Patent ID: 12259538

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

DETAILED DESCRIPTION

FIG.1illustrates a sample chamber1known from the prior art, which surrounds a sample space2in which a sample3can be positioned. The sample space2is filled with a sample medium4, into which an objective5of an imaging optical unit of an optical apparatus (not illustrated in more detail) can be immersed. The sample medium4, which acts as immersion medium14at the same time, has a first refractive index n1and is in direct contact with the objective5. If the sample medium4is a chemical or a mixture of chemical components, as are used to clear the sample3, there is a risk of the objective5being damaged on account of the often aggressive properties thereof. The sample space2is surrounded on three sides by a wall6, 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 sample3can be illuminated with illumination radiation along an illumination beam path7, which coincides with an optical axis OA of the objective5. Detection radiation effected in the sample3is captured by the objective5along a detection axis DA of a detection beam path8, with the optical axis OA and the detection axis DA coinciding.

A first exemplary embodiment of a sample chamber1is shown inFIG.2ain a lateral sectional illustration. The sample space2is delimited by a wall6, wherein an outer side6.1of the wall6has the shape of a spherical segment (FIG.2a). The spherical segment covers a circular area9with a center10(see alsoFIG.2b). The sample space2is delimited by a further wall11, which extends in the plane of the circular area9. If used as intended, the sample chamber1is provided to allow the illumination beam path7to pass through the outer side6.1and into the sample space2, and to allow the detection beam path8likewise to pass through the outer side6.1. Illumination beam path7and detection beam path8may coincide or, as indicated inFIG.2a, be separated from one another.

The spherical sample chamber1surrounds the sample3, for example, a brain to be imaged, as tightly as possible. The sample medium4used for clearing is situated in the sample chamber1. Detection radiation can be captured by means of an objective5embodied as an air objective (seeFIGS.4ato4c, for example).

In a further exemplary embodiment, the sample chamber1is placed on a carrier surface12of a sample carrier13and surrounded by a wall15in the form of a hollow cylinder standing upright on the carrier surface12(FIG.3). Together, sample carrier13and carrier surface12can also form an open vessel in further embodiments. In further embodiments, the wall15or cylinder wall can have a different shape in a plan view, for example, a polygonal or oval shape. A space16present above the sample chamber1is filled with air or with an immersion medium14, into which the objective5is immersed or can be immersed.

The immersion medium14has a second refractive index n2and serves to increase the numerical aperture (NA) of the objective5. A nonaggressive chemical compound or such a substance mixture, which is usually also cost-effective, can be selected as immersion medium14.

The first and second refractive indices n1, n2and 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 wall6can be as low as possible and/or the wall6can include a material whose refractive index is close to the refractive index n1or n2.

The immersion medium14can also be mixed from two or more suitable components, and hence the refractive indices n1and n2can be matched to one another to the best possible extent. Such an adjustment of the composition of the immersion medium14can also be implemented dynamically in further embodiments by virtue of components of the immersion mixture14or an already mixed immersion medium14being supplied to the space16in 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.

FIGS.4ato4cschematically show the course of one example process. InFIG.4a, a sample chamber1is 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 area9extends parallel to a plane spanned by axes x and y. Along an illumination beam path7and an illumination axis BA, illumination radiation is radiated through the outer side6.1and into the sample space2at an angle of incidence α. On account of the differences in the refractive indices of surroundings and sample medium4, the illumination radiation is refracted upon passage through the wall6and subsequently extends approximately horizontally through the sample space2and the sample3. The positioning of the refracted section of the illumination beam path7along the Z-axis z specifies a first relative z-position. The objective5is focused on the refracted section of the illumination beam path7, which can be embodied as a light sheet17in particular.

An optical axis OA of the objective5used to capture detection radiation is directed perpendicular to the refracted section of the illumination beam path7and through the center10. Image data of a part of the illuminated regions, in particular, of an illuminated plane, of the sample3can now be captured by means of the objective5. To capture a volume of the sample3along the optical axis OA, a so-called z-stack18(seeFIG.5) of object planes is captured in different relative z-positions (symbolized by the vertical double-headed arrow) and subsequently put together (see alsoFIG.5). Corrections of the angle of incidence α of the illumination beam path7possibly required in the process are explained in relation toFIG.9.

Using this procedure, it is not possible to fully image the volume of a relatively large sample3since 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 inFIGS.4band4c. However, if the sample chamber1were moved laterally relative to the objective5for complete image capture of the sample3, significant aberrations would occur on account of the arching of the sample chamber1. Therefore, the sample chamber1can be pivoted and/or rotated about the center10, as shown inFIGS.4band4c. Thus, as shown inFIG.4b, the sample chamber1has been brought into a further relative angular position and into a further relative z-position. At this relative angular position, a z-stack18can again be captured (FIG.4c), stored, and finally put together to form a representation of the sample3. For complete capture of the sample3, it is possible to set, in freely selectable fashion, corresponding relative angular positions and capture z-stacks18.

This procedure is shown in simplified fashion in partial figures,FIGS.5ato5c. In a first relative angular position of the sample chamber1, a first z-stack18is captured along a detection beam path8in 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 chamber1is pivoted and/or rotated through a certain angle about the center10. A z-stack18of image data of the sample3that 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-stacks18, the entire volume of the sample space2and of the sample3(not illustrated) is captured and the stored individual z-stacks18can be put together for a representation of the overall volume of the sample3, as is symbolized schematically inFIG.5c.

Occurring overlaps between the individual z-stacks18can 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).

The image data obtained by the techniques described herein can be represented in different coordinates. Purely in representative fashion,FIGS.6and7show a transformation of a tilted z-stack18into 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-stack18are 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).

A schematic representation of an exemplary embodiment of an imaging system that has the option of choosing different imaging methods depending on the sample3(FIG.8) includes a beam shaping unit19for providing and shaping illumination radiation to form a light sheet17and/or a laser scanning microscope LSM (also abbreviated LSM below).

Optionally, an adaptive optical unit20can be present in the illumination beam path7of the beam shaping unit19and/or of the LSM. By way of example, at least one optical lens23and a beam splitter21are arranged in the illumination beam path7of the LSM. As a result of the effect of the beam splitter21, the illumination radiation is steered to the objective5and radiated into the sample space2of the sample chamber1. Detection radiation generated in the sample3passes through the objective5and the beam splitter21, which is transmissive to the detection radiation, along the detection beam path8to a tube lens22and, from there, to a detector25, for example a camera. Optionally, the beam splitter21can be inserted or pivoted into the illumination beam path7and/or the detection beam path8.

The sample3can be selectively illuminated by a light sheet17, 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 unit19serve as a light source.

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 array26. To correct aberrations that may occur, an appropriately controllable adaptive optical unit20can be arranged in the detection beam path8. The microlens array26can optionally be inserted or pivoted into the detection beam path8using appropriate actuators (not shown).

The sample chamber1can 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 stage27. A rotation about the axis Z is possible. The sample chamber1can be pivoted about the center10(seeFIGS.4ato5c) and optionally rotated about an axis that extends through the center10and extends in the direction of the Z-axis Z (see, e.g.,FIGS.4ato5). In addition to, or as an alternative to, a feed movement along the Z-axis z, the objective5is embodied to be positioned in the direction of the Z-axis z by means of a controllable drive30.

Optionally, it may be possible for the sample chamber1to 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 stage27, for example, in order to position the sample chamber1relative to the illumination beam path7. The movement option along the Z-axis z represents an option for capturing the z-stacks18.

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 objective5. 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 objective5and sample stage27.

To control the sample stage27and the various actuators or at least one drive30there is a control unit28, for example, a computer or an appropriately configured part of a computer, and said control unit can be connected to the sample stage27, the drives30(only one illustrated in exemplary fashion), and optionally the camera25(only shown in indicated fashion) in a manner that is suitable for transferring data and control commands.

If the illumination/detection is implemented through the objective5, the z-stacks18(see, e.g.,FIGS.5ato5c) can be recorded by moving the sample3and/or by moving the objective5and altering the respective focal plane as relative z-position. The above-described rotation of the sample chamber1has no optical effect in this case if the point of rotation and the center10coincide (see, e.g.,FIG.5).

Particularly when illuminating the sample3with a light sheet17, 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 inFIG.9.

The inserted figure inFIG.9schematically shows various angles of a sample chamber1, of an illumination beam path7, and of a light sheet17. Here, the sample chamber1and the illumination beam path7are 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 chamber1, the illumination radiation passes through a medium with the first refractive index na. The sample chamber1with the sample medium4includes a second refractive index nb, with nb>na. The respective angle of incidence αn is measured between the respective illumination beam path7and a normal on the outer side6.1. Since the wall6, in particular, the outer side6.1thereof, is formed as a spherical segment with a radius R, an extension of the normal extends through the center10and includes an angle γn with a further wall11that acts as a base of the sample chamber1or with the plane of the circular area9.

In the first relative Z-position, the illumination radiation along the illumination beam path7is directed at the outer side6.1of the spherically shaped wall6of the sample chamber1at a first angle of incidence α1. The extension of the normal passes through the center10at an angle γ1. The illumination beam path7is refracted in accordance with the differences in the refractive indices na and nb. Here, the angle of incidence α1is chosen in such a way that the refracted section of the illumination beam path7extends parallel to the plane of the circular area9and image data can be captured along the detection axis DA, for example, by means of an objective5. The refracted section of the illumination beam path7includes an angle β1with the extension of the normal.

If the sample chamber1is 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 objective5if an angle of incidence α2is set. In the process, the extension of the normal now passes through the center10at an angle γ2. The refracted section of the illumination beam path7includes an angle β2with the extension of the normal, where β1>β2.

Without such a correction of the angle of incidence αn, the refracted section of the illumination beam path7would no longer extend parallel to the plane of the circular area9in the second relative Z-position.

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 unit19, in which illumination radiation provided by a light source (not illustrated in more detail) is shaped to form a light sheet17. At least one optical lens23, a first scanner S1, a scanning optical unit24, a tube lens22and an objective5, which acts as illumination objective, follow in the illumination beam path7.

The first scanner S1is located in, or in the vicinity of, a pupil P of the illumination beam path7and serves for displacing the location. By way of example, the first scanner S1can include a galvanometric scanning mirror. An angle in the pupil P corresponds to a location in the sample3, which is why an angle change of the first scanner S1leads to displacement of the light sheet17in the direction of the z-axis z.

For the purposes of updating the angle, a second scanner S2is arranged between the beam shaping unit19and the first scanner S1. It is located in the vicinity of an intermediate image ZB. The two scanners S1and S2deflect the illumination beam path7in 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 path7to the outer side6.1of the sample chamber1, the pivot point of the second scanner S2is imaged onto the outer side6.1by means of the scanning optical unit24. The second scanner S2alters an angle in the intermediate image ZB, which corresponds to a change in location in the pupil P and on the first scanner S1and to an angle in the sample3. Since the angle should be set just in front of the sample3, an angle deviation can be corrected accordingly by means of an axial displacement of the optical lens23.

A correction of the focusing along the propagation direction of the light sheet17can be implemented, for example, by displacing the objective5, the tube lens22, or by other focus optical units. A correction of the focusing may be required since the optical path length in the sample medium4is different at different relative Z-positions and therefore changes when capturing a z-stack18.

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 S3brings about a fast movement of the light sheet17perpendicular 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 sheet17.

By way of example, to simplify an automated capture of image data, a plurality of sample chambers1may have been arranged or can be arranged on a sample carrier13. The sample carrier13having a plurality of holding areas for sample chambers1can have a planar embodiment (FIG.10). A sample chamber1to be observed in each case is fed to the objective5by an appropriate control of the sample stage27and/or the objective5is fed to the respective sample chamber1. A first z-stack18can be captured in a first relative angular position. To capture further z-stacks18, the objective5can be pivoted and/or the sample carrier13can be pivoted about the center10of the sample chamber1to be captured at this time.

In a further embodiment of a sample carrier13for a number of sample chambers1, the former has carrier surfaces12which have an angular offset with respect to one another and on which a sample chamber1is arranged or can be arranged in each case. By way of example, the carrier surfaces12can 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 perFIG.10.

A sample carrier13according to any one of the preceding exemplary embodiments can include at least one channel29, which opens into one of the holding areas (FIG.11). The channel29serves to supply and/or remove sample medium4to and from the sample space2.

In further embodiments there can be at least one channel29as a media supply line and at least one channel29as a media removal line in each holding area or in each sample chamber1. To allow a supply or removal of media, the sample chamber1is delimited in the plane of the circular area9by the carrier surface12or openings (indicated) corresponding to some or all channels are correspondingly present in a further wall11. In further embodiments, these openings can be provided with a valve or a seal in order to allow the sample chamber1to be removed from the carrier surface12.

It is also possible for the sample chamber1to include the wall6and a closure in the plane of the circular area9to be formed by a surface of the carrier surface12as a further wall11. The sample space2can be supplied with a sample medium4via the channel29or via the channels29. In this case, the sample medium4might be, for example, a compound for clearing the sample3, a nutrient solution, a buffer, or a compound for assisting the storage of the sample3.

The exemplary embodiment illustrated inFIG.11shows carrier surfaces12which have an angular offset with respect to one another and on each of which a sample chamber1is placed onto a holding area. In each case, a carrier surface12with the sample chamber1present thereon can be brought into a capture position, the mid-position in the example shown, in which the carrier surface12located in the capture position is aligned orthogonal to the optical axis OA of the objective5. In order to capture a number of z-stacks18, as already described above, the objective5can be pivoted relative to the sample chamber1and different focal planes can be captured such that sample chamber1and objective5can be brought into different relative angular positions and/or relative z-positions relative to one another.

The angled arrangement of the carrier surfaces12which have an angular offset with respect to one another facilitates a better accessibility of the respective sample chamber1to be captured since the respectively adjacently arranged sample chambers1are pivoted out of the plane of the sample chamber1to be captured. The control of the current alignment of the sample carrier13and of the supply and/or removal of media via the channels29is implemented by means of the control unit28and by means of drives30and/or pumps30.