilted accordingly.

[0029] The configuration with an SLM divided into two areas with different illumination can be applied analogously to the illumination beam path, with one area being designed to generate light sheets and the other area for manipulation illumination. In addition, the SLM can also function as a correction element in both areas.

[0030] Particularly advantageously, the light sources, wavelengths, and intensities of the manipulation light are designed to excite two or more photon transitions, allowing for particularly gentle manipulation of the sample in very spatially confined areas. For axial localization of the intensity of the light used for manipulation along the axis of the manipulation beam path, for example, the detection objective, a temporal focusing mechanism is preferably provided, which preferably comprises a femtosecond pulse laser with a controller for controlling the group velocity dispersion.

[0031] It is understood that the features mentioned above and those to be explained below can be used not only in the combinations indicated, but also in other combinations or on their own, without departing from the scope of the present invention. Short description of the drawings

[0032] The invention is explained in more detail below, for example, with reference to the accompanying drawings, which also disclose essential features of the invention. They show: Fig. 1 a general structure for sample-protecting light sheet microscopy, Fig. 2 a first non-inventive embodiment of manipulation by the detection objective, Fig. 3a), b) two further non-inventive embodiments for manipulation by the detection objective, Fig. 4 a fourth non-inventive embodiment of manipulation by the detection objective, Fig. 5 a fifth non-inventive embodiment of manipulation by the detection objective, Fig. 6a)-c) inventive embodiments for manipulation by a separate manipulation objective, Fig. 7a), b) two possible cases of non-inventive manipulation by the illumination objective, Fig. 8 a first embodiment of a light sheet microscope with non-inventive manipulation by the illumination objective, Fig. 9 a second non-inventive embodiment of manipulation by the illumination objective, Fig.Fig. 10 shows a third embodiment of the manipulation not according to the invention using the illumination lens, Fig. 11 shows the use of temporal focusing with the light sheet, Fig. 12 shows a basic representation of the manipulation from two directions, Fig. 13 shows an arrangement for multi-photon manipulation, and Fig. 14 shows the procedure for manipulation and imaging. Detailed description of the drawings

[0033] Fig. 1first shows the general setup as it can be used for sample-friendly light sheet microscopy with simultaneous manipulation. A sample 1 is arranged on a sample carrier 2. The light sheet microscopy observation takes place here in an inverse configuration, with an illumination objective O1 and a detection objective O2 being arranged beneath the sample carrier 2, which is part of an interface system. The sample carrier 2 is aligned parallel to a flat reference surface 3, which can, for example, correspond to the underside of the sample carrier 2. The sample 1 can be embedded in a medium, which is not shown separately here. Via the illumination objective O1, the sample 1 is illuminated via an illumination beam path with a light sheet, the light sheet plane of which, here corresponding to the optical axis 4 of the illumination objective O1, encloses an illumination angle other than zero with the normal of the reference surface.The detection objective O2 is part of a detection optics system with a detection beam path whose optical axis 5, in the region of the sample carrier 2, also encloses a detection angle other than zero with the normal of the reference surface 3. The optical axis 5 here corresponds to the optical axis of the detection objective O2. To correct or reduce aberrations that arise due to the oblique passage of light to be detected or of light for illuminating the sample 1 through interfaces of the separation layer system—comprising the sample carrier 2 and the air layer below it—the light sheet microscope comprises at least one correction element; in this case, there are several correction elements.Firstly, the illumination objective O1 comprises a correction element in the form of a wavefront manipulator 6, and the detection objective O2 comprises a correction element in the form of a further wavefront manipulator 7. The wavefront manipulators 6 and 7 are formed from plates with freeform surfaces that can be displaced laterally relative to the optical axes 4 and 5, respectively; these are also referred to as Alvarez plates. Furthermore, between the objective and the underside of the sample carrier 2, a further correction element in the form of a virtual relay, i.e., an optical transmission system 8, is arranged. For the manipulation, a separate manipulation objective O3 is used here as an example for clarification.With this—or with the other two objectives—means for exposing the sample to light intensity, preferably exclusively in essentially point-like regions of the light sheet plane or in a predetermined volume encompassing the light sheet plane at least temporarily, can be realized via at least one manipulation beam path, preferably entirely independent of the light sheet generation. Right in . Fig. 1 In the section shown in the dashed box, two manipulation beams 9 are indicated, the ends of which lie in the light sheet and thus in the focal plane of the detection objective O2, and which are to be exposed to a predetermined intensity.

[0034] The means for applying light intensity to sample 1 will be explained in more detail below using various exemplary embodiments.

[0035] Fig. 2shows a non-inventive embodiment of a light sheet microscope in which manipulation is performed by the detection objective O2, i.e., in which the manipulation objective is identical to the detection objective O2. The sample stage with the sample carrier 2 is provided here with a sample stage control 10 for spatially positioning the sample 1 in all three spatial directions. Illumination is achieved by means conventional in light sheet microscopes. An illumination laser 11 emits light at an illumination wavelength. A beam is formed in a beam-shaping unit 12, which is fed to a light sheet scanner 13, which serves for the quasi-static generation of the light sheet and for positioning the light sheet. The light sheet is imaged onto the sample 1 via a tube lens 14 and the objective O1.The beam shaping unit 12 serves to imprint a desired intensity profile on the light beam, for example according to a Gaussian distribution or a Bessel beam, which is then also imprinted on the light sheet in cross-section perpendicular to the light sheet plane by scanning.

[0036] The light to be detected passes through the detection lens O2, passes through a dichroic beam splitter 15, and is imaged via a tube lens 16 onto a planar detector, which may, for example, be part of a camera 17. The detected signals are then further processed and / or displayed.

[0037] A stimulation laser 18 is used for manipulation, the light of which is fed to a stimulation scanner 19, which is designed here as a point scanner. A deflection element in the form of a mirror 20 is interposed, which serves only to ensure the compact design of the arrangement. The manipulation light is coupled into the detection beam path via the dichroic beam splitter 15 via a further lens 21 and a tube lens 22 corresponding to lens 16.

[0038] With the Fig. 2In the version of a light sheet microscope shown, a point-like manipulation of the sample takes place. The focus can be set to the light sheet plane, but also to areas above and below the light sheet plane, so that effectively a spatial manipulation is possible, but in principle only for individual points one after the other. For this focus shift to areas outside the light sheet plane, another adjustable optic, for example a collimator, can be inserted between the stimulation laser 18 and the stimulation scanner 19 in the beam path, so that the manipulation focus can be adjusted independently of the detection focus. With appropriate design of the stimulation laser 18 and the manipulation optics, the Fig. 2The embodiment shown can also be used for multi-photon excitation. By introducing one or more additional optical elements, for example a microlens array between the stimulation laser 18 and the stimulation scanner 19, whereby the laser beam is expanded in front of the microlens array and then collimated again, the Fig. 2 Convert the used point scanner into a multipoint scanner. This is not shown here.

[0039] Fig. 3 shows in figures a) and b) two further embodiments of a light sheet microscope not according to the invention, in which the manipulation is carried out by the detection objective. Fig. 3b ) can be connected to the module shown in Fig. 3a) shown module, both are coupled at the same point in the beam path, characterized by the dichroic beam splitter 15. The setup does not necessarily have to be modular, two independent light sheet microscopes can also be generated, which are not interchangeable. While in the Fig. 2 In the case of a point scanner, essentially point-shaped areas of the sample are sequentially exposed to light intensity in the arrangement shown, or in the case of a multi-point scanner, a plurality of such point-shaped areas are sequentially exposed to light intensity in the arrangement shown, whereby a number of such point-shaped areas are exposed to light intensity simultaneously, the Fig. 3a), b) enable the spatial generation of intensity patterns in the sample, ie they enable the simultaneous exposure of a number of essentially point-shaped areas to light intensity, whereby each point can be exposed independently of the other points and the points can be distributed not only in a plane but spatially in a volume.

[0040] At the Fig. 3a), the means for manipulation comprise a first spatial light modulator 23 for applying a predetermined intensity distribution to the sample. The stimulation laser 18 again serves as the light source. Since the phase of the light is changed by means of the spatial light modulator 23, the spatial light modulator (SLM) 23 must be arranged in a pupil plane; therefore, an additional lens 24 is necessary for imaging. In addition, the arrangement also comprises a telescope 25, with which the illumination of the pupil or the SLM 23 can be adjusted. To adjust the polarization, a polarization element 26, for example a polarizer or a λ / 2 plate, can optionally be arranged in the beam path. In the case of Fig. 3b) a digital micromirror array (DMD) 27 is used instead of the SLM 23; in this case the lens 24 can be omitted since the intensity is manipulated directly; for this purpose the DMD 27 is arranged in an intermediate image plane of the manipulation beam path. In this case too the stimulation laser 18 can be used as the light source, but in principle incoherent light sources 28 can also be used, for example conventional lamps - particularly in the UV range - or LEDs. The DMD 27 is used to generate an intensity pattern, i.e. a predetermined intensity distribution in a plane, preferably a plane parallel to the light sheet plane or in the light sheet plane.

[0041] In addition to Fig. 3a ) the means for manipulation may comprise a second spatial light modulator, not shown here, for applying a predetermined amplitude distribution to the predetermined intensity distribution.

[0042] Fig. 4shows a further embodiment of a light sheet microscope not according to the invention, in which the manipulation takes place through the detection beam path. In this embodiment, the spatial light modulator 23 has a first region 29, which is designed to apply a predetermined intensity or amplitude distribution to the sample and as a correction element, and a second region 30, which is designed as a correction element in the detection beam path. Deflecting means are provided for directing the light to be detected to the second region 30 and the light used for manipulation to the first region 29. The wavefront manipulators 7 in the detection objective O2 can be omitted in this case.In this case, a phase pattern is imprinted on the second region 30 of the SLM 23. This phase pattern serves exclusively to correct the aberrations caused by the oblique passage of the light through the interface system with the sample carrier 2. The light used for manipulation does not strike this second region 30, but is deflected past it. The first region 29 of the SLM 23 is imprinted with the phase pattern for the spatial intensity distribution to be placed in the sample, as well as the phase pattern for correcting the oblique light passage. The beam path is shown in . Fig. 4shown. The light to be detected is shown in dashed lines, the manipulation light of the stimulation laser 18 with a solid line. The deflection means here comprise a dichroic element 31, whose active optical surfaces, aligned parallel to one another, are preferably arranged perpendicular to the active optical surface of the spatial light modulator 23. By means of two plane mirrors 32, 33, whose mirror surfaces are also arranged parallel to the active optical surfaces of the dichroic element 31, the beam path is guided in such a way that the light used for the manipulation and the light to be detected are each deflected twice onto the dichroic element 31. By means of the dichroic element 31 - the thickness of which is determined based on the necessary beam offset of the partial beam paths on the SLM, whereby the beam offset itself in turn depends on the size of the active surfaces of the SLM - the deflection to the first region 29 orreaches the second region 30. In the detection beam path, there are also two further lenses 34 and 35, which produce a 4f image, thus creating space for the accommodation of the additional components, ie the deflection means.

[0043] A further embodiment of a light sheet microscope not according to the invention, in which the manipulation is carried out by the detection optics, is described in Figure 5The means for manipulation here comprise both an SLM 23 with stimulation laser 18 and a DMD 27 with two light sources 28, 28a, which can be selectively coupled into the beam path for illuminating the DMD 27 via a light source switching mirror 36. In the case of the DMD beam path, the telescopic system 25 is symbolized only by a lens, which also symbolizes a condenser. Between the DMD 27 and the tube lens 22, a further lens 37 is arranged, which images the DMD 27 into an intermediate image plane located between the lens 37 and the tube lens 22. In the embodiment according to Fig.5The light sheet microscope also includes switching means, in particular a switching mirror 38, which serves as a central element for selecting the manipulation means through appropriate position / rotation in the beam path: Either exclusively the DMD 27, or exclusively the SLM 23 is switched on as the manipulation means, or both in combination. In particular in the latter embodiment, in which the SLM 23 and DMD 27 are coupled into the beam path simultaneously using a beam combiner 38a, the manipulation speed can be increased, since DMDs with frequencies of approximately 1 kHz are faster than efficient LCOS SLMs, which can be switched at approximately 60 Hz. With an appropriate phase pattern, the SLM 23 can then, for example, define several spatially delimited areas as ROIs (regions of interest), which can then be quickly and individually switched on and off by the DMD 27.

[0044] Another possibility, not shown, is to generate any pattern by one SLM or DMD or multiple SLMs and DMDs and to use a scanning system to quickly switch between the patterns.

[0045] In the Fig. 5In the example shown, one of the two light sources with which the DMD 27 can be illuminated is selected using a light source switching mirror 36. If only two light sources, for example a laser and a conventional lamp, are to be selected, such a light source switching mirror 36 can be dispensed with when using a DMD 27 to generate the intensity distribution if the light sources and the DMD 27 are arranged in specific positions relative to one another: The DMD 27 has an array of micromirrors that are switched between two fixed angles, for example between an angle of -12° for a first "on" state and +12° for a second "off" state, each measured from the normal to the base surface of the DMD 27 on which the micromirrors are arranged. If the light sources orIf the beam path is now adjusted so that the DMD 27 is illuminated by one light source in the "on" state and by the other light source in the "off" state, and the further manipulation beam path initially follows the direction of the normal to the base surface of the DMD 27, the light source switching mirror 36 is not necessary. A combination with one or more light source switching mirrors is nevertheless possible in order to increase the selection of possible light sources, just as in the case of the embodiment shown in . Fig. 5 shown variant.

[0046] According to the invention, the means for manipulation comprise a separate manipulation lens, which is then arranged above the sample, ie on the other side of the sample carrier 2. Examples of this are shown in Fig. 6a )-c) are shown in schematic diagrams. A manipulation lens O3 is positioned with its optical axis parallel to the normal of reference surface 3.

[0047] Fig. 6a) shows the use of a proprietary manipulation lens O3 with a point scanner analogous to Fig. 2 , the statements made regarding the design shown there can essentially be transferred to this configuration. Analogously, the Fig. 6b ) shown version with a SLM 23 to the version in Fig. 3a ), which in Fig. 6c ) shown version with a DMD 27 corresponds to Fig. 3b ). Here, too, a combination of several manipulation options is possible, similar to that used in connection with Fig. 5 The combination described for the detection objective is possible. Correction elements can be omitted for manipulation from above, unless a sample container sealed with a coverslip is used and the manipulation objective is positioned at an angle other than zero to the normal of the reference surface.

[0048] For manipulation, two cases must be fundamentally distinguished, which are described below using the inventive manipulation from above by means of a separate manipulation lens O3, but also apply to the non-inventive manipulation by means of the detection or illumination lens.

[0049] With rapid imaging of the volume in which the essentially point-like areas are exposed to manipulation intensity by the light sheet system, the sequence of manipulation and imaging can be defined across the volume. In this case, the effect of the light distribution generated by the manipulation device in the sample volume can also be observed on the timescale of the imaging of this volume. Then, the manipulation can be performed both in the light sheet plane and in the volume region analogously to the manipulation described in connection with the manipulation by the detection objective O2.However, if the time scale on which the effect of the manipulation radiation is observed is so short that the manipulation has to be observed directly within a camera frame, i.e. within the light sheet generated by the illumination objective O1 and detected by the detection objective O2, it must be ensured that the intensity distribution impressed on the sample by the manipulation objective O3 lies in the light sheet plane, as shown in the dashed box in . Fig. 6a ) is indicated. In the case of Fig. 6a) This can be achieved by quickly focusing the point scanner, for example, by internally focusing the manipulation lens O3, by moving the tube lens 22, by using a rapidly deformable lens, such as the EL-10-30 lens from Optotune, or by using an adaptive optical element, such as a deformable mirror. In the case of an SLM, a corresponding spatial intensity distribution lying in the plane of the light sheet can be specified.

[0050] Manipulation can also be performed through the illumination objective O1, whereby the numerical aperture of the illumination objective is usually lower than that of the detection objective O2. However, some methods, such as FRAP (Fluorescence recovery after photobleaching), do not require a larger NA and can use the illumination objective O1 for manipulation. Just as with manipulation using a dedicated manipulation objective O3, two cases must be distinguished. In the first case, the volume imaging by the light sheet system is so fast that the sequence of manipulation and imaging can be defined across the volume. The effect of the light distribution generated by the manipulation device in the sample volume can also be observed on the time scale of the imaging of this volume. This case is in Fig. 7a). In a selected sample volume 39, spatially distributed, essentially point-shaped areas of the sample are exposed to light intensity at a given time, for example, with a given intensity distribution, which is converted into a phase pattern using an inverse Fourier transform algorithm, which is then impressed on a spatial light modulator. This can be achieved, for example, with a light sheet microscope, as is used in connection with Fig. 8 described.

[0051] In the second case, the time scale on which the effect of the manipulation radiation is observed is so short that it must be observed within one camera frame, i.e. within the light sheet generated by the illumination lens O1 and detected by the detection lens O2. This is Fig. 7b ), the point-like areas are shown here as dashed lines on the light sheet, while in Figure 7a ) are distributed as crosses in the volume.

[0052] Fig. 8 As already indicated, FIG. 1 shows a light sheet microscope not according to the invention, in which the manipulation lens is identical to the illumination lens O1. The manipulation means here again comprise an SLM 23 along with the associated light generation and imaging optics. Coupling into the illumination beam path is achieved via a dichroic beam splitter 40.

[0053] Spatial light modulators are expensive and technically complex components. For this reason, when manipulation is performed through the illumination lens O1, it is advantageous to use the same SLM for both generating the light sheet and specifying an intensity distribution using wide-field holographic methods. In preferred Fig. 9 and Fig. 10In the embodiments shown, in which the manipulation lens is identical to the illumination lens O1, the illumination optics comprises a spatial light modulator 41, to which a phase pattern for exposing the sample to a predetermined intensity distribution or amplitude distribution is imprinted in a manipulation region 42, and a phase pattern for the light sheet formation is imprinted in a light sheet generation region 43. Both regions can be controlled independently of each other, ie the light sheet generation continues to occur independently of the manipulation, even if - not necessarily - the same element is used. In analogy to the one described in connection with Fig. 4In the arrangement described above, the SLM 41 can also function as a correction element, i.e., it can additionally be imprinted with a corresponding phase pattern to correct aberrations resulting from oblique light passage. To ensure that light for manipulation is directed exclusively to the manipulation area (42) and light for light sheet generation exclusively to the light sheet generation area (43), the illumination optics comprise corresponding deflection means, which can also perform spectral decomposition, since different wavelengths are generally used for light sheet generation on the one hand and manipulation on the other.

[0054] Two examples of light sheet microscopes not according to the invention, in which an SLM 41 is used to generate both the light sheet and the intensity distribution, are shown in the Figs. 9 and 10The prerequisite is that the stimulation laser 18 emits a different wavelength than the illumination laser 11, as this is used for beam separation. First, the light from both light sources is combined via a dichroic beam combiner 44 and then split into Fig. 9 or a dichroic beam splitter 46 in Fig. 10 divided, whereby the Fig. 9 shown configuration analogous to that used in connection with Fig. 4 described arrangement, the thickness of the dichroic beam splitter 45 is used for deflection. The light used for illumination is directed to the light sheet generation area 43, the manipulation light of the stimulation laser 18 used to generate the predetermined intensity distribution is directed to the manipulation area 42 of the SLM 41. Subsequently, both light beams are either combined, as in Fig. 9 shown, or parallel, as in Fig. 10shown, guided to a further dichroic beam splitter 47. When illuminating the SLM 41, the laser beams are expanded by corresponding lens combinations (not shown) so that they homogeneously illuminate the respective area on the SLM 41. The area of ​​the SLM 41 that has the phase pattern for light sheet formation, i.e. the light sheet generation area 43, must lie in an intermediate image plane 48 of the image of the illumination objective O1 during further imaging. The manipulation area 42, on the other hand, must lie in a pupil plane 49 of the image of the illumination objective O1. Both areas of the SLM 41 must therefore be imaged differently, which can be achieved with the help of the further dichroic beam splitter 47, which once again splits the beam paths for manipulation light and illumination light. By means of a first optics 50, the manipulation area 42 is imaged into the pupil plane 49 of the illumination objective O1.The light sheet generation region 43 is imaged into the intermediate image plane 48 of the illumination objective O1 by a second optics 51, which also images the manipulation region 42, via the light sheet scanner 13 and a scanning lens 52. The two wavelengths are superimposed again using a dichroic beam combiner 53.

[0055] The statements according to Fig. 9 and Fig. 10 differ essentially only in the type of color division. In Fig. 9 is a solution analogous to Fig. 4, in which the beams are always superimposed, but which requires a dichroic beam splitter 45 on a relatively thick substrate in order to achieve sufficient separation on the spatial light modulator 41. The different wavelengths of the lasers can, for example, be guided through a common optical fiber 54, whereby more than one light wavelength can be used for manipulation and / or light sheet generation. In contrast, in Fig. 10 the otherwise usual distribution of the different wavelengths into different paths along the optical beam path is shown.

[0056] The imaging of the manipulation area 42 into the pupil plane 49 can also be omitted, i.e., the SLM 41 imaged into the intermediate image plane 48 can also be used for manipulation. This requires that the manipulation area 42 be spectrally separated from the light-sheet generation area 43. Another possibility is to run the manipulation quickly and sequentially; in this case, switching between the specified intensity distribution and the pattern for light-sheet generation only needs to be performed synchronously with the switching of the respective laser wavelength. However, this significantly limits the number of intensity distributions that can be generated in the sample.

[0057] In principle, manipulation by the detection objective O2 is also possible using an SLM 41 divided into two areas with a light sheet generation area 43 and a manipulation area 42, although the beam guidance here is somewhat more complex than in the case of manipulation by the illumination objective O1.

[0058] As in the case of Fig. 2 for the non-inventive manipulation by the detection lens O2 or in the case of Fig. 6a) In the case of manipulation according to the invention by means of a separate manipulation lens O3, the means for manipulation can also comprise a point scanner for the temporally sequential exposure of individual, essentially point-shaped areas of the sample with light intensity in the event that the illumination lens is identical to the manipulation lens. However, such a point scanner operates sequentially and, in the case of very fast light sheet imaging, can only move to a very limited number of positions in the light sheet within an exposure sequence, in particular since the positioning of the intensity distribution must be axial, i.e. along the optical axis 4 of the illumination lens O1 and thus by refocusing. It is therefore advantageous when examining samples to first sequentially apply the manipulation radiation, i.e.to deposit the intensity distribution along the optical axis at the specified points sequentially along the light sheet and then quickly image this essentially flat volume of sample 1 with the light sheet. Since the light sheet scanner 13 and the stimulation scanner 19 are operated independently of each other, manipulation can also be performed in front of or behind the light sheet at desired temporal and / or spatial intervals during light sheet imaging.

[0059] In particular, it is advantageous if the light sources, the wavelengths and the intensities of the manipulation light are designed to excite two- and multi-photon transitions. In this way, the exposure of the sample to the corresponding intensity can be limited to the respective focus, since the Gaussian beam of the point scanner, with linear excitation, completely and possibly interactively penetrates the sample 1 along the optical axis 4 of the illumination objective O1.

[0060] When using a point scanner with multiphoton excitation, axial restriction along the manipulation axis can also be achieved by temporal focusing. For a light sheet microscope, this is exemplified in Fig. 11The temporal focusing takes place in the light sheet, which is generated by scanning. The control over the location to which an intensity is applied for manipulation is carried out laterally by the scan in the light sheet, which is Fig. 11 from above. The location of the focus along the light sheet axis, ie along the optical axis 4 of the illumination objective O1, is determined by means of the SLM and by temporal focusing using the group velocity dispersion control of the stimulation laser 18. In this way, Fig. 11 from the light sheet shown above, for example, deposit intensities one after the other at the three focus points 55 shown.

[0061] Of course, it is also possible to apply intensity to the sample not only from one direction, but also from several directions, as in Fig. 12As indicated, all three objectives—manipulation objective O3, detection objective O2, and illumination objective O1—are used to apply light intensity to the sample. Different manipulation methods and / or different wavelengths can then be used from different directions. For example, two-dimensional regions can be generated simultaneously by the detection objective O2 using a DMD and a first wavelength, and spatially distributed regions can be generated sequentially by the manipulation objective O3 using an SLM. The DMD can handle rapid manipulation in the light sheet plane; this is also feasible with incoherent light sources with wavelengths, for example, in the UV range, which are rarely available as laser sources. The SLM, whose phase pattern is imposed on the sample as an intensity distribution by the manipulation objective O3, can then sequentially manipulate spatial regions with other wavelengths.

[0062] The light sheet arrangement with two or more manipulation lenses also offers the possibility of coherently superimposing the manipulation radiation, i.e. the radiation for exposing the sample to light intensity in essentially point-like areas from two or three directions. In this case, which is not covered by the claimed invention, it can be advantageous if, for example, the detection lens O2 and the manipulation lens O3 have a common optical axis, since then the opposing wavefronts can interfere to the smallest possible extents of the intensity distribution in the light sheet plane. For this purpose, the manipulation lens O3 can, for example, be immersed in a sample solution in which the sample is stored (in Fig. 12not shown). For coherent superposition, the jointly used stimulation laser 18 must be divided into the corresponding beam paths for the detection objective O2 and the manipulation objective O3.

[0063] If a pair of lenses that do not share a common optical axis is used, a pump-probe technique such as CARS can also be used. Here, the pump radiation and the probe radiation are guided through different lenses of the array. Since the desired interaction only occurs in the overlap region of the pump radiation and the probe radiation, this can be achieved by further narrowing the areas.

[0064] As already mentioned, multiphoton excitation can be used to very advantageously restrict the intensity distribution along the optical axis of the objective used for manipulation, especially when using a point scanner to impinge on the sample with the appropriate light intensity, since a focused beam is always present. Wide-field microscopy approaches based on the use of a spatial light modulator and / or a DMD produce a less focused beam than when using a point scanner, depending on the desired manipulation pattern. However, temporal focusing can also be used to restrict the manipulation and intensity distribution in the propagation direction in the wide-field.In this way, the depth discrimination effect, which occurs in point-scanning two-photon microscopy due to the quadratic intensity dependence of the excitation in combination with strong focusing, can also be achieved in a wide-field image.

[0065] Fig. 13shows a corresponding arrangement in which multiphoton excitation is used for manipulation by the detection objective O2, which is not according to the invention. A femtosecond short-pulse laser 56 is used as the manipulation light source here. For example, a titanium-sapphire laser can be used. Its beam is expanded accordingly by means of the telescope 25 in order to illuminate the SLM 23 as completely as possible. A corresponding spatial phase pattern is impressed on the SLM 23 to generate a predetermined intensity distribution in the sample. A first Fourier transform of the phase pattern is imaged onto a correspondingly designed diffraction grating 58 via a lens 57. The diffraction grating 58 is located in an intermediate image plane in the detection beam path and is imaged into the sample by the tube lens 22 and the detection objective O2 using the correction elements, here the wavefront manipulators 7.Due to the spectral splitting of the pulses at the diffraction grating 58, the temporal focusing results in axial restriction of the illumination, limited to the desired point-like areas in the sample.

[0066] Instead of a single lens 57, the phase pattern of the SLM 23 can also be imaged onto the grating via a 4f array 59 with two lenses 60 and 61. A phase element 62 in a pupil plane of this 4f array 59 results in a phase shift of the zeroth diffraction order by π relative to the diffracted radiation, or vice versa. Thus, the phase pattern after the 4f imaging is directly converted into an intensity distribution on the diffraction grating 58.

[0067] Imaging with the light sheet microscopes described above can generally be performed in two ways: In the so-called sample scan, the illumination objective O1 and the detection objective O2 are stationary, and the light sheet is positioned in the focal plane of the detection objective O2 via the light sheet scanner 13. The sample is then scanned via the sample stage control 10 in the direction shown in the dashed image section. Fig. 1 shown direction.

[0068] In the so-called light-sheet scan, sample 1 remains stationary, but the light sheet must then be scanned for imaging. To prevent the light sheet from moving out of focus, the detection objective O2 must be tracked, as must the illumination objective O1, so that the beam waist of the light sheet remains in the center of the image field. Alternatively, the beam waist can also be tracked using another type of focusing, such as internal focusing.

[0069] Fig. 14 shows different temporal and spatial sequences of manipulation and depiction. In Fig. 14a ) shows the simultaneous manipulation and imaging in the light sheet plane. The light intensity used for manipulation is deposited in the light sheet plane at a given time t, and its effect can be observed immediately in this plane. This procedure is best implemented for sample scanning and light sheet scanning with planar manipulation, for example, using a DMD through the detection objective O2, for example, with arrangements as shown in Fig. 3 or Fig. 4 are shown.

[0070] Fig. 14b) shows the sequential sequence of manipulation and imaging, both spatially and temporally. First, the specified spatial intensity distribution is deposited in the desired, essentially point-like areas—marked by crosses—in the observation volume. Then, this observation volume, represented by the rectangular box in the sample, is imaged, if necessary multiple times, in order to observe the temporal course of the effect of the manipulation. This procedure can be implemented for both sample scanning and light-sheet scanning with, in principle, all of the previously mentioned variants that allow spatial manipulation, since the sequential sequence imposes no boundary conditions on the manipulation.

[0071] At the Fig. 14cIn the mixed approach shown in Figure 1, the manipulation radiation is introduced during the imaging light sheet scan at desired times and / or at desired locations in the light sheet, in front of and / or behind it. Furthermore, it is also possible to continuously adjust the spatial manipulation volume while the volume is being imaged, for example, to compensate for very rapid shape changes in the sample or to create two different volumes for the deposition of manipulation intensity for light sheet positions.

[0072] The light sheet microscopy arrangements described above allow particularly gentle observation of the sample while simultaneously manipulating it. List of reference symbols

[0073] 1 Sample 2 Sample carrier 3 Flat reference surface 4, 5 Optical axis 6, 7 Wavefront manipulator 8 Optical transmission system 9 Manipulation beam 10 Sample stage control 11 Illumination laser 12 Beam shaping unit 13 Light sheet scanner 14 Tube lens 15 Dichroic beam splitter 16 Tube lens 17 Camera 18 Stimulation laser 19 Stimulation scanner 20 Mirror 21 Laser 22 Tube lens 23 First spatial light modulator (SLM) 24 Lens 25 Telescope 26 Polarization element 27 Digital micromirror array (DMD) 28, 28a Light source 29 First region 30 Second region 31 Dichroic element 32, 33 Plane mirror 34, 35 Lens 36 Light source switching mirror 37Lens 38Switching mirror 38aBeam combiner 39Sample volume 40Dichroic beam splitter 41Spatial light modulator 42Manipulation area 43Light sheet generation area 44Dichroic beam combiner 45,46dichroic beam splitter 47further dichroic beam splitter 48intermediate image plane 49pupil plane 50first optic 51second optic 52scan lens 53dichroic beam combiner 54optical fiber 55focus point 56femtosecond laser 57lens 58diffraction grating 594f array 60, 61lens 62phase element , O1Illumination lens O2Detection lens O3Manipulation lens

Claims

1. A light sheet microscope, comprising: - illumination optics for illuminating a sample (1) which is located on a sample carrier (2) in a medium, the sample carrier (2) being aligned parallel to a flat reference surface (3), via an illumination beam path with a light sheet whose light sheet plane encloses a non-zero illumination angle with the normal of the reference surface (3), - detection optics with a detection beam path with an optical axis (5) which encloses a non-zero detection angle, in the region of the sample carrier (2), with the normal of the reference surface (3), - a separating layer system with at least one layer of a predetermined material having a predetermined thickness, which separates the medium from the illumination and detection optics, wherein the separating layer system is in contact with the medium, at least in the region accessible for illumination and detection, via a base surface aligned parallel to the reference surface (3), wherein - the light sheet microscope comprises means, independent of the light sheet generation, for applying light intensity to the sample (1) in substantially point-shaped areas in the light sheet plane or in a predetermined volume at least temporarily encompassing the light sheet plane via at least one manipulation beam path, characterised in that - the means for manipulation comprise a separate manipulation objective (O3) in the manipulation beam path, which is arranged with its optical axis parallel to the normal of the reference surface (3), - and the light sheet microscope comprises at least one correction element in the illumination and / or detection beam path for reducing such aberrations which arise due to the oblique passage of light to be detected or of light for illuminating the sample (1) through interfaces of the separating layer system.

2. The light sheet microscope according to claim 1, characterised in that the illumination optics comprise an illumination objective (O1) and the detection optics comprise a detection objective (O2).

3. The light sheet microscope according to claim 2, characterised in that the illumination objective (O1) is identical to the detection objective (O2).

4. The light sheet microscope according to any one of claims 2 or 3, characterised in that the means for manipulation comprise a point scanner for the temporally sequential application of light intensity to individual substantially point-shaped areas of the sample (1) or a multipoint scanner for the temporally sequential application of light intensity to a plurality of substantially point-shaped areas of the sample (1), to a number of which light intensity can be simultaneously applied in each case.

5. The light sheet microscope according to any one of claims 2 to 4, characterised in that the means for manipulation comprise a first spatial light modulator (23, 41) adapted for photostimulation in spatially extended areas for applying a predetermined intensity distribution to the sample.

6. The light sheet microscope according to claim 5, characterised in that the means for manipulation comprise a second spatial light modulator for applying a predetermined amplitude distribution to the predetermined intensity distribution.

7. The light sheet microscope according to any one of claims 2 to 6, characterised in that the means for manipulation comprise a digital micromirror array (27) for generating a predetermined intensity distribution in a plane, preferably parallel to the light sheet plane, particularly preferably in the light sheet plane.

8. The light sheet microscope according to claim 7, if referring back to claim 5 or 6, comprising a switching means, preferably a switching mirror (38) for switching between the first spatial light modulator (23), the digital micromirror array (27) or a combination of both.

9. The light sheet microscope according to any one of claims 1 to 8, characterised in that the light sources, wavelengths and intensities of the manipulation light are designed to excite two-photon and multiphoton transitions.