Microscope and method for scanning image acquisition by means of scanning microscope

Abstract

The invention provides a microscope with rapid quasi-confocal detection. The simultaneous acquisition of multiple images in a microscope should be possible with greater flexibility. In addition, simultaneous acquisition should be made possible with less effort. In a microscope (1) having a main beam splitter (22) and an adjustable beam deflection unit (21) for moving illumination light through a sample space (P), the beam deflection unit (21) is optically arranged for this purpose between a light source (12) and the main beam splitter (22) such that sample light away from the beam deflection unit (21) passes to a sensor (28), both the illumination light and the sample light passing through the same intermediate image (ZB). The invention also provides a method for scanning image acquisition by means of a scanning microscope.

Description

Field of the Invention

[0001] 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 includes a sample space, a microscope objective, a tube lens, an intermediate image generated by the tube lens, and a two-dimensional spatially resolving optoelectronic sensor which has a detection optical unit for imaging the intermediate image onto the sensor, and wherein the illumination beam path includes a light source and an adjustable beam deflection unit for moving ("scanning", "raster scanning") illumination light through the sample space, wherein the illumination beam path and the detection beam path are optically coupled to form a common beam path by means of the main beam splitter such that illumination light from the light source is transmitted through the microscope objective into the sample space via the main beam splitter, and sample light from the sample space passes through the microscope objective and is transmitted to the sensor via the main beam splitter. Background Art

[0002] Confocal fluorescence imaging provides high-contrast, high-resolution images of biological samples. As in DE 19702 753 A1, a laser beam is used to irradiate the sample point by point, and the resulting fluorescence is detected confocal ly through a pinhole. This aperture very effectively blocks out-of-focus light, resulting in high contrast. However, the relatively long acquisition time due to point-by-point scanning is disadvantageous. Due to the excitation light, this also inevitably results in a high phototoxic sample load.

[0003] The disadvantages of point-by-point imaging can be reduced by parallelization. Examples of this are confocal microscopes with a Nipkow disk and light sheet microscopes, the latter requiring additional illumination optics. As an alternative, parallelization can be achieved by means of linear illumination as in EP 1 617258 A1. To achieve confocal ity at least in one dimension (transverse to the row), linear detection using a slit-type aperture is also required here.

[0004] Instead of a mechanical slit diaphragm, a sensor with an electronic slit diaphragm can be used, in particular a sensor in the form of a rolling shutter, and the beam deflection unit is synchronized with this sensor. Such an electronic slit diaphragm is known, for example, from "Virtual slit scanning microscopy" by R. Fiolka et al. in "Histochemistry and Cell Biology", Vol. 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 "Frontiers in Optics 2008" conference (DOI 10.1364 / FIO.2008.FWW2). The sample light is transmitted to the sensor away from the beam deflection unit; this can be referred to as non-descanned detection.

[0005] Regarding further parallelization, as in DE 10 2021 34 427 A1, the sample light can be divided by means of a beam splitter such that different sample planes or different spectral ranges are imaged simultaneously on non-overlapping regions of the sensor. This reduces the acquisition time by the corresponding integer factor. However, this arrangement has the disadvantage of occupying two ports of the microscope, one port for the illumination beam path and one port for the detection beam path. This limits the flexibility of the microscope. In addition, two separate modules are required for illumination and detection, and this is expensive.

[0006] Another disadvantage thereof is that at least one in the image field is shifted relative to the optical axis of the detection. On the other hand, when the central region of the sample is excited on the object side, the linear illumination is centered. This disruption of the symmetry between excitation and detection causes the image of the aggregation volume to be tilted on the sensor. In the case of a large field, the image can extend beyond the region of the slit diaphragm, in particular the region of the "rolling shutter". In this case, line confocal detection is no longer possible because it is inevitably achieved in a manner that is strictly aligned along the pixel rows of the sensor. Summary of the Invention

[0007] The problem solved by the present invention is: to improve a microscope of the type described at the beginning such that more flexible and less complex use becomes possible. In addition, it should be possible to record a larger field in a special embodiment.

[0008] This problem is solved by a microscope having the features specified below.

[0009] The invention provides a microscope having an illumination beam path, a detection beam path, and a main beam splitter, wherein the detection beam path includes a sample space, a microscope objective, a tube lens, an intermediate image generated by the tube lens, and a two-dimensional spatially resolving optoelectronic sensor having a detection optical unit for imaging the intermediate image onto the sensor, and wherein the illumination beam path includes a light source and an adjustable beam deflection unit for moving the illumination light through the sample space, wherein the illumination beam path and the detection beam path are optically coupled by the main beam splitter to form a common beam path such that illumination light from the light source is transmitted through the microscope objective via the main beam splitter into the sample space, and sample light from the sample space reaches the sensor via the main beam splitter through the microscope objective, the beam deflection unit being optically arranged between the light source and the main beam splitter such that the sample light remote from the beam deflection unit is transmitted to the sensor, and both the illumination light traveling to the microscope objective and the sample light traveling to the sensor pass through the same intermediate image generated by the tube lens.

[0010] Advantageous configurations of the invention are given below.

[0011] In one embodiment, the common beam path includes an optical unit that is optically located between the intermediate image and the main beam splitter and is configured to generate a pupil plane conjugate to the pupil of the microscope objective, the pupil plane being located on or near the beam deflection unit, in particular wherein the conjugate pupil plane is formed by reflection away from the main beam splitter, in particular wherein the conjugate pupil plane is transmitted into the detection beam path near a secondary beam splitter.

[0012] In one embodiment, the detection optical unit is arranged such that its optical axis is inclined at a first non-zero angle with respect to the optical axis of the microscope objective, and thus the image of the sample space on the sensor is offset from the optical axis by an offset length, and the beam deflection unit is inclined at a second non-zero angle about an axis perpendicular to its scanning rotation axis, in particular a second non-zero angle having approximately the same magnitude, such that a first aberration generated by the inclination of the detection optical unit and a second aberration generated by the inclination of the beam deflection unit at least partially compensate each other, in particular in the case where the optical axis of the detection optical unit is vertically arranged with respect to the surface of the sensor.

[0013] In one embodiment, the tilt angle corresponds to arctan(Δx / f), where Δx is the offset length on the sensor and f is the (sensor-side) focal length of the detection optical unit.

[0014] In one embodiment, the illumination beam path includes a beam shaper for generating a linear distribution of the illumination light, and wherein the sensor has a linear aperture that is confocal with the intermediate image, in particular an electronic aperture, in particular an electronic aperture in the form of a rolling shutter, in particular having the ability to synchronize the adjustment of the beam deflection unit with the dynamic position of the aperture, in particular via a control unit electrically connected to the sensor and the beam deflection unit.

[0015] In one embodiment, the detection beam path between the main beam splitter and the detection optical unit includes a secondary beam splitter and at least one reflector, wherein the secondary beam splitter divides the sample light into two components and guides the two components to non-overlapping regions of the sensor or to corresponding sensors, such that two non-overlapping images of the sample space are generated, in particular with the same offset length for the two images, wherein only one of the components is transmitted to the associated sensor via the reflector, in particular using an embodiment of the secondary beam splitter for separating colors, in particular in the form of a dichroic beam splitter or a neutral intensity beam splitter.

[0016] In one embodiment, the illumination beam path includes a second light source having an emission wavelength different from that of the first light source, and wherein the secondary beam splitter is a dichroic filter, in particular a high-pass filter or a low-pass filter.

[0017] In one embodiment, the difference between the number of times the first component is reflected to the sensor and the number of times the second component is reflected to the sensor is odd.

[0018] In one embodiment, the detection beam path includes a second detection optical unit, and the first component is transmitted to the sensor away from the reflector through the first detection optical unit, and the second component is transmitted to the sensor through the second detection optical unit via the reflector.

[0019] In one embodiment, the beam deflection unit includes a MEMS mirror and is particularly capable of deflecting the illumination light in a one-dimensionally adjustable manner only.

[0020] In one embodiment, the beam deflection unit rotates about its scanning rotation axis such that the illumination light is transmitted to the beam deflection unit in a plane different from the plane spanned by the optical axis of the microscope objective and the longitudinal direction of the pixel rows of the sensor.

[0021] In one embodiment, the microscope comprises a support on which the microscope objective is arranged, in particular within an objective turret, wherein the support comprises a first port and at least one additional port, the tube lens and the intermediate image being arranged in the region of the first port, the at least one additional port having an additional tube lens and an additional intermediate image, wherein the sample light can be guided to the two ports simultaneously or sequentially by means of at least one beam splitter, in particular a removably repeatable beam splitter, or by means of a mirror, and wherein the main beam splitter, the detection optical unit, the sensor, the beam deflection unit and the scanning unit are arranged within a module that is optically and mechanically detachably connected to one of the ports.

[0022] In one embodiment, the light source comprises three or more selectively emit disjoint spectral bands, and the main beam splitter is implemented as a spectral N-fold notch filter (N = 3, 4, 5, 6), and each notch of the notch filter corresponds spectrally to a corresponding spectral band of the spectral bands from the light source, the light source being particularly equipped with a control unit configured to perform the method according to any one of the following claims.

[0023] The present invention also provides a method for acquiring a scanned image by means of a scanning microscope having an illumination beam path, a detection beam path, a main beam splitter and a secondary dichroic beam splitter, the illumination beam path comprising at least one light source having three or more selectively emit disjoint spectral bands, the detection beam path comprising a microscope objective and a two-dimensional spatially resolving sensor for recording light from a sample space, the main beam splitter being implemented as a spectral N-fold notch filter (N = 3, 4, 5, 6) that optically couples the illumination beam path and the detection beam path to each other, wherein each notch of the notch filter corresponds spectrally to a corresponding spectral band of those spectral bands from the light source, the secondary dichroic beam splitter being designed as a high-pass or low-pass filter having a spectral edge and being optically arranged between the main beam splitter and the sensor, wherein each position to be scanned in the sample space is illuminated sequentially with a first spectral band and a second spectral band, and wherein there is also illumination with a third spectral band during illumination with the first spectral band, wherein the first spectral band and the second spectral band are located on a first spectral side of the spectral edge of the secondary dichroic beam splitter, and the third spectral band is located on a second spectral side different from the first spectral side.

[0024] In one embodiment, the selection of the simultaneously illuminated spectral bands and the change of the sequentially illuminated spectral bands are achieved without mechanical movement, in particular by means of one or more acousto-optic elements.

[0025] In one embodiment, a microscope having four illumination light spectral bands and a four-notch filter having these spectrally corresponding notches is used as the main beam splitter, or a microscope having six or more illumination light spectral bands and a plurality of interchangeable notch filters having notches spectrally corresponding to different subgroups of the spectral bands is used as the main beam splitter, in particular two four-notch filters on a filter wheel or filter slider or a four-notch filter and a double-notch filter on a filter wheel or filter slider, in particular each having an electric drive in each case.

[0026] In one embodiment, each position to be scanned in the sample space is illuminated sequentially with pairs of spectral bands, wherein in each of these pairs, one of the spectral bands is located on a first spectral side of the spectral edge of the secondary dichroic mirror and the other spectral band is located on a second spectral side of the spectral edge of the secondary dichroic mirror, in particular during each constant position of the beam deflection unit, the pairs of spectral bands are irradiated immediately and continuously at each position to be scanned in the sample space.

[0027] In the method according to one embodiment, a microscope according to the above is used.

[0028] According to the invention, it is provided that the beam deflection unit is optically arranged between the light source and the main beam splitter such that the sample light remote from the beam deflection unit is transmitted to the sensor, and both the illumination light traveling to the microscope objective and the sample light traveling to the sensor pass through the same intermediate image generated by the tube lens. The sample light is transmitted to the sensor remote from the beam deflection unit; this can be referred to as non-descanned detection.

[0029] Advantageously, as a result of sharing the same intermediate image for illumination and detection, only a single port of the microscope is occupied. Possible other ports can be used for other purposes, so the microscope has greater flexibility than known microscopes while maintaining a short acquisition time. In particular, fast confocal microscopes can be combined in a multimode manner with other methods that require their own ports. Illumination and detection can be advantageously integrated into a single module, thereby reducing manufacturing and installation costs and enabling a more compact and stable design. In addition, compared to light sheet microscopes, this only requires a single illumination optical unit in the form of a microscope objective.

[0030] In an advantageous embodiment, an optical system (preferably a scanning lens) for generating a pupil plane conjugate to the pupil (rear focal plane) of the microscope objective and located on or near the beam deflection unit is included in the common beam path, optically between the intermediate image and the main beam splitter. As a result, on the one hand, the main beam splitter is arranged in collimated light and thus possible contamination has only a small impact on its transmission quality. On the other hand, this allows for a compact coupling of the illumination and detection beam paths without the need for an additional collimation as in the prior art. In particular, this eliminates the need for an additional relay optical unit.

[0031] Particularly advantageously, the conjugate pupil plane can be generated due to reflection off the main beam splitter in this embodiment, and thus the beam deflection unit is located in or near the reflected conjugate pupil plane, in particular, the detection beam path passing through the conjugate pupil plane is transmitted near the secondary beam splitter, and thus the secondary beam splitter is located in or near the passed conjugate pupil plane. In this way, no additional relay optical unit is required in the detection beam path. The transmission into the detection beam path (compared to the arrangement in reflection) results in an improved extinction of the excitation light and thus an improved contrast between the measurement signal and the excitation signal at the detector. In particular, the secondary beam splitter can thus be arranged in collimated light and thus possible contamination has only a small impact on its transmission quality.

[0032] Preferably, the detection optical unit is arranged such that its optical axis is tilted by a non-zero (planar) angle with respect to the optical axis of the microscope objective (optionally deflected by at least one reflection), and thus the image of the sample space on the sensor is offset from the optical axis by an offset length, for example, a quarter of the sensor width, and the beam deflection unit (in particular its scanning rotation axis, around which the adjustable deflection mirror can rotate) is tilted by a second (planar) non-zero angle around an axis perpendicular to its scanning rotation axis (in the plane spanned by the sensor row and the optical axis of the microscope objective) such that the first aberration (in particular distortion) generated by the tilt of the detection optical unit and the second aberration (in particular distortion) generated by the tilt of the beam deflection unit at least partially compensate each other (such that there is a better correspondence between the illumination aggregation volume and the detection aggregation volume), and in particular, the illumination focused volume is imaged on the sensor in a manner parallel to the sensor row over a larger sample area). This can reduce aberrations such that a larger field can be acquired. In particular, the first tilt angle and the second tilt angle can have the same magnitude.

[0033] In order to minimize the aberration, a vertical arrangement of the optical axis of the detection optical unit relative to the surface of the sensor can be provided. At the main beam splitter, the incident angle of the illumination light from the beam deflection unit is configured such that the optical axis of the illumination beam path and the optical axis of the microscope objective coincide in the common (coupling) beam path (between the main beam splitter and the microscope objective). Usually, the incident angle is 45° - as in a conventional reflected light scanning microscope - although the beam deflection unit is tilted. For this purpose, a preferably adjustable deflection mirror is suitably arranged between the light source and the beam deflection unit. A small incident angle of less than 15° or less than 10° can be provided as an alternative to the 45° incident angle at the main beam splitter.

[0034] In the case where there is no tilt of the beam deflection unit, tilting the detection optical unit will result in an image representation of the illuminated aggregate volume on the sensor according to the following extension: - that is, tilted in a y - position - dependent manner - in a manner dependent on the position x (where x is along the sensor row and parallel to the direction of the line confocal slit - type aperture) and on the sensor - side focal length f of the detection optical unit (see Figure 3 a), where y is transverse to the sensor row and transverse to the direction of the line confocal slit - type aperture, and y0 is the distance from the optical axis along this direction, and Δx is the offset length generated by the tilt. This aberration (tilt, distortion) is equivalent to the aberration that occurs when using a scanning mirror far from the optical axis (i.e., when the scanning rotation axis of the scanning mirror is not perpendicular to the plane spanned by the normal vector of the scanning mirror and the propagation vector (average k - vector) of the incident illumination light). In more pictorial terms, the tilted beam deflection unit causes the illuminated aggregate volume to be tilted in the sample space. Due to the tilted detection optical unit, the sensor points at the sample space with the same y - position - dependent tilt, so that the illumination line is parallel to its pixel row in its reference frame. 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 (average k - vector) of the incident illumination light in order to obtain maximum compensation for the aberration. Then, the incident angle of the illumination light at the scanning mirror in the zero position of the scanning mirror (the neutral position where no artificial force acts on the scanning mirror) is equal to the tilt angle of the detection optical unit.

[0035] The (especially the same) tilt angles of the detection optical unit and the beam deflection unit are preferably approximately arctan(Δx / f), where Δx is the offset length and f is the (sensor - side) focal length of the detection optical unit. The aberration can be compensated to a large extent by this tilt angle. To compensate for the residual aberration of the detection optical unit, the tilt angle can also deviate from its specified measure. For example, line warping can be adjusted by the distortion of the detection optical unit such that it has only a minimal impact on the sensor.

[0036] In particular, the beam deflection unit can be rotated about its scanning axis of rotation - in addition to tilting - such that the illumination light is transmitted to the beam deflection unit without intersecting the 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 in such a way that the illumination light is guided past the main beam splitter in a plane that is truly parallel to the plane spanned by the optical axis of the microscope objective and the longitudinal direction of the pixel rows of the sensor (i.e., outside the aforementioned plane), so that the illumination beam path and the detection beam path are arranged compactly, especially in a common module.

[0037] The beam deflection unit can deflect the illumination light one-dimensionally or two-dimensionally. In the two-dimensional case, this preferably involves point-by-point scanning of the sample with a point-like illumination spot or illumination volume.

[0038] In contrast, embodiments with linear illumination and detection are preferred; in these, the illumination beam path advantageously includes a beam shaper (e.g., a cylindrical lens) for generating a linear distribution of the illumination light, and the sensor includes a linear aperture, where the aperture is arranged confocal with the intermediate image and can in particular be an electronic aperture, especially in the form of a "rolling shutter". Linear illumination allows shorter acquisition times. In particular, the adjustment of the beam deflection unit can be synchronized with the dynamic position (movement) of the aperture, especially by a control unit electrically connected to the sensor and the beam deflection unit. This allows minimization of the acquisition time. For this purpose, the sensor can preferably have a light sheet readout mode (as mentioned by Hamamatsu) and have a port at which information about the position and / or movement of the aperture is present, especially a signal indicating the start of an image ("frame") on the sensor. Commercially, sensors with this mode and corresponding port can be obtained from: Hamamatsu: https: / / www.hamamatsu.com / eu / en / product / cameras / cmos-cameras / lightsheet-readod-mode.html. This port is called "external trigger output" by Hamamatsu.

[0039] Advantageously, the detection beam path between the main beam splitter and the detection optical unit may include a secondary beam splitter and at least one reflector, in particular a reflector in the form of a prism, wherein the secondary beam splitter divides the sample light into two components and guides the latter to non-overlapping regions of the sensor or to corresponding sensors, such that two non-overlapping images of the sample space are produced, in particular with the same offset length for the two images (and the same angle between the optical axes of the images and the optical axis of the detection optical unit, in particular with point symmetry with respect to the optical axis of the detection optical unit between the components), wherein only one component is transmitted via the reflector to the associated sensor. Thus, two images can be acquired simultaneously in order to shorten the acquisition duration, in particular in different spectral ranges, by forming the secondary beam splitter to separate colors, in particular in the form of a dichroic separator. In the case of the same offset length (and the same angle with the optical axis of the detection optical unit, in particular point symmetry with respect to the optical axis of the detection optical unit), the aberration caused by the tilt of the detection optical unit is compensated to the greatest extent in the two images.

[0040] In such an embodiment, the illumination beam path may preferably include a second light source having an emission wavelength different from that of the first light source and / or the secondary beam splitter may be a dichroic beam splitter, in particular a high-pass filter or a low-pass filter. In this way, two spectral ranges associated with the emission wavelength, in particular two spectral ranges of two different fluorescent dyes, can be acquired simultaneously in a short time with few image errors. The first light source and the second light source may 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.

[0041] In an advantageous method for acquiring an image by scanning (raster scanning) with a scanning microscope, the scanning microscope has an illumination beam path, a detection beam path, a main beam splitter, and a secondary dichroic filter. The illumination beam path includes at least one light source having three or more selectively emittable non-overlapping spectral bands. The detection beam path includes a microscope objective and a two-dimensional spatially resolving sensor for recording light from the sample space. The main beam splitter is implemented as a spectral N-notch filter (with N = 3, 4, 5, 6 spectral notches), which optically couples the illumination beam path and the detection beam path to each other (such that the illumination light from the light source, in particular the spectral bands, is transmitted through the objective to the sample space, and the sample light, in particular the fluorescence emission, is transmitted from there through the objective to the sensor), where each notch of the notch filter corresponds spectrally to the corresponding spectral bands from the light source, and the secondary dichroic filter is designed as a high-pass or low-pass filter with a spectral edge and is optically arranged between the main beam splitter and the sensor. During illumination with the first spectral band, each position to be scanned in the sample space is sequentially illuminated by the first spectral band and the second spectral band and also by the third spectral band, where the first spectral band and the second spectral band are located on a first spectral side of the spectral edge of the secondary dichroic filter, and the third spectral band is located on a second spectral side different from the first spectral side. In this way, three or more fluorescent dyes can be excited in a short time, and their fluorescence emissions can be recorded in separate images. The spectral bands can particularly have the relevant widths of the corresponding individual emission lines of a laser. For example, the N-notch filter can be designed as in US 2014 / 0092460A1.

[0042] The selection of the simultaneously illuminated spectral bands and the change of the sequentially illuminated spectral bands can preferably be achieved without mechanical movement, in particular by means of one or more acousto-optic elements, such as by means of an acousto-optic tunable filter (AOTF). Combining the multiple-notch filter of the main dichroic filter and the splitting of the spectrum into two parts by the secondary dichroic filter allows three or more spectral bands to be guided to each position to be scanned in the sample space in a very short time, in particular for recording the corresponding number of fluorescent dyes in separate images.

[0043] Particularly preferably, a microscope with four illumination light spectral bands and a four-notch filter with spectrally corresponding notches is used as the main beam splitter or a multi-order notch filter with six or more illumination light spectral bands and interchangeable notches with different subgroups, where the different subgroups of notches correspond spectrally to the spectral bands, such as two four-notch filters on a filter wheel or a filter slider or a four-notch filter and a two-notch filter on a filter wheel or a filter slider, preferably each with an electric drive in each case.

[0044] Preferably, each position to be scanned in the sample space of these microscopes is irradiated successively with pairs of spectral bands (irradiating the first and third spectral bands simultaneously, before or after which the second and fourth spectral bands are irradiated simultaneously), where one spectral band of each pair of spectral bands lies on a first spectral side of the spectral edge of the dichroic beam splitter and the other spectral band lies on a second spectral side of the spectral edge of the dichroic beam splitter. In this way, color splitting can be optimally used to record an even number of fluorescent dyes as quickly as possible.

[0045] These pairs (in particular the pair consisting of the first and third spectral bands) can be radiated continuously immediately at each position to be scanned in the sample space during each (almost) constant position of the beam deflection unit, or alternately after a complete scan operation over all positions to be scanned in the sample space.

[0046] Such a method can advantageously be used in conjunction with a microscope according to the invention having an inclined detection optical unit and an inclined beam deflection unit in order to record 2n + 2 (n = 1, 2, …) spectrally different images of the sample space, in particular n different fluorescent dyes, in a very short time. However, this sequential-simultaneous method can also be used with conventional scanning microscopes. The wavelength pairs must be switched as quickly as possible. This is achieved, for example, using acousto-optic tunable filters (AOTF) or digital micromirrors (DMD), in particular using microelectromechanical systems (MEMS). Such a method requires one or more light sources having a total corresponding number of selectively emittable spectral bands. In an alternative, broadband or white light sources can be used. In the case of only one light source, it is advantageous to use an AOTF which simultaneously directs a plurality of spectral bands to the microscope objective in a programmable manner. However, a corresponding AOTF can be provided for each light source or for a group of light sources.

[0047] A particularly advantageous embodiment is an embodiment in which the difference between the number of times the first component is reflected onto the sensor and the number of times the second component is reflected onto the sensor (including the reflections leaving the dichroic beam splitter itself) is odd, where the optical axes of the two components preferably lie in the same plane and pass through the detection optical unit at the same distance and at the same angle from the optical axis of the detection optical unit, although on opposite sides of this axis. Due to the odd number of reflections, the beam of the first component is precisely mirror-inverted into the beam of the second component. Since the detection optical unit is preferably rotationally symmetric, the two components are subject to the same aberrations and thus their images on the sensor have comparable characteristics. The aberrations of the two components can thus be compensated to a large extent by tilting the beam deflection unit about an axis perpendicular to its scanning rotation axis, as described above. Then, one of the images is vertically mirrored (with respect to the y-axis) and must be inverted accordingly before further processing.

[0048] In a possibly more complex embodiment, the detection beam path includes a second detection optical unit, and the first component is transmitted away from the reflector through the first detection optical unit to the sensor, and the second component is transmitted through the second detection optical unit via the reflector to the same sensor or a separate sensor. The optical axis of each component can coincide here with the respective optical axis of one of the two detection optical units, resulting in fewer aberrations. In particular, it can be managed in this way without tilting the beam deflection unit and the detection optical unit.

[0049] Advantageously, the beam deflection unit may include a MEMS mirror and in particular deflect the illumination light only in a one-dimensionally adjustable manner. This enables a compact structure and a high deflection frequency. The beam deflection unit including a two-dimensionally adjustable MEMS mirror alternatively has the advantage that scanning along the longitudinal direction of the illumination line is possible in order to either expand or homogenize the illumination of the sample space or to minimize coherence effects. For this purpose, the scanning along the longitudinal direction of the rows in the sample space is faster than the scanning across the longitudinal direction of the lines. A resonant quasi-static MEMS scanner is particularly advantageous for this. In an alternative, a MEMS scanner that is quasi-static on two axes can be used to only minimize the coherence interference effects in the illumination. Then, it may be advantageous to control the axis for scanning along the longitudinal direction of the illumination line with white noise of a predetermined amplitude. In an alternative, the beam deflection unit may include one or more galvanometer scanners. The longitudinal direction of the illumination line extends in the direction of its longest extent.

[0050] A particularly preferred embodiment is an embodiment in which the microscope includes a support, a microscope objective is arranged on the support, in particular within an objective turret, wherein the support includes a first port and at least one additional port, a tube lens and an intermediate image are arranged in the region of the first port, and the at least one additional port has an additional tube lens and an additional intermediate image, wherein the sample light can be guided to the two ports simultaneously or sequentially by means of at least one beam splitter, in particular a removably repeatable beam splitter, or by means of a mirror, and the main beam splitter, the detection optical unit, the sensor, the beam deflection unit, and the scanning unit are arranged in a module that is optically and mechanically detachably connected to one of the ports. Thereby, a flexible microscope system with few aberrations and a short acquisition time can be provided. Description of the Drawings

[0051] The present invention will be explained in more detail below based on exemplary embodiments.

[0052] In the drawings:

[0053] Figure 1 a microscope is shown,

[0054] Figure 2A and 2B the beam path of the microscope is shown, and

[0055] Figure 3 Shows the geometric extent of the image of the detection focus on the sensor.

[0056] In all the figures, matching parts have the same reference numerals. Detailed Description

[0057] Figure 1 Shows a schematic view of the microscope 1. It consists of a support 2, a scanning module 3 and a laser module 4. The support 2 has a microscope objective 5 with a tube lens 6, and the tube lenses 6 each produce an intermediate image ZB in the region of two ports 7. The support also includes a beam splitter 9 that can be pivoted in, such as a neutral intensity beam splitter or a dichroic beam splitter, in order to proportionally guide light to the ports and / or the eyepiece 10 and / or to guide light from the lamp 11 to the microscope objective 5 and from there into the sample space P in a selectively configurable manner.

[0058] Merely by way of example, the laser module 4 includes four lasers 12 having different emission wavelengths, and their respective intensities can be adjusted by means of corresponding AOTFs 13. In an alternative (not shown), one or more of the lasers can be directly modulated. Their illumination light is input-coupled into the optical fiber 15 via a coupling optical unit 14 and guided to the scanning module 3, where the illumination light is collimated, for example, by means of a longitudinally displaceable collimator 16. The collimator 16 can be used to compensate for longitudinal chromatic aberration and / or to focus the illumination light at different depths in the sample space P. In an alternative (not shown), different emission wavelengths can, for example, already be combined in the laser module, such that only a single optical fiber 15 is required and it can be managed without a collimator 16. Via a mirror 17 and a beam combiner 18, the combined illumination light is thus transmitted to a deflecting mirror 19, which deflects the illumination light such that, after the illumination light has passed through a cylindrical lens 20, the illumination light is incident on a beam deflection unit 21 at an incident angle α, and the beam deflection unit is in turn inclined by the same angle α about an axis perpendicular to its scanning rotation axis with respect to the optical axis OA of the microscope objective 5, and the optical axis OA of the microscope objective 5 is deflected by means of a main beam splitter 22. From the main beam splitter 22, the illumination light is transmitted via a scanning lens 23, an intermediate image ZB and one of the tube lenses 6 to the microscope objective 5 and from there into the sample space P. Since the cylindrical lens focuses the illumination light in one dimension into a plane that is conjugate to the pupil of the microscope objective 5 and lies on the beam deflection unit 21, a substantially linear illumination focus volume appears in the sample space. Due to the inclination of the beam deflection unit 21, the linear illumination focus volume becomes increasingly inclined in the sample space P as the distance from the optical axis of the microscope objective 5 increases.

[0059] The sample light, and in particular also the fluorescence excited by the illumination light in the sample, is transmitted via the intermediate image ZB to the main beam splitter 22 in the opposite manner. The component of the illumination light reflected in the sample and on its way there is reflected back to the beam deflection unit 21 by the main beam splitter 22, which is designed, for example, as a dichroic notch filter. In particular due to the Stokes shift, the fluorescence contained in the sample light is transmitted through the main beam splitter 22 to the secondary beam splitter 24, which is designed, for example, as a dichroic low-pass filter. The scanning lens 23 is designed such that the plane conjugate to the pupil of the microscope objective 5 is located first on the beam deflection unit 21 and second in the region of the secondary beam splitter 24. The secondary beam splitter 24 divides the remaining sample light (usually the fluorescence emission) at this point into two components by its spectral low-pass effect. The first component A1 is transmitted directly to the detection optical unit 25. The second component A2 is initially reflected away from the two inner surfaces 26 of the prism 27, so that the second component undergoes an odd number of reflections before it also enters the detection optical unit 25. However, relative to the optical axis OA of the microscope objective 5, the detection optical unit 25 is inclined, for example, by the same angle α as the beam deflection unit 21 in the plane spanned by the pixel rows of the sensor and the optical axis OA of the microscope objective 5, whereby the two components A1, A2 separated at the secondary beam splitter 24 enter the detection optical unit 25 at equal angles with opposite signs and thus undergo symmetrically opposite aberrations in this way. However, these precisely compensate for the aberrations imposed on the illumination light by the inclination of the beam deflection unit 21. In an alternative embodiment (not shown), the inclination angles of the detection optical unit 25 and the beam deflection unit 21 can be different from each other in order to compensate for another aberration. However, in all cases, the two inclination angles are not zero. The two components are transmitted to the sensor 28 as non-overlapping, largely error-corrected images (corresponding "component images"), the sensor 28 having a "rolling shutter" as a line confocal slit diaphragm, the movement of the beam deflection unit 21 being synchronized with this diaphragm, for example by starting the scanner movement based on the signals emitted by the sensor 28 operating in the light sheet readout mode and indicating the start of a new image on the sensor 28. By means of said compensation, the linearly detected aggregation volume at a position-dependent angle in the sample space P is imaged parallel to the pixel rows and thus has minimized distortion on the corresponding active straight lines of the "rolling shutter". A control unit (not depicted here) reads the sensor, for example, after acquiring a complete sensor frame, assigns its data to two corresponding digital images and vertically reflects the data of the digital image corresponding to the second sample light component, such that the two digital images have the same viewing angle.

[0060] The inclination angle α of the detection optical unit 25 and the beam deflection unit 21 corresponds to α = arctan(Δx / f), where Δx is the offset length of the image on the sensor (projected relative to the optical axis of the detection optical unit 25) and f is the sensor-side focal length of the detection optical unit 25.

[0061] The scanning module can be designed in a particularly compact and efficient manner because (apart from the intermediate image plane ZB at the port of the support 2) the image plane is arranged only on the sensor 28, and in each branch of the detection beam path downstream of the main beam splitter 22 in the detection direction, exactly one pupil plane PE' conjugate to the microscope objective 5 is arranged.

[0062] Figure 2A and 2B Schematically depicts the beam path of the microscope 1. Part Figure 2A Shows them in a plan view, part Figure 2B Shows them in a side view. The pupil plane PE of the microscope objective 5, the pupil plane PE' conjugate thereto, and the intermediate image plane ZB are easily recognizable. In this regard, the arrangement shown corresponds to Figure 1 the arrangement in . Here, the detection beam path also includes only exactly one intermediate image ZB between the microscope objective 5 and the sensor 28 and only exactly one conjugate pupil plane PE' in each branch between the tube lens 6 and the sensor 28. In an alternative embodiment (not shown here), additional conjugate pupil planes and / or intermediate image planes can be present via additional relay optical units.

[0063] Deviating from Figure 1 , the illumination light is spatially guided around the main beam splitter 22 to the beam deflection unit 21 - the laser 12 is arranged above the optical axis OA of the microscope objective 5, as is obvious from the side view. For this purpose, the beam deflection unit 21 is additionally tilted around its scanning rotation axis. Via a deflection mirror (not depicted here), for example, a deflection mirror that deflects the illumination light in such a way that the illumination light is incident on the beam deflection unit 21 at an incident angle α after passing through the cylindrical lens 20, the illumination light initially passes obliquely through the plane spanned by the optical axis OA of the microscope objective 5 and the longitudinal direction x of the sensor row, where the illumination light is incident on the beam deflection unit 21. The beam deflection unit 21 is precisely twisted such that the reflection of the illumination light to the microscope objective 5 is achieved within the said plane.

[0064] Figure 3 Schematically shows the detection aggregate volume imaged on the sensor in two component images by beam splitting at different times during the scanning process in three different exemplary configurations to visually show the effect of the present invention.

[0065] Sub - figure a) shows the result of a virtual configuration where the detection optical unit, rather than the beam deflection unit, is tilted with respect to the optical axis of the microscope objective 5. The scanned illumination of the sample space P is here conventionally centered on the optical axis of the microscope objective. Due to the tilt of the detection optical unit, the tilt of the detection aggregation volume DV towards the two vertical image edges (i.e., in the y - direction) increases, resulting in distortion. Also plotted is the current position of the electronic shutter "rolling shutter" RS. It is clearly evident that due to the tilt, only a small part of the detection aggregation volume can be detected by the active area of the sensor. It is indeed possible to capture the entire detection focus volume by increasing the width of the RS electron slit - type aperture. However, in order to achieve greater suppression of out - of - focus light, a narrower RS electron slit - type aperture is also required, especially one with a width of only one pixel row.

[0066] Sub - figure b) is the result of a configuration where both the detection optical unit and the beam deflection unit are tilted. However, in this case, after the secondary beam splitting, the right - hand component image is exposed to an even number of reflections, while the left - hand component image is not reflected at all after the secondary beam splitting. This does not compensate for the tilt and the resulting distortion, but rather tends to increase them.

[0067] Finally, sub - figure c) shows the result of a configuration where both the detection optical unit and the beam deflection unit are tilted, and the right - hand component image is exposed to an odd number of reflections after the secondary beam splitting, while the left - hand component image is again not reflected at all after the secondary beam splitting. This largely compensates for the tilt and thus the previously existing distortion.

[0068] List of reference numerals

[0069] 1 Microscope

[0070] 2 Bracket

[0071] 3 Scanning module

[0072] 4 Laser module

[0073] 5 Microscope objective

[0074] 6 Tube lens

[0075] 7 Port

[0076] 9 Beam splitter

[0077] 10 Eyepiece

[0078] 11 Lamp

[0079] 12 Laser

[0080] 13 AOTF

[0081] 14 Input coupling optical unit

[0082] 15 Optical fiber

[0083] 16 Collimator

[0084] 17 Mirror

[0085] 18 Beam combiner

[0086] 19 Deflecting mirror

[0087] 20 Cylindrical lens

[0088] 21 Beam deflection unit

[0089] 22 Main beam splitter

[0090] 23 Scanning lens

[0091] 24 Sub-beam splitter

[0092] 25 Detection optical unit

[0093] 26 Inner prism surface

[0094] 27 Prism

[0095] 28 Sensor

[0096] P Sample space

[0097] OA Optical axis

[0098] ZB Intermediate image

[0099] PE Pupil plane

[0100] PE' Conjugate pupil plane

[0101] DV Detection aggregate volume

[0102] RS Rolling shutter

[0103] α Tilt angle

[0104] A1 First component

[0105] A2 Second component

Claims

1. A microscope (1) comprising an illumination beam path, a detection beam path and a primary beam splitter (22), wherein the detection beam path comprises a sample space (P), a microscope objective (5), a tube lens (6), an intermediate image (ZB) generated by the tube lens and a two-dimensional spatial resolution photoelectric sensor (28), the two-dimensional spatial resolution photoelectric sensor (28) having a detection optical unit (25) for imaging the intermediate image (ZB) on the sensor (28), and wherein the illumination beam path comprises 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 through the main beam splitter (22) to form a common beam path, so that the illumination light from the light source (12) is transmitted through the microscope objective (5) via the main beam splitter (22) into the sample space (P), and the sample light from the sample space (P) is transmitted through the microscope objective (5) via the main beam splitter (22) to reach the sensor (28), characterized in that the beam deflection unit (21) is optically arranged between the light source (12) and the main beam splitter (22), so that the sample light away from the beam deflection unit (21) is transmitted to the sensor (28), and both the illumination light traveling to the microscope objective (5) and the sample light traveling to the sensor (28) pass through the same intermediate image (ZB) generated by the tube lens (6).

2. The microscope according to claim 1, wherein: The common beam path comprises an optical unit which is optically located between the intermediate image and the primary beam splitter and is used to generate a pupil plane which is conjugate with a pupil of the microscope objective, the pupil plane being located on or near the beam deflection unit, in particular wherein the conjugate pupil plane is formed by reflection away from the primary beam splitter, in particular wherein the conjugate pupil plane is transmitted through the conjugate pupil plane into the detection beam path near the secondary beam splitter.

3. A microscope according to any one of the preceding claims, wherein: The detection optical unit is arranged so that its optical axis is tilted by a first non-zero angle relative to the optical axis of the microscope objective, and thus the image of the sample space on the sensor deviates from the optical axis by an offset length, and the beam deflection unit is tilted by a second non-zero angle about an axis perpendicular to its scanning rotation axis, in particular a second non-zero angle of almost the same size, so that a first aberration resulting from the tilt of the detection optical unit and a second aberration resulting from the tilt of the beam deflection unit at least partially compensate each other, in particular in the case of a vertical arrangement of the optical axis of the detection optical unit relative to the surface of the sensor.

4. The microscope according to claim 3, wherein: The tilt angle corresponds to arctan(Δx / f), where Δx is the offset length on the sensor and f is the (sensor-side) focal length of the detection optical unit.

5. A microscope according to any one of the preceding claims, wherein: The illumination beam path comprises a beam shaper for generating a linear distribution of the illumination light, and wherein the sensor has a linear aperture, in particular an electronic aperture, in particular in the form of a rolling shutter, arranged confocally with the intermediate image, in particular with the ability to synchronize an adjustment of the beam deflection unit with a dynamic position of the aperture, in particular by means of a control unit electrically connected to the sensor and the beam deflection unit.

6. A microscope according to any one of the preceding claims, wherein: The detection beam path between the main beam splitter and the detection optical unit comprises a secondary beam splitter and at least one reflector, wherein the secondary beam splitter splits the sample light into two components and directs the two components to non-intersecting areas of the sensor or to corresponding sensors, so that two non-intersecting images of the sample space are generated, in particular for both images to have the same offset length, wherein only one of the components is passed via the reflector to the relevant sensor, in particular using an embodiment of the secondary beam splitter for separating colors, in particular in the form of a dichroic beam splitter or a neutral intensity beam splitter.

7. The microscope according to claim 6, wherein: The illumination beam path comprises 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.

8. The microscope according to claim 6 or 7, wherein: A difference between the number of times the first component is reflected to the sensor and the number of times the second component is reflected to the sensor is an odd number.

9. Microscope according to any of the preceding claims, provided that these claims do not refer back to claim 3 or 4, wherein: The detection beam path comprises a second detection optical unit, and the first component is transferred through the first detection optical unit away from the reflector to the sensor, and the second component is transferred through the second detection optical unit via the reflector to the sensor.

10. The microscope according to any one of the preceding claims, wherein: The beam deflection unit comprises a MEMS micromirror and is in particular capable of deflecting the illumination light in a manner that is adjustable in only one dimension.

11. The microscope according to any one of the preceding claims, wherein: The beam deflection unit rotates about its scanning rotation axis so that the illumination light is delivered to the beam deflection unit in a plane different from a plane spanned by the optical axis (OA) of the microscope objective (5) and the longitudinal direction of the pixel rows of the sensor.

12. The microscope according to any one of the preceding claims, wherein: The microscope comprises a support, the microscope objective is arranged on the support, in particular in an objective turret, wherein the support comprises a first port and at least one further port, the tube lens and the intermediate image are arranged in the region of the first port, the at least one further port having a further tube lens and a further intermediate image, wherein the sample light can be directed to two ports 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 optical unit, the sensor, the beam deflection unit and the scanning unit are arranged in a module which is optically and mechanically detachably connected to one of the ports.

13. The microscope according to any one of the preceding claims, wherein: The light source comprises three or more non-overlapping spectral bands that can be selectively emitted, and the main beam splitter is implemented as a spectral N-fold notch filter (N=3, 4, 5, 6), and each notch of the notch filter spectrally corresponds to a corresponding spectral band of the spectral band from the light source. The light source is particularly equipped with a control unit, which is configured to perform a method according to any one of the following claims.

14. A method for scanning image acquisition by means of a scanning microscope, the scanning microscope having an illumination beam path, a detection beam path, a primary beam splitter and a secondary color splitter, the illumination beam path comprising at least one light source having three or more non-overlapping spectral bands that can be selectively emitted, the detection beam path comprising a microscope objective and a two-dimensional spatial resolution sensor for recording light from a sample space, the primary beam splitter being implemented as a spectral N-fold notch filter (N=3, 4, 5, 6) that optically couples the illumination beam path and the detection beam path to each other, wherein each notch of the notch filter has a wavelength of 1 / 400 nm. The secondary color splitter is designed as a high-pass or low-pass filter with a spectral edge and is optically arranged between the primary beam splitter and the sensor, wherein each of the positions to be scanned in the sample space is sequentially illuminated with a first spectral band and a second spectral band, and during the illumination with the first spectral band there is also illumination with a third spectral band, wherein the first spectral band and the second spectral band are spectrally located on a first spectral side of a spectral edge of the secondary color splitter, and the third spectral band is located on a second spectral side different from the first spectral side.

15. The method according to claim 13, wherein: The selection of the simultaneously illuminated spectral bands and the changing of the sequentially illuminated spectral bands are achieved without mechanical movement, in particular by means of one or more acousto-optic elements.

16. The method according to claim 13 or 14, wherein: A microscope with four illumination light spectral bands and a quad-notch filter with these spectrally corresponding notches is used as a main beam splitter, or a microscope with six or more illumination light spectral bands and an interchangeable plurality of notch filters with notches spectrally corresponding to different subsets of the spectral bands are used as a main beam splitter, in particular two quad-notch filters on a filter wheel or a filter slider or a quad-notch filter and a double notch filter on a filter wheel or a filter slider, in particular with an electric drive in each case.

17. The method according to claim 13, 14 or 15, wherein: Each position to be scanned in the sample space is sequentially illuminated with pairs of spectral bands, wherein, in each of these pairs, one of the spectral bands is located on a first spectral side of a spectral edge of the sub-color separator and the other spectral band is located on a second spectral side of a spectral edge of the sub-color separator, and in particular during each constant position of the beam deflection unit, the pairs of spectral bands are immediately and continuously illuminated at each position to be scanned in the sample space.

18. The method according to any one of claims 13 to 16, wherein: Use of a microscope according to any one of claims 1 to 12.

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