Microscope with fast quasi-confocal detection

Abstract

2.1. The simultaneous acquisition of multiple images in the microscope should be made possible with greater flexibility. Furthermore, simultaneous acquisition should be made possible with less effort. 2.2. In a microscope (1) with a main beam splitter (22) and an adjustable beam deflection unit (21) for moving the illuminating light through the sample space (P), the beam deflection unit (21) is arranged optically between the light source (12) and the main beam splitter (22) for this purpose, so that the sample light reaches the sensor (28) away from the beam deflection unit (21), wherein both the illuminating light and the sample light pass through the same intermediate image (ZB).

Description

The invention relates to a microscope having an illumination beam path, a detection beam path and a main beam splitter, wherein the detection beam path has a sample space, a microscope objective, a tube lens, an intermediate image generated by the tube lens and a two-dimensionally spatially resolving optoelectronic sensor having a detection optics for imaging the intermediate image onto the sensor, and wherein the illumination beam path has a light source and an adjustable beam deflection unit for moving ("scanning", "scanning") the illumination light through the sample space, wherein the illumination beam path and the detection beam path are optically coupled by means of the main beam splitter to a common beam path in such a way that illumination light from the light source reaches the sample space via the main beam splitter through the microscope objective and sample light from the sample space reaches the sensor through the microscope objective via the main beam splitter.Confocal fluorescence imaging provides high contrast, high resolution images of biological samples. In this case, the sample is scanned point by point by means of laser illumination as in DE 197 02 753 A1, and the generated fluorescent light is detected confocally by means of a pinhole aperture (pinhole). By this aperture, extra-focal light is highly effectively dimmed, causing high contrast. However, the comparatively high recording duration due to the point-by-point scanning is disadvantageous. This inevitably also results in a high phototoxic sample load due to the excitation light.The disadvantages of pointwise imaging can be reduced by parallelization. Examples of these are confocal microscopes with a Nipkow disk and light sheet microscopes, the latter requiring additional illumination optics. A parallelization can alternatively take place via linear illumination as in EP 1 617 258 A1. In order to achieve confocality at least in one dimension (transversely to the line), linear detection with a slit diaphragm is also necessary here.Instead of a mechanical slit diaphragm, a sensor with an electronic slit diaphragm, in particular in the form of a rolling shutter (rolling shutter), can be used, with which the beam deflection unit is synchronized. Such electronic slit diaphragms are known, for example, from "Virtual slit scanning microscopy" by R. Fiolka et al. in "Histochemical and Cell Biology", Volume 128 (6), 2007 (DOI 10.1007 / s00418-007-0342-2) and from "Inexpensive and Flexible Slit-Scanning Confocal Imaging Using a Rolling Electronic Aperture" by M. S. Muller et al. in "Microscopy Instrument and Software Developments (FWW)" at the conference "Frontiers in Optics 2008" (DOl 10.1364 / FIO.2008.FWW2). The sample light reaches the sensor away from the beam deflection unit, which can be referred to as non-scanned (non-scanning) detection.For further parallelization, the sample light can be divided by means of beam splitters, as in DE 10 2021 134 427 A1, in such a way that different sample planes or different spectral ranges are simultaneously mapped onto disjunctive regions of the sensor. This reduces the recording duration by a corresponding integer factor. However, this arrangement has the disadvantage of occupying two outputs (ports) of the microscope, one for the illumination beam path and one for the detection beam path. This limits the flexibility of the microscope. In addition, two separate modules are required for illumination and detection, which is expensive.A further disadvantage of this is that at least one of the image fields is displaced with respect to the optical axis of the detection. The linear illumination takes place, on the other hand, centered, since a central region of the sample is excited on the object side. This symmetry break between excitation and detection leads to tilting of the image of the focus volume on the sensor. In the case of large fields, the image can thus project out of the region of a slit diaphragm, in particular of a "rolling shutter". Line confocal detection is then no longer possible since it is necessarily effected aligned strictly along a pixel line of the sensor.The object of the invention is to improve a microscope of the type mentioned at the beginning, so that a more flexible, less complicated use is made possible.Moreover, in particular embodiments, larger fields are intended to be receivable.The object is achieved by a microscope which has the features specified in claim 1.Advantageous embodiments of the invention are specified in the dependent claims.According to the invention, it is provided that the beam deflection unit is arranged optically between the light source and the main beam splitter, so that the sample light reaches the sensor away from the beam deflection unit, and that both the illumination light on its path to the microscope objective and the sample light on its path to the sensor run through the same intermediate image generated by the tube lens. The sample light reaches the sensor away from the beam deflection unit, which can be referred to as non-scanned (non-scanning) detection.By sharing the same intermediate image for illumination and detection, advantageously only a single output of the microscope is occupied. Possible further outputs are available for other uses, so that the microscope-while maintaining a short duration of exposure-has greater flexibility than known microscopes. In particular, fast quasi-confocal microscopy can be combined with other methods requiring its own output in a multimodal manner. Illumination and detection can be advantageously integrated into a single module, which reduces the manufacturing and installation outlay and enables a more compact and stable design. Compared to light sheet microscopes, only a single illumination optical unit in the form of the microscope objective is also required.An embodiment is advantageous in which the common beam path optically between the intermediate image and the main beam splitter has an optical unit, preferably a scanning optical unit ("scan lens"), for generating a pupil plane conjugate to a pupil (rear focal plane) of the microscope objective on or in the vicinity of the beam deflection unit. As a result, on the one hand, the main beam splitter is arranged in collimated light, so that possible soiling has only a slight influence on its transmission quality. On the other hand, in this way, a compact coupling of the illumination and detection beam paths can be effected without requiring additional collimation, as in the prior art. In particular, this makes it possible to dispense with additional relay optics.In this embodiment, the conjugate pupil plane can particularly advantageously arise by reflection at the main beam splitter, such that the beam deflection unit lies in or in the vicinity of the reflected conjugate pupil plane, in particular with transmission of the conjugate pupil plane into the detection beam path in the vicinity of a secondary beam splitter, such that the secondary beam splitter lies in or in the vicinity of the transmitted conjugate pupil plane. In this way, no additional relay optics are required in the detection beam path. The transmission into the detection beam path leads (compared to an arrangement in reflection) to better extinction of the excitation light, so that an improved contrast of measurement signal to excitation signal is achieved at the detector. In particular, the secondary beam splitter can thereby be arranged in collimated light, so that possible soiling has only a slight influence on its transmission quality.Preferably, the detection optics is arranged such that their optical axis is tilted by a (planar) angle different from zero with respect to an optical axis of the microscope objective (optionally deflected by at least one reflection), such that an image of the sample space on the sensor is offset away from the optical axis by an offset width, for example by a quarter of the sensor width, and the beam deflection unit (in particular its scanning rotational axis, about which an adjustable deflection mirror is rotatable) is tilted by a second (planar) angle different from zero about an axis perpendicular to its scanning rotational axis (within a plane spanned by the sensor lines and the optical axis of the microscope objective), such that a first aberration resulting from the tilting of the detection optics, in particular a distortion, and a second imaging error resulting from the tilting of the beam deflection unit, in particular likewise a distortion, at least partially compensate one another (such that the illumination focus volume corresponds better to the detection focus volume and in particular the illumination focus volume is imaged onto the sensor in a larger sample region parallel to the sensor lines). This can reduce the aberrations, so that larger fields can be recorded. In particular, the first tilting angle and the second tilting angle can be of the same size.In order to minimize imaging errors, a perpendicular arrangement of the optical axis of the detection optics to a surface of the sensor can be provided. The angle of incidence of the illumination light on the main beam splitter from the beam deflection unit is set up in such a way that the optical axis of the illumination beam path and that of the microscope objective coincide in the common (coupled) beam path (between the main beam splitter and the microscope objective). The angle of incidence is typically 45°-as in the conventional incident light scanning microscope, despite the tilting of the beam deflection unit. For this purpose, a preferably adjustable deflecting mirror is expediently arranged between the light source and the beam deflection unit. Alternatively to an angle of incidence of 45° on the main beam splitter, small angles of incidence of less than 15° or less than 10° can also be provided.Without tilting the beam deflection unit, as a result of the tilting of the detection optics, the imaging of the illumination focus volume onto the sensor would run obliquely as a function of the position x (x is the direction along the sensor lines and parallel to the line-confocal slit diaphragm) and as a function of the sensor-side focal length f of the detection optics according to-i.e. y-position-(see FIG. 3 a), wherein y is the direction transversely to the sensor lines and transversely to the line-confocal slit diaphragm and y 0 indicates the distance from the optical axis along this direction and Δx indicates the offset width resulting from the tilting. This aberration (inclination, distortion) is equivalent to an aberration which arises when a scanning mirror ("scan mirror") is used away from the optical axis, i.e. when the scanning rotational axis of the scanning mirror is not arranged perpendicular to the plane spanned by the normal vector of the scanning mirror and the propagation vector (medium k vector) of the incident illumination light. Expressed more graphically, the illumination focus volume lies obliquely in the sample space by the tilted beam deflection unit. Due to the tilted detection optics, the sensor looks at the sample space in an equally oblique manner depending on the y-position, whereby it sees the illumination line parallel to its pixel lines. According to the invention, the scanning rotation axis of the beam deflection unit is preferably arranged parallel to the plane spanned by the normal vector of the scanning mirror and the propagation vector (medium k vector) of the incident illumination light, in order to achieve maximum compensation of the aberrations. Then, the angle of incidence of the illumination light on the scanning mirror in its zero position (neutral position without artificial force acting on the scanning mirror) is equal to the tilting angle of the detection optics.The (in particular identical) tilt angle of the detection optics and of the beam deflection unit is preferably approximately arctan(Δx / f), wherein Δx is the offset width and f is the (sensor-side) focal length of the detection optics. This tilting angle substantially compensates for the imaging errors. In order to compensate for residual errors of the detection optics, the tilting angle can also deviate therefrom to a predefined extent. For example, line curvatures can be mediated by a distortion of the detection optics in such a way that they have only a minimal effect on the sensor.In particular, the beam deflection unit can be rotated about its scanning rotational axis-in addition to the tilting-in such a way that the illumination light reaches the beam deflection unit without intersecting an optical axis of the common beam path. In this way, the illumination light can be guided spatially around the main beam splitter to the beam deflection unit by guiding the illumination light past the main beam splitter in a plane which is spanned truly parallel to a plane spanned by the optical axis of the microscope objective and the longitudinal direction of the pixel lines of the sensor (i.e. outside this plane), which enables a compact arrangement of the illumination beam path and the detection beam path, in particular in a common module.The beam deflection unit may deflect the illumination light one-dimensionally or two-dimensionally. In the two-dimensional case, this is preferably a point-by-point scanning of the sample with a point-by-point illumination spot or illumination volume.In contrast, embodiments with linear illumination and detection are preferred in which the illumination beam path advantageously has a beam former (for example a cylindrical lens) for generating a linear distribution of the illumination light and the sensor has a linear aperture, wherein the aperture is arranged confocally with the intermediate image and can in particular be an electronic aperture, in particular in the form of a "rolling shutter". The linear illumination enables a shorter recording duration. In this case, in particular, an adjustment of the beam deflection unit can be synchronized with a dynamic position (movement) of the aperture, in particular by a control unit which is electrically connected to the sensor and the beam deflection unit. This enables a minimization of the recording duration. For this purpose, the sensor can preferably have a light sheet read-out mode (referred to by Hamamatsu as "Lighsheet Readout Mode") and have an output at which information about the position and / or movement of the aperture is present, in particular a signal which indicates a start of an image ("frame") on the sensor. Commercially available are sensors with such a mode and a corresponding output from Hamamatsu: https: / / www.hamamatsu.com / eu / en / product / cameras / cmos cameras / liqhtsheet-reading-mode.html. The output is designated by Hamamatsu as "external trigger output.".Advantageously, the detection beam path between the main beam splitter and the detection optics can have a secondary beam splitter and at least one reflector, in particular in the form of a prism, wherein the secondary beam splitter divides the sample light into two portions and directs it onto disjunctive regions of the sensor or onto a respective sensor, so that two disjunctive images of the sample space are produced, in particular with identical offset widths for both images (and identical angles between the optical axes of the images and the optical axis of the detection optics, in particular with point symmetry between the portions with respect to the optical axis of the detection optics), wherein only one of the portions reaches the relevant sensor via the reflector. In this way, two images can be recorded simultaneously in order to shorten the recording duration, in particular in different spectral ranges by forming the secondary beam splitter for color division, in particular in the form of a dichroite. With identical offset widths (and identical angles to the optical axis of the detection optics, in particular with point symmetry with respect to the optical axis of the detection optics), the imaging errors arising as a result of the tilting of the detection optics are compensated for at the maximum in both images.In such an embodiment, the illumination beam path can preferably have a second light source with a different emission wavelength than the first light source and / or the secondary beam splitter can be a color splitter, in particular a high-pass filter or a low-pass filter. In this way, two spectral ranges belonging to the emission wavelengths, in particular of two different fluorescent dyes, can be recorded simultaneously with low image errors in a short time. The first and the second light source can be the same light source, for example in the form of a multi-line laser or a broadband light source, in particular a white light source.In an advantageous method for scanning (rastering) image recording by means of a scanning microscope having an illumination beam path which has at least one light source having three or more selectively emitable disjunctive spectral bands, a detection beam path which has a microscope objective and a two-dimensionally spatially resolving sensor for recording light from a sample space, a main beam splitter which is designed as an N-fold spectral notch filter (having N=3.4,5,6 spectral notches) and which optically couples the illumination beam path and the detection beam path to one another (such that illumination light from the light source, in particular the spectral bands, passes through the objective into the sample space and sample light, in particular fluorescence emissions, passes from there through the objective to the sensor), wherein each notch (notch) of the notch filter corresponds spectrally to a respective one of the spectral bands of the light source, and a secondary color splitter configured as a high-pass or low-pass filter having a spectral edge and arranged optically between the main beam splitter and the sensor, each of the locations to be scanned in the sample space is sequentially illuminated with the first spectral band and the second spectral band and, during the illumination with the first spectral band, is also illuminated with the third spectral band, wherein the first and the second spectral band are spectrally on a first spectral side and the third spectral band are on a second spectral side, different from the first side, of the spectral edge of the secondary color splitter. In this way, three or more fluorescent dyes can be excited in a short time and their fluorescent emission recorded in separate images. A spectral band can in particular have the relevant width of a respective individual emission line of a laser. The N-fold notch filter can be configured, for example, as in US 2014 / 0092460 A1.The selection of the simultaneously illuminating and the change of the sequentially illuminating spectral bands can preferably be effected free of mechanical movements, in particular by means of one or more acousto-optical elements, for example by means of an acousto-optical, adjustable filter (AOTF). In this way, in combination with the multiple notch filter of the main color splitter and the spectral splitting by the secondary color splitter, three or more spectral bands can be passed to each location in the sample space to be scanned in a very short time, in particular for recording a corresponding number of fluorescent dyes in separate images.Particular preference is given to using microscopes having four illumination light spectral bands and a quadruple notch filter having these spectrally corresponding notches as main beam splitters or microscopes having six or more illumination light spectral bands and mutually exchangeable multiple notch filters having different subgroups of the notches spectrally corresponding to the spectral bands as main beam splitters, for example two quadruple notch filters on a filter wheel or filter slide or one quadruple notch filter and one double notch filter on a filter wheel or filter slide, preferably each with an electric drive.Preferably, each location of the sample space to be scanned is illuminated sequentially in these microscopes with pairs of the spectral bands (first and third spectral bands simultaneously, before or after this second and fourth spectral bands simultaneously), wherein in each of these pairs one of the spectral bands is respectively on the first spectral side and the other spectral band is respectively on the second spectral side of the spectral edge of the secondary color splitter. In this way, the color division can be optimally utilized for the quickest possible recording of an even number of fluorescent dyes.The pairs (in particular that consisting of the first and the third spectral band) can be irradiated either at each of the locations to be scanned in the sample space during a respectively (almost) constant position of the beam deflection unit in a direct succession or else alternately over all the locations to be scanned of the sample space after a complete scanning operation.Such methods can advantageously be used with the microscope according to the invention with tilted detection optics and tilted beam deflection unit in order to record 2n+2(n=1,2,...) spectrally different images of the sample space, in particular of n different fluorescent dyes, in a very short time. However, such a sequential-simultaneous method can also be used with conventional scanning microscopes. The switching of the wavelength pairs must take place as quickly as possible. This is achieved, for example, with acousto-optical filters (AOTF) or micromirrors (DMD), in particular with microelectromechanical systems (MEMS). For such methods, one or more light sources having a total corresponding number of selectively emittable spectral bands are required. Alternatively, a broadband or white light source may be used. In the case of only one light source, the use of an AOTF is expedient, which programmably directs a plurality of spectral bands simultaneously to the microscope objective. However, a respective AOTF can also be provided for each light source or for groups of light sources.Embodiments are particularly advantageous in which a difference between a number of reflections of the first component up to the sensor and a number of reflections of the second component up to the sensor-including reflections at the secondary beam splitter itself-is an odd number, wherein the optical axes of the two components through the detection optics preferably run in the same plane and at the same distance from and at the same angle to the optical axis of the detection optics, but on opposite sides of this axis. Due to the odd number of reflections, the beam of the first component is mirror-reversed even to the beam of the second component. Since the detection optics are preferably designed rotationally symmetrically, both components are then subject to the same imaging errors, so that their images on the sensor have comparable properties. The aberrations of both components can therefore be largely compensated for by tilting the beam deflection unit about an axis perpendicular to its scanning rotational axis, as described above. Then, one of the images is mirrored vertically (about the y-axis) and must be appropriately reversed before it can be further processed.In a possible more complex embodiment, the detection beam path comprises a second detection optics and the first portion reaches the sensor by the first detection optics away from the reflector and the second portion reaches the same sensor or a separate sensor by the second detection optics via the reflector. The optical axes of each component can coincide here with the respective optical axis of one of the two detection optics, as a result of which fewer aberrations occur. In particular, tilting of the beam deflection unit and of the detection optics can thus be dispensed with.Advantageously, the beam deflection unit can comprise MEMS micromirrors and in particular deflect the illumination light in a manner adjustable only one-dimensionally. This allows a compact construction and high deflection frequencies. A beam deflection unit comprising two-dimensionally adjustable MEMS micromirrors alternatively has the advantage that scanning along the longitudinal direction of the illumination line is possible in order either to expand, to homogenize or to minimize coherent effects the illumination of the sample space. For this purpose, the scanning along the longitudinal direction of the line in the sample space takes place more quickly than that transverse to the longitudinal direction of the line. Resonant-quasistatic MEMS scanners are particularly advantageous for this purpose. Alternatively, a quasi-static MEMS scanner can be used in both axes in order to minimize only the coherent interference effects in the illumination. It can then be advantageous to control the axis for scanning along the longitudinal direction of the illumination line with white noise of predetermined amplitude. Alternatively, the beam deflection unit may comprise one or more galvanometer scanners. The longitudinal direction of the illumination line extends in the direction of its longest extension.Embodiments are particularly preferred in which the microscope has a stand on which the microscope objective is arranged, in particular in an objective turret, wherein the stand has a first output, in the region of which the tube lens and the intermediate image are arranged, and at least one further output having a further tube lens and a further intermediate image, wherein the sample light can be guided to both outputs simultaneously or sequentially by means of at least one beam splitter, in particular a repeatedly removable beam splitter, or by means of a mirror, wherein the main beam splitter, the detection optics, the sensor, the beam deflection unit and a scanning unit are arranged within a module which is detachably mechanically and optically connected to one of the outputs. A flexibly usable microscope system with low imaging errors with a short duration of exposure can thus be provided.The invention is explained in more detail below with reference to exemplary embodiments.In the drawings, there are shown: FIG. 1 shows a microscope, FIG. 2 shows optical paths of a microscope, and FIG. 3 shows geometric profiles of the images of the detection focus on the sensor.In all drawings, corresponding parts bear like reference numerals.FIG. 1 shows a schematic representation of a microscope 1. it consists of a stand 2, a scanning module 3 and a laser module 4. stand 2 has a microscope objective 5 with tube lenses 6, which each generate an intermediate image ZB in the region of two outputs 7. The stand also has beam splitters 9 which can be pivoted in, for example neutral splitters or colour splitters, in order to selectively guide configurable light proportionally to the outputs and / or to the eyepiece 10 and / or light from a lamp 11 to the microscope objective 5 and from there into the sample space P.The laser module 4 comprises, purely by way of example, four lasers 12 having different emission wavelengths, the respective intensity of which can be adjusted by means of a respective AOTF 13. Alternatively (not shown), one or more of the lasers may be directly modulatable. Its illumination light is coupled into optical fibers 15 via coupling optics 14 and conducted to the scanning module 3, where it is collimated by means of, for example, longitudinally displaceable collimators 16. The collimators 16 can serve to compensate for longitudinal chromatic aberrations and / or to focus the illumination light into different depths of the sample space P. Alternatively (not shown), the different emission wavelengths can be combined, for example, already in the laser module, so that only a single optical fiber 15 is required and collimators 16 can be dispensed with. Via a mirror 17 and a beam combiner 18, the illumination light combined in this way reaches a deflection mirror 19 which deflects the illumination light such that, after it has passed through a cylindrical lens 20, it falls at an angle of incidence α onto the beam deflection unit 21, which in turn is tilted by an identical angle α about an axis perpendicular to its scanning rotational axis with respect to an optical axis OA of the microscope objective 5, which is deflected by means of a main beam splitter 22. From the main beam splitter 22, the illumination light reaches the microscope objective 5 via the scanning optics 23, the intermediate image ZB and one of the tube lenses 6 and from there into the sample space P. Since the cylindrical lens focuses the illumination light in one dimension into the plane lying on the beam deflection unit 21 and conjugate with the pupil of the microscope objective 5, a basically linear illumination focus volume results in the sample space. Due to the tilting of the beam deflection unit 21, the linear illumination focus volume is increasingly obliquely situated in the sample space P as the distance from the optical axis of the microscope objective 5 increases.Sample light, in particular also fluorescence excited in the sample by the illumination light, reaches the main beam splitter 22 on the reverse path via the intermediate image ZB. The portion of the illumination light reflected in the sample and on the way there is reflected back to the beam deflection unit by the main beam splitter 22, which is configured, for example, as a dichroic notch filter (notch filter). Fluorescence contained in the sample light is transmitted, in particular due to the Stokes shift, through the main beam splitter 22 to a secondary beam splitter 24, which is designed, for example, as a dichroic low-pass filter. The scanning optics 23 are designed such that on the one hand on the beam deflection unit 21 and on the other hand in the region of the secondary beam splitter 24 a plane conjugate to the pupil of the microscope objective 5 lies. The secondary beam splitter 24 splits the sample light remaining at this point (typically fluorescence emission) into two portions by its spectral low-pass effect. The first portion A 1 passes directly into a detection optics 25 by transmission. the second portion A 2 is first mirrored on two inner surfaces 26 of a prism 27, so that the second portion experiences an odd number of reflections before it likewise enters the detection optics 25. However, the detection optics 25 is tilted with respect to the optical axis OA of the microscope objective 5 within a plane spanned by the pixel lines of the sensor and the optical axis OA of the microscope objective 5 by, for example, the same angle α as the beam deflection unit 21, whereby the two portions A 1, A 2 separated at the secondary beam splitter 24 fall into the detection optics 25 at an angle of opposite equal size and thus experience symmetrically opposite aberrations. However, these compensate precisely for an aberration imposed on the illumination light by the tilting of the beam deflection unit 21. In alternative embodiments (not shown), the tilt angles of the detection optics 25 and the beam deflection unit 21 can differ from one another in order to compensate for another imaging error. In all cases, however, the two tilting angles are different from zero. The two components pass as disjunctive, largely error-corrected images (a respective "component image") onto the sensor 28, which has a "rolling shutter" as a line-confocal slit diaphragm, with which the movement of the beam deflection unit 21 is synchronized, for example by starting the scanner movement as a function of a signal emitted by the sensor 28 operated in a light sheet read-out mode, which signal indicates the start of a new image on the sensor 28. By means of the compensation, the linear detection focus volume, which is oblique depending on position in the sample space P, is imaged pixel-line-parallel and thus with minimized distortion on the respectively active, rectilinear line of the rolling shutter. A control unit, not shown, reads out the sensor, for example after the recording of a complete sensor frame, assigns its data to two respective digital images and vertically mirrors the data of the digital image corresponding to the second sample light portion, so that both digital images have the same perspective.The tilting angle α of the detection optics 25 and of the beam deflection unit 21 corresponds here to α=arctan(Δx / f) with Δx as the offset distance of the images on the sensor (projected relative to the optical axis of the detection optics 25) and f as the sensor-side focal length of the detection optics 25.The scanning module can be designed to be particularly compact and efficient because (in addition to the intermediate image planes ZB at the outputs of the stand 2) an image plane is arranged only on the sensor 28 and, in the detection direction, behind the main beam splitter 22, only exactly one pupil plane PE' conjugate to the microscope objective 5 is arranged in each branch of the detection beam path.FIG. 2 schematically shows the beam paths of a microscope 1. FIG. 2A shows it in plan view, and FIG. 2B in side view. The pupil plane PE of the microscope objective 5, the pupil planes PE' conjugated thereto and the intermediate image plane ZB are readily recognizable. In this respect, the arrangement shown corresponds to that in FIG. 1. Here too, the detection beam path between the microscope objective 5 and the sensor 28 comprises only exactly one intermediate image ZB and between the tube lens 6 and the sensor 28 only exactly one conjugate pupil plane PE' in each branch. In alternative embodiments (not shown), further conjugate pupil planes and / or intermediate image planes can be present by means of additional relay optics.As a variation from FIG. 1, the illumination light is guided spatially around the main beam splitter 22 to the beam deflection unit 21-the lasers 12 are arranged above the optical axis OA of the microscope objective 5, as can be seen in the lateral view. For this purpose, the beam deflection unit 21 is additionally tilted about its scanning rotational axis. By means of a deflection mirror (not shown), for example the one which deflects the illumination light such that, after it has passed through the cylindrical lens 20, it falls onto the beam deflection unit 21 at an angle of incidence α, the illumination light first passes obliquely into the plane which is spanned by the optical axis OA of the microscope objective 5 and the longitudinal direction x of the sensor lines, where it impinges on the beam deflection unit 21. The beam deflection unit 21 is rotated precisely in such a way that the reflection of the illumination light takes place within the said plane towards the microscope objective 5.FIG. 3 schematically shows, for visual clarification of the effect of the invention, the detection focus volume imaged onto the sensor by beam splitting into two component images at different times of a scanning operation in three different, exemplary configurations.The result of a fictitious configuration is shown in partial figure a), in which although the detection optics, but not the beam deflection unit, are tilted with respect to the optical axis of the microscope objective 5. The scanning illumination of the sample space P is centered here in a conventional manner about the optical axis of the microscope objective. Due to the tilting of the detection optics, an increasing inclination, as a result a distortion, of the detection focus volume DV results towards both vertical image edges, i.e. in the y direction. Also shown is the instantaneous position of the electronic shutter of a rolling shutter RS. It can be clearly seen that, owing to the inclination, only small parts of the detection focus volume can be detected by the active region of the sensor. The entire detection focus volume could be detected by increasing the width of the electronic slit aperture RS. In order to achieve a stronger suppression of out-of-focus light, however, it would be necessary to also use narrower electronic slit diaphragms RS, in particular with a width of only one pixel row.Partial figure b) is the result of a configuration in which both the detection optics and the beam deflection unit are tilted, but the right-portion image after the side-beam splitting is subjected to an even number of reflections, while the left-portion image after the side-beam splitting is not reflected at all. As a result, the inclination and the resulting distortions are not compensated for, but even tend to be increased more.Finally, FIG. c ) shows the result of a configuration in which both the detection optics and the beam deflection unit are tilted and the right portion image after the side beam splitting is subjected to an odd number of reflections, while the left portion image after the side beam splitting is again not reflected at all. As a result, the inclination and thus the distortions previously present are compensated for to the greatest possible extent.List of reference characters1 Microscope 2 Stand 3 Scanning module 4 Laser module 5 Microscope objective 6 Tube lenses 7 Output 8 9 Beam splitter 10 Eyepiece 11 Lamp 12 Laser 13 AOTF 14 Input coupling optical system 15 Optical fiber 16 Collimator 17 Mirror 18 Beam combiner 19 Deflection mirror 20 Cylindrical lens 21 Beam deflection unit 22 Main beam splitter 23 Scanning optical system 24 Secondary beam splitter 25 Detection optical system 26 Prism inner surface 27 Prism 28 Sensor 8 P Sample space OA Optical axis ZB Intermediate image PE Pupil plane PE' Conjugate pupil plane DV Detection focus volume RS "Rolling shutter" α Tilting angle A1 First portion A2 Second portionReferences included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedDE 197 02 753 A1

[0002] EP 1 617 258 A1

[0003] DE 10 2021 134 427 A1

[0005] US 2014 / 0092460 A1

[0024] Cited Non-Patent LiteratureVirtual slit scanning microscopy" by R. Fiolka et al. in "Histochemical and Cell Biology", Volume 128 (6), 2007 (DOI 10.1007 / s00418-007-0342-2

[0004] Inexpensive and Flexible Slit-Scanning Confocal Imaging Using a Rolling Electronic Aperture" by M. S. Muller et al. in "Microscopy Instrument and Software Developments (FWW)" at the conference "Frontiers in Optics 2008" (DOl 10.1364 / FIO.2008.FWW2

[0004]

Claims

Microscope (1) having an illumination beam path, a detection beam path and a main beam splitter (22), wherein the detection beam path has a sample space (P), a microscope objective (5), a tube lens (6), an intermediate image (ZB) generated by the tube lens and a two-dimensionally spatially resolving optoelectronic sensor (28) having a detection optical unit (25) for imaging the intermediate image (ZB) onto the sensor (28), and wherein the illumination beam path has a light source (12) and an adjustable beam deflection unit (21) for moving the illumination light through the sample space (P), wherein the illumination beam path and the detection beam path are optically coupled in such a way to a common beam path by means of the main beam splitter (22), that illumination light from the light source (12) reaches the sample space (P) via the main beam splitter (22) through the microscope objective (5) and sample light from the sample space (P) reaches the sensor (28) through the microscope objective (5) via the main beam splitter (22), characterized in that the beam deflection unit (21) is arranged optically between the light source (12) and the main beam splitter (22), so that the sample light reaches the sensor (28) away from the beam deflection unit (21), and that both the illumination light on its path to the microscope objective (5) and the sample light on its path to the sensor (28) extend through the same intermediate image (ZB) generated by the tube lens (6).Microscope according to the preceding claim, wherein the common beam path optically between the intermediate image and the main beam splitter has an optical system for generating a pupil plane conjugate to a pupil of the microscope objective on or in the vicinity of the beam deflection unit, in particular with the formation of this conjugate pupil plane by reflection at the main beam splitter, in particular with transmission of the conjugate pupil plane into the detection beam path in the vicinity of a secondary beam splitter.Microscope according to one of the preceding claims, wherein the detection optics is arranged such that their optical axis is tilted with respect to an optical axis of the microscope objective by a first angle different from zero, such that an image of the sample space on the sensor is offset away from the optical axis by an offset distance, and the beam deflection unit is tilted by a second angle different from zero, in particular of approximately the same size, about an axis perpendicular to its scanning rotational axis, such that a first aberration resulting from the tilting of the detection optics and a second aberration resulting from the tilting of the beam deflection unit at least partially compensate one another, in particular with a perpendicular arrangement of the optical axis of the detection optics to a surface of the sensor.Microscope according to the preceding claim, wherein the tilting angle corresponds to arctan(Δx / f), wherein Δx is the offset distance on the sensor and f is the (sensor-side) focal length of the detection optics.Microscope according to one of the preceding claims, wherein the illumination beam path has a beam former for generating a linear distribution of the illumination light and wherein the sensor has a linear aperture which is arranged confocally with respect to the intermediate image, in particular an electronic aperture, in particular in the form of a rolling shutter, in particular with synchronizability of an adjustment of the beam deflection unit with a dynamic position of the aperture, in particular by a control unit which is electrically connected to the sensor and the beam deflection unit.Microscope according to one of the preceding claims, wherein the detection beam path between the main beam splitter and the detection optics has a secondary beam splitter and at least one reflector, wherein the secondary beam splitter splits the sample light into two portions and directs it onto disjunctive regions of the sensor or onto a respective sensor, so that two disjunctive images of the sample space are produced, in particular with identical offset widths for both images, wherein only one of the portions reaches the relevant sensor via the reflector, in particular with the formation of the secondary beam splitter for color splitting, in particular in the form of a dichroite or a neutral splitter.Microscope according to the preceding claim, wherein the illumination beam path has a second light source having a different emission wavelength than the first light source, and wherein the secondary beam splitter is a color splitter, in particular a high-pass filter or a low-pass filter.The microscope according to any one of claims 6 or 7, wherein a difference between a number of reflections of the first portion to the sensor and a number of reflections of the second portion to the sensor is an odd number.Microscope according to one of the preceding claims, insofar as they are not dependent on claim 3 or 4, wherein the detection beam path comprises a second detection optical unit and the first portion reaches the sensor away from the reflector through the first detection optical unit and the second portion reaches the sensor through the second detection optical unit via the reflector.Microscope according to one of the preceding claims, wherein the beam deflection unit comprises MEMS micromirrors and in particular can deflect the illumination light in a manner adjustable only one-dimensionally.Microscope according to one of the preceding claims, wherein the beam deflection unit is rotated about its scanning rotational axis such that the illumination light reaches the beam deflection unit in a plane which is different from a plane which is spanned by the optical axis (OA) of the microscope objective (5) and a longitudinal direction of a pixel line of the sensor.Microscope according to one of the preceding claims, wherein the microscope has a stand on which the microscope objective is arranged, in particular in an objective turret, wherein the stand has a first output, in the region of which the tube lens and the intermediate image are arranged, and at least one further output having a further tube lens and a further intermediate image, wherein the sample light can be guided to both outputs simultaneously or sequentially by means of at least one beam splitter, in particular a repeatedly removable beam splitter, or by means of a mirror, wherein the main beam splitter, the detection optics, the sensor, the beam deflection unit and a scanning unit are arranged within a module which is detachably mechanically and optically connected to one of the outputs.Microscope according to one of the preceding claims, wherein the light source has three or more selectively emitable disjunctive spectral bands and the main beam splitter is designed as an N-fold spectral notch filter (N=3, 4, 5, 6) and each notch of the notch filter corresponds spectrally to a respective one of the spectral bands of the light source, in particular equipped with a control unit which is set up to carry out a method according to one of the following claims.Method for scanning image recording by means of a scanning microscope having an illumination beam path which has at least one light source having three or more selectively emitable disjunctive spectral bands, a detection beam path which has a microscope objective and a two-dimensionally spatially resolving sensor for recording light from a sample space, a main beam splitter which is designed as an N-fold spectral notch filter (N=3, 4, 5, 6) and which optically couples the illumination beam path and the detection beam path to one another, each notch of the notch filter corresponding spectrally to a respective one of the spectral bands of the light source, and a secondary colour splitter which is designed as a high-pass filter or low-pass filter having a spectral edge and is arranged optically between the main beam splitter and the sensor, wherein each of the locations to be scanned in the sample space is sequentially illuminated with the first spectral band and the second spectral band and during the illumination with the first spectral band is also illuminated with the third spectral band, wherein the first and the second spectral band are spectrally on a first spectral side and the third spectral band are on a second spectral side, different from the first side, of the spectral edge of the auxiliary color splitter.Method according to claim 13, wherein the selection of the simultaneously illuminating and the change of the sequentially illuminating spectral bands takes place free of mechanical movements, in particular by means of one or more acousto-optical elements.Method according to claim 13 or 14, wherein microscopes with four illumination light spectral bands and a quadruple notch filter with these spectrally corresponding notches are used as main beam splitters or microscopes with six or more illumination light spectral bands and mutually exchangeable multiple notch filters with different subgroups of the notches spectrally corresponding to the spectral bands are used as main beam splitters, in particular two quadruple notch filters on a filter wheel or slide or one quadruple notch filter and one double notch filter on a filter wheel or slide, in particular each with an electric drive.Method according to claim 13, 14 or 15, wherein each location of the sample space to be scanned is illuminated sequentially with pairs of the spectral bands, wherein in each of these pairs one of the spectral bands lies on the first spectral side and the other spectral band lies on the second spectral side of the spectral edge of the secondary colour splitter, in particular with irradiation of the pairs at each of the locations to be scanned in the sample space during a respective constant position of the beam deflection unit immediately following one another.The method according to any one of claims 13 to 16, wherein a microscope according to any one of claims 1 to 12 is used.

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