DE102019214929B4 - Compact light sheet microscope and use of a finitely corrected objective in a light sheet microscope - Google Patents

Abstract

Light sheet microscope (1) for observing an object (41) arranged in a sample volume (31), comprising an illumination objective (5) for transmitting illumination light (21) from an illumination side (23) to a sample side (25) into the sample volume (31) , and a detection objective (7) for the transmission of scattered and/or fluorescent light (55) from the sample volume (31) to a detector side (53) of the detection objective (7), the illumination objective (5) having a distance (47) between the sample volume (31) and the main plane (43b) of the illumination lens (5) on the sample volume side, which is greater than the focal length of the illumination lens (5) and/or that the detection lens (7) has a distance (47) between the sample volume (31) and the having the main plane (43b) of the detection objective (7) on the sample volume side, which is greater than the focal length of the detection objective (7), characterized in that on the illumination side (23) of the illumination object ivs (5) and/or on the detector side (53) of the detection objective (7) there is a scanning device (59) which can be moved and/or tilted with respect to an optical axis (9a, 9b) of the respective objective (5, 7). , and that the scanning device (59) is arranged in the illumination-side focal length (45a) of the illumination objective (5) and/or in the detector-side focal length (45c) of the detection objective (7).

Description

The invention relates to a light sheet microscope for observing an object arranged in a sample volume, comprising an illumination objective for transmitting illumination light from an illumination side to a sample side into the sample volume, and a detection objective for transmitting scattered and/or fluorescent light from the sample volume to a detector side of the detection objective , wherein the illumination objective has a distance between the sample volume and the main plane of the illumination objective on the sample volume side, which is greater than the focal length of the illumination objective and/or the detection objective has a distance between the sample volume and the main plane of the detection objective on the sample volume side, which is greater than the focal length of the detection lens.

Light sheet microscopes are known from the prior art, for example from publications DE 10 2016 103 182 A1 , DE 10 2018 204 940 A1 , WO 2018 / 089 839 A1 , DE 10 2012 109 577 A1 and DE 20 2016 008 115 U1 . Furthermore, the DE 10 2007 015 063 A1 and the WO 2017 / 032 805 A1 optical assemblies for use in light sheet microscopes. In light sheet microscopes, illuminating light is focused into a sample volume by means of an illuminating objective, with a resulting focus preferably not being a one-dimensional focal point or light spot, but rather a two-dimensionally extended focal area. This can be achieved, for example, by using a cylindrical lens. In this case one speaks of a static light sheet. The light sheet can also be generated by scanning a focused beam of the illumination light along one direction, so that a so-called virtual light sheet is formed. The light sheet extends along a longitudinal direction, which is oriented parallel to the optical axis of the illumination lens, along a width direction, which is oriented perpendicular to the optical axis of the illumination lens, and along a height direction, which is also oriented perpendicular to the optical axis of the illumination lens. The extent of the light sheet is generally many times larger in the longitudinal and width directions than the extent in the vertical direction. The latter is also referred to as the thickness of the light sheet.

An object that is located in the sample volume is therefore essentially illuminated in a locally limited area. If the object is transparent or semi-transparent, a cross-section is illuminated. Opaque objects are only illuminated along the perimeter and may cast shadows in the direction of illumination.

A detection lens is arranged in such a way that its optical axis is oriented perpendicular to the optical axis of the illumination lens. Scattered and/or fluorescent light, which was scattered from the illuminated plane of the object or emitted in the illuminated plane of the object (fluorescence light), is picked up by the detection lens and forwarded to a detector and/or to an observation device.

The illumination and detection objectives of the light sheet microscopes from the prior art have the disadvantage that tube lenses are used in their structures. However, such a structure, in which the tube lens and lens form at least approximately a 4f system, is very long and access to the rear focal plane or pupil of the illumination lens and the detection lens is not possible, since these focal planes lie in the lenses. Solutions from the prior art that allow access to the rear focal plane or pupil are, for example, telecentric 4f optics: these require a great deal of space. A scanning system from the prior art typically has an overall length of approximately 0.5 m. Lens-free scanning systems from the prior art use additional mirrors and/or expensive and complex parabolic mirrors, which also have to be adjusted precisely and in a time-consuming manner.

The object of the present invention is therefore to create a compact light sheet microscope that is easy to adjust and in which the rear focal plane of the illumination and/or detection objective is directly accessible.

The light sheet microscope according to the invention mentioned at the outset achieves the above objects in that a scanning device is arranged on the illumination side of the illumination objective and/or on the detector side of the detection objective, which can be moved and/or tilted with respect to an optical axis of the respective objective, and that the scanning device in the focal length of the illumination lens on the illumination side and/or in the focal length of the detection lens on the detector side.

Furthermore, the invention relates to the use of an objective corrected to be finite as an illumination and/or detection objective in a light sheet microscope for observing an object arranged in a sample volume.

Under a lens here is a light-collecting optical system comprising a Arrangement of refractive optics, especially lenses, and / or mirrors to understand. In this case, the arrangement preferably has a common housing.

The present invention can be further improved on the basis of exemplary configurations that are each advantageous in and of themselves. Technical features of the configurations can be combined with one another and/or omitted as desired, provided that the technical effect produced by the omitted technical feature is not important.

In one embodiment, the illumination objective and/or the detection objective can be an objective corrected to be finite (also referred to as a “finite objective”). Such a finitely corrected lens can be used as an illumination and/or detection lens in a light sheet microscope for observing an object arranged in a sample volume, with the illumination lens having a distance between the sample volume and the main plane of the illumination lens on the sample volume side that is greater than the focal length of the illumination objective and/or the detection objective has a distance between the sample volume and the main plane of the detection objective on the sample volume side which is greater than the focal length of the detection objective.

The sample volume can be viewed as a delimited area that is delimited, for example, by a cuvette or a corresponding volume element for receiving a sample or an object. It is also conceivable that characteristics of the light sheet define the sample volume. The scan area can define a width of the sample volume, the extension of the light sheet along the optical axis over twice the Rayleigh length can, for example, define a length of the sample volume and the thickness of the light sheet in the vertical direction can define a height of the sample volume.

The respective main plane on the sample volume side is to be understood as meaning the main plane of the illumination objective or of the detection objective which is arranged closer to the sample volume. The focal length of the corresponding lenses is measured from the corresponding principal planes. According to the invention, each point in space of the sample volume is further away from the main plane on the sample volume side than the focal length.

A finitely corrected lens (finite lens) is to be understood as meaning that this is optimized for the imaging of finitely distant conjugate planes. This means that an object to be imaged and the real image of the object are at a finite distance from the respective object-side or image-side main plane. Equivalent to this is the concept of finitely corrected optics (finite optics). In contrast, with so-called infinity optics or an infinity lens, a conjugate plane is infinitely far away from the optics or lens. A finite objective or finite optic is optimized with regard to the shape and the material of the lenses used for the even distribution of the refraction on all surfaces or boundary surfaces. Finite optics and finite lenses are therefore not optimized to focus collimated light at the focal point or to collimate light from a punctiform light source arranged at the focal point.

One of the conjugate planes that is at a finite distance from the respective main plane preferably lies in the sample volume. This conjugate plane is preferably located centrally in the sample volume or defines the center of the sample volume. Starting from the corrected plane, the sample volume can extend towards and away from the respective objective. The associated second conjugate plane results from imaging the first conjugate plane through the lens.

The second conjugate plane is on the side of the respective lens facing away from the sample volume and is located at a distance from the main plane on the light source side in the case of the illumination lens and at a distance from the main plane on the detector side in the case of the detection lens, with the respective distance being greater than the focal length of the corresponding illumination lens or detection lens .

In a further embodiment of the light sheet microscope according to the invention, it is advantageous if the illumination objective and/or the detection objective is a mirror objective. Mirror lenses are free of any refractive optics and thus chromatic aberrations can be avoided. Objectives that are based on refractive optics can have correction or compensation elements that minimize or correct chromatic aberrations, but these increase the technical effort and costs. In particular, if a wide spectral range is desired, which is intended to cover, for example, the UV range, the visible range and the near-infrared range (NIR), an adequate correction over this entire range cannot be implemented. Furthermore, the use of refractive optics in the ultraviolet or infrared requires materials that do not absorb in these spectral ranges.

Catadioptric lenses can also be used. These include reflective and refractive optical elements. The latter are preferably chromatically corrected.

Another advantage of mirror lenses is a typically larger numerical aperture (NA) compared to refractive lenses with the same free working distance and overall length. The free working distance is the distance between the front optical element (lens or mirror) facing the sample volume and the plane to be imaged. A large free working distance is desirable for light sheet microscopy.

The illumination objective and/or detection objective configured as a mirror objective is particularly preferably an optic corrected for conjugate planes at a finite distance. Furthermore, the mirror lens can have aspheric surfaces for correcting spherical aberrations.

In a further configuration, the illumination objective and/or the detection objective can be an immersion objective. The lenses used can preferably be immersion lenses with a finite structure, more preferably mirror lenses in addition.

In this case, the sample volume can be located in a sample chamber which is filled with an immersion medium. In particular, the objective or the objectives can have a transparent boundary surface which faces the sample volume and is located in the immersion medium and which is arranged spherically or cylindrically in the center point of the curvature of a mirror of the mirror objective. The transparent interface can be, for example, a thin membrane made of fluoroethylene propylene (FEP) or made of glass.

In the beam path of the illumination lens, the curvature of the boundary surface can be optimized for ideal beam quality in the focal plane of the detection lens. In the beam path of the detection objective, the curvature of the interface can also be optimized for ideal image quality for regions to be imaged that do not lie on the optical axis of the detection objective. The use of an aspherical curvature or an additional lens can be advantageous for this. Likewise, an additional meniscus lens can be provided on the side of the objective facing the sample or the object, so that a dispersion-free use of the objective in any immersion media is possible.

The magnification of the image can be adjusted by shifting the lens system, i.e. the illumination and/or detection lens and the light source (in the case of an illumination beam path) or the detector (in the case of a detection beam path). Furthermore, the use of an adjustable mirror (DM) for the correction of aberrations can be advantageous here. It is also conceivable that correction optics can be pivoted into the beam path, for example static phase plates.

In a further embodiment of the light sheet microscope according to the invention, an optical waveguide can be arranged on the illumination side of the illumination objective, via which illumination light can be coupled into the illumination objective. The coupling of the illumination light via an optical waveguide has the advantage that a flexible connection is possible via this. Furthermore, a coupling-out facet of the optical waveguide, via which the illumination light is coupled out of the optical waveguide, i.e. the end of the optical waveguide, can be movable, in particular displaceable. A further advantage of an optical waveguide is that the light source, which emits the illuminating light, can be arranged at a distance from the light sheet microscope.

Using the scanning device, a beam path of illumination light to be coupled into the illumination lens can be varied with respect to its distance from the optical axis and/or its angle to the optical axis. Two separate scanning devices can thus be provided on the detector side and the illumination side. A shared scanning device can also be provided on the detector side and the illumination side.

A tilting of the beam path of the illuminating light, i.e. a tilting of the illuminating beam path, is converted into an offset by focusing optics (or the focusing optical system of the lens) and correspondingly an offset into a tilting.

Since the use of finite lenses does not require a collimated illumination beam path, the beam diameter of the illumination beam can advantageously be selected to be smaller. Consequently, the scanning device can also be selected to be smaller, which, in addition to cost savings, can also lead to an increase in the speed of the scanning process.

A preferably combined variation of the offset and the tilting of the illumination beam path by means of the scanning device allows the position of the light sheet in the sample volume, ie to vary in the object and, for example, to scan through the object without changing the sample volume or the illumination lens must be moved.

In particular, the light sheet can be displaceable in the width and height direction and thus enable the illumination of objects which (for example because of their size) cannot be fully illuminated using a single light sheet. The width direction can be defined as a direction that lies within the focal plane of the detection objective or a plane parallel to it, i.e. perpendicular to the detection axis and parallel to the illumination axis. Correspondingly, the height direction can be defined as a direction that is perpendicular to the illumination plane but is oriented parallel to the detection axis of the optical axis of the detection lens. The width and height directions are preferably oriented perpendicular to one another.

The offset and/or the tilting of the illumination beam path can be implemented in a simple manner when using the optical light waveguide by displacing and/or tilting the movable end of the light waveguide using a corresponding device. It is also possible to use a tilting mirror or several tilting mirrors in order to vary the illumination, ie the position of the light sheet. Alternatively or additionally, the illumination lens can contain one or more electrically tunable lenses (ETL), the adjustment of which also allows the position of the light sheet to be varied. In all of these variants, both the illumination objective and the detection objective can be designed to be static in relation to their spatial position and do not require any mobility in relation to this when scanning.

A displacement of the light sheet is advantageously associated with the fact that the position of the area that is illuminated by the light sheet and detected by the detection lens can be readjusted using a scanning device. In particular, when the light sheet is displaced along the optical axis of the detection lens, ie along the height direction, a sharp image of the illuminated plane on a detector can always be ensured by readjusting the detection lens. In general, as in the case of the scanning device, this scanning device can be implemented in various ways in the illumination beam path, which can be equivalent to the variants already mentioned above for the illumination beam path. A displacement of the light sheet along the width direction is also possible. In this case, a scanning device in the detection beam path can readjust the detection beam path. Various combinations of ETL+galvo or galvo+ETL can be used for both readjustment options, with the effect of an ETL being advantageously achieved as described above by shifting the lens and/or detector or light source. The displacement preferably takes place perpendicularly to the illumination axis, so that a z-stack can be recorded.

The scanning device is arranged in the illumination-side focal length (focal plane) of the illumination lens and/or in the detector-side focal length of the detection lens. This arrangement of the scanning device has the advantage that, for example, a tilting of the scanning device corresponds to a change in the position (parallel offset) of the beam path, for example a light source or to the detector. It should be noted that only the scanning device is arranged in the focal length, but not a point light source or the focus of a light source, since this would lead to collimation of the illumination light.

The tilting and the offset can be performed by a scanner system of the scanning device. A first scanning mirror and a second scanning mirror can be provided, it being possible for the first scanning mirror to be smaller than the second scanning mirror, thus enabling higher scanning frequencies. In this way, a faster tilting can be generated, for example, during the slow displacement of the beam. Such a scanner system can enable a parallel offset of the beam path. Furthermore, the distance between the scanner system and the lens can be arbitrary and/or variable. Compared to the light sheet microscopes of the prior art, this embodiment does not require a so-called scanning lens which generates a real intermediate image, in which case a scanning device is arranged at the position of the real intermediate image of the scanning lens. Thus, in particular, the optically particularly complex scanning lens can be dispensed with. Compact MEMS mirrors (microelectromechanical systems) can preferably be used for a scanner system of the scanning device.

For example, with such a scanner system, the center of rotation of the beam bundle can be adjusted along the optical axis. This also means that if, for example, the lens is moved to change the magnification, this shift can be compensated for by the scanner system, so that in this case the center of rotation of the beam is again in the (illuminating-side or detector-side) focal plane of the lens.

Furthermore, in the light sheet microscope according to the invention, a spatial light modulation element can be arranged on the illumination side of the illumination lens and/or on the detector side of the detection lens, which has at least two spatially separate areas with different optical transmission and/or reflection properties.

Purely by way of example, micromirror actuators (digital mirror device), DMD for short, deformable mirrors (DM for short), or spatial light modulators (SLM) for short, can be used as the spatial light modulation element.

A DMD consists of an array of individually switchable mirrors, preferably micromirrors, with which patterns can be generated. The switching positions of the individual mirrors are discrete and can be binary or trinary, for example. A DMD allows, for example, the display of a ring aperture.

A DM has a continuous mirrored surface that can be deformed by actuators. Depending on the number of actuators, almost any surface curves of the mirrored surface are possible with a DM, so that it allows errors in the optics of the corresponding lens to be compensated for. Furthermore, a DM makes it possible to change the convergence or divergence of the illumination beam path and/or the detection beam path, i.e., for example, to focus or defocus the illumination light.

An SLM works, for example, with birefringent liquid crystals in order to apply phase differences to the transmitted or reflected light, depending on the location. A focusing or defocusing phase plate can be formed in the SLM by appropriate control.

The spatial light modulation element can preferably be illuminated homogeneously and allows flexible illumination patterns to be generated through the configuration of the individual areas. In the detection beam path, the spatial light modulation element can preferably be used to compensate for any errors in the detection lens or to vary the position of the plane that is sharply imaged on the detector by changing the beam path through the detection lens. The position of this plane can thus be readjusted, for example, to a change in position of the light sheet away from the detection lens or towards it. Furthermore, the spatial light modulation element in the detection beam path can be used to manipulate the scattered and/or fluorescent light, for example to filter spatial frequencies.

The possible focusing or defocusing in the illumination beam path makes it possible to displace the light sheet that is being formed along or counter to the longitudinal direction, that is to say parallel to the optical axis of the illumination lens.

In a further embodiment of the light sheet microscope according to the invention, an optical element with variable focal length can be arranged on the illumination side of the illumination objective and/or on the detector side of the detection objective. As described above, the position of the light sheet along or against the optical axis of the illumination lens can be varied by changing the divergence or convergence in the illumination beam path and/or the position of the plane that is sharply imaged on the detector can be varied in the detection beam path and, in particular, the detection lens can be adjusted to a change in position of the light sheet away from the detection lens or towards it.

In particular, the variable focal length optical element can be configured as an electrically tunable lens, ETL for short.

In a particularly advantageous embodiment, the light sheet microscope according to the invention can have both an optical element with variable focal length and a scanning device. Such a combination makes it possible to shift the light sheet to be formed in the sample volume in all three spatial directions and, according to this shift, to readjust the position of the plane that is sharply imaged onto the detector by the detection objective, the change in position of the light sheet, so that the sharply imaged plane coincides with the Level in which the light sheet is formed matches.

An effect similar to that of an ETL can also be achieved without a lens by pushing the fiber back and forth.

Furthermore, a scan mode is advantageous in which the fiber is not in the back focal plane but performs a wobbling motion. A tumbling movement is to be understood as a lateral displacement coupled with a corresponding tilting, with this combined movement in the conjugate plane approximately corresponding to a parallel displacement of the beam.

Furthermore, a further embodiment of the light sheet microscope according to the invention can provide a light source arrangement which emits light of at least one wavelength. The light source arrangement can have at least one light source, ie also two, three or more light sources. The light sources can be discrete or continuous, or have discrete and continuous spectral components. Furthermore, the at least one light source can be a free-beam laser or a fiber-coupled laser.

If a fiber-coupled laser is used, the beam can be expanded directly by a collimator, and the occurrence of possible chromatic aberrations can be prevented by using achromatic collimators. It is also possible to use reflective collimators.

If free-beam lasers are used, devices for expanding the beam can be provided. These can be corrected for chromatic aberrations or use reflective optics.

The light source arrangement can have an ultra-short-pulse laser or an extremely broadband light source, for example in the form of a white-light laser.

It is also possible for the light source arrangement to provide light from at least two spectral ranges from one or different lasers simultaneously or in an alternating or otherwise clocked sequence. The at least two spectral ranges can differ greatly from one another, for example if wavelengths from the ultraviolet spectral range (for example around 405 nm) and wavelengths from the near-infrared range (for example 1300 nm) are used.

In such applications, the use of mirror lenses is particularly advantageous since they have no dispersion and consequently no chromatic aberrations.

In the light sheet microscope according to the invention, no optical elements with a refractive power are preferably arranged along the optical axis of the illumination objective that shift a sample volume-side focal plane of the illumination objective into the sample volume and/or along the optical axis of the detection objective no optical elements with a refractive power are arranged that shift the sample volume-side focal plane of the detection objective into the sample volume.

In other words, there are no lenses whose use would modify the optical structure of the illumination lens and/or the detection lens in such a way that a finite structure becomes an infinite structure.

Likewise, there is preferably no further lens that can be referred to as a tube lens or acts as such.

The present invention is explained in more detail below using exemplary configurations. The specific configurations are each advantageous in and of themselves, it being possible for the technical features of the configurations shown to be combined with one another and/or omitted as desired. Identical technical features and technical features with the same function or technical effect are provided with the same reference symbols. Duplicate explanations are omitted for the sake of clarity.

• 1a eine erste Ausgestaltung des erfindungsgemäßen Lichtblattmikroskops;
• 1b die schematische Darstellung einer Taumelbewegung der Lichtquelle;
• 2a eine zweite Ausgestaltung des erfindungsgemäßen Lichtblattmikroskops;
• 2b das Lichtblattmikroskop der 2a mit einem Kippspiegelsystem;
• 3 eine dritte Ausgestaltung des erfindungsgemäßen Lichtblattmikroskops;
• 4 eine vierte Ausgestaltung des erfindungsgemäßen Lichtblattmikroskops;
• 5 eine fünfte Ausgestaltung des erfindungsgemäßen Lichtblattmikroskops; und
• 6 eine sechste Ausgestaltung des erfindungsgemäßen Lichtblattmikroskops.

Show it:

• 1a a first embodiment of the light sheet microscope according to the invention;
• 1b the schematic representation of a wobbling movement of the light source;
• 2a a second embodiment of the light sheet microscope according to the invention;
• 2 B the light sheet microscope 2a with a tilting mirror system;
• 3 a third embodiment of the light sheet microscope according to the invention;
• 4 a fourth embodiment of the light sheet microscope according to the invention;
• 5 a fifth embodiment of the light sheet microscope according to the invention; and
• 6 a sixth embodiment of the light sheet microscope according to the invention.

1a shows a light sheet microscope 1 according to the invention in a first embodiment. In the 1a until 5 only the main optical components 3 of the light sheet microscope 1 are shown in each case. Mechanical structures and electrical control or evaluation are not shown for the sake of clarity. The lenses 5, 7 are special configurations of lens systems 6.

The light sheet microscope 1 comprises an illumination objective 5 and a detection objective 7, each of which has an optical axis 9. An optical axis of the illumination lens 9a is oriented essentially perpendicular to an optical axis of the detection lens 9b.

For the sake of simplicity, both objectives 5, 7 are shown schematically with a biconvex lens 11, but in real configurations they can comprise a multiplicity of lenses of different shapes (biconvex, plano-convex, convex-concave, plano-concave, biconcave).

In the 1a Lenses 5, 7 shown are designed as refractive lenses 13, but can also be designed in the form of a mirror lens 15. 1 shows such a mirror objective 15, which can replace the illumination objective 5 shown in the light sheet microscope 1 and/or the detection objective 7 shown.

Analog of the 1a can the refractive lenses 13 of the 2 , 3 and 4 be replaced by mirror lenses 15 and the mirror lenses 15 of the configurations of the light sheet microscope 1 can be correspondingly exchanged for refractive lenses 13 .

In 1a an optical waveguide 17 in the form of an optical fiber 19 is also shown, via which illumination light 21 is guided to an illumination side 23 of the illumination lens 5 . The illumination light 21 is coupled into the illumination objective 5 and, as in FIG 1a shown schematically focused by the biconvex lens 11 to a sample side 25.

The illumination beam path 27 shown is purely schematic and greatly simplified. The same applies to a detection beam path 29 of the detection objective 7.

The illumination light 21 is focused in a sample volume 31 and forms a light sheet 33 in it. The sample volume 31 extends in a longitudinal direction 35, a width direction 37 and a height direction 39, with the extension in the length 35 and width direction 37 being significantly greater than in the height direction 39.

An object 41 is arranged in the sample volume 31 .

Both the illumination lens 5 and the detection lens 7 have two main planes 43, which are only shown schematically in 1 are drawn. The illumination objective 5 has a main plane 43a on the illumination side and a main plane 43b on the sample volume side, which can differ in their position in the longitudinal direction 35 . Analogously, the main plane 43b on the sample volume side and a main plane 43c on the detector side are shown for the detection objective 7 . Starting from the corresponding main planes 43a, 43b and 43c, a focal length 45a on the illumination side, a focal length 45b on the sample volume side and a focal length 45c on the detector side are measured. The focal lengths 45a and 45b of the illumination lens 5 are always identical for the illumination lens 5 and also in 1 identical to the focal lengths 45b and 45c of the detection lens 7. In other configurations, the focal lengths 45a, 45b of the illumination lens 5 can differ from the focal lengths 45b, 45c of the detection lens.

In general, the illumination lens 5 has a focal length and the detection lens has a focal length.

How out 1a It can be clearly seen that a distance 47 between the main plane 43b on the sample volume side of both lenses 5, 7 and the sample volume 31 is greater than the respective focal length 45b on the sample volume side.

In 1a a filter 49 and a detector 51 are also shown, which are located on a detector side 53 of the detection objective 7 . The detection objective 7 collects scattered and/or fluorescent light 55 from the sample volume 31 and transmits it to the detector side 53, where it is focused onto the detector 51.

1a also shows that the optical fiber 19, in particular one end 57 of the optical fiber 19, is arranged on a scanning device 59, which is movable along the width direction 37 and/or the height direction 39. This is represented schematically by a shift 61 . The shift 61 is shown schematically and in perspective, in contrast to the sample volume 31 and the lenses 5, 7, and corresponds to a shift 61 along the width direction 37. A shift in the width direction results in a shift 61b on the sample volume side against the width direction 37 and vice versa. This is indicated by differently drawn arrows. Such a shift 61 makes it possible to shift the light sheet 33 along or counter to the width direction 37, or to form a virtual light sheet (not shown) in the sample volume 31 through a high-frequency shift 61 due to the sample volume-side shift 61b.

The lenses 5, 7 of the 1a are finitely corrected lenses 63, respectively. These finitely corrected lenses 63 are optimized for conjugate planes 65 located at finite distance, which means that lens errors (not shown) are minimized for conjugate planes 65 so located. The conjugate planes 65 are in for clarity 2 , but not in 1 drawn. It should be noted here that the conjugate planes of the illumination lens 65a are arranged in a folded beam path 67 are, the conjugate planes of the detection lens 65b, however, in a non-folded beam path 69.

In the prior art (not shown), the lenses 5, 7 in light sheet microscopes have a beam guide in which the light runs approximately collimated between a first lens system (a lens 5, 7) and a second lens system (tube lens).

1b shows schematically how a wobbling movement 70 of the end 57 of the fiber 19 is composed of an offset 70a and a tilting 70b. This wobbling movement 70 is implemented by the biconvex lens 11 in a parallel offset 70c. the 1b also shows in a schematically simplified representation these two degrees of freedom 129, the offset 70a (up and down) and the tilting 70b (vertical) as well as the front-to-back offset 70d, the left-right offset 70e and the horizontal tilting 70f.

In 2a a second embodiment of the light sheet microscope 1 is shown.

In the following 2a until 6 are, for the sake of clarity, technical features that are 1 are shown, such as the optical axes 9, the main planes 43, the sample volume 31, the object 41 and the like are only drawn in if they are necessary to explain the configuration shown.

In the 2a The second embodiment shown has a second scanning device 71 which is designed in the form of a tilting mirror 73 . In the 2a The configuration shown comprises a single tilting mirror 73, wherein in further configurations of the light sheet microscope 1 according to the invention, several, for example two, tilting mirrors 73 (see 2 B) can be provided, wherein each tilting mirror 73 can be used for tilting about one of two mutually perpendicular axes 75.

in the in 2a In the configuration shown, both axes of rotation 75, which are perpendicular to one another, lie in tilting mirror 73, with a first rotation 77a about a first axis of rotation 75a leading to a movement of the light sheet 41 along the width direction 37, and a second rotation 77b about a second axis of rotation 75b leading to a displacement of the light sheet 41 along the vertical direction 39. It should be noted here that a second counterclockwise rotation 77b about the second axis of rotation 75b leads to a displacement of the light sheet 41 counter to the vertical direction 39. A movement of the illumination light 21 on the illumination side 23 is thus inverted on the sample side 25 .

In 2a the focal length 45a on the illumination side and the focal length 45b on the sample volume side are also drawn in, with the second scanning device configured as a tilting mirror 73 being located in the focal length 45a of the illumination objective 5 on the illumination side. This position is particularly advantageous since tilting the tilting mirror 73 in this position offsets the illumination light 21 laterally and parallel on the sample side 25 .

The optical fiber 19 in 2 The second embodiment shown has a facet 81a, which lies in the conjugate plane of the illumination lens 65a facing the fiber 19.

In 2 B is the light sheet microscope 1 of 2a shown with a tilting mirror system 74 made up of two tilting mirrors 73 . The tilting mirror system 74 makes it possible to rotate each tilting mirror 73 independently about the respective two rotation axes 75a, 75b and to combine the rotations 77a and 77b.

Such a combination allows, apart from the front-back offset 70d, all degrees of freedom 129 that 1b are shown.

the 3 shows a third embodiment of the light sheet microscope 1 according to the invention, in which embodiment the illumination light 21 fed in by the optical fiber 19, ie by its facet 81a, falls over a large area on a spatial light modulation element.

This in 3 The spatial light modulation element shown is a micromirror actuator 87, hereinafter abbreviated to DMD. The DMD 87 has at least two, generally a plurality of mirror areas (not shown), which can be controlled separately and can vary the illumination beam path 27 in the exemplary embodiment shown such that a first illumination beam path 27a and a second illumination beam path 27b can be obtained separately or simultaneously .

In 3 It is shown that the first 27a and the second illumination beam path 27b are focused in the sample volume 31 in such a way that foci 81 are offset from one another both in the width direction 37 and in the height direction 39 . In the longitudinal direction 35, the foci 81 are not offset from one another. When presented in 3 it should be noted that both the DMD 87 and the sample volume 31 are schematically sketched in a perspective view. The illumination objective 5 and the detection objective 7, on the other hand, are in a simple schematic side view shown. The perspective representation is illustrated by a corresponding coordinate system 89 .

In further configurations of the light sheet microscope 1, the spatial light modulation element can also be configured as a deformable mirror 91 or as a spatial light modulator 93. Both elements 91, 93 are outlined in a sectional view. The deformable mirror 91 is characterized by a steady and continuously running mirror surface 92 with which the illumination light 21 can be modified as desired. In the configuration shown, the spatial light modulator 93 comprises a polarizer 95, a pixel electrode 97, a liquid crystal layer 99, a backside electrode 101 and a reflection element 103.

Both elements 91, 93 are known from the prior art, so that a more detailed explanation of their mode of operation will not be given at this point.

The scanning device 59 of 1 , the tilting mirror 73 of 2 , as well as the spatial light modulation element of 3 can be arranged in other configurations of the light sheet microscope 1 in addition to or as an alternative to the filter 49 between the detection objective 7 and the detector 51 in the detection beam path 29 . It is also possible for the elements 59, 73 and 87 to be arranged in any combination in the beam paths 27, 29 at the positions described above.

Both the spatial light modulator 93 and the deformable mirror 91 allow the focus 81 of the illumination beam path 27 to be varied along the longitudinal direction 35 to a certain extent.

In 4 another possibility is shown for realizing this displacement along the longitudinal direction 35 . In the fourth embodiment shown of the light sheet microscope 1 according to the invention, an optical element 105 with variable focal length is arranged on the optical fiber 19 . The variable focal length optical element 105 is designed as an electrically tunable lens 107 . In the following, this is abbreviated to ETL (Electrically Tunable Lens).

The ETL 107 has a first focal length 109a for the first illumination beam path 27a and a second focal length 109b for the second illumination beam path 27b, the first focal length 109a being smaller than the second focal length 109b, so that there is a first convergence for the illumination beam paths 27a, 27b 111a and a second convergence 111b which differ from each other.

The differing convergences 111a, 111b cause the foci 81 to shift along the longitudinal direction 35 in the sample volume 31. A first focus 81a forms further in the longitudinal direction 35 at the greater first convergence 111a. In 4 it should be noted that the sample volume 31 is shown in side view, which is illustrated by the corresponding coordinate system 89 . The ETL 107, as well as the lenses 5 and 7 and a deflection mirror 113 are also shown in the side view.

In a further refinement, the ETL 107 can be combined as desired with elements from the group consisting of the scanning device 59, the tilting mirror 73 and the spatial light modulation element. For example, the fixed deflection mirror 113 by the tilting mirror 73 of 2 be replaced.

The ETL 107 can also be arranged in the detection beam path 29 in order to vary the position of a detection focus 81c along or counter to the vertical direction 39 and preferably to adapt it to the position of the light sheet 33 . The position of the light sheet 33 can be varied along the height direction 39 by the elements described above, such as the scanning device 59, tilting mirror 73 or spatial light modulation element. If the detection focus 81c is not adapted to such a variation, the light sheet 33 is imaged out of focus in the detector 51.

the 5 shows the light sheet microscope 1 according to the invention in a fifth embodiment, which is shown in 1 shown first embodiment is similar. However, both the illumination lens 5 and the detection lens 7 are mirror lenses 15. Furthermore, the mirror lenses 15 are finitely corrected lenses 63.

Both the illumination beam path 27 and the detection beam path 29 are varied due to the use of mirror lenses 15. For example, the paraxial rays are hidden in these. the 5 also shows the main plane 43a of the illumination lens 5 on the illumination side, the main planes 43b of the illumination lens 5 and detection lens 7 on the sample volume side, and the main plane 43c of the detection lens 7 on the detector side is as the distance 47 to the sample volume 31.

The fifth embodiment shown also has an end 57 of the optical fiber 19 that can be moved via the scanning device 59 so that a displacement 61 of the end 57 of the fiber 19 leads to a displacement 61b of the light sheet 33 in the sample volume 31 on the sample volume side. The directions of the displacements 61, 61b of the fifth embodiment correspond to those of the first embodiment of FIG 1 .

the 5 FIG. 11 further shows a light source arrangement 114, which in the embodiment shown comprises a single light source 115. FIG. The light source 115 is in the form of a laser 117 and the illumination light 21 emitted by it is coupled into the optical fiber 19 and transmitted by it.

In 6 a sixth embodiment of the light sheet microscope 1 according to the invention is shown. Both the illumination lens 5 and the detection lens 7 are mirror lenses 15, finitely corrected lenses 63 and immersion lenses 119.

The immersion objectives 119 have a transparent interface 121 which is immersed in an immersion liquid 123 of a sample chamber 125 . The transparent boundary surfaces 121 can be spherical or cylindrical, with the center point of the curvature being able to be arranged in the focus 81 of the respective lens.

The transparent boundary surfaces 121 shown can be produced as a thin membrane 127, for example made of FEP or glass.

The technical characteristics of the 1 until 6 Configurations shown can be combined with one another in any way, so that, for example, the scanning device 59 of 1 with the ETL 107 of 4 and the mirror 15 and immersion 119 objectives of 6 can be provided in an embodiment not shown.

Reference List

1

light sheet microscope

3

optical main components

5

lighting lens

6

lens system

7

detection lens

9

optical axis

9a

optical axis of the illumination lens

9b

optical axis of the detection lens

11

biconvex lens

13

refractive lens

15

mirror lens

17

optical fiber

19

optical fiber

21

illumination light

23

lighting side

25

sample page

27

illumination beam path

27a

first illumination beam path

27b

second illumination beam path

29

detection beam path

31

sample volume

33

light sheet

35

longitudinal direction

37

latitude direction

39

height direction

41

object

43

main level

43a

lighting-side main level

43b

sample volume side main level

43c

detector-side main level

45a

illumination side focal length

45b

sample volume side focal length

45c

detector-side focal length

47

distance

49

filter

51

detector

53

detector side

55

Scattered and/or fluorescent light

57

end

59

scanning device

61

shift

61b

sample-volume shift

63

on finitely corrected lens

65

conjugate plane

65a

conjugate plane of the illumination lens

65b

conjugate plane of the detection lens

67

folded beam path

69

unfolded beam path

70

tumbling motion

70a

offset

70b

tilting

70c

parallel offset

70d

Offset front-back

70e

offset left-right

70f

Horizontal tilt

71

second scanning device

73

tilting mirror

74

tilting mirror system

75

axis of rotation

75a

first axis of rotation

75b

second axis of rotation

77a

first turn

77b

second rotation

81

focus

81a

facet

81c

detection focus

83

opening angle

87

micromirror actuator

89

coordinate system

91

deformable mirror

93

spatial light modulator

92

steady and continuous mirror surface

95

polarizer

97

pixel electrode

99

liquid crystal layer

101

rear electrode

103

reflection element

105

variable focal length optical element

107

electrically tunable lens (ETL)

109a

first focal length

109b

second focal length

111a

first convergence

111b

second convergence

113

deflection mirror

114

light source arrangement

115

light source

117

laser

119

immersion lens

121

transparent interface

123

immersion fluid

125

sample chamber

127

thin membrane

129

degrees of freedom

Claims (10)

Light sheet microscope (1) for observing an object (41) arranged in a sample volume (31), comprising an illumination objective (5) for transmitting illumination light (21) from an illumination side (23) to a sample side (25) into the sample volume (31) , and a detection objective (7) for the transmission of scattered and/or fluorescent light (55) from the sample volume (31) to a detector side (53) of the detection objective (7), the illumination objective (5) having a distance (47) between the sample volume (31) and the main plane (43b) of the illumination lens (5) on the sample volume side, which is greater than the focal length of the illumination lens (5) and/or that the detection lens (7) has a distance (47) between the sample volume (31) and the having the main plane (43b) of the detection lens (7) on the sample volume side, which is greater than the focal length of the detection lens (7), characterized in that on the illumination side (23) of the illumination object tivs (5) and/or on the detector side (53) of the detection objective (7) there is a scanning device (59) which can be moved and/or tilted with respect to an optical axis (9a, 9b) of the respective objective (5, 7). , and that the scanning device (59) is arranged in the illumination-side focal length (45a) of the illumination lens (5) and/or in the detector-side focal length (45c) of the detection lens (7). Light sheet microscope (1) after claim 1 , characterized in that the illumination lens (5) and/or the detection lens (7) is a finitely corrected lens (63). Light sheet microscope (1) after claim 1 or 2 , characterized in that the illumination objective (5) and/or the detection objective (7) is a mirror objective (15). Light sheet microscope (1) according to one of Claims 1 until 3 , characterized in that the illumination objective (5) and/or the detection objective (7) is an immersion objective (119). Light sheet microscope (1) according to one of Claims 1 until 4 , characterized in that an optical waveguide (17) is arranged on the illumination side (23) of the illumination objective (5), via which illumination light (21) can be coupled into the illumination objective (5). Light sheet microscope (1) according to one of Claims 1 until 5 , characterized in that a spatial light modulation element (87, 91, 93) is arranged on the illumination side (23) of the illumination lens (5) and/or on the detector side (53) of the detection lens (7), which has at least two spatially separate areas having different optical transmission and/or reflection properties. Light sheet microscope (1) according to one of Claims 1 until 6 , characterized in that on the illumination side (23) of the illumination lens (5) and/or on the detector side (53) of the detection lens (7) an optical element (105) with variable focal length is arranged. Light sheet microscope (1) according to one of Claims 1 until 7 , characterized in that a light source arrangement (114) is provided which emits light of at least one wavelength. Light sheet microscope (1) according to one of Claims 1 until 8th , characterized in that along the optical axis (9a) of the illumination lens (5) there are no optical elements with a refractive power that shift a sample volume-side focal plane (45b) of the illumination lens (5) into the sample volume (31) and/or along the Optical axis (9b) of the detection objective (7) no optical elements are arranged with a refractive power, which move the sample volume side focal plane (45b) of the detection objective (7) in the sample volume (31). Use of a finitely corrected objective (63) as an illumination (5) and/or detection objective (7) in a light sheet microscope (1) for observing an object (41) arranged in a sample volume (31), the illumination objective (5) having a distance (47) between the sample volume (31) and a main plane (43b) of the illumination lens (5) on the sample volume side, which is greater than the focal length of the illumination lens (5) and/or the detection lens (7) has a distance (47) between the Sample volume (31) and the main plane (43b) of the detection objective (7) on the sample volume side, which is greater than the focal length of the detection objective (7), and wherein on the illumination side (23) of the illumination objective (5) and/or on the detector side ( 53) of the detection objective (7), a scanning device (59) is arranged, which can be moved and/or tilted with respect to an optical axis (9a, 9b) of the respective objective (5, 7), and that the Sc is arranged on device (59) in the illumination-side focal length (45a) of the illumination objective (5) and/or in the detector-side focal length (45c) of the detection objective (7).

Images