EP3610314A1

EP3610314A1 - Light sheet microscope

Info

Publication number: EP3610314A1
Application number: EP18724461.1A
Authority: EP
Inventors: Florian Fahrbach; Werner Knebel
Original assignee: Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2017-04-10
Filing date: 2018-04-10
Publication date: 2020-02-19
Also published as: DE102017107733A1; WO2018189187A1; US20200064608A1; US11287624B2; DE102017107733B4; CN110520778A

Abstract

The invention relates to a light sheet microscope (10), comprising a detection lens (32) for imaging a target region (58) of a sample (44), which is located in a focal plane (54) of the detection lens (32), an illumination lens (20) for focusing an illumination light bundle (50) into the sample (44), wherein the detection lens (32) and the illumination lens (20) lie opposite each other by the optical axes thereof (O₂, O₁), aligned perpendicular to the focal plane, (54) and define a sample space (38) therebetween containing the focal plane (54), a sample holder (40) having at least one carrier surface (42) arranged in the sample space (38), on which the sample can be attached (44), and a light deflection device (46) having a deflection surface (48), which is arranged in the sample space (38) laterally offset relative to the optical axis (O₁) of the detection lens (32) and is designed to deflect the illumination light bundle (50) focused through the illumination lens (20) in a direction perpendicular to the optical axis (O₁) of the detection lens (32) such that the deflected illumination light bundle (50) forms a light-sheet-like illumination light distribution (52) focused in the focal plane (54), wherein the light deflection device (46) has a collision portion (86) facing the carrier surface (42), which defines a mechanical stop for the carrier surface (42) on the light deflection device (46), preventing a coplanar arrangement of the carrier surface (42) in the focal plane (54). According to the invention, the carrier surface (42) is inclined relative to the focal plane (54) at a predetermined inclination angle such that a portion of the carrier surface (42) is arranged in the focal plane (54).

Description

Light sheet microscope

The invention relates to a light-beam microscope comprising a detection objective for imaging a target region of a sample located in a focal plane of the detection objective, an illumination objective for focusing an illumination light beam into the sample, the detection objective and the illumination objective being aligned with their optical axes oriented perpendicular to the focal plane lie opposite and define between them a sample plane containing the focal plane, a sample holder with at least one disposed in the sample space support surface on which the sample is attachable, and a Lichtumlenkvorrichtung with a deflection surface laterally offset in the sample chamber to the optical axis of the detection lens and is formed, the deflected by the illumination lens - preferably focused - illuminating light beam in a direction perpendicular to the optical axis of the detection lens such that the deflected Bele The light deflection device has a collision section facing the carrier surface, which defines a mechanical stop for the carrier surface on the light deflection device, which defines a coplanar arrangement of the light beam Carrier surface prevented in the focal plane. Furthermore, the present invention relates to a retrofit kit for a light sheet microscope.

In the recent past, especially in fluorescence microscopy so-called light-sheet or optical disc microscopes are used, in which only a thin layer is illuminated in the sample. Compared with conventional fluorescence microscopes, light-sheet microscopes thus enable higher resolution and lower light exposure, thereby reducing undesirable effects due to bleaching or light-induced stress in biological samples. Light sheet microscopes are therefore particularly useful for fluorescence studies on living organisms.

Various optical arrangements for the realization of a light-sheet microscope are known from the prior art. In the context of the present invention, particular mention should be made here of arrangements in which illumination and detection take place via two separate objectives. In this case, the illumination objective and the detection objective are usually arranged perpendicular to one another. However, this vertical arrangement of the lenses has the particular disadvantage that they do not readily exist in already existing microscope systems, e.g. Integrate confocal or scanning microscopes.

To avoid this disadvantage, an arrangement was proposed by the applicant with DE 10 2012 109 577 AI, in which the In contrast to the above-mentioned right-angled arrangement, the illumination objective and the detection objective face one another along the vertical axis, for example implemented in an inverted microscope stand. In order to be able to produce a light sheet perpendicular to the detection axis in this arrangement, the illuminating light bundle passing through the illuminating objective along the vertical tripod axis is directed onto a mirror system which deflects the illuminating light bundle at a right angle to the sample in the horizontally lying focal plane of the detection objective to illuminate in the usual way with a light sheet-like illumination light distribution. The target area of the sample lying in the focal plane is then imaged onto a camera sensor by the detection objective.

The aforementioned mirror system consists of two mirror elements, which are arranged at an angle of 45 ° on both sides of the optical axis of the detection objective. The dimension of the respective mirror element dimensioned along the optical axis is determined by the beam cross section which the illumination light beam has at the location of the mirror element. Since the illumination objective is designed to focus the illumination light bundle so that it has its smallest extension along the optical axis of the detection objective in the target region of the sample, the beam cross section of the illumination light bundle at the location of the respective mirror element is still significantly larger than in the target region of the sample. Thus, the mirror element parallel to the optical axis of the detection lens has an extension which corresponds at least to the extent of the beam cross-section, which is a makes comparatively expensive sample preparation required. Thus, namely, the support surface of the sample holder used, which is usually designed as a transparent cover glass, not exactly positioned in the illuminated with the light sheet focus plane of the detection lens, since the cover glass collides with its approach to the detection lens with the attached to the detection lens mirror system before it reaches the focal plane. In order to compensate for the resulting axial offset between the support surface of the sample holder and the focal plane, the sample must be prepared on a base arranged on the support surface. Although this base is only a few 100 μηη high, it still makes the sample preparation difficult, especially when switching from a socket-free preparation, as is customary in wide-field microscopy or in confocal microscopy.

For further prior art reference is made to the publications Chen, B.-C. et al., Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014); Planchon, TA et al., Rapid three-dimensional isotropic imaging ofliving cells using Besse I beam plane illumination., Nat. Methods 8, 417 (2011); and Wu, Y. et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineage and neurodevelopmental imaging in Caenorhabditis elegans. PNAS 108, 17708 (2011). Arrangements are disclosed from these opening recesses, in which the illumination objective and the detection objective each lie on the same side of the cover glass and are arranged at an angle of 45 ° with respect to it. DE 10 2013 107 297 A1 discloses a light-sheet microscope in which the illumination objective and the detection objective are likewise arranged on one side of the cover glass, the respective angle at which the illumination objective or the detection objective is aligned with respect to the cover glass being determined on the basis of FIG numerical aperture of the respective lens is predetermined.

Finally, from DE 10 2012 110 077 AI a light sheet microscope is known in which the illumination lens and the detection lens are on opposite sides of the cover glass. As usual, the detection objective is oriented perpendicular to the cover glass. In contrast, the illumination lens is inclined relative to the cover glass.

The object of the invention is to develop a light sheet microscope of the type described above so that it allows a high-resolution imaging with a simple sample preparation.

The invention achieves this object by a light-beam microscope comprising a detection objective for imaging a target region of a sample located in a focal plane of the detection objective, an illumination objective for focusing an illumination light beam into the sample, the detection objective and the illumination objective being aligned with their plane perpendicular to the focal plane optical axes face each other and define a sample space containing the focal plane, a sample holder with at least one support surface arranged in the sample space on which the sample can be attached, and a light deflection device with a deflection surface which is arranged and formed laterally offset from the optical axis of the detection objective in the sample space; to deflect the illuminating light bundle - preferably focused - in a direction perpendicular to the optical axis of the detection lens such that the deflected illuminating light beam forms a focused in the focal plane light sheet-like illumination light distribution, wherein the Lichtumlenkvorrichtung has a the carrier surface facing collision section, which is a mechanical surface for the support surface Defined stop on the Lichtumlenkvorrichtung, which prevents a coplanar arrangement of the support surface in the focal plane. According to the invention, the carrier surface is inclined relative to the focal plane at a predetermined angle of inclination such that a part of the carrier surface is arranged in the focal plane.

The invention therefore provides for tilting the support surface of the sample holder relative to the focal plane of the illumination objective and / or the detection objective coinciding with the illumination plane formed by the light-beam-like illumination light distribution in order to cover part of the support surface carrying the sample in the focal plane of the illumination objective and / or or the detection lens. This makes it possible to prepare or arrange the sample without base directly on the support surface of the sample holder. This considerably facilitates the work with the light-sheet microscope, in particular if the microscope at the same time as a wide-field microscope and / or as Confocal microscope is used, whereby usually a sample preparation takes place anyway directly on the support surface or a slide or a coverslip and without a pedestal.

The light deflection device according to the invention is realized for example in the form of a single mirror element, which is laterally offset from the optical axis of the detection objective and thus arranged laterally offset from the target area of the sample to be imaged by the detection objective. In this case, the light deflection device can also be designed so that it has a plurality of mirror facets, in order to be able to vary the illumination direction within the illumination plane and thus the focal plane as desired, for example to illuminate the sample from slightly different directions.

One advantage of the invention is that the sample illumination according to the invention can be realized in a simple manner on already existing microscope systems without the aid of a sample socket. Required are only an inclined support surface and a trained light deflection device, which are easy to retrofit.

Preferably, the support surface of the sample holder is at least partially transparent, wherein the illumination objective directs the illumination light bundle through the transparent part of the support surface on the deflection surface. In particular, it is possible, the sample holder apart from the inclination of its support surface in the manner of a Conventional coverslip, ie perform in its entirety transparent. As a result of the at least partially transparent design of the carrier surface, it is possible to guide the illuminating light bundle emerging from the illumination objective next to the sample resting on the carrier surface through the sample holder onto the deflecting surface of the light deflecting device, then perpendicularly to the latter for generating the light sheet from the side to direct the optical axis of the detection lens in the focal plane.

Alternatively or additionally, the carrier surface-optionally in cooperation with an immersion medium and / or a sample medium-is designed in such a way that the smallest possible part of the illumination light bundle is reflected on the carrier surface. This is in particular intended for the fact that after the illumination light bundle has passed through the carrier surface and was deflected at the deflection surface, it again strikes the carrier surface. This could be achieved by coating the support surface, for example with an anti-reflection coating, which is provided or optimized for the wavelengths of the illumination light used. Also, the use of immersion medium and / or a sample medium having a refractive index which is largely adapted to the refractive index of the sample holder (or vice versa, a sample holder whose refractive index is adapted to the sample and the immersion medium), can reduce the reflection components of the illumination light beam. In a preferred embodiment, the illumination objective is designed to direct the illuminating light bundle onto the transparent part of the carrier surface in a perpendicular direction of incidence upon a first impingement of the illuminating light bundle on the sample holder or on the carrier surface. This embodiment takes into account the fact that, in the event that aberrations occur, the illumination light beam passes obliquely through the support surface of the sample holder. These are even more pronounced if a sample medium has a different refractive index between the detection objective and the carrier surface than a medium present between the carrier surface and the illumination objective. By the vertical incidence of light on the carrier surface provided in this embodiment, such aberrations can be reduced or avoided.

The sample holder is designed for example as a microscope stage insert, which is adapted in its dimensions or substantially identical to other, provided for an existing microscope stage inserts and thus easily replaceable. Such an insert has at its bottom, for example, one or more transparent, inclined support sections on which the samples can be arranged. It is also possible to carry out only a part of the respective support section transparent and to guide the illumination light bundle through this transparent part to the deflection surface. The remaining part of the support section, in particular that part on which the sample is located, can then be opaque and, for example, provided with markings or mold elements, for example to position, guide and / or guide the sample in the correct position To prevent slippage of the sample on the inclined support surface. Preferably, the support surface is provided in cooperation with the sample holder and / or the microscope stage with calibration or Kodiermarkeirungen so that a control device of the light sheet microscope knows the relative position of the support surface to the illumination lens and / or the detection lens and from this can calculate the current height of the cover glass or knows. Thus, the control device knows on the one hand the position of the sample and on the other hand, a collision between deflecting mirror and support surface (and / or the illumination objective or the detection objective) can be avoided.

The light deflection device is preferably attached to a part of the detection objective facing the sample. Thus, the light-deflecting device is e.g. executed in the form of a so-called mirror cap, which attach as in the prior art named mirror caps on the detection lens and can be solved by this. In contrast to conventional mirror caps, which have two mirror elements laterally offset from the optical axis of the detection objective, however, in this development, the mirror cap has only a single mirror element in order to allow a certain inclination of the carrier surface without abutment against the mirror cap.

The illumination objective and the deflection surface preferably cooperate in such a way that the light-sheet-like illumination light distribution formed on the optical axis of the detection objective by the focused illumination light beam in the focal plane has its minimum axial extent having. The smaller the axial extent of the light sheet along the optical axis of the detection objective, the higher the resolution achievable in the imaging.

Preferably, the predetermined inclination angle at which the support surface of the sample holder is inclined with respect to the focal plane is within an angular range ranging from a lower limit angle at which the support surface of the sample holder is in abutment with the collision portion of the light deflection to an upper limit angle. The upper limit angle is also determined, for example, by a stop of the carrier surface on the light deflection device.

Preferably, the vertex of the angle of inclination, under which the support surface intersects the focal plane, is located on an edge of an image field facing the deflection surface, which defines the target area imaged by the detection objective. The vertex can be marked accordingly in the above-mentioned calibration or coding of the support surface or the sample holder, so that the position of the vertex of the control unit of the light sheet microscope is known or can be made known.

The extent of the deflection surface is preferably dimensioned such that the projection of the deflection surface onto the optical axis of the detection objective is substantially equal to the beam cross section or the beam diameter which the illumination light bundle in the projection at the deflection surface having. This ensures that the axial extent of the deflection surface is limited to the beam cross section of the illumination light beam before deflection at the deflection surface, whereby the inclination angle at which the support surface of the sample holder is inclined relative to the focal plane, can be kept relatively low.

In a preferred embodiment, the light-sheet microscope includes a lighting unit upstream lighting unit with a light source and a light sheet generator, which is formed to generate from the light source bundle generated by the illumination light beam in cooperation with the illumination lens, the light sheet-like illumination light distribution.

In a particularly preferred embodiment, the light sheet generator comprises a scanning unit. This scanning unit is designed to deflect the illumination light beam generated by the light source in such a way that it performs a scanning movement in the focal plane of the detection objective through which the light beam is built up. For this purpose, the scanning unit has one or more scanning mirrors, which deflect the illumination light beam in the desired manner. For example, a confocal scanner with three scanning mirrors can be used as the scanning unit, of which a first and a second scanning mirror provide, for example, for a tilting of the illumination light beam in the x direction and a third scanning mirror for a tilting in the y direction. In this case, the first and the second x-scanning mirror are used to tilt the illuminating light bundle about a point which is located on the y-axis. Scanning mirror is located. Thus, the second x scanning mirror partially compensates for tilting by the first x scanning mirror so that the illuminating light beam on the third scanning mirror, which is tilted in the y direction, remains at a fixed position, typically on the optical axis. In the context of the present invention, the mirrors can also cooperate in such a way that the illumination beam is offset both along the x-direction and tilted about the y-direction and perpendicular to the first upon impact of the illumination beam on the carrier surface or the sample holder opposite the optical axis of the illumination lens tilted support surface hits, (possibly below:

In a preferred embodiment, the optical axis of the illumination objective is laterally offset with respect to the optical axis of the detection objective. Thus, it is possible, for example, to move the illumination objective laterally by means of a suitable adapter in a nosepiece.

The abovementioned lateral offset of the illumination objective takes into account the fact that without such an offset, the magnification of the illumination objective must be comparatively small in order to ensure that the illumination objective with the illumination light beam strikes the laterally offset deflection surface. If, on the other hand, a lateral offset is made possible for the illumination objective, the requirements for enlarging the illumination objective can thus be significantly reduced. The fact that with a laterally offset Illumination lens whose entrance pupil can not be fully illuminated with the illumination beam, is problematic in the preferred application, since preferably an under-illumination of the entrance pupil takes place, so that it comes to the desired tilting of the illumination light beam with respect to the optical axis of the illumination objective.

Accordingly, the light sheet generator is preferably configured to direct the illumination light beam on a partial area of an entrance pupil of the illumination objective, which is laterally offset from the optical axis of the illumination objective from the center of the entrance pupil.

Furthermore, the light-leaf generator is preferably designed to tilt the illuminating light bundle with respect to the optical axis of the illumination objective onto the said subarea.

In an advantageous embodiment, the carrier surface is movable along an axis in order to displace the focal plane within the sample, said axis being defined by the optical axis of the detection objective, by an axis perpendicular to the optical axis of the detection objective, or by an axis perpendicular to the carrier surface can. It is particularly preferred in this case to move the support surface along an axis which is perpendicular to the support surface. This displacement movement can be achieved by a superposition of an axial displacement and a lateral displacement. It leads to image volumes that have a trapezoidal cross-section. One Another advantage of the displacement of the support surface along the propagation direction of the illumination light beam is that the illumination light beam always strikes the same point of the deflection surface. This also applies if the carrier surface is moved perpendicular to the propagation direction of the illumination light beam.

The above object is further achieved by a retrofit kit for a light sheet microscope having a sample holder insert and a Lichtumlenkvorrichtung. The sample holder insert and the Lichtumlenkvorrichtung are in this case according to the above embodiments and in particular according to one of claims 1 to 15 formed and are such in a microscope or in a light sheet microscope retrofitted that the support surface relative to the focal plane at a predetermined inclination angle is inclined so that a part the support surface is arranged in the focal plane of the detection objective (and / or the illumination objective). Finally, the retrofit kit according to the invention is suitable for forming an existing conventional microscope or light-sheet microscope into a light-sheet microscope according to the invention. The person skilled in the art will form such a retrofit kit in accordance with the above statements, so that reference is made to avoid repetition of the preceding part of the description.

The invention will be explained in more detail below with reference to FIGS. Show: Figure 1 is a schennatic representation of a light sheet microscope according to the invention.

FIG. 2 shows a light deflection device in cooperation with a support surface of the sample holder oriented horizontally in a conventional manner; FIG.

FIG. 3 shows a representation corresponding to FIG. 2 with the support surface of the sample holder inclined in accordance with the invention; FIG.

4 is a schematic representation of the carrier surface in a conventional 45 ° arrangement;

5 shows a representation corresponding to FIG. 4 with the support surface tilted according to the invention;

6 shows a schematic representation for illustrating how a lateral offset of the illuminating light bundle in the objective pupil leads to a tilting of the illuminating light bundle in the focal plane;

7 shows a schematic representation for illustrating how a tilting of the illumination light beam in the objective pupil leads to a lateral offset of the illumination light bundle in the focal plane; Fig. 8 is a representation corresponding to Figure 7, wherein the illumination lens and the detection lens are aligned coaxially to each other;

9 shows a schematic illustration for illustrating the image volumes which are generated by displacing the support surface arranged at a minimal inclination angle;

Fig. 10 is a representation corresponding to Figure 9, wherein the support surface is set at a maximum inclination angle;

11 shows a schematic representation for illustrating the various possibilities of displacing the carrier surface for volume imaging;

12 shows an embodiment of the sample holder according to the invention, and

13 each show an embodiment of a conventional and a sample holder according to the invention with a microscope stage insert.

Figure 1 shows a light-sheet microscope 10, which represents an embodiment of the present invention.

The light-sheet microscope 10 includes a lighting unit 12, which includes a light source 14, a scanning unit 16, a tube lens 18 and a Illumination lens 20 includes. The scanning unit 16 is formed of three scanning mirrors 22, 24 and 26 and a Abtastlinse 28.

The light-sheet microscope 10 further includes a detection unit 30, which has a detection objective 32, an eyepiece 34 and a camera 36.

Between the detection objective 32 and the illumination objective 20, which are arranged coaxially opposite one another with their optical axes Oi and O ₂ , a sample space 38 is defined, in which a sample holder 40 is located. The sample holder 40, which is embodied for example as a plane-parallel, transparent plate, has a support surface 42 facing the detection objective 32, on which a sample 44 to be imaged is arranged.

The light-sheet microscope 10 further comprises e.g. designed as a mirror cap light deflecting device 46 which is attached to the detection objective 32, that its deflecting surface 48 formed in the form of a mirror offset laterally relative to the optical axis Oi of the detection objective 32. The light deflection device 46 serves to deflect an illumination light bundle 50, which irradiates the illumination unit 16 into the sample space 38 in the manner described below.

The illumination light beam 50 generated by the light source 14 is first supplied to the scanning unit 16. In the embodiment of Figure 1, the scanning unit 16 forms a confocal scanner, which is formed from the three scanning mirrors 22, 24 and 26. The two scanning mirrors 22 and 24 serve to to deflect the illumination light beam 50 in a first direction, which is parallel to the x-axis with reference to the coordinate system of Figure 1. On the other hand, the scanning mirror 26 arranged downstream of the two x scanning mirrors 22, 24 in the light propagation direction deflects the illuminating light beam 50 in a direction which is parallel to the y axis. The three scanning mirrors 22, 24 and 26 cooperate such that the two x scanning mirrors 22, 24 tilt the illuminating light beam 50 by a point located on the y scanning mirror 26. In this way, the second x-scanning mirror 24 compensates to some extent for the tilting of the illuminating light beam 50 caused by the first x-scanning mirror 22, so that the latter is tilted on the y-scanning mirror 26, which is tilted in the y-direction (perpendicular to the plane of the drawing). remains at a fixed position. This position is not necessarily on the optical axis 0 ₂ .

The y-scanning mirror 26 directs the illuminating light beam 50 onto the scanning lens 28, which directs the illuminating light beam 50 onto the tube lens 18. The illumination light beam 50 in this exemplary embodiment does not illuminate completely when it enters the illumination objective 20 of its entrance pupil. In addition, the illumination light beam 50 is laterally offset relative to the optical axis O _{2 of} the illumination objective 20 and tilted in the entrance pupil of the illumination objective 20 with respect to the optical axis O ₂ .

The illuminating light beam 50 incident as a parallel beam into the illumination objective 20 is focused by the latter and directed onto the deflection surface 48 of the light deflection device 46. The side of the the optical axis Oi of the detection lens 32 arranged deflection surface 48 then directs the illumination light beam 50 in the sample space 38 substantially perpendicular to the optical axis Oi inwardly. As a result of the focusing effect of the illumination objective 20, a light-sheet-like illumination light distribution 52 perpendicular to the optical axis Oi of the detection objective 32, in particular in cooperation with the scanning mirrors 22, 24, is produced within the sample space 38, which is also referred to simply as a light sheet. The light sheet 52 defines an illumination plane which coincides with the focal plane of the detection objective 32. A target area of the sample 44 illuminated with the light sheet 52 is thus imaged sharply on the camera 36 by the detection objective 32. In this connection, it should be pointed out that, for reasons of a clearer representation, the light sheet 52 in FIG. 1 lies outside the sample 44. However, it will be understood that the light sheet 52 in the actual imaging is focussed into the sample 44 to illuminate the target area to be imaged.

As shown in FIG. 1, the sample holder 40 is positioned in the light-sheet microscope 10 such that its carrier surface 42 is inclined with respect to the illumination plane and thus the focal plane of the detection objective 32. This tendency of the support surface 42 represents an essential aspect of the present invention. To illustrate this aspect, however, reference is first made to the schematic representation of Figure 2, which illustrates the hitherto conventional in the prior art horizontal orientation of the support surface 42 and the associated disadvantages. FIG. 2 shows the light deflection device 46 with its mirror surface 48, which is offset laterally relative to the optical axis Oi of the detection objective 32 (not shown in FIG. 2). The mirror flap 46 has an end face 86 which faces the carrier surface 42 of the sample holder 40 shown purely schematically in FIG. 2 as a dashed line. The end face 86 forms a stop for the support surface 42, which prevents a coplanar positioning of the support surface 42 in the focal plane 54 of the detection objective 32 in the case of horizontal alignment of the support surface 42.

FIG. 2 also shows an image field 56 which has a width d _F ov perpendicular to the optical axis Oi of the detection objective 32. Within the image field 56, the target region of the specimen designated by 58 in FIG. 1 is located in the focal plane 54 and thus imaged by the detection objective 32 onto the camera 36 shown in FIG. The distance which the laterally offset deflection mirror 48 has from the center of the image field 56 lying on the optical axis Oi is denoted by d _S i </ 2 in FIG. In addition, in FIG. 2, the quantity AA indicates the working distance of the detection objective 32 from the focal plane 54.

Since the deflection mirror 48 has the function of reflecting the illumination light beam 50 as possible without loss of light in the direction of the center of the image field 56, its axial extent, ie its extension in projection onto the optical axis Oi of the detection objective 32, must not be smaller than the beam cross section the illumination light beam 50 at the location of Deflecting mirror 48 has. However, since the illumination light beam 50 is focussed into the center of the image field 56, the beam cross section of the illumination light beam 50 at the Unnlenkspiegel 48 is significantly larger than in the center of the image field 56. This has the consequence that defined by the focused illumination light beam 50 and the focal plane 54 coincident Beleuchtungsebne along the optical axis Oi of the detection lens 32 relative to the horizontal end face 86 of the Lichtumlenkvorrichtung 46 (in Figure 2 upwards) is offset. Since the end face 86 of the light deflection device 46 defines the next approach of the carrier surface 42 to the focal plane 54, this results in an axial offset between the carrier surface 42 and the focal plane 54, which is designated C _S K in FIG. In the prior art, the offset C _S K in the context of the sample preparation is compensated for by means of a base (not shown) arranged on the support surface 42 whose height in the direction of the optical axis Oi corresponds exactly to the axial offset C _S K.

The schematic representation according to FIG. 3 illustrates how, by means of the oblique position of the support surface 42 according to the invention, a socket-free sample preparation is made possible in an arrangement otherwise identical to FIG.

As shown in Figure 2, the support surface 42 is tilted relative to the focal plane 54 by an inclination angle that is within an angular range of from a lower limit angle Ymin to an upper limit angle Y _ma x ranges. The said angle of inclination with respect to the focal plane 54 is in this case a Vertex B, which is defined by the intersection between the support surface 42 and the focal plane 54. The minimum angle of inclination Y _m i _n is defined by a marked in Figure 2 with A stop the support surface 42 to the end face 86 of the Lichtumlenkvorrichtung 46. In contrast, the maximum angle of inclination Y _{max is} determined by a stop C in FIG. 3 of the carrier surface 42 against a section of the light deflection device 46 which is located on the side of the light deflection device 46 facing away from the deflection mirror 48. This means that the support surface 42 can be tilted relative to the optical axis Oi of the detection objective 32 within an angular range which is limited by the two mechanical stops A and C to the light deflection device 46.

From the illustration in FIG. 3, in particular with a view to the stop C, it becomes immediately clear that the light deflection device 46 according to the invention has only one deflection element, namely the deflection mirror 48 arranged on the left of FIG. 3 on the optical axis Oi. Only then is it possible to remove the stop C located on the other side of the optical axis Oi as far from the focal plane 54, that a sufficient maximum inclination angle may be Y _m a _x provided. In addition, it is indicated in FIG. 3 that the illuminating light bundle 50 is preferably inclined with respect to the optical axis Oi in such a way that it falls perpendicularly onto the inclined support surface 42. Accordingly, the illuminating light beam 50 is shown in Figure 3 in two different directions of incidence, one to the minimum inclination angle Y _m i _n and the other to the maximum Tilt angle Y _m a _{x is} related. This tilting of the illumination light beam 50 serves to avoid aberrations that always occur when the illumination light beam 50 otherwise impacts on the carrier surface 42, in particular when the medium between the detection objective 32 and the carrier surface 42 has a refractive index that differs from the refractive index of the medium, which is located between the support surface 42 and the illumination objective 20.

In order for the illumination light bundle 50, as explained above, to be inclined perpendicular to the support surface 42 in order to avoid aberrations with respect to the optical axis Oi of the detection objective, the deflection mirror 48 must be tilted accordingly in order to ensure that the aberration is applied to the deflection mirror 48 reflected illumination light beam 50 is irradiated perpendicular to the optical axis Oi in the direction of the image field 56. This is illustrated in FIGS. 4 and 5.

FIG. 4 shows, purely schematically, the alignment of the deflection mirror 48 in a conventional arrangement, in which the illumination light beam 50 falls onto the deflection mirror 48 parallel to the optical axis Oi. In this arrangement, the deflection mirror 48 is inclined by 45 ° relative to the optical axis Oi.

In contrast, Figure 5 shows an arrangement corresponding to Figure 3, in which the illumination light beam 50 by the angle γ, which is equal to the inclination angle of the support surface 42, relative to the optical axis Oi is inclined. This jet inclination by the angle γ has the consequence that the deflecting mirror 48 is inclined by the angle γ / 2 in relation to the usual 45 ° orientation.

In the following, certain requirements for the illumination objective 20 to be used are illustrated purely by way of example for the variables indicated in FIG.

The minimum inclination angle Ymin is given by: Si nYmin = CSK / ((dsK-d _F0V ) / 2)

Assuming for example the value 500 μηη for C _S K, the value 330 μηη for d _F ov and the value 6 mm for d _S k, the result for Y _min is 9.1 °. If, on the other hand, d _S K is about 3.5 mm, the result for Y _{min is} 15.1 °.

For the numerical aperture NA | ₀ and the working distance AA of the illumination lens follow the following conditions:

= arcsin (0.15 / l, 33) = 6.5 °, NA _ro >0; 27 m = arcsin (0,15 / l, 33) = 6,5 ° is NAio> 0, 37

AA> dsK / 2 + x with x = dsi / 2 siny

DSK ~ y = y _m = 9.1 ° is 6mm x = 0,47mm - AA> 3mm + 0,47mm ~ 3.5mm at dsK ~ 3.5mm, y _m i _n = 15.1 °, x = 0.46mm - AA> 1, 75mm + 0.46mm ^ 2.25mm

Accordingly, suitable illumination objectives have a relatively large free working distance and can have a nominal aperture of up to 0.4 or greater.

In addition, the illumination objective 20 should have a comparatively small magnification, in order to ensure that the illuminating light bundle 50 emerging from the illumination objective 20 strikes the deflection mirror 48 of the light deflection device 46. Assuming again purely by way of example that the field of view number of the confocal scanner or the scanning unit 16 (see Figure 1) is about 20 mm, then mirrors at a distance of d _S K = 8 mm or more can only be hit by the illumination beam, if the illumination lens 20 has a magnification of 2.5 or less. Illumination objectives having a magnification of about 2.5 typically have only small numerical apertures of, for example, 0.07. On the other hand, lenses with higher apertures of 0.3 typically also have higher magnifications and therefore, with a given field of view number of the confocal scanner, can only meet light deflecting devices that are at a smaller distance d _S K from each other. Therefore, it is provided in the present case that the illumination objective 20 is laterally displaced relative to the optical axis Oi of the detection objective 32, for example by means of a suitable adapter, in order to reduce the requirement for the objective magnification. A corresponding embodiment is shown purely schematically in FIG. In the arrangement according to FIG. 6, the optical axis O _{2 of} the illumination objective 20 is laterally offset with respect to the optical axis Oi of the detection objective 32. Thus, an entrance pupil 84 of the illumination objective 20 has illuminating illumination light bundles 50 opposite the optical axis O ₂ having a lateral offset, which is indicated in FIG. 6 by the arrow PI. This lateral offset in the entrance pupil 84, which is located in the rear focal plane of the illumination objective 20, leads to a tilting of the illumination light bundle 50 in the image plane and thus the focal plane of the detection objective 32. This beam tilting is indicated by the arrow P2 in FIG. The tilt of the illumination light beam 50 is just adjusted so that the illumination light beam 50 falls perpendicular to the sample holder 40.

In order to ensure that the illumination light beam 50 strikes the deflection mirror 48 of the light deflection device 46, which according to the representation of FIG. 3 is typically located at the edge of the image field 56, it is favorable to additionally tilt the illumination light beam 50 in the entrance pupil of the illumination objective 20. This additional beam tilting in the objective pupil is illustrated in FIG. 7 by an arrow P3. It leads in the focal plane to a lateral offset of the illumination light beam 50, which is indicated in Figure 7 by an arrow P4.

The tilting shown in Figure 7 with offset of the illumination light beam 50 is of course not bound to the offset of the optical axis 0 ₂ against Oi. Whether the illumination objective 20 is offset relative to the detection objective 32 must be, as described above, significantly determined by the fact that the deflection 48 must be in the field of view of the illumination lens 20 in conjunction with the scanning unit 16. FIG. 8 shows an alternative arrangement which manages without lateral offset of the illumination objective 20 relative to the detection objective 32. This is achieved in that the beam offset indicated by the arrow PI and the beam tilting indicated by the arrow P3 in the objective pupil 84 are chosen to be correspondingly larger than in the arrangement according to FIG. The arrangement shown in Figure 8 corresponds to that of the embodiment of Figure 1, wherein the there shown, formed from the three scanning mirrors 22, 24 and 26 scanning unit 16 ensures that the illumination light beam 50 according to Figure 8 in the desired manner in the entrance pupil or the objective pupil 84 of the illumination objective 20 is laterally displaced and tilted.

FIGS. 9 and 10 show how the sample holder 40 can be displaced in the sample space 38 in order to successively record a series of individual images for the purpose of volume imaging. With reference to FIG. 3, an arrangement is shown in FIG. 9 in which the carrier surface 42 is inclined at a minimum angle of inclination Y min with respect to the focal plane 54. In this arrangement, therefore, the stop A also shown in Figure 3 comes into effect. In contrast, in Figure 10, the support surface 42 is positioned below the maximum inclination angle Y _ma x with respect to the focal plane 54, whereby the stop C is effective. In FIGS. 9 and 10, reference is made in each case to a coordinate system whose x ^' axis is oriented parallel and whose z ^' axis is oriented perpendicular to the carrier surface 42 of the sample holder 40. Accordingly, Figures 9 and 10 illustrate on the one hand a displacement of the support surface 42 along the x ^' axis, ie parallel to the support surface 42, and secondly a displacement along the z ^' axis, ie perpendicular to the support surface 42 (respectively indicated by the two arrows). The displacement of the support surface 42 along the x ^' axis results in individual images which in their entirety result in a captured image volume 60. Accordingly, the displacement of the support surface 42 along the z ^' axis results in individual images which in their entirety result in an image volume 62.

FIG. 11 is a purely schematic representation in which four different possibilities for recording a stack of individual images 64 are illustrated. FIG. 11 shows in the partial image a) an arrangement in which the support surface 42 in the z-direction, i. along the optical axis Oi of the detection lens 32 is moved. The area of the image volume lying above the support surface 42 is 66 and the region of the image volume below the support surface 42 is designated 68.

In the partial image b) of FIG. 11, a displacement of the carrier surface 42 in the x direction, ie, parallel to the focal plane 64, is shown. Such a shift is not preferred because it can not gain volume data through it. In the arrangement shown in the partial image c) of FIG. 11, the carrier surface 42 is displaced in the z ^' direction, ie in a direction perpendicular to the carrier surface 42. As a result, an image stack lying perpendicularly to the carrier surface 42 can be produced, from the data of which data can be obtained by means of a suitable data processing, for example using a so-called reslice method, images lying parallel to the carrier surface 42 whose position along the x-axis differs from the case shown in the drawing a) does not depend on z ^' .

In the partial image d), finally, a displacement of the support surface 42 along the x ^' axis is shown. This type of displacement is particularly advantageous if the concretely present geometric relationships are such that a collision of the sample arranged on the carrier surface 42 with the light deflection device 46 is not to be feared.

FIG. 12 shows a sample holder 70 which, for example, forms an insert for a microscope stage and has a plurality of inclined carrier surfaces 72, 74, 76. On each of these support surfaces 72, 74, 76, a sample 78, 80 and 82 can be arranged. The sample holder 70 is designed, for example, so that it can be easily inserted into an existing microscope stage. In order to allow the passage of the illumination light beam 50, the sample holder 70 is formed in its entirety or even partially transparent. For example, it is possible to make transparent only that part of the respective slanted support surface 72, 74, 76, through which the illumination light beam 50 passes, while the remaining part is opaque. This makes it possible to apply to the opaque part of the sample holder 70, for example, markings or moldings through which the respective sample 78, 80, 82 is guided mechanically in order to avoid slippage of the sample on the inclined support surface.

Fig. 13 shows in part a) a conventional microscope stage insert 90 for a sample holder in the center in a plan view, left in a sectional view along the line A-A shown in plan view and right in a perspective view. In part b) of FIG. 13, a microscope stage insert 90 according to the invention for a sample holder according to the invention (not shown) is shown in the middle in a plan view, left in a sectional view along the line A-A shown in plan view and on the right in a perspective view.

In the conventional microscope stage insert 90 shown in FIG. 13 a), a slide or a cover glass - with standard dimensions - can be inserted into the recessed edge area 92 and easily exchanged. The slide is on the slide or coverslip. The microscope stage insert 90 has a side wall 94, which serves, for example, to add an aqueous sample medium in the interior of the microscope stage insert 90, so that the sample (not shown in FIG. 13) is in a preferably lifelike environment during the microscopic examination. The recessed edge region 92 is formed essentially parallel to the surface of the part-circular insert region 96. The part-circular insert area 96 serves for inserting the microscope stage insert 90 in one correspondingly formed recess on the microscope stage (not shown in FIG. 13).

In the microscope stage insert 90 according to the invention shown in FIG. 13 b), a slide or a cover glass, on which the sample is located, can likewise be inserted into the recessed edge region 92. Also, this microscope stage insert 90 has a sidewall 94 which serves, e.g. To add an aqueous sample medium in the interior of the microscope stage insert 90, so that the sample (not shown in Fig. 13) is in the closest possible environment during the microscopic examination. The recessed edge portion 92 is formed substantially at an inclination angle γ with respect to the surface of the part-circular insert portion 96. In that regard, an inserted into the recessed edge portion 92 coverslip or slide with a support surface relative to the focal plane of the detection lens at the predetermined inclination angle γ is arranged.

The two lateral regions 98 of the microscope stage insert 90, which project beyond the circumference of the part-circular insert region 96, can ensure that the microscope stage insert 90 is always inserted in a predetermined orientation into a microscope stage with a corresponding recess. Preferably, the microscope stage and the microscope stage insert 90 is suitably calibrated so that the height of the cover glass of a control device of the light-sheet microscope is known at a predetermined microscope stage position, in particular to avoid collisions between deflecting mirror and cover glass. Finally, it should be particularly noted that the embodiments discussed above are merely for the purpose of describing the claimed teaching, but do not limit it to the exemplary embodiments. In particular, all features contained in this description and / or their functions, effects and properties are to be considered in isolation and / or in combination with each other as disclosed herein, which one skilled in the art may, individually or in combination thereof, possibly taking into account his or her expertise to solve the objective task or related problems.

Luminous sheet microscope

lighting unit

light source

scanning

tube lens

lighting lens

scanning

detection unit

detection objective

eyepiece

camera

sample space

sample holder

support surface

sample

optical deflecting

deflection

Illumination light beam

light sheet

Focal plane of (32) 6 image field

8 target area

0 image volume

2 image volumes

64 frames

66 part of the image volume

68 part of the image volume

70 sample holders

72 carrier surface

74 carrier surface

76 carrier surface

78 sample

80 sample

82 sample

84 entrance pupil

86 face of (46)

90 Microscope table insert

92 border area of (90)

94 sidewall of (90)

96 application range of (90)

98 lateral area of (90)

0 ₁ Optical axis

0 ₂ Optical axis

A stop

B vertex attack

Working distance of (32)

Claims

claims

A light-sheet microscope (10) comprising:

a detection objective (32) for imaging a target region (58) of a sample (44) located in a focal plane (54) of the detection objective (32),

an illumination objective (20) for focusing an illumination light beam (50) into the sample (44),

wherein the detection objective (32) and the illumination objective (20) with their perpendicular to the focal plane (54) aligned optical

Axes (0 ₂ , Oi) lie opposite each other and between them one

Define the sample plane (38) containing the focal plane (54),

a sample holder (40) with at least one in the sample space (38) arranged support surface (42) on which the sample is attachable (44), and

a light deflection device (46) having a deflection surface (48) which is arranged and formed laterally offset from the optical axis (Oi) of the detection objective (32) in the sample space (38), illuminating light bundle (50) through the illumination objective (20) Direction perpendicular to the optical axis (Oi) of the detection lens (32) deflect such that the deflected illumination light beam (50) forms a concentrated in the focal plane (54) light sheet-like illumination light distribution (52),

wherein the light deflection device (46) has a collision section (86) facing the carrier surface (42), which for the carrier surface (42). defines a mechanical abutment against the light deflection device (46), which prevents a coplanar arrangement of the support surface (42) in the focal plane (54),

characterized in that the support surface (42) relative to the focal plane (54) is inclined at a predetermined inclination angle such that a part of the support surface (42) in the focal plane (54) is arranged.

Light-sheet microscope (10) according to claim 1, characterized in that the support surface (42) is at least partially transparent, wherein the illumination objective (20) passes the illumination light bundle (50) through the transparent part of the support surface (42) onto the deflection surface (48). directed and / or that the support surface (42) - optionally in cooperation with an immersion medium and / or a sample medium - is designed such that the smallest possible part of the illumination light beam (50) on the support surface (42) is reflected, for example by an anti - Reflection coating.

Light-sheet microscope (10) according to claim 2, characterized in that the illumination objective (20) is adapted to direct the illumination light beam (50) in the direction of vertical incident on the transparent part of the support surface (42).

4. Light sheet microscope (10) according to claim 3, characterized in that the sample holder is designed as a microscope stage insert.

5. light sheet microscope (10) according to any one of the preceding claims, characterized in that the Lichtumlenkvorrichtung (46) on one of the sample (44) facing part of the detection objective (32) or on the illumination objective (20) is mounted.

6. light-sheet microscope (10) according to any one of the preceding claims, characterized in that the illumination objective (20) and the deflection surface (48) cooperate such that the formed by the focused illuminating light beam (50) in the focal plane (54) light sheet-like illumination light distribution (52 ) has its minimum axial extent on the optical axis (Oi) of the detection objective (32).

The light sheet microscope (10) of any one of the preceding claims, wherein the predetermined inclination angle is within an angular range ranging from a lower limit angle at which the support surface (42) abuts the collision portion (86) to an upper limit angle ,

8. light-sheet microscope (10) according to claim 7, characterized in that the apex of the angle of inclination, under which the support surface (42) intersects the focal plane (54), at one of Deflection surface (48) facing edge of an image field (56) which defines the by the detection objective (32) imaged target area (58).

9. light-sheet microscope (10) according to any one of the preceding claims, characterized in that the extension of the deflection surface (48) in projection on the optical axis (Oi) of the detection objective (32) is substantially equal to the beam cross-section, the illumination light beam (50) in said projection on the deflection surface (48).

10. Light sheet microscope (10) according to one of the preceding claims, characterized by a lighting objective (20) upstream lighting unit (12) having a light source (14) and a light sheet generator (16) which is formed from that of the light source (14). generated illumination light beam (50) in cooperation with the illumination objective (20) to generate the light sheet-like illumination light distribution (52).

11. Light sheet microscope (10) according to claim 10, characterized in that the light sheet generator comprises a scanning unit (16).

12. Light sheet microscope (10) according to claim 10 or 11, characterized in that the optical axis (0 ₂ ) of the illumination objective (20) relative to the optical axis (Oi) of the detection objective (32) is laterally offset.

13. Light sheet microscope (10) according to any one of the preceding claims, characterized in that the light sheet generator (16) is adapted to direct the illumination light beam (50) on a partial region of an entrance pupil (84) of the illumination objective (20), with respect to the optical axis (0 ₂ ) of the illumination objective (20) from the center of the entrance pupil (84) is laterally offset.

14 light-sheet microscope (10) according to claim 13, characterized in that the light sheet generator (16) is formed, the illumination light beam (50) relative to the optical axis (0 ₂ ) of the illumination lens (20) tilted on said portion of the entrance pupil (84). to judge.

15. light-sheet microscope (10) according to any one of the preceding claims, characterized in that for moving the focal plane (54) within the sample (44) the support surface (42) along an axis is movable, which through the optical axis of the detection objective (32) is defined by an axis perpendicular to the optical axis (Oi) of the detection objective (32) or by an axis perpendicular to the support surface (42).

16. Retrofit kit for a light sheet microscope (10) with a sample holder insert and a Lichtumlenkvorrichtung (46), wherein the sample holder insert and the Lichtumlenkvorrichtung (46) after a of the preceding claims is formed and can be retrofitted into a microscope or in a light sheet microscope, that the support surface (42) relative to the focal plane (54) at a predetermined inclination angle is inclined so that a part of the support surface (42) in the focal plane (54 ) is arranged.