METHOD FOR OPERATING A SAMPLE CHAMBER FOR MICROSCOPIC IMAGING, APPARATUS, AND SAMPLE CHAMBER

Abstract

A sample chamber encloses a sample space for positioning a sample and includes a wall that delimits the sample space and has an outer side facing away from the sample space. An illumination beam path is directed through the outer side into the sample space. Detection radiation is detected along a detection axis extending from the sample chamber through the wall of the sample chamber. The sample chamber has an outer surface with a shape of a spherical section with a circular disc as the base surface. The sample chamber and the detection axis are rotated and/or pivoted relative to one another about the center point of the circular disc so that different angular positions of the sample chamber relative to the detection axis are adjusted and image data is recorded at different angular positions of the sample chamber relative to the detection axis.

Claims

1-15. (canceled)

16. A method for operating a sample chamber for microscopic imaging, the method comprising: providing the sample chamber in an illumination beam path, wherein the sample chamber surrounds a sample space for positioning a sample and includes a wall that delimits the sample space and has an outer side facing away from the sample space, the outer side having a shape of a spherical segment with a circular disk as a base, and wherein the illumination beam path is directed through the outer side and into the sample space along an illumination axis; capturing detection radiation, which was caused by illumination radiation directed into the sample space along the illumination axis, along a detection axis that extends from the sample space through the wall of the sample chamber; rotating and/or pivoting the sample chamber relative to the detection axis about a center of the circular disk, so that different relative angular positions of sample chamber and detection axis are set; and capturing image data at the different relative angular positions of the sample chamber and the detection axis.

17. The method of claim 16, further comprising: displacing the sample chamber and/or a focal plane of an imaging optical unit relative to one another along the detection axis to set at least two respective relative z-positions; and capturing image data in at least two of the respective relative z-positions in one of the set relative angular positions.

18. The method of claim 16, further comprising: shaping the illumination radiation into a light sheet; and radiating the light sheet into the sample space.

19. The method of claim 18, further comprising producing the light sheet from a non-diffractive beam of the illumination radiation.

20. The method of claim 18, further comprising: displacing the sample chamber and/or a focal plane of an imaging optical unit relative to one another along the detection axis to set at least two respective relative z-positions; capturing image data in at least two of the respective relative z-positions in one of the set relative angular positions; and selecting an angle of incidence (α) of the light sheet radiated into the sample space based on the respective relative z-position.

21. The method of claim 20, wherein the angle of incidence (α) is selected on the basis of a relationship α(z)=arcsin((n.sub.b/n.sub.a*R)z), where n.sub.a is a refractive index of a medium outside of the sample chamber, n.sub.b is a refractive index of a medium within the sample chamber, R is the radius of the spherical segment of the outer side, and z is a respective position along the detection axis.

22. The method of claim 18, further comprising capturing the image data by plenoptic detection.

23. The method of claim 16, further comprising capturing image data of adjoining or overlapping portions of the sample with different relative angular positions of sample chamber and detection axis.

24. The method of claim 23, further comprising evaluating image data of overlapping portions of the sample by means of a multi-view fusion algorithm.

25. A sample chamber for microscopy, wherein the sample chamber surrounds a sample space for positioning a sample, the sample chamber comprising: a wall that delimits the sample space and that includes an outer side facing away from the sample space, wherein the outer side of the wall has a shape of a spherical segment with a circular disk as a base; and a sample holder configured for holding the sample in a plane of the circular disk.

26. A sample carrier comprising: a plurality of carrier surfaces arranged with angular offsets with respect to one another, each of the carrier surfaces being configured to receive, in a respective holding area, a sample chamber that surrounds a sample space for positioning a sample and that includes a wall that delimits the sample space and that includes an outer side facing away from the sample space, wherein the outer side of the wall has a shape of a spherical segment with a circular disk as a base and wherein each sample chamber includes a sample holder configured for holding the sample in a plane of the circular disk.

27. The sample carrier of claim 26, further comprising channels for supplying and/or removing media to and from each of the sample spaces surrounded by the sample chambers.

28. The sample carrier of claim 26, wherein at least one of the carrier surfaces includes a circumferential wall standing upright on the carrier surface, wherein the wall surrounds a holding area of a received sample chamber as a lateral boundary.

29. An apparatus for capturing image data, the apparatus comprising: a plurality of carrier surfaces arranged with angular offsets with respect to one another, each of the carrier surfaces being configured to receive, in a respective holding area, a sample chamber that surrounds a sample space for positioning a sample and that includes a wall that delimits the sample space and that includes an outer side facing away from the sample space, wherein the outer side of the wall has a shape of a spherical segment with a circular disk as a base and wherein each sample chamber includes a sample holder configured for holding the sample in a plane of the circular disk; a beam shaping unit configured for providing illumination radiation along an illumination beam path and for shaping the illumination radiation to form a light sheet; a first scanner in, or in a vicinity of, a pupil of the illumination beam path, and configured for deflecting the illumination radiation; and a second scanner that is movable about a pivot point and that is arranged in a vicinity of an intermediate image in the illumination beam path, wherein the pivot point of the second scanner is imaged on an entry point of the light sheet on the outer side of sample chambers received by the carrier surfaces, wherein the first scanner and the second scanner are configured to oscillate in a common plane of movement.

30. The apparatus of claim 29, further comprising a third scanner arranged in the illumination beam path and configured for moving the illumination radiation in a plane that is orthogonal to a plane in which the illumination radiation is moved by deflection due to the first scanner.

31. The apparatus of claim 29, further comprising channels for supplying and/or removing media to and from each of the sample spaces surrounded by the sample chambers.

32. The apparatus of claim 29, wherein at least one of the carrier surfaces includes a circumferential wall standing upright on the carrier surface, wherein the wall surrounds a holding area of a received sample chamber as a lateral boundary.

33. An apparatus for capturing image data, the apparatus comprising: a sample chamber that surrounds a sample space for positioning a sample, wherein the sample chamber includes a wall that delimits the sample space and that includes an outer side facing away from the sample space, wherein the outer side of the wall has a shape of a spherical segment with a circular disk as a base, and a sample holder configured for holding the sample in a plane of the circular disk; a beam shaping unit configured for providing illumination radiation along an illumination beam path and for shaping the illumination radiation to form a light sheet; a first scanner in, or in a vicinity of, a pupil of the illumination beam path, and configured for deflecting the illumination radiation; and a second scanner that is movable about a pivot point and that is arranged in a vicinity of an intermediate image in the illumination beam path, wherein the pivot point of the second scanner is imaged on an entry point of the light sheet on the outer side of the sample chamber, wherein the first scanner and the second scanner are configured to oscillate in a common plane of movement.

34. The apparatus of claim 33, further comprising a third scanner arranged in the illumination beam path and configured for moving the illumination radiation in a plane that is orthogonal to a plane in which the illumination radiation is moved by deflection due to the first scanner.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The invention is explained in more detail below on the basis of exemplary embodiments and figures. In the drawings:

[0050] FIG. 1 shows a schematic illustration of an exemplary embodiment of a sample chamber as per the prior art;

[0051] FIG. 2 shows a schematic illustration of a first exemplary embodiment of a sample chamber, in a sectional illustration (FIG. 2a) and in a plan view from below (FIG. 2b);

[0052] FIG. 3 shows a schematic illustration of an exemplary embodiment of a sample chamber and of a sample carrier;

[0053] FIG. 4 shows a schematic illustration of a first configuration of the method, with FIG. 4a showing the capture of image data in a first relative angular position and in a first relative Z-position; FIG. 4b showing the capture of image data in a second relative angular position and in a second relative Z-position; and FIG. 4c showing the capture of image data in the second relative angular position and in a third relative Z-position;

[0054] FIGS. 5a to 5c show a schematic illustration of a sequential capture of z-stacks at different relative angular positions and a resultant coverage of the sample with a number of z-stacks;

[0055] FIG. 6 shows a schematic illustration of a coordinate transformation of a z-stack into a horizontally aligned coordinate system;

[0056] FIG. 7 shows a schematic illustration of a coordinate transformation of a z-stack into a spherical coordinate system;

[0057] FIG. 8 shows a schematic illustration of an exemplary embodiment of an imaging system having the option of choosing different imaging methods depending on the sample;

[0058] FIG. 9 shows a schematic illustration of an exemplary embodiment of an optical arrangement for updating location and angle in the case of light sheet illumination on a sample chamber and a schematic illustration of the angle update (inserted figure);

[0059] FIG. 10 shows a schematic illustration of a first exemplary embodiment of a sample carrier with a number of sample chambers, and

[0060] FIG. 11 shows a schematic illustration of a second exemplary embodiment of a sample carrier with a number of sample chambers and channels for supplying and/or removing media.

[0061] The exemplary embodiments are illustrated schematically. Identical technical elements are labeled by the same reference signs.

DETAILED DESCRIPTION

[0062] FIG. 1 illustrates a sample chamber 1 known from the prior art, which surrounds a sample space 2 in which a sample 3 can be positioned. The sample space 2 is filled with a sample medium 4, into which an objective 5 of an imaging optical unit of an optical apparatus (not illustrated in more detail) can be immersed. The sample medium 4, which acts as immersion medium 14 at the same time, has a first refractive index n1 and is in direct contact with the objective 5. If the sample medium 4 is a chemical or a mixture of chemical components, as are used to clear the sample 3, there is a risk of the objective 5 being damaged on account of the often aggressive properties thereof. The sample space 2 is surrounded on three sides by a wall 6, formed substantially from planar partial areas in the prior art, wherein the partial areas need not be optically effective, for example, need not be transparent. The sample 3 can be illuminated with illumination radiation along an illumination beam path 7, which coincides with an optical axis OA of the objective 5. Detection radiation effected in the sample 3 is captured by the objective 5 along a detection axis DA of a detection beam path 8, with the optical axis OA and the detection axis DA coinciding.

[0063] A first exemplary embodiment of a sample chamber 1 is shown in FIG. 2a in a lateral sectional illustration. The sample space 2 is delimited by a wall 6, wherein an outer side 6.1 of the wall 6 has the shape of a spherical segment (FIG. 2a). The spherical segment covers a circular area 9 with a center 10 (see also FIG. 2b). The sample space 2 is delimited by a further wall 11, which extends in the plane of the circular area 9. If used as intended, the sample chamber 1 is provided to allow the illumination beam path 7 to pass through the outer side 6.1 and into the sample space 2, and to allow the detection beam path 8 likewise to pass through the outer side 6.1. Illumination beam path 7 and detection beam path 8 may coincide or, as indicated in FIG. 2a, be separated from one another.

[0064] The spherical sample chamber 1 surrounds the sample 3, for example, a brain to be imaged, as tightly as possible. The sample medium 4 used for clearing is situated in the sample chamber 1. Detection radiation can be captured by means of an objective 5 embodied as an air objective (see FIGS. 4a to 4c, for example).

[0065] In a further exemplary embodiment, the sample chamber 1 is placed on a carrier surface 12 of a sample carrier 13 and surrounded by a wall 15 in the form of a hollow cylinder standing upright on the carrier surface 12 (FIG. 3). Together, sample carrier 13 and carrier surface 12 can also form an open vessel in further embodiments. In further embodiments, the wall 15 or cylinder wall can have a different shape in a plan view, for example, a polygonal or oval shape. A space 16 present above the sample chamber 1 is filled with air or with an immersion medium 14, into which the objective 5 is immersed or can be immersed.

[0066] The immersion medium 14 has a second refractive index n2 and serves to increase the numerical aperture (NA) of the objective 5. A nonaggressive chemical compound or such a substance mixture, which is usually also cost-effective, can be selected as immersion medium 14.

[0067] The first and second refractive indices n1, n2 and possibly also the Abbe number v1 and v2, respectively, thereof can be chosen to be close to one another or identical to one another. Then, the thickness of the wall 6 can be as low as possible and/or the wall 6 can include a material whose refractive index is close to the refractive index n1 or n2.

[0068] The immersion medium 14 can also be mixed from two or more suitable components, and hence the refractive indices n1 and n2 can be matched to one another to the best possible extent. Such an adjustment of the composition of the immersion medium 14 can also be implemented dynamically in further embodiments by virtue of components of the immersion mixture 14 or an already mixed immersion medium 14 being supplied to the space 16 in controlled fashion by way of supply and removal lines (not shown). In this way, such a “dipping chamber” can be flexibly adapted to different operating conditions.

[0069] FIGS. 4a to 4c schematically show the course of one example process. In FIG. 4a, a sample chamber 1 is brought into a first relative angular position. This means that it is inclined at a certain angle in relation to a reference coordinate system, for example, the illustrated Cartesian coordinate system with mutually orthogonal axes x, y, and z. In the example shown, the circular area 9 extends parallel to a plane spanned by axes x and y. Along an illumination beam path 7 and an illumination axis BA, illumination radiation is radiated through the outer side 6.1 and into the sample space 2 at an angle of incidence α. On account of the differences in the refractive indices of surroundings and sample medium 4, the illumination radiation is refracted upon passage through the wall 6 and subsequently extends approximately horizontally through the sample space 2 and the sample 3. The positioning of the refracted section of the illumination beam path 7 along the Z-axis z specifies a first relative z-position. The objective 5 is focused on the refracted section of the illumination beam path 7, which can be embodied as a light sheet 17 in particular.

[0070] An optical axis OA of the objective 5 used to capture detection radiation is directed perpendicular to the refracted section of the illumination beam path 7 and through the center 10. Image data of a part of the illuminated regions, in particular, of an illuminated plane, of the sample 3 can now be captured by means of the objective 5. To capture a volume of the sample 3 along the optical axis OA, a so-called z-stack 18 (see FIG. 5) of object planes is captured in different relative z-positions (symbolized by the vertical double-headed arrow) and subsequently put together (see also FIG. 5). Corrections of the angle of incidence α of the illumination beam path 7 possibly required in the process are explained in relation to FIG. 9.

[0071] Using this procedure, it is not possible to fully image the volume of a relatively large sample 3 since it is only possible to capture a restricted lateral extent (=field of view) and vertical extent (=depth of field) of the focal plane—in fact of a focal volume—as are schematically elucidated by the rectangle in FIGS. 4b and 4c. However, if the sample chamber 1 were moved laterally relative to the objective 5 for complete image capture of the sample 3, significant aberrations would occur on account of the arching of the sample chamber 1. Therefore, the sample chamber 1 can be pivoted and/or rotated about the center 10, as shown in FIGS. 4b and 4c. Thus, as shown in FIG. 4b, the sample chamber 1 has been brought into a further relative angular position and into a further relative z-position. At this relative angular position, a z-stack 18 can again be captured (FIG. 4c), stored, and finally put together to form a representation of the sample 3. For complete capture of the sample 3, it is possible to set, in freely selectable fashion, corresponding relative angular positions and capture z-stacks 18.

[0072] This procedure is shown in simplified fashion in partial figures, FIGS. 5a to 5c. In a first relative angular position of the sample chamber 1, a first z-stack 18 is captured along a detection beam path 8 in the direction of the Z-axis z and is stored together with data relating to the respective relative angular position and relative z-position (FIG. 5a). Subsequently, the sample chamber 1 is pivoted and/or rotated through a certain angle about the center 10. A z-stack 18 of image data of the sample 3 that has been brought into a second or further relative angular position in this way is captured (FIG. 5b). After a number N of such captures of z-stacks 18, the entire volume of the sample space 2 and of the sample 3 (not illustrated) is captured and the stored individual z-stacks 18 can be put together for a representation of the overall volume of the sample 3, as is symbolized schematically in FIG. 5c.

[0073] Occurring overlaps between the individual z-stacks 18 can be used to improve the data quality by virtue of, for example, an evaluation being carried out by means of a multi-view fusion algorithm. Remaining aberrations can be corrected by means of adaptive optical elements (see, e.g., FIG. 8).

[0074] The image data obtained by the techniques described herein can be represented in different coordinates. Purely in representative fashion, FIGS. 6 and 7 show a transformation of a tilted z-stack 18 into a horizontal alignment (FIG. 6) and a transformation of a tilted z-stack into spherical coordinates (FIG. 7), respectively. By way of example, a weighted interpolation can be used to transform the coordinates. By way of example, captured intensity values Ix,y,z of four points of a z-stack 18 are shown, which are interpolated to a transformed intensity value Ix′,y′,z′ of a point (coordinate) of the horizontal coordinate system (FIG. 6) or of the spherical coordinate system (FIG. 7).

[0075] A schematic representation of an exemplary embodiment of an imaging system that has the option of choosing different imaging methods depending on the sample 3 (FIG. 8) includes a beam shaping unit 19 for providing and shaping illumination radiation to form a light sheet 17 and/or a laser scanning microscope LSM (also abbreviated LSM below).

[0076] Optionally, an adaptive optical unit 20 can be present in the illumination beam path 7 of the beam shaping unit 19 and/or of the LSM. By way of example, at least one optical lens 23 and a beam splitter 21 are arranged in the illumination beam path 7 of the LSM. As a result of the effect of the beam splitter 21, the illumination radiation is steered to the objective 5 and radiated into the sample space 2 of the sample chamber 1. Detection radiation generated in the sample 3 passes through the objective 5 and the beam splitter 21, which is transmissive to the detection radiation, along the detection beam path 8 to a tube lens 22 and, from there, to a detector 25, for example a camera. Optionally, the beam splitter 21 can be inserted or pivoted into the illumination beam path 7 and/or the detection beam path 8.

[0077] The sample 3 can be selectively illuminated by a light sheet 17, by means of a point scanner of an LSM or by means of a multipoint scanner of an LSM. The LSM or the beam shaping unit 19 serve as a light source.

[0078] The detection radiation can be captured in confocal fashion, in the wide field or in plenoptic fashion. The latter variant can be implemented using a microlens array 26. To correct aberrations that may occur, an appropriately controllable adaptive optical unit 20 can be arranged in the detection beam path 8. The microlens array 26 can optionally be inserted or pivoted into the detection beam path 8 using appropriate actuators (not shown).

[0079] The sample chamber 1 can be pivoted about each of the axes X, Y and Z and can be displaced along these axes by means of a controlledly drivable sample stage 27. A rotation about the axis Z is possible. The sample chamber 1 can be pivoted about the center 10 (see FIGS. 4a to 5c) and optionally rotated about an axis that extends through the center 10 and extends in the direction of the Z-axis Z (see, e.g., FIGS. 4a to 5). In addition to, or as an alternative to, a feed movement along the Z-axis z, the objective 5 is embodied to be positioned in the direction of the Z-axis z by means of a controllable drive 30.

[0080] Optionally, it may be possible for the sample chamber 1 to be able to be displaced in a controlled fashion along the x and y axes and, in particular, in the direction of the Z-axis z by means of the sample stage 27, for example, in order to position the sample chamber 1 relative to the illumination beam path 7. The movement option along the Z-axis z represents an option for capturing the z-stacks 18.

[0081] In further possible embodiments, the relative angular positions and/or the relative z-positions can be set by way of an appropriate movement of the objective 5. It is also possible, for example, for the settings of the relative angular positions and/or the relative z-positions to be set by combinations of the movements of objective 5 and sample stage 27.

[0082] To control the sample stage 27 and the various actuators or at least one drive 30 there is a control unit 28, for example, a computer or an appropriately configured part of a computer, and said control unit can be connected to the sample stage 27, the drives 30 (only one illustrated in exemplary fashion), and optionally the camera 25 (only shown in indicated fashion) in a manner that is suitable for transferring data and control commands.

[0083] If the illumination/detection is implemented through the objective 5, the z-stacks 18 (see, e.g., FIGS. 5a to 5c) can be recorded by moving the sample 3 and/or by moving the objective 5 and altering the respective focal plane as relative z-position. The above-described rotation of the sample chamber 1 has no optical effect in this case if the point of rotation and the center 10 coincide (see, e.g., FIG. 5).

[0084] Particularly when illuminating the sample 3 with a light sheet 17, it may be necessary to adapt the angle of incidence αn on the basis of a respectively current relative Z-position within the sense of angle tracking. Additionally, a spatial displacement may be necessary in order to correct focal displacements caused by aberrations, which occur in practice. On part of the apparatus, angle tracking and spatial displacement are possible using an apparatus as shown in FIG. 9.

[0085] The inserted figure in FIG. 9 schematically shows various angles of a sample chamber 1, of an illumination beam path 7, and of a light sheet 17. Here, the sample chamber 1 and the illumination beam path 7 are illustrated in a first relative Z-position using continuous solid lines and in a second relative Z-position using broken solid lines. Outside of the sample chamber 1, the illumination radiation passes through a medium with the first refractive index na. The sample chamber 1 with the sample medium 4 includes a second refractive index nb, with nb>na. The respective angle of incidence αn is measured between the respective illumination beam path 7 and a normal on the outer side 6.1. Since the wall 6, in particular, the outer side 6.1 thereof, is formed as a spherical segment with a radius R, an extension of the normal extends through the center 10 and includes an angle γn with a further wall 11 that acts as a base of the sample chamber 1 or with the plane of the circular area 9.

[0086] In the first relative Z-position, the illumination radiation along the illumination beam path 7 is directed at the outer side 6.1 of the spherically shaped wall 6 of the sample chamber 1 at a first angle of incidence α1. The extension of the normal passes through the center 10 at an angle γ1. The illumination beam path 7 is refracted in accordance with the differences in the refractive indices na and nb. Here, the angle of incidence α1 is chosen in such a way that the refracted section of the illumination beam path 7 extends parallel to the plane of the circular area 9 and image data can be captured along the detection axis DA, for example, by means of an objective 5. The refracted section of the illumination beam path 7 includes an angle β1 with the extension of the normal.

[0087] If the sample chamber 1 is displaced along a distance Δz in the direction of the Z-axis z into the second relative Z-position, image data can be captured with an unchanged focal position of the objective 5 if an angle of incidence α2 is set. In the process, the extension of the normal now passes through the center 10 at an angle γ2. The refracted section of the illumination beam path 7 includes an angle β2 with the extension of the normal, where β1>β2.

[0088] Without such a correction of the angle of incidence αn, the refracted section of the illumination beam path 7 would no longer extend parallel to the plane of the circular area 9 in the second relative Z-position.

[0089] The optical arrangement for tracking location and angle in the case of light sheet illumination for a sample chamber in accordance with the illustrated exemplary embodiment can include the beam shaping unit 19, in which illumination radiation provided by a light source (not illustrated in more detail) is shaped to form a light sheet 17. At least one optical lens 23, a first scanner S1, a scanning optical unit 24, a tube lens 22 and an objective 5, which acts as illumination objective, follow in the illumination beam path 7.

[0090] The first scanner S1 is located in, or in the vicinity of, a pupil P of the illumination beam path 7 and serves for displacing the location. By way of example, the first scanner S1 can include a galvanometric scanning mirror. An angle in the pupil P corresponds to a location in the sample 3, which is why an angle change of the first scanner S1 leads to displacement of the light sheet 17 in the direction of the z-axis z.

[0091] For the purposes of updating the angle, a second scanner S2 is arranged between the beam shaping unit 19 and the first scanner S1. It is located in the vicinity of an intermediate image ZB. The two scanners S1 and S2 deflect the illumination beam path 7 in the same movement planes. This distinguishes them from a scanner arrangement of a typical LSM, in which the movement planes are orthogonal to one another. To displace the location of the point of incidence of the illumination beam path 7 to the outer side 6.1 of the sample chamber 1, the pivot point of the second scanner S2 is imaged onto the outer side 6.1 by means of the scanning optical unit 24. The second scanner S2 alters an angle in the intermediate image ZB, which corresponds to a change in location in the pupil P and on the first scanner S1 and to an angle in the sample 3. Since the angle should be set just in front of the sample 3, an angle deviation can be corrected accordingly by means of an axial displacement of the optical lens 23.

[0092] A correction of the focusing along the propagation direction of the light sheet 17 can be implemented, for example, by displacing the objective 5, the tube lens 22, or by other focus optical units. A correction of the focusing may be required since the optical path length in the sample medium 4 is different at different relative Z-positions and therefore changes when capturing a z-stack 18.

[0093] In a further embodiment of the optical arrangement, a third scanner S3 (not shown) can be arranged near the pupil and, for example, close to the first scanner S1. The effect of the third scanner S3 brings about a fast movement of the light sheet 17 perpendicular to the plane of the drawing. Such an arrangement can be used if a beam shape requiring lateral “smearing” (e.g., a Bessel beam) is chosen for producing the light sheet 17.

[0094] By way of example, to simplify an automated capture of image data, a plurality of sample chambers 1 may have been arranged or can be arranged on a sample carrier 13. The sample carrier 13 having a plurality of holding areas for sample chambers 1 can have a planar embodiment (FIG. 10). A sample chamber 1 to be observed in each case is fed to the objective 5 by an appropriate control of the sample stage 27 and/or the objective 5 is fed to the respective sample chamber 1. A first z-stack 18 can be captured in a first relative angular position. To capture further z-stacks 18, the objective 5 can be pivoted and/or the sample carrier 13 can be pivoted about the center 10 of the sample chamber 1 to be captured at this time.

[0095] In a further embodiment of a sample carrier 13 for a number of sample chambers 1, the former has carrier surfaces 12 which have an angular offset with respect to one another and on which a sample chamber 1 is arranged or can be arranged in each case. By way of example, the carrier surfaces 12 can be circumferential faces of a carousel or a turret. Such an embodiment increases the accessible angular range, within which relative angular positions can be set, in relation to an embodiment as per FIG. 10.

[0096] A sample carrier 13 according to any one of the preceding exemplary embodiments can include at least one channel 29, which opens into one of the holding areas (FIG. 11). The channel 29 serves to supply and/or remove sample medium 4 to and from the sample space 2.

[0097] In further embodiments there can be at least one channel 29 as a media supply line and at least one channel 29 as a media removal line in each holding area or in each sample chamber 1. To allow a supply or removal of media, the sample chamber 1 is delimited in the plane of the circular area 9 by the carrier surface 12 or openings (indicated) corresponding to some or all channels are correspondingly present in a further wall 11. In further embodiments, these openings can be provided with a valve or a seal in order to allow the sample chamber 1 to be removed from the carrier surface 12.

[0098] It is also possible for the sample chamber 1 to include the wall 6 and a closure in the plane of the circular area 9 to be formed by a surface of the carrier surface 12 as a further wall 11. The sample space 2 can be supplied with a sample medium 4 via the channel 29 or via the channels 29. In this case, the sample medium 4 might be, for example, a compound for clearing the sample 3, a nutrient solution, a buffer, or a compound for assisting the storage of the sample 3.

[0099] The exemplary embodiment illustrated in FIG. 11 shows carrier surfaces 12 which have an angular offset with respect to one another and on each of which a sample chamber 1 is placed onto a holding area. In each case, a carrier surface 12 with the sample chamber 1 present thereon can be brought into a capture position, the mid-position in the example shown, in which the carrier surface 12 located in the capture position is aligned orthogonal to the optical axis OA of the objective 5. In order to capture a number of z-stacks 18, as already described above, the objective 5 can be pivoted relative to the sample chamber 1 and different focal planes can be captured such that sample chamber 1 and objective 5 can be brought into different relative angular positions and/or relative z-positions relative to one another.

[0100] The angled arrangement of the carrier surfaces 12 which have an angular offset with respect to one another facilitates a better accessibility of the respective sample chamber 1 to be captured since the respectively adjacently arranged sample chambers 1 are pivoted out of the plane of the sample chamber 1 to be captured. The control of the current alignment of the sample carrier 13 and of the supply and/or removal of media via the channels 29 is implemented by means of the control unit 28 and by means of drives 30 and/or pumps 30.