DE102019214929A1 - Compact light sheet microscope - Google Patents

Abstract

The invention relates to a light sheet microscope (1) for observing an object (41) arranged in a sample volume (31), comprising an illuminating objective (5) for transmitting illuminating light (21) from an illuminating side (23) to a specimen 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 invention also relates to the use of an objective (63) corrected to finite in a light sheet microscope (1). Light sheet microscopes (1) have the disadvantage that their structure is identical and does not allow access to the conjugate planes (65). The light sheet microscope (1) according to the invention improves the solutions from the prior art in that the illumination objective (5) has a greater distance (47) between the sample volume (31) and the main plane (45b) of the illumination objective (5) on the sample volume side than the focal length (46b) of the illumination objective (5) and / or that the detection objective (7) has a distance (47) between the sample volume (31) and the main plane (45b) of the detection objective (7) on the sample volume side which is greater than the Focal length (46b) of the detection lens (7).

Description

The invention relates to a light sheet microscope for observing an object arranged in a sample volume, comprising an illumination objective for the transmission of illumination light from an illumination side to a sample side into the sample volume, and a detection objective for the transmission of scattered and / or fluorescent light from the sample volume to a detector side of the detection objective .

Light sheet microscopes are known from the prior art. In these, illuminating light is focused into a sample volume by means of an illuminating objective, a resulting focus preferably not being a one-dimensional focal point or light spot, but a two-dimensionally extended focal area. This can be achieved, for example, by using a cylinder 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 illuminating light along a direction, so that a so-called virtual light sheet is formed. The light sheet extends along a longitudinal direction that is oriented parallel to the optical axis of the illumination objective, along a width direction that is oriented perpendicular to the optical axis of the illumination objective, and along a height direction that is also oriented perpendicular to the optical axis of the illumination objective. The expansion of the light sheet is generally many times greater in the longitudinal and width directions than the expansion in the height direction. The latter is also referred to as the thickness of the light sheet.

An object which is located in the sample volume is consequently illuminated essentially 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 can cast shadows in the direction of illumination.

A detection objective is arranged in such a way that its optical axis is oriented perpendicular to the optical axis of the illumination objective. Scattered and / or fluorescent light that has been scattered from the illuminated plane of the object or emitted in the illuminated plane of the object (fluorescent light) is picked up by the detection lens and passed on 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 objective form at least approximately a 4f system, is very long and, in addition, access to the rear focal plane or pupil of the illumination objective and the detection objective is not possible, since these focal planes lie in the objectives. 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 which 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 beginning achieves the above objects in that 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 that the detection objective has a distance between the sample volume and the main plane on the sample volume side of the detection lens which is greater than the focal length of the detection lens.

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

An objective is to be understood here as an optical system that collects light, comprising an arrangement of refractive optics, in particular lenses and / or mirrors. The arrangement here preferably has a common housing.

The present invention can be further improved on the basis of exemplary configurations that are advantageous per se. 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.

Thus, in one embodiment, the illumination objective and / or the detection objective can be a finitely corrected objective (also referred to as a “finite objective”). Such a finite-corrected objective can be used as an illumination and / or detection objective in a light sheet microscope can be used to observe an object arranged in a sample volume, the illumination objective having 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 sample volume Has detection lens which is greater than the focal length of the detection lens.

The sample volume can be viewed as a delimited area which is limited, 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 define, for example, 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 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 respective lenses is measured from the respective main 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 objective (finite objective) is to be understood as meaning that it 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. The term “finite optics” is to be understood as equivalent to this. In contrast to this, in so-called infinite optics or an infinite lens, a conjugate plane is infinitely far away from the optics or the lens. The optimization of a finite lens or a finite optic takes place with regard to the shape and the material of the lenses used for the uniform distribution of the refraction on all surfaces or interfaces. Finite optics and finite lenses are therefore not optimized to focus collimated light in the focal point or to collimate light from a point-shaped light source arranged in the focal point.

One of the conjugate planes finitely distant from the respective main plane is preferably located in the sample volume. This conjugate plane is preferably located centrally in the sample volume or defines the center of the sample volume. The sample volume can extend from the corrected plane towards and away from the respective objective. The associated second conjugate plane results from the imaging of the first conjugate plane through the objective.

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

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 based on refractive optics can indeed have correction or compensation elements which minimize or correct chromatic imaging errors, but these increase the technical effort and costs. In particular, if a broad spectral range, which is intended to cover the UV range, the visible range and the near infrared range (NIR), for example, is desired, 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.

The use of catadioptric lenses is also possible. These include reflective and refractive optical elements. The latter are preferably corrected chromatically.

Another advantage of mirror objectives is a typically larger numerical aperture (NA) compared to refractive objectives 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.

Particularly preferably, the illumination objective and / or detection objective configured as a mirror objective is an optical system corrected to finitely distant conjugate planes. Furthermore, the Mirror lens have aspherical surfaces for correcting spherical aberrations.

In a further embodiment, the illumination objective and / or the detection objective can be an immersion objective. The objectives used can preferably be immersion objectives in the finite structure, more preferably also mirror objectives.

Here, the sample volume can be located in a sample chamber which is filled with an immersion medium. In particular, the objective or objectives can have a transparent interface, facing the sample volume and located in the immersion medium, which is arranged spherically or cylindrically in the center 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 objective, the curvature of the interface can be optimized for an ideal beam quality in the focal plane of the detection objective. In the beam path of the detection objective, the curvature of the boundary surface can also be optimized for ideal image quality for areas 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. An additional meniscus lens can also be provided on the side of the objective facing the sample or the object, so that 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 objective and the light source (in the case of an illumination beam path) or the detector (in the case of a detection beam path). The use of an adjustable mirror (DM) for correcting aberrations can also 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 light waveguide can be arranged on the illumination side of the illumination objective, via which the illumination light can be coupled into the illumination objective. Coupling in the illuminating light via an optical light wave guide has the advantage that a flexible connection is possible via this. Furthermore, a decoupling facet of the optical light waveguide, via which the illuminating light is decoupled from the optical light waveguide, i.e. the end of the optical light waveguide, can be movable, in particular displaceable. Another advantage of an optical light waveguide is that the light source which emits the illuminating light can be arranged at a distance from the light sheet microscope.

The light sheet microscope according to the invention can be further improved by arranging a scanning device on the illumination side of the illumination objective and / or on the detector side of the detection objective, which is movable and / or tiltable with respect to an optical axis of the respective objective. By means of the scanning device, a beam path of illuminating light to be coupled into the illuminating objective can be varied with regard to its distance to 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 a focusing optics (or the focusing optical system of the objective) and, accordingly, an offset is converted into a tilt.

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 tilting of the illumination beam path by means of the scanning device allows the position of the light sheet in the sample volume, i.e. in the object, to be varied and, for example, to be scanned through the object without the sample volume or the illumination objective having to be moved.

In particular, the light sheet can be displaced in the width and height direction and thus enable the illumination of objects which (for example due to their size) cannot be completely illuminated by means of a single light sheet. The width direction can be defined as a direction which lies within the focal plane of the detection objective or a plane parallel to it, ie is oriented perpendicular to the detection axis and parallel to the illumination axis. Correspondingly, the height direction can be defined as a direction which is perpendicular to the plane of illumination but which is oriented parallel to the detection axis of the optical axis of the detection objective. 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 fiber-optic cable in that the movable end of the optical fiber is displaced and / or tilted by means of a corresponding device. It is also possible to use one tilting mirror or several tilting mirrors in order to vary the lighting, that is to say the position of the light sheet. Alternatively or additionally, the lighting objective can contain one or more electrically focusable lenses (electrically tunable lenses, ETL), the adjustment of which also enables the position of the light sheet to be varied. In all of these variants, both the illumination objective and the detection objective can be configured statically in relation to their spatial position and do not require any mobility with respect to this during scanning.

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

The scanning device can preferably be arranged in the illumination-side focal length (focal plane) of the illumination objective and / or in the detector-side focal length of the detection objective. 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 the collimation of the illuminating light.

The tilting and the offset can be done by a scanner system of the scanning device. A first scanning mirror and a second scanning mirror can be provided, wherein the first scanning mirror can be smaller than the second scanning mirror and thus enables higher scanning frequencies. In this way, for example, even faster tilting can be generated 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 objective can be arbitrary and / or variable. In comparison to the light sheet microscopes from 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. In particular, the optically particularly complex scan lens can thus 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 fulcrum of the beam can be adjusted along the optical axis. This also means that, for example, if the objective is shifted in order to change the magnification, this shift can be compensated by the scanner system, so that in this case, too, the pivot point of the beam is again in the focal plane (on the illumination side or on the detector side) of the objective.

Furthermore, a spatial light modulation element can be arranged in the light sheet microscope according to the invention on the illumination side of the illumination objective and / or on the detector side of the detection objective, which has at least two spatially separated areas of 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 spatial light modulation elements.

A DMD consists of an arrangement 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 representation of a ring diaphragm.

A DM has a continuous mirrored surface that can be deformed by actuators. Depending on the number of actuators, almost any surface curve of the mirrored surface is possible with a DM, so that it allows errors in the optics of the corresponding lens to be compensated. Furthermore, a DM enables the illuminating beam path and / or the detection beam path to be changed with regard to its convergence or divergence, i.e., for example, to focus or defocus the illuminating 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 means of a corresponding control.

The spatial light modulation element can preferably be illuminated homogeneously and, through the configuration of the individual areas, allows flexible lighting patterns to be generated. In the detection beam path, the spatial light modulator 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, for example, be readjusted for a change in position of the light sheet away from the detection lens or towards it. Furthermore, the spatial light modulator element in the detection beam path can be used to manipulate the scattered and / or fluorescent light, for example to filter spatial frequencies.

Due to the possible focusing or defocusing in the illumination beam path, it is possible to shift the light sheet that is being formed along or against the longitudinal direction, that is to say parallel to the optical axis of the illumination objective.

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, by changing the divergence or convergence in the illumination beam path, the position of the light sheet along or against the optical axis of the illumination objective can be varied and / or the position of the plane sharply imaged on the detector can be varied in the detection beam path, and in particular the detection objective can be varied in response to a change in position of the light sheet can be readjusted away from the detection lens or towards this.

In particular, the focal length variable optical element can be configured as an electrically tunable lens, or 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 adjust the position of the plane that is sharply imaged by the detection lens on the detector, the change in position of the light sheet, so that the sharply mapped plane with Plane in which the light sheet is formed, coincides.

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 located in the rear focal plane, but performs a wobbling motion. A tumbling movement is to be understood as a lateral offset coupled with a corresponding tilt, this combined movement in the conjugate plane approximately corresponding to a parallel offset 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, i.e. 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, whereby the occurrence of possible chromatic imaging errors can be prevented by using achromatic collimators will. It is also possible to use reflective collimators.

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

The light source arrangement can have an ultrashort 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 at the same time 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 objectives is particularly advantageous, since they have no dispersion and consequently no chromatic imaging errors.

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

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

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

The present invention is explained in more detail below on the basis of exemplary configurations. The specific configurations are each advantageous in themselves, with the technical features of the configurations shown being able to be combined with one another and / or omitted as desired. The same technical features and technical features with the same function or technical effect are provided with the same reference symbols. For the sake of clarity, no duplicate explanation is made.

• 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 tumbling movement of the light source;
• 2a a second embodiment of the light sheet microscope according to the invention;
• 2 B the light sheet microscope of 2a with a tilting mirror system;
• 3 a third embodiment of the light sheet microscope according to the invention;
• 4th 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
• 6th a sixth embodiment of the light sheet microscope according to the invention.

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

The light sheet microscope 1 includes an illumination lens 5 and a detection lens 7th each having an optical axis 9 exhibit. An optical axis of the illumination lens 9a is essentially perpendicular to an optical axis of the detection lens 9b oriented.

Both lenses are for simplicity 5 , 7th schematically with a biconvex lens 11 shown, can, however, comprise a multiplicity of lenses of different shapes (biconvex, plano-convex, convex-concave, plano-concave, biconcave) in real configurations.

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

Analogous to the 1a can use the refractive lenses 13th of the 2 , 3 and 4th by Mirror lenses 15th to be replaced and the mirror lenses 15th the configurations of the light sheet microscope 1 can accordingly against refractive lenses 13th be replaced.

In 1a is also an optical fiber 17th in the form of an optical fiber 19th shown about what illuminating light 21 to a lighting side 23 of the lighting lens 5 is directed. The illuminating light 21 gets into the lighting lens 5 coupled and as in 1a shown schematically by the biconvex lens 11 to a sample page 25th focused.

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

The illuminating light 21 is in a sample volume 31 focuses and forms a sheet of light in it 33 out. The sample volume 31 extends in a longitudinal direction 35 , a width direction 37 and an altitude direction 39 , where the expansion in longitudinal 35 and width direction 37 is significantly larger than in the height direction 39 .

In the sample volume 31 is an object 41 arranged.

Both the lighting lens 5 , as well as the detection lens 7th has two main levels 43 which are only shown schematically in 1 are shown. The lighting lens 5 has a main level on the lighting side 43a and a main plane on the sample volume side 43b that are in their position lengthways 35 can distinguish. The same applies to the detection lens 7th the main plane on the sample volume side 43b and a main plane on the detector side 43c drawn. From the relevant main levels 43a , 43b and 43c The starting point is an illumination-side 45a, a sample volume-side 45b and a detector-side focal length 45c measured. The focal lengths 45a and 45b of the lighting lens 5 are for the lighting lens 5 always identical and also in 1 identical to the focal lengths 45b and 45c of the detection lens 7th . In other configurations, the focal lengths 45a , 45b of the lighting lens 5 of the focal lengths 45b , 45c of the detection lens.

In general, the lighting lens 5 a focal length 46a and the detection lens has a focal length 46b on.

How out 1a Clearly recognizable is a distance 47 between the main plane on the sample volume side 43b both lenses 5 , 7th and the sample volume 31 greater than the respective focal length on the sample volume side 45b .

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

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

The lenses 5 , 7th of the 1a are each to finitely corrected lenses 63 . These on finally corrected lenses 63 are for conjugate planes arranged at finite distance 65 optimized, which means that lens errors (not shown) for conjugate planes arranged in this way 65 are minimized. The conjugate planes 65 are in. for the sake of clarity 2 but not in 1 drawn. It should be noted here that the conjugate planes of the illumination objective 65a in a folded beam path 67 are arranged, the conjugate planes of the detection lens 65b on the other hand, in a non-folded beam path 69 .

In the prior art (not shown) the objectives 5 , 7th in light sheet microscopes a beam guidance in which the light is between a first lens system (an objective 5 , 7th ) and a second lens system (tube lens) runs approximately collimated.

1b shows schematically how a tumbling motion 70 of the end 57 the fiber 19th from an offset 70a and a tilt 70b is composed. This tumbling motion 70 is from the biconvex lens 11 in a parallel offset 70c implemented. The 1 b also shows in a schematic simplified representation of these two degrees of freedom 129 , the offset 70a (up and down) and the tilt 70b (vertical) as well as the forward-back offset 70d, the left-right offset 70e and the horizontal tilt 70f.

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

In the following 2a to 6th are, for the sake of clarity, technical features that are included in 1 are shown, such as the optical axes 9 , the main levels 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 on that in the form of a tilting mirror 73 is designed. In the 2a The embodiment shown comprises a single tilting mirror 73 , wherein in further refinements of the light sheet microscope according to the invention 1 several, for example two tilting mirrors 73 (please refer 2 B) can be provided, each tilting mirror 73 for tilting around one of two mutually perpendicular axes 75 can be used.

In the in 2a The embodiment shown are both mutually perpendicular axes of rotation 75 in the tilting mirror 73 , with a first rotation 77a around a first axis of rotation 75a to a movement of the light sheet 41 along the width direction 37 leads and a second turn 77b around a second axis of rotation 75b to a shift of the light sheet 41 along the height direction 39 . It should be noted that a second rotation 77b around the second axis of rotation 75b counterclockwise to shift the light sheet 41 against the height direction 39 leads. A movement of the illuminating light 21 on the lighting side 23 is thus on the sample side 25th inverted.

In 2a are also the focal length on the lighting side 45a and the focal length on the sample volume side 45b shown, with the as tilting mirror 73 configured second scanning device in the illumination-side focal length 45a of the lighting lens 5 is located. This position is particularly advantageous because the tilting mirror is tilted 73 in this position the illuminating light 21 on the sample side 25th laterally offset parallel.

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

In 2 B is the light sheet microscope 1 of the 2a with a tilting mirror system 74 from two tilting mirrors 73 shown. The tilting mirror system 74 allows any tilting mirror 73 independently around the respective two axes of rotation 75a , 75b to spin and the twists 77a and 77b to combine.

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

The 3 shows a third embodiment of the light sheet microscope according to the invention 1 , in this embodiment that of the optical fiber 19th , ie from its facet 81a , fed-in illumination light 21 over a large area on a spatial light modulation element 85 falls.

This in 3 spatial light modulation element shown 85 is a micromirror actuator 87 , which is abbreviated as DMD in the following. The DMD 85 has at least two, generally a large number of mirror areas (not shown) that can be controlled separately and the illumination beam path 27 in the embodiment shown can vary in such a way that a first illumination beam path 27a and a second illumination beam path 27b can be obtained separately or simultaneously.

In 3 is shown that the first 27a and the second illumination beam path 27b in the sample volume 31 are so focused that foci 81 both in the width direction 37 , as well as in height direction 39 are formed offset from one another. In the longitudinal direction 35 are the foci 81 not offset to each other. When shown in 3 it should be noted that both the DMD 87 , as well as the sample volume 31 are sketched schematically in a perspective view. The lighting lens 5 and the detection lens 7th are, however, shown in a simple schematic side view. The perspective representation is based on a corresponding coordinate system 89 made clear.

In further configurations of the light sheet microscope 1 can the spatial light modulation element 85 also as a deformable mirror 91 or as a spatial light modulator 93 be designed. Both elements 91 , 93 are sketched in a sectioned view. The deformable mirror 91 is through a steady and continuously running mirror surface 92 marked with the illuminating light 21 can be modified at will. The spatial light modulator 93 comprises a polarizer in the embodiment shown 95 , a pixel electrode 97 , a liquid crystal layer 99 , a back electrode 101 and a reflective element 103 .

Both elements 91 , 93 are known from the prior art, so that a more detailed explanation of their mode of operation is dispensed with at this point.

The scanning device 59 of the 1 , the tilting mirror 73 of the 2 , as well as the spatial light modulation element 85 of the 3 can be used in other configurations of the light sheet microscope 1 in addition to or as an alternative to the filter 49 between the detection lens 7th and the detector 51 in the detection beam path 29 be arranged. It is also possible that the elements 59 , 73 and 85 in any combination in the beam paths 27 , 29 are arranged in the positions described above.

Both the spatial light modulator 93 , as well as the deformable mirror 91 allow focus to some extent 81 of the illumination beam path 27 along the longitudinal direction 35 to vary.

In 4th Another possibility is shown, this shift along the longitudinal direction 35 to realize. In the fourth embodiment of the light sheet microscope according to the invention shown 1 is on the optical fiber 19th a focal length variable optical element 105 arranged. The focal length variable optical element 105 is available as an electrically tunable lens 107 designed. This is abbreviated as ETL in the following (English: electrically tunable lens).

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

The differing convergences 111a , 111b cause the foci 81 along the longitudinal direction 35 in the sample volume 31 move. In doing so, convergence is formed at the larger first 111a a first focus 81a further in the longitudinal direction 35 out. In 4th it should be noted that the sample volume 31 is shown in side view, what by the corresponding coordinate system 89 is made clear. Also the ETL 107 , as well as the lenses 5 and 7th and a deflecting mirror 113 are shown in the side view.

In a further embodiment, the ETL 107 arbitrarily with elements of the group consisting of the scanning device 59 , the tilting mirror 73 and the spatial light modulation element 85 , be combined. For example, the fixed deflecting mirror 113 through the tilting mirror 73 of the 2 be replaced.

Also the ETL 107 can in the detection beam path 29 be arranged to the location of a detection focus 81c along or against the vertical direction 39 to vary and preferably to the position of the light sheet 33 adapt. The location of the light sheet 33 can through the elements described above such as scanning device 59 , Tilting mirror 73 or spatial light modulation element 85 along the height direction 39 can be varied. Unless the detection focus 81c is not adapted to such a variation, a blurred image of the light sheet occurs 33 in the detector 51 .

The 5 shows the light sheet microscope according to the invention 1 in a fifth embodiment, which is the in 1 shown first embodiment is similar. However, both the lighting lens are 5 , as well as the detection lens 7th Mirror lenses 15th . Furthermore, the mirror lenses 15th on finally corrected lenses 63 .

Both the illumination beam path 27 , as well as the detection beam path 29 are due to the use of mirror lenses 15th varies. For example, the paraxial rays are masked out in these. The 5 also shows the main plane on the lighting side 43a of the lighting lens 5 , the main planes on the sample volume side 43b of the lighting 5 and detection lens 7th and the main plane on the detector side 43c of the detection lens 7th . It can also be seen that in this embodiment, too, the focal lengths on the sample volume side 45b both lenses 5 , 7th is smaller than the distance 47 to the sample volume 31 .

The fifth embodiment shown also has an over the scanning device 59 moving end 57 the optical fiber 19th on, making a shift 61 of the end 57 the fiber 19th for shifting the sample volume 61b of the light sheet 33 in the sample volume 31 leads. The directions of the shifts 61 , 61b of the fifth embodiment correspond to those of the first embodiment of FIG 1 .

The 5 also shows a light source arrangement 114 , which in the embodiment shown is a single light source 115 includes. The light source 115 is in the form of a laser 117 designed and the illuminating light emitted by this 21 gets into the optical fiber 19th coupled and transmitted from this.

In 6th is a sixth embodiment of the light sheet microscope according to the invention 1 shown. Both the lighting lens 5 , as well as that Detection lens 7th are mirror lenses 15th , on finally corrected lenses 63 and immersion objectives 119 .

The immersion objectives 119 exhibit a transparent interface 121 on that in an immersion liquid 123 a sample chamber 125 are immersed. The transparent interfaces 121 can be spherical or cylindrical with the center of curvature in focus 81 of the respective lens can be arranged.

The transparent interfaces shown 121 can as a thin membrane 127 , for example made of FEP or glass.

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

List of reference symbols

1

Light sheet microscope

3

main optical components

5

Lighting lens

6th

Lens system

7th

Detection lens

9

optical axis

9a

optical axis of the illumination lens

9b

optical axis of the detection lens

11

Biconvex lens

13th

refractive lens

15th

Mirror lens

17th

optical fiber optic cable

19th

optical fiber

21

Illuminating light

23

Lighting side

25th

Sample side

27

Illuminating 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

Width direction

39

Height direction

41

object

43

Main level

43a

main level on the lighting side

43b

main plane on the sample volume side

43c

main level on the detector side

45a

focal length on the lighting side

45b

focal length on the sample volume side

45c

focal length on the detector side

46a

Focal length of the lighting lens

46b

Focal length of the detection lens

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 finally corrected lens

65

conjugate plane

65a

conjugate plane of the illumination lens

65b

conjugate plane of the detection objective

67

folded beam path

69

non-folded beam path

70

Tumbling motion

70a

Offset

70b

Tilt

70c

Parallel offset

70d

Offset front-back

70e

Left-right offset

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

85

spatial light modulation element

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

Back electrode

103

Reflective element

105

focal length variable 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 liquid

125

Sample chamber

127

thin membrane

129

Degrees of freedom

Claims (12)

Light sheet microscope (1) for observing an object (41) arranged in a sample volume (31), comprising an illumination objective (5) for transmitting illuminating 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), characterized in that the illumination objective (5) is at a distance (47) between the sample volume (31) and the sample volume-side main plane (43b) of the illumination objective (5) which is greater than the focal length (46a) of the illumination objective (5) and / or that the detection objective (7) has a distance (47) between the Sample volume (31) and the sample volume-side main plane (43b) of the detection objective (7) which is greater than the focal length (46b) of the detection objective (7). Light sheet microscope (1) Claim 1 , characterized in that the illumination objective (5) and / or the detection objective (7) is a finitely corrected objective (63). Light sheet microscope (1) 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 the Claims 1 to 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 the Claims 1 to 4th , characterized in that an optical light 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 the Claims 1 to 5 , characterized in that a scanning device (59) is arranged on the illumination side (23) of the illumination objective (5) and / or on the detector side (53) of the detection objective (7) which, with respect to an optical axis (9a, 9b) of the respective Objective (5, 7) is movable and / or tiltable. Light sheet microscope (1) Claim 6 , characterized in 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). Light sheet microscope (1) according to one of the Claims 1 to 7th , characterized in that on the lighting side (23) of the lighting lens (5) and / or on the detector side (53) of the Detection objective (7) a spatial light modulation element (85) is arranged, which has at least two spatially separated areas of different optical transmission and / or reflection properties. Light sheet microscope (1) according to one of the Claims 1 to 8th , characterized in that an optical element (105) with variable focal length is arranged on the illumination side (23) of the illumination objective (5) and / or on the detector side (53) of the detection objective (7). Light sheet microscope (1) according to one of the Claims 1 to 9 , 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 the Claims 1 to 10 , characterized in that no optical elements with a refractive power are arranged along the optical axis (9a) of the illumination objective (5) which shift a sample volume-side focal plane (45b) of the illumination objective (5) into the sample volume (31) and / or along the optical axis (9b) of the detection objective (7) no optical elements with a refractive power are arranged, which shift the sample volume-side focal plane (45b) of the detection objective (7) into the sample volume (31). Use of an objective (63) corrected to finite 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) Distance (47) between the sample volume (31) and a sample volume-side main plane (43b) of the illumination objective (5) which is greater than the focal length (46a) of the illumination objective (5) and / or the detection objective (7) has a distance (47) ) between the sample volume (31) and the sample volume-side main plane (43b) of the detection objective (7) which is greater than the focal length (46b) of the detection objective (7).

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