Arrangement for light sheet microscopy

Abstract

Arrangement for light sheet microscopy, comprising - a sample vessel (1) for receiving a sample (3) located in a medium (2), wherein the sample vessel (1) has a cover and is aligned with respect to a flat reference surface, - an illumination optics with an illumination objective (6) for illuminating the sample (3) with a light sheet, wherein the optical axis (7) of the illumination objective (6) and the light sheet lie in a plane which encloses an illumination angle β different from zero with the normal of the reference surface, - a detection optics with a detection lens (8), the optical axis (9) of which encloses a detection angle δ different from zero with the normal of the reference surface, characterized in that - at least one bulge transparent to illumination and detection light is formed on the cover, with an inner boundary surface (12) and an outer boundary surface (13) for receiving the sample (3) in this bulge, and - the shape of the at least one bulge, its position during observation and the position of the optical axes (7, 9) of the illumination objective (6) and the detection objective (9) are coordinated with one another in that the optical axes (7, 9) enclose a minimal angle with the normals of the boundary surfaces (12, 13) at least in the region of the passage of the optical axes (7, 9) through the boundary surfaces in order to avoid or minimize aberrations.

Description

[0001] The invention relates to an arrangement for light sheet microscopy. Such an arrangement comprises a sample vessel for receiving a sample located in a medium, wherein the sample vessel is aligned with respect to a flat, usually horizontal reference surface. The arrangement also comprises illumination optics with an illumination objective for illuminating the sample with a light sheet, wherein the optical axis of the illumination objective and the light sheet lie in a plane that encloses an illumination angle β different from zero with the normal of the reference surface. Finally, the arrangement for light sheet microscopy also comprises detection optics with a detection objective, whose optical axis encloses a detection angle δ different from zero with the normal of the reference surface. The illumination objective and detection objective can also be designed as a so-called double objective, as described, for example, in EP 0 866 993 B1.Both lenses are then combined in a common unit, the respective optics - ie lenses with associated beam paths and optical elements arranged therein - then share some elements.

[0002] Such an arrangement is used particularly in 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 to the detection direction, which generally corresponds to the optical axis of the detection objective. 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 sections combined with relative movement in a direction perpendicular to the sectioning plane, a spatially extended visual representation of the sample is possible.

[0003] The SPIM technique is preferably used in fluorescence microscopy, where it is also referred to as LSFM (Light Sheet Fluorescence Microscopy). Compared to other established techniques, such as confocal laser scanning microscopy or two-photon microscopy, the LSFM technique has several advantages: Since detection can be carried out 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 with the LSFM technique because the penetration depth is greater. Furthermore, the light exposure of the samples is lowest with this technique, which, among other things, reduces the risk of sample bleaching because the sample is only illuminated by a thin light sheet at a non-zero angle to the detection direction.

[0004] Instead of using a purely static light sheet, a quasi-static light sheet can also be created by rapidly scanning the sample with a light beam. The light sheet-like illumination is created by subjecting the light beam to very rapid relative movement to the sample to be observed, and thereby repeatedly and sequentially sequentially. The integration time of the camera, on whose sensor the sample is ultimately imaged, is selected so that the scan is completed within the integration time. Instead of a camera with a 2D array, a line sensor can also be used in combination with a rescan in the detection optics. Detection can also be performed confocally.

[0005] The SPIM technique has been described many times in the literature, for example in DE 102 57 423 A1 and the subsequent WO 2004 / 053558 A1, as well as 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.

[0006] DE 10 2012 108 158 A1 discloses a capillary cell, an arrangement, and a method for receiving, positioning, and examining a microscopic sample, in particular a brightened, fluorescent sample, using a light-sheet fluorescence microscope. The capillary cell is suitable for positioning in a chamber volume and contains a capillary section comprising a wall. The wall encloses a sample volume and is, at least in sections, flat and transparent. Furthermore, the capillary cell comprises an upper and a lower closure section, which are connected to the capillary section and close the capillary section. The sample volume is separated from the chamber volume by the capillary section, the upper closure section, and the lower closure section.

[0007] US 2010 / 0067104 A1 discloses a sample holder for a microscope. The sample holder comprises a sample chamber filled with an immersion liquid and containing a sample. The sample chamber has an upper opening. It further comprises means for displacing the sample relative to a detection objective of the microscope and means for rotating the sample about an axis of rotation extending in a substantially horizontal plane that forms a non-zero angle with the optical axis of the detection objective. In the sample holder, the sample is embedded in a transparent embedding medium, which at least partially has a higher strength than the immersion liquid. Furthermore, the sample chamber has means for horizontally supporting the embedded sample against gravity.

[0008] One of the main applications of light-sheet microscopy is the imaging of medium-sized organisms ranging in size from a few hundred µm to a few mm. These organisms are typically embedded in an agarose gel, which in turn is contained within a glass capillary. The glass capillary is inserted from above or below into a water-filled sample chamber, and the sample is pushed slightly out of the capillary. The sample in the agarose is illuminated with a light sheet, and the fluorescence is imaged onto a camera using a detection objective positioned perpendicular to the light sheet and thus also perpendicular to the light sheet optics.

[0009] This method of light-sheet microscopy has three major disadvantages. First, the samples to be examined are relatively large, originating from developmental biology. Furthermore, due to the sample preparation and the dimensions of the sample chamber, the light sheet is relatively thick, thus limiting the achievable axial resolution. Furthermore, the sample preparation is complex and incompatible with standard sample preparations and sample holders commonly used in fluorescence microscopy for single cells.

[0010] To at least partially circumvent these limitations, an SPIM setup has been developed in recent years. The illumination objective and the detection objective are perpendicular to each other and each directed at a 45° angle from above onto the sample. If the reference surface is, for example, the sample stage on which the sample holder is fixed, or another horizontal plane, the illumination angle β and detection angle δ are both 45°. Such a setup is described, for example, in WO 2012 / 110488 A2 and WO 2012 / 122027 A2.

[0011] In such a setup, the sample is located, for example, on the bottom of a Petri dish. The Petri dish is filled with water, and the illumination objective and detection objective are immersed in the liquid, which also acts as an immersion medium. This approach offers the advantage of higher resolution in the axial direction because a thinner light sheet can be generated. Due to the higher resolution, even smaller samples can be examined. Sample preparation has also become significantly easier. The major disadvantage is that both the sample preparation and the sample holder do not yet meet the aforementioned standards. The Petri dish must be relatively large so that the two objectives can be immersed in the dish without hitting the edge of the dish.Microtiter plates – also known as multi-well plates – which are standard in many areas of biology and in the fluorescence microscopic analysis of single cells, cannot be used with this method because the lenses cannot penetrate the very small wells of the plate. Furthermore, this method has the disadvantage that the analysis of many samples in a very short time (high-throughput screening) is not easily possible, as the lenses must be cleaned when changing samples to avoid contamination of the different samples.

[0012] The object of the invention is to further develop an arrangement for light sheet microscopy of the type described above in such a way that a high-throughput analysis of samples is simplified by simplifying the use of microtiter plates, ie sample holders that can accommodate a large number of samples.

[0013] This object is achieved for a light-sheet microscopy arrangement as described above in that at least one bulge, at least partially transparent to illumination and detection light, is formed on the cover to accommodate the sample, wherein the bulge has an inner and an outer boundary surface. This significantly simplifies the access of the objectives to the sample. In particular, microtiter plates—including rotatable microtiter plates—can be used, whose wells can then be configured with smaller lateral dimensions than if the sample were located at the bottom of the vessel, especially when an upright microscope configuration is used for analysis.

[0014] It is essential to coordinate the shape of the bulge, its position during observation, and the position of the optical axes of the illumination and detection objectives in order to avoid or minimize aberrations that would occur with an oblique beam path through the interfaces, resulting in oblique light incidence and exit from the sample vessel. The coordination is achieved in such a way that the optical axes of the illumination and detection objectives enclose a minimal angle with the normals of the inner and outer interfaces, at least in the area where the optical axes pass through the interfaces, i.e. an angle that is zero or deviates by only a few degrees, up to approximately 5°. If the optical axes and the interfaces are perpendicular to one another, only spherical aberrations occur, which can be corrected as with known microscope objectives adapted to cover glasses.

[0015] The shape of the bulge is essentially arbitrary, as long as the aforementioned condition is met. For example, the bulge can take the shape of a half-barrel or hemisphere, with the optical axes of the two lenses then coinciding with normals of tangents to the surface of the half-barrel in the coordinated arrangement in the best possible configuration.

[0016] In a particularly preferred embodiment, the at least one bulge comprises two plate-shaped elements protruding from the cover and the sample vessel and having parallel interfaces which are in contact at at least one point at the point on the bulge which is the furthest away from the rest of the sample vessel - in the case of a bulge designed as a depression, this is the deepest point of the depression, in the case of a bulge designed as an elevation, this is the highest point of the elevation - and at this point close off the depression or the elevation or the sample vessel or the vessel lid at the bottom or top. The normals of the interfaces of a first plate-shaped element coincide with the optical axis of the illumination objective in the region of the passage thereof, in the sense that the normals and the optical axis are parallel to the optical axis of the illumination objective at every point of the interfaces of the plate-shaped element.Accordingly, the normals of the interfaces of a second plate-shaped element coincide with the optical axis of the detection objective, i.e., they are parallel to it at every point of the interface of the second plate-shaped element. This allows for greater flexibility in coordinating the position of the bulge in relation to the two objectives. The plate shape, which implies a parallel position of the inner and outer interfaces to one another, is not mandatory, however, and particularly in the area where the two plate-shaped elements are in contact, this area can be provided with a small bulge on the inside, so that, for example, in the case of a depression, on the one hand, its strength is reinforced at its lowest point and, on the other hand, the stubborn adhesion of contaminants is prevented, as is the case with two flat plates that meet at an angle, i.e.would be the case with a depression with at least a partial V-shaped cross-section.

[0017] The sum of the illumination angle β and the detection angle δ is preferably 90°, which facilitates the positioning of a detector in the beam path. For other angles, care must be taken to ensure that the image plane (i.e., the plane in which the detector is located), the object plane (i.e., the plane irradiated by the light sheet), and the object-side principal plane of the detection lens intersect in a straight line.

[0018] The at least one bulge can be groove-shaped, whereby several grooves can be arranged one behind the other in the sample vessel, for example in the vessel base. In a particularly preferred embodiment, the bulge is pyramid-shaped, so that the two plate-shaped elements have a triangular shape and are supplemented by two further plate-shaped elements. This enables analysis of a sample located in the bulge from four different sides, which can be advantageous if the sample is deposited on one side. In addition, pyramid-shaped bulges can be arranged in a grid pattern on the vessel base or in the vessel lid, so that the sample vessel can also be designed as a microtiter plate with a plurality of such pyramid-shaped bulges.The groove-shaped design can also be used for the design of a microtiter plate if the individual grooves are divided into individual sections by separating elements such as struts.

[0019] A sample vessel with such a depression can be made of glass, but preferably in a cheaper version of plastic, for example by deep drawing if the depression is trough-shaped.

[0020] At least one portion of the inner interface of the protrusion is functionalized for cell growth on this interface, i.e., coated with a special structure to which the surface structures of the cells bind and anchor themselves. For individual cells that are to be analyzed in high throughput using the present light-sheet microscopy setup, the growth conditions can be even better adapted to a natural growth environment by filling at least one protrusion, such as a groove or pyramid-shaped depression, with a gel or alginate, which can be used to simulate a spatial matrix.

[0021] As already indicated, in a preferred embodiment, in which the illumination objective and detection objective are arranged in an inverse configuration below the sample vessel, the cover is designed as the vessel bottom, and the bulge is designed as a depression in the vessel bottom. This significantly simplifies the objective's access to the sample; in particular, microtiter plates with a plurality of wells can also be used, with each of these wells having a depression for a sample. The use of a depression or depressions for the examination of many cell samples makes it possible to increase the number of wells in a sample vessel, since the lateral dimensions—in the plane of the reference surface—can be reduced.

[0022] Instead of the vessel bottom, for an upright configuration of the light sheet microscope - i.e. for observation from above - the vessel lid can also be adapted as a cover in a similar way to that described for the vessel bottom, and the analysis of the cells can be carried out using an upright light sheet microscopy arrangement. In this embodiment of the invention, the illumination and detection objectives are therefore arranged above the sample vessel. Instead of a depression, at least one elevation is formed in the vessel lid. The shape of the elevation, its position during observation, and the position of the optical axes of the illumination and detection objectives are then also coordinated with one another in that the optical axes of the illumination and detection objectives enclose a minimal angle with the normals of the interfaces, at least in the region where the optical axes pass through the interfaces.The elevations, like the depressions, can be trough-shaped, pyramid-shaped, half-barrel-shaped or hemispherical.

[0023] Since in microtiter plates the sample usually sinks to the depth due to gravity or is deposited at the deepest point, observation is not easily possible if the illumination and detection objectives are arranged above the sample vessel. For this reason, if the elevations are formed in the vessel lid, additional means for positioning the sample are arranged in the upper area of the sample vessel, relative to its depth, within the working distance of the objectives. The positioning means can also be arranged in the vessel lid, likewise within the working distance of the objectives. The working distance for typical objectives with a high numerical aperture is usually in a range of a few 100 µm to a few mm.

[0024] Depressions and elevations can be composed of plate-shaped elements, and the inner interfaces can be functionalized. Another possible design for the sample vessel is to use a rotatable microtiter plate in which the elevations in the lid initially point downwards. The sample is placed into this elevation, which corresponds to a depression in the loading position, and fixed there using the means for positioning the sample in the at least one elevation, for example using a stamp. The base is then placed onto the rotatable microtiter plate from above and the plate is closed. The plate is then rotated for analysis if it is to be used with an upright light sheet microscope. Rotation is not necessary if it is to be used with an inverted light sheet microscope.

[0025] The means for positioning the sample in the upper quarter or in the at least one elevation in the vessel lid advantageously comprise a membrane permeable to nutrient solutions, a platform with a plurality of openings, or a ridge. It is important that the sample is in contact with the nutrient solution in all cases, but must not sink into it due to gravity. The membrane, platform, or ridge can also be made of a gel.

[0026] Due to the fact that the light passes through three different media or two interfaces between each objective and the sample, spherical aberrations occur with respect to the interfaces even when the objectives are aligned vertically. These aberrations can be corrected for the bulge in the form of a depression in the vessel bottom or in the form of a projection in the vessel lid, and for known thicknesses, at least for plate-shaped elements with parallel interfaces, in a way that is commonly used for microscope objectives. In some cases, they are corrected with respect to a given cover glass thickness made of a given material. Such corrections are particularly preferred for the detection objective, which generally has a larger numerical aperture than the illumination objective.

[0027] In a preferred embodiment, the illumination optics and / or detection optics comprise not only correction means for reducing the above-mentioned aberrations, but also those which arise from the passage of illumination light and / or light to be detected through the interfaces at an angle different from 90°.

[0028] Therefore, special correction lenses are preferably arranged in the illumination objective and / or the detection objective. These can also include cylindrical lenses, tilted lenses, or lenses not arranged on the optical axes if the objectives form an angle other than zero with the normals of the boundary surfaces. Correction elements with aspherical or freeform surfaces can also be used for correction. Alternatively or additionally, correction means in the form of adaptive optical elements for manipulating the phase fronts of the illumination and / or detection light can be arranged in the illumination beam path. Deformable mirrors, spatial light modulators, or phase plates are preferably used for this purpose.

[0029] Another way to reduce aberrations is to use specially adapted materials for the cover or the elevations in the vessel lid or the depressions in the vessel bottom.

[0030] In a particularly preferred embodiment, materials with a refractive index that differs by less than 5% from the refractive index of the medium in which the sample is located are used as the material for the depression or elevation in the vessel bottom or in the vessel lid. If, for example, water is used as the medium in which the sample is located, which has a refractive index n d = 1.33 at a wavelength λ d = 578.56 nm, PTFE (polytetrafluoroethylene, n d = 1.35), CYTOP ® (n d = 1.34), or PFEP (perfluoroethylenepropylene, n d = 1.34). Perfluorodioxolane polymers can also be used, whose refractive index is also usually between 1.33 and 1.36. Another particularly suitable material is Teflon. ® AF, which usually has a refractive index n d= 1.32. This material is an amorphous polymer, and its glass transition temperature can be adjusted so that the polymer, when cooled, has the refractive index of the medium in which the sample is located. Other amorphous polymers with adjustable glass transition temperatures can, of course, also be used.

[0031] If the refractive indices do not exactly match, aberrations will still occur, albeit to a lesser extent. To further reduce these aberrations, the bulge should be configured as thin as possible and no thicker than a few hundred µm. If the cover also serves as the base of the sample vessel, as is the case with an inverse arrangement, it is of course necessary to ensure sufficient stability against the pressure exerted by the medium in which the sample is located. This is not necessary if the cover serves as the lid of the sample vessel for upright observation; here the material can be formed much thinner, with thicknesses of less than 100 µm.

[0032] In a further step, which is particularly easy to implement with upright light-sheet microscopy, immersion objectives can be used again. If the same medium as that used for the sample is used as the immersion medium, for example, water with the refractive indices of water, and a material with a refractive index almost identical to that of water is used for the elevations / depressions in the vessel lid or vessel base, the interfaces are not noticeable through scattering or refraction, and the objectives do not require further correction.

[0033] A nanostructured mixed material consisting of a first and a second component can also be used as the material for the elevations or depressions in the vessel lid and vessel base, whereby the refractive index of the first component is smaller and the refractive index of the second component is greater than the refractive index of the medium for receiving the sample. If the average structure size of the material of the first component is smaller than the wavelengths of the light used for illumination and the light to be detected, this results in an effective refractive index for the mixed material, which, depending on the size and number of the regions, can also be adapted to the refractive index of the medium, so that it lies within 5% of the refractive index of the medium for embedding the sample. For example, nanoporous silicon dioxide can be used; here the first component is air and the second component is silicon dioxide.Such nanostructured materials are described, for example, in the article "Optical thin-film materials with low refractive index for broadband elimination of Fresnel reflection" by J.-Q. Xi et al., published in 2007 in Nature Photonics, Vol. 1, pp. 176-179 in connection with the production of antireflection coatings.

[0034] 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.

[0035] 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 an arrangement for light sheet microscopy, Fig. 2 an example of a sample vessel with depressions, Fig. in example for a vessel lid or vessel bottom, Fig. 4 a)-c) different ways of arranging a sample in the upper part of a well of a microtiter plate, and Fig. 5 the use of a rotatable microtiter plate.

[0036] In Fig. Figure 1 initially shows the basic structure of a setup for light sheet microscopy, which allows easy access to the sample to be examined and thus fulfills the requirements for use in high-throughput analyses of individual cells. The setup is configured here as an inverted light sheet microscope, but can easily be transferred to an upright light sheet microscope. A sample 3 is located in a medium 2 in a sample vessel 1. The sample vessel 1 is aligned with respect to a flat reference surface, which is defined here by the horizontal surface of a sample stage 4. The setup also includes illumination optics with a light source 5 and an illumination objective 6 for illuminating the sample 3 with a light sheet. The light sheet and the optical axis 7 of the illumination objective 6 lie in a plane which encloses an illumination angle β different from zero with the normal to the reference surface.Light coming from the sample is imaged onto a detector 10 via a detection optics with a detection objective 8, whose optical axes 9 form a non-zero detection angle δ with the normal of the reference surface. The detector 10 converts the recorded intensity into image data for further processing. The illumination angle β and detection angle δ are the same here, but this is not mandatory. For example, if the two objectives have different apertures, the angles may also be set differently due to space requirements.

[0037] The illumination objective 6 and the detection objective 8 are arranged below the sample vessel 1. The sample vessel 1 has a vessel bottom 11 that is transparent to the illumination and detection light and has an inner boundary surface 12 and an outer boundary surface 13. At least one depression 14 that is transparent to the illumination and detection light is formed on the vessel bottom 11 for depositing the sample 3 into the depression. It is sufficient if the sample vessel 1 is transparent in the area of the depression 14, but manufacturing it from a uniform material such as glass or deep-drawn plastic is generally easier. The deposition of the sample 3 in this depression 14 makes the sample 3 more easily accessible for the optical arrangement of the light sheet microscope, the illumination objective 6, and the detection objective 8.A sample vessel 1 with a plurality of such wells 14 is therefore better suited for high-throughput analysis of individual cells than a vessel with a flat bottom, since the deposition of the sample in the well allows the individual wells of such a multi-well plate or microtiter plate to be designed with smaller laterally dimensioned wells. The microtiter plates then do not need to be changed as frequently.

[0038] The shape of the depression 14, its position during observation, and the positions of the optical axes 7 and 9 of the illumination objective 6 and the detection objective 8 are coordinated with one another in that these optical axes 7, 9 of the illumination objective 6 and the detection objective 8 form a minimal angle with the normals of the inner boundary surface 12 and the outer boundary surface 13, at least in the region where the optical axes 7 and 9 pass through the boundary surfaces 12 and 13. In this way, the occurrence of aberrations due to oblique light incidence or oblique light exit through the boundary surfaces can be minimized. The angle is preferably zero.

[0039] In Fig. 1, the at least one depression 14 comprises a first and a second plate-shaped element 15 and 16, respectively, protruding from the vessel bottom 11. In each of the plate-shaped elements 15 and 16, the inner boundary surface 12 is arranged parallel to the outer boundary surface 13. At the deepest point of the depression 14, the two plate-shaped elements 15 and 16 are in contact at least at one point, wherein the normals of the boundary surfaces 12, 13 of the first plate-shaped element 15 coincide with the optical axis 7 of the illumination objective 6, and the normals of the boundary surfaces 12, 13 of the second plate-shaped element 16 coincide with the optical axis 9 of the detection objective 8. The sum of the illumination angle β and the detection angle δ is 90° here, but it can also deviate from this. This arrangement has the great advantage that aberrations, such as those that occur when light passes obliquely through the interfaces 12, 13, can be completely avoided.For the illumination objective 6, which generally has a small numerical aperture on the order of 0.3, since the light sheet to be generated should be as thin as possible, further corrections are then no longer absolutely necessary. However, for the detection objective 8, which generally has a high numerical aperture on the order of 1.0, further corrections are advantageous, in special cases also for the illumination optics. The correction means can, for example, comprise corrective lenses in the illumination objective 6 or in the detection objective 8, or also adaptive optical elements for manipulating the phase fronts of the illumination and / or detection light, which are arranged in the illumination beam path or in the detection beam path and are preferably designed as deformable mirrors, spatial light modulators, or phase plates.

[0040] In order to completely avoid scattering and refraction at the interfaces 12, 13, the vessel bottom 11 can also be made of a material having a refractive index that differs by less than 5% from the refractive index of the medium 2 in which the sample 3 is located. Particularly suitable for this purpose are amorphous polymers, whose glass transition temperature can be adjusted so that the material has exactly the desired refractive index upon cooling. A nanostructured mixed material, for example, made of nanoporous silicon dioxide, i.e. silicon dioxide with a multitude of cylindrical openings, can also be used as the material for the vessel bottom 11. In any case, the thickness of the vessel bottom 11 should be selected as thin as possible in order to suppress aberrations as much as possible. Fig. The example shown in Figure 1 can also be transferred in an equivalent manner for an upright arrangement of illumination objective 6 and detection objective 8; instead of a depression 14 in the vessel bottom 11, the vessel lid here has a corresponding elevation.

[0041] In Fig. Figure 2 shows an example of a sample vessel 1 suitable for high-throughput cell analysis. It shows two groove-shaped depressions 14 arranged parallel to each other in a section of a sample vessel 1. Each of these depressions 14 is divided into individual wells by struts 17, which make it possible to arrange multiple samples side by side in one depression 14 without any possibility of mutual contamination.

[0042] Instead of the vessel bottom 11, a corresponding vessel lid can also be designed in this way. Fig. 3 shows a section of a vessel lid 18 on which a plurality of pyramid-shaped elevations 19 are arranged. Each of these elevations 19 covers a depression in the sample vessel 1. The vessel bottom 1 can also be designed in this manner.

[0043] The inner interfaces 13 in the depressions 14 or elevations 19 can be functionalized to allow cell growth on this interface, so that, for example, cells can attach to the elevations 19 without additional aids. The depression 14 or elevation 19 can also be filled with a gel or alginate to immobilize the sample.

[0044] In order to facilitate the observation of the samples and to be able to design the wells of a microtiter plate with a small lateral diameter, such sample vessels intended for upright observation preferably have means for positioning the sample in the upper area of the sample vessel 11, based on its depth, within the working distance of the illumination and detection objective, or for corresponding positioning within the working distance in the elevation 19 in the vessel lid 18. Such means are described in the Fig. 4 a) - c). The box-shaped element symbolizes a well 20 of a multi-well plate in a sample vessel 1 for upright observation. In Fig. 4 a) a permeable membrane 21 is arranged in the upper area, on which the sample 3 is stored and which ensures contact with a comparatively large volume of nutrient fluid to enable cell growth. The membrane 21 enables both the diffusion of nutrients and support of the sample 3. Instead of a membrane 21, a flat platform 22 with openings can also be used, for example, this is shown in Fig. 4 b). The platform can be made of glass, for example, allowing sample preparation to proceed essentially according to standard protocols. The cell culture can also be immobilized in a matrix gel. Fig. 4 a) shows a protrusion 19 of a vessel lid 18 in the form of a groove or a cut-out of a groove on the depression 20. The use of a flat cover, for example a film 23, as in Fig. 4 b) is conceivable in principle. The film 23 can be glued or welded to the sample container. Another configuration is shown in Fig. 4 c). Here, a web 24 is shown, which protrudes into the center of the recess. The elevation 19 has a half-barrel shape. The aforementioned support elements, membrane 21, platform 22, and web 24, can also be made of gel, provided it has sufficient rigidity.

[0045] The use of rotatable microtiter plates is also conceivable, as in Fig.5. First, the sample 3 is placed in a well 20 of the microtiter plate—here, for example, a funnel-shaped well. The well 20 is filled with the medium 2. Subsequently, a funnel-shaped element 25, at the end of which a membrane 21 with a smaller diameter is arranged, is inserted into the funnel-shaped well 20. The microtiter plate is then sealed with the vessel base 11. The plate is then inverted, and the sample can then be observed using an upright arrangement for light sheet microscopy. List of reference symbols 1 sample container 2 Medium 3 Sample 4 Sample table 5 Light source 6 Illumination lens 7 optical axis 8 Detection lens 9 optical axis 10 Detector 11 Vessel bottom 12 inner interface 13 outer boundary surface 14 depression 15 first plate-shaped element 16 second plate-shaped element 17 Strut 18 vessel lids 19 Survey 20 Deepening 21 Membran 22 Platform 23 Slide 24 jetty 25 funnel-shaped element

Claims

[1] Arrangement for light sheet microscopy, comprising - a sample vessel (1) for receiving a sample (3) located in a medium (2), wherein the sample vessel (1) has a cover and is aligned with respect to a flat reference surface, - an illumination optics with an illumination objective (6) for illuminating the sample (3) with a light sheet, wherein the optical axis (7) of the illumination objective (6) and the light sheet lie in a plane which encloses an illumination angle β different from zero with the normal of the reference surface, - a detection optics with a detection lens (8), the optical axis (9) of which forms a detection angle δ different from zero with the normal of the reference surface, characterized by , that - at least one bulge transparent to illumination and detection light is formed on the cover, with an inner boundary surface (12) and an outer boundary surface (13) for receiving the sample (3) in this bulge, and - the shape of the at least one bulge, its position during observation and the position of the optical axes (7, 9) of the illumination objective (6) and the detection objective (9) are coordinated with one another in that the optical axes (7, 9) enclose a minimal angle with the normals of the boundary surfaces (12, 13) at least in the region of the passage of the optical axes (7, 9) through the boundary surfaces in order to avoid or minimize aberrations. [2] Arrangement for light sheet microscopy according to claim 1, characterized byin that the at least one bulge comprises two plate-shaped elements (15, 16) projecting from the cover and from the sample vessel and having parallel boundary surfaces (12, 13) which are in contact at least one point at that point on the bulge which is at the greatest distance from the rest of the sample vessel (1), wherein the normals of the boundary surfaces (12, 13) of a first plate-shaped element (15) coincide with the optical axis (7) of the illumination objective (6) and the normals of the boundary surfaces (12, 13) of a second plate-shaped element (16) coincide with the optical axis (9) of the detection objective (8), and wherein the sum of the illumination angle β and the detection angle δ is preferably 90°. [3] Arrangement for light sheet microscopy according to claim 1 or 2, characterized by that at least one bulge is groove-shaped or pyramid-shaped. [4] Arrangement for light sheet microscopy according to one of claims 1 to 3, characterized bythat the inner interface (12) of the at least one bulge is functionalized for the growth of cells. [5] Arrangement according to one of claims 1 to 4, characterized by that the illumination objective (6) and detection objective (8) are arranged below the sample vessel (1), the cover is designed as a vessel bottom (11) and the at least one bulge is designed as a depression (14). [6] Arrangement for light sheet microscopy according to claim 5, characterized by that the sample vessel (1) is designed as a microtiter plate with a plurality of wells, and a pyramid-shaped depression (14) is formed on each well. [7] Arrangement according to one of claims 5 or 6, characterized by that the at least one depression (14) is filled with a gel or alginate. [8] Arrangement for light sheet microscopy according to one of claims 1 to 4, characterized bythat the illumination objective (6) and detection objective (8) are arranged above the sample vessel (1), the cover is designed as a vessel lid (18), the at least one bulge is designed as an elevation (19), and in the sample vessel (1) means for positioning the sample (3) in the upper region of the sample vessel (1), based on its depth, within the working distance of the illumination objective (6) and detection objective (8) and / or for corresponding positioning in the at least one elevation (19) are arranged. [9] Arrangement for light sheet microscopy according to claim 8, characterized by that the sample vessel (1) is designed as a microtiter plate with a plurality of pyramid-shaped elevations (19) in the vessel lid (18). [10] Arrangement for light sheet microscopy according to claim 9, characterized by that the microtiter plate is designed to be rotatable. [11] Arrangement for light sheet microscopy according to one of claims 8 to 10, characterized by that the means for positioning the sample (1) in the upper region of the sample vessel or in the at least one elevation (19) comprise a membrane (21) permeable to nutrient solutions, a platform (22) with a plurality of openings or a web (24). [12] Arrangement for light sheet microscopy according to claim 11, characterized by that the membrane (21), the platform (22) or the bridge (24) are made of a gel. [13] Arrangement for light sheet microscopy according to one of claims 1 to 12, characterized by that the illumination optics and / or the detection optics comprise correction means for reducing such aberrations which arise from oblique passage of illumination light and / or light to be detected through the interfaces. [14] Arrangement for light sheet microscopy according to claim 13 characterized bythat the correction means comprise correction lenses in the illumination objective (6) and / or in the detection objective (8), preferably cylindrical lenses, tilted or non-axially arranged lenses or correction elements with aspherical surfaces or with free-form surfaces, or comprise adaptive optical elements arranged in the illumination beam path and / or in the detection beam path for manipulating the phase fronts of the illumination and / or detection light, preferably deformable mirrors, spatial light modulators or phase plates. [15] Arrangement for light sheet microscopy according to one of claims 1 to 14, characterized by that the vessel bottom (11) and / or the vessel lid (18) consists of a material which has a refractive index which differs by less than 5% from the refractive index of the medium (2) in which the sample (3) is located. [16] Arrangement for light sheet microscopy according to claim 15, characterized bythat the material is a nanostructured mixed material of a first and a second component, wherein the refractive index of the first component is smaller and the refractive index of the second component is greater than the refractive index of the medium (2), and wherein the average structure sizes of the regions made of material of the first component have an average diameter which is smaller than the light wavelengths of the light used for illumination and of the light to be detected.

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