MICROSCOPE AND METHOD FOR IMAGING A SAMPLE

Description

[0001] The invention relates to a microscope for imaging a sample, comprising an illumination unit for emitting illumination light onto the sample, a detector for capturing detection light originating from the sample, optics for focusing the illumination light emitted by the illumination unit into the sample and for focusing the detection light originating from the sample onto the detector, and a scanning unit for scanning the sample with the illumination light. Furthermore, the invention relates to a method for microscopically imaging a sample.

[0002] Particularly in fluorescence microscopy, microscopy techniques have recently been used in which the sample is illuminated with a flat or linear light distribution. Examples of these are microscopy techniques known as SPIM (Single Plane Illumination Microscopy), OPM (Oblique Plane Microscopy) and SCAPE (Swept Confocally-Aligned Planar Excitation). In an SPIM microscope, for example, an illuminating light beam is focused in only one direction using a cylindrical lens, for example, and thus widened. This illuminates the sample with a light sheet or disk that only illuminates one sample plane within the sample. An SPIM microscope has two separate objectives on the sample side for illumination and detection, whose optical axes are perpendicular to each other. The sample plane to be imaged is perpendicular to the optical axis of the detection objective.The illumination of this sample plane is provided by the light sheet, which the illumination objective radiates into the sample perpendicular to the optical axis of the detection objective.

[0003] In contrast, the SCAPE method uses a single sample-side objective for both illumination and detection. Illumination is achieved with a light sheet positioned at an angle to the optical axis of the objective. Due to this inclined position of the light sheet, a SCAPE microscope typically features an erecting optics system that interacts with the objective, with tilted sub-optics positioned at an angle to each other. These optics ensure, via intermediate imaging, that the sample area illuminated by the tilted light sheet is imaged correctly onto the detector.

[0004] For a more detailed explanation of the aforementioned SPIM, OPM and SCAPE methods, reference is made to the publications Kumar, S. et al., High-speed 2D and 3D fluorescence microscopy of cardiac myocytes. Opt. Express 19, 13839 (2011); Dunsby, C., Optically sectioned imaging by oblique plane microscopy, Opt. Express 16, 20306-20316 (2008) and Bouchard, MB et al., Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms, Nat. Photonics 9, 113-119 (2015) as well as to the patent documents US 8582203 B2 and US 8619237 B2.

[0005] A related microscopy technique that does not require an upright lens is the so-called HILO (Highly Inclined and Laminated Optical Sheet) technique. For more information, see Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K., "Highly inclined thin illumination enables clear single-molecule imaging in cells.", Nat. Methods 5, 159-161 (2008).

[0006] In DE 10 2011 000 835 B4, the light sheet intended for oblique illumination of the sample is generated using a scanning unit located in a plane conjugate to the rear focal plane of the illumination lens. The fluorescent light to be detected is coupled out between the lens and the scanning unit.

[0007] Finally, regarding the prior art, reference is made to WO 2015 / 109323 A2, which shows, among other things, a microscope setup in which a dichroic mirror for separating the fluorescent light to be detected is arranged downstream of a scanning mirror in the light direction, so that the fluorescent light is directly descanned by the scanning mirror. This setup largely corresponds to the setup of a point-scanning confocal microscope. However, it differs from this in that the illumination beam is weakly focused and directed obliquely into the sample. Secondly, the fluorescent light collected from the oblique line focus running counter to the focal plane of the objective is erected using an erecting lens. This allows image information along the illuminated line to be acquired with the help of a line sensor.Since the scanning mirror is used both to scan the sample with the illumination beam and to descant the detected fluorescent light, a stationary detector can be used.

[0008] The methods described above, known from the prior art, only provide for the detection of a single sample plane at a given time. This makes it comparatively complex to image an extended sample volume. Furthermore, the concentration of the illumination light on a single sample plane can lead to undesirable saturation of fluorophores in some of these methods.

[0009] WO 2015 / 184124 A1 discloses a light-sheet microscope comprising two diametrically opposed illumination objectives that illuminate the sample in two offset sample planes. Two detector units are located on opposite sides of the sample, each consisting of a detection objective, a tube lens, and an adjacent detector. Of the two illuminated sample planes, one is imaged onto one detector and the other onto the other detector.

[0010] Document 2015 / 177506 A1 describes a light-sheet microscope designed to illuminate a sample with multiple parallel, offset light sheets. For this purpose, the light-sheet microscope has either a cylindrical lens array, a phase modulator, a prism, a light guide, and connected collimator lenses, or several separate light sources.

[0011] The publication by KAVYA MOHAN ET AL: "Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy," PLOS ONE, Vol. 9, No. 6, June 9, 2014 (2014-06-09), page e96551, XP055277114, DOI: 10.1371 / Journal.pone.0096551, discloses a light-sheet microscope that focuses multiple, parallel, offset light sheets onto a sample. The sample planes illuminated by the light sheets are imaged onto a CCD camera via a detection optics arranged perpendicular to the illumination beam path. To image the various sample planes, the sample or the detection optics is moved along the detection axis.

[0012] The publication PAUL A. DALGARNO ET AL: "Multiplane imaging and three dimensional nanoscale particle tracking in biological microscopy", OPTICS EXPRESS, Vol. 18, No. 2, January 18, 2010 / (2010-01-18), page 877, XP055123803, ISSN: 1094-4087, DOI: 10.1364 / OE.18.000877 discloses a detection arrangement with a square-distorted grating that makes it possible to simultaneously capture parallel, offset focal planes on a single detection surface of a camera.

[0013] From the subsequently published patent application WO 2016 / 189012 A1, a microscope according to the preamble of claim 1 is known. Regarding the prior art, reference is also made to document WO 2015 / 109323 A2. This document discloses a system in which the illumination light beam and the detection light beam for light-sheet microscopy imaging are guided through the same objective.

[0014] The object of the invention is to provide a microscope and a method for imaging a sample that enable efficient and gentle volume imaging.

[0015] The invention solves this problem by the subject matter of the independent claims. Advantageous further developments are specified in the dependent claims.

[0016] The invention makes it possible to simultaneously illuminate and spatially separate multiple sample regions. For this purpose, the detector has separate detection channels assigned to the spatially separated sample regions. The separation of the illumination light beams within the sample is selected such that crosstalk of the detection light from its assigned detection channel to another detection channel is reliably avoided. Such crosstalk would increase the interfering signal background in the individual images and thus reduce image contrast.If it is assumed that the optics according to the invention, which focuses the detection light originating from the sample onto the detector, defines a detection volume in the form of a point spread function, PSF for short, for each illuminated sample area, the desired beam separation within the sample can be suitably determined taking this PSF into account.

[0017] The microscope according to the invention can be operated both like a conventional SPIM microscope and like a conventional SCAPE or OPM microscope, with the difference that at a given time, not just a single contiguous sample area, but several spatially separated sample areas are illuminated. In particular, the illuminating light beams can also be used to create multiple light sheets that simultaneously illuminate the sample in different sample planes. For example, it is conceivable to create the light sheet sequentially within a sample plane under consideration using the scanning unit. In the sample plane under consideration, the detector therefore "sees" the moving illuminating light beam in the form of a contiguous light distribution that forms the light sheet.In this case, a spatially separated recording of the detection light occurs only with respect to the different sample planes, but not within a sample plane under consideration.

[0018] The distribution of the illumination light according to the invention can be used advantageously for gentle sample illumination, in particular to avoid undesired saturation of the fluorophores.

[0019] Preferably, the illumination unit, the scanning unit and the optics interact in such a way that the illumination light beams in the sample have a mutual parallel offset.

[0020] This parallel offset can include a first parallel offset along a first axis perpendicular to the optical axis of a detection objective included in the optics, and / or a second parallel offset along a second axis perpendicular to the optical axis of the detection objective, wherein the first and second axes are aligned perpendicular to each other. The illumination light beams offset parallel to each other in the sample allow the detector to be designed in a comparatively simple manner such that a desired, spatially separated detection of the detection light beams is possible.

[0021] In an advantageous embodiment, the scanning unit is configured to scan the sample with the illuminating light beams along a first axis perpendicular to the optical axis of a detection objective contained in the optics, and / or along a second axis perpendicular to the optical axis of the detection objective, wherein the first axis and the second axis are aligned perpendicular to one another. In this embodiment, it is possible, for example, to build up a light sheet within a predetermined sample plane in the manner described above using the scanning movement along the first axis, while the thus-built light sheets are successively displaced within the sample by the scanning movement along the second axis. This enables particularly efficient volume imaging.

[0022] In a preferred embodiment, the scanning unit comprises a scanning mirror, wherein the scanning mirror is tiltable about the second axis in order to scan the sample with the illumination light beams along the first axis, and / or the scanning mirror is tiltable about a first axis in order to scan the sample with the illumination light beams along the second axis. For example, a galvanometer mirror (for example with gimbal suspension) or a microelectromechanical mirror (MEMS) can be used as a 1D or 2D scanner. Alternatively, instead of a single scanning mirror, a combination of two or more mirrors can also be used. However, the invention is not limited to this type of scanning unit. For example, the use of an acousto-optical deflector (AOD) is also conceivable. Combinations of the aforementioned techniques are of course also feasible.

[0023] The optics contain a single objective lens, which simultaneously forms an illumination objective for focusing the illumination light beam emitted by the illumination unit onto the sample and a detection objective for focusing the detection light beam originating from the sample onto the detector. This allows the microscope according to the invention to be operated in the manner of a SCAPE or OPM microscope.

[0024] Preferably, the scanning unit is additionally configured to descan the detection light beams focused on the detector. "Descanning" here means that the detection light originating from the sample is returned through the single sample-side objective lens to the scanning unit, which is already acting on the illumination light, before it falls on the detector. By returning the detection light to the scanning unit, the scanning movement imparted to the illumination light by the scanning unit is counteracted, resulting in a particularly advantageous result in the use of a stationary detector to capture the detection light.

[0025] Preferably, the illumination unit and the scanning unit interact such that the illumination light beams enter an entrance pupil of the objective, tilted relative to one another about the second axis, in order to cause the first parallel offset along the first axis in the sample, and / or the illumination unit and the scanning unit interact such that the illumination light beams enter the entrance pupil of the objective, tilted relative to one another about the second axis, in order to cause the second parallel offset along the second axis in the sample. The tilting of the illumination light beams in the objective pupil therefore leads to a displacement of the illumination light beams exiting the objective transversely to the optical axis of the objective.

[0026] In a preferred embodiment, the illumination unit and the scanning unit interact in such a way that the illumination light beams each illuminate only an off-center portion of an entrance pupil of the objective. This off-center illumination of the objective pupil results in an oblique position of the illumination light beam exiting the objective relative to the optical axis of the objective.

[0027] Preferably, an erecting optics system is provided downstream of the scanning unit in the propagation direction of the detection light. It comprises a first partial optics system for generating an intermediate image and a second partial optics system, positioned at an angle relative to the first partial optics system, for projecting the intermediate image onto the detector in the correct position. The erecting optics system thus ensures the erection of the sample area imaged by the objective lens under oblique illumination.

[0028] In a particularly preferred embodiment, the sample regions illuminated by the illumination light beams are located in several sample planes, preferably offset parallel to one another. By simultaneously acquiring image data from the different sample planes, volume imaging becomes particularly efficient.

[0029] The detector comprises several sub-detectors, each of which is assigned to one of the sample planes, whereby the respective sub-detector detects the detection light beam which is emitted from the sample plane assigned to this sub-detector.

[0030] The partial detectors form separate detection channels, which enable the spatially separated capture of the detection light beams.

[0031] The partial detectors are arranged within a depth of field of a detection optics arranged upstream of the detector. In the SCAPE application, the detection optics are formed, for example, by the erection optics. In this embodiment, it is possible to design the detector in the form of a one-piece area detector comprising several linear or strip-shaped detector regions that form the partial detectors.

[0032] In an alternative embodiment, at least one element arranged upstream of the detector is provided to influence the optical path length, which compensates for the offset of the sample planes.

[0033] The element mentioned is, for example, a glass block that compensates for the offset of the illumination light beams along the optical axis of the detection objective to enable a sharp image of the illuminated sample plane. The use of a one-piece area detector is also possible in this embodiment.

[0034] In a further preferred embodiment, the detector comprises a plurality of line detectors, which form the sub-detectors. This embodiment allows the line detectors to be arranged along the optical axis of the detection objective in such a way that the axial offset of the illumination light beams within the sample is taken into account.

[0035] It is also possible to provide a suitable optics system for each individual illumination light beam, ensuring a sharp image on the associated line detector. It should be noted that the axial offset of the illumination light beams within the sample is typically in the range of only a few micrometers, which is so small that correction of imaging errors, especially spherical aberrations, is generally not necessary.

[0036] In an alternative embodiment, not according to the claims, the sample areas illuminated by the illumination light beams can also lie only in a single sample plane. In this case, the sample is scanned with the illumination light beams only in one sample plane. This makes it possible to generate images of this single sample plane in very rapid succession. The time between the individual images of this sample plane can be significantly shorter than the time a single camera would need to image the entire image field several times in succession. The time between the individual images is even much shorter than the time required to expose a single sample plane. In this embodiment, for example, image sequences can be acquired that are intended to depict extremely fast-running processes.For example, technically very complex double-pulse systems are known from PIV microscopy (Particle Image Velocimetry). These systems use an expensive laser that emits two extremely powerful light pulses in quick succession. A camera then records two images. However, the system consisting of the camera and laser requires a long pause after each of these double pulses before another pair of images can be recorded. This technique is very valuable for the analysis of flow profiles, which, however, must be in flow equilibrium, since the entire flow profile can only be calculated from a large number of image pairs. In the aforementioned embodiment, the microscope according to the invention can be used, for example, for the corresponding observation of extremely fast processes in biomedical research.

[0037] The illumination unit may comprise a single light source that emits the separate illumination beams. However, it is also possible to provide a separate light source for each illumination beam, thus avoiding coherence between the individual illumination beams.

[0038] The illumination unit is preferably configured to emit illumination light beams with different wavelengths. These different wavelengths can be separated accordingly from a single broadband light source or from different light sources. In this embodiment, it is conceivable, for example, to shift the wavelengths of the illumination light beams relative to one another so strongly that the illumination light beams excite different fluorophores, whose fluorescent light can be spectrally separated using simple filters. In this case, the illumination light beams do not necessarily have to be offset along the scanning direction (in the sample plane perpendicular to the propagation direction of the illumination beams) or run in different sample planes.

[0039] In an advantageous embodiment, the detector comprises a TDI (Time Delay Integration) line scan camera. Such a TDI line scan camera consists of several adjacent sensor lines and enables time-delayed multiple exposure of a moving object. At the end of each exposure period, the charges generated so far are shifted line by line to the next higher line. During subsequent exposure periods, further charges are added, summed, transported line by line, and finally output as a signal after a number of exposures corresponding to the number of sensor lines. In the present context, such a TDI line scan camera can be used to perform line-like capture of the detection light beams without relying on a single-line sensor.For example, a detection light beam is usually wider than a single pixel row of a single-line sensor, so that this pixel row is significantly outshone by the detection light beam. Unlike a single-line sensor, a TDI line scan camera can capture the entire width of the detection light beam without blurring the detection signal. In particular, this does not require step-by-step scanning. Instead, a TDI line scan camera allows continuous scanning.

[0040] Preferably, the scanning unit and the optics form a telecentric system.

[0041] According to a further aspect of the invention, a method for microscopically imaging a sample is provided. This method is particularly intended to be applied in the manner of a SPIM, OPM, or SCAPE method. It can also be combined with pulsed lasers for two-photon excitation.

[0042] The invention is explained in more detail below using exemplary embodiments with reference to the figures. Fig. 1a conventional SCAPE microscope as a comparison example; Fig. 2the conventional SCAPE microscope according to Figure 1 from a different viewing direction; Fig. 3 a microscope according to the invention, which operates in the manner of a SCAPE microscope, as a first embodiment; Fig. 4 the first embodiment of the microscope according to the invention from a different viewing direction; Fig. 5 a schematic representation to illustrate how, in the first embodiment, the sample areas illuminated with the illumination light beams are imaged onto the detector; Fig. 6 a schematic representation corresponding Figure 5from a different viewing direction; Fig. 7 a schematic representation to illustrate how, in a second embodiment of the microscope according to the invention, the sample areas illuminated with the illuminating light beams are imaged onto the detector; Fig. 8 a schematic representation corresponding Figure 7 from a different viewing direction; Fig. 9 a schematic representation to illustrate how, in a third example not according to the claims, the sample areas illuminated with the illumination light beams are imaged onto the detector; Fig. 10 a schematic representation corresponding Figure 9 from a different perspective; Fig. 11 a schematic representation corresponding Figure 9 with a different geometric representation of the light beams; Fig. 12 a schematic representation corresponding Figure 10with a different geometric representation of the light beams; Fig. 13 shows a block diagram with exemplary functional components for implementing a microscope according to the invention that operates in the manner of a SCAPE microscope; and Fig. 14 shows a block diagram with exemplary functional components for implementing a microscope according to the invention that operates in the manner of a SPIM microscope.

[0043] First, the Figures 1 and 2 the structure of a conventional SCAPE microscope 10 is explained, to which reference is made below to explain the embodiments according to the invention. Figures 1 and 2 and all other figures refer to an orthogonal coordinate system with the axes x, y and z.

[0044] As in the Figures 1 and 2As shown, the SCAPE microscope 10 contains an illumination unit 12 with a light source 14 that emits an illumination light beam 16. The illumination light beam 16 strikes a mirror 18, which reflects the illumination light beam 16 towards a 2D scanning mirror 20. The scanning mirror 20, which is designed, for example, as a galvanometer mirror or MEMS mirror, is rotatable both around the x-axis (in Figure 1 perpendicular to the plane of the drawing) and around the y-axis (in Figure 2 perpendicular to the plane of the drawing) by means of a drive not shown in the figures. The illumination light beam 16 reflected by the scanning mirror 20 enters an optics system 22, which is formed from a scanning lens 24, a tube lens 26, and an objective 28. The scanning mirror 20 is located in a plane conjugate to the rear focal plane of the objective 28. The scanning mirror 20 and the optics system 22 form a telecentric system.

[0045] As in Figure 1As shown, the illuminating light beam 16 falls on the scanning mirror 20 in such a way that it is irradiated into the objective 28 offset along the y-axis. The illuminating light beam 16 therefore only illuminates an off-center portion of the entrance pupil of the objective 28, whereby it propagates within the sample 28 tilted relative to the optical axis of the objective 28. The illuminating light beam 16 thus illuminates the sample 28 along a line- or strip-shaped area which Figure 1 labeled A, and excites a sample 30 to emit fluorescent radiation. To simplify the illustration, the sample 30 is only Figure 1 shown.

[0046] The light given by the fluorescence radiation and in the Figures 1 and 2The detection light beam, designated 32, in turn enters the objective lens 30, which thus functions as both an illumination objective lens and a detection objective lens. After passing through the tube lens 26 and the scanning lens 24, the detection light beam 32 falls onto the scanning mirror 20, which reflects the detection light beam 32 such that it passes the mirror 18 and enters an erecting optics 34. The erecting optics 34 comprises a second objective lens 36, a third objective lens 38, and a tube lens 40, followed by a line detector 42. By being returned to the scanning mirror 20, the detection light beam 32 is descanned, similar to a confocal microscope. In this way, the illuminated sample area A is imaged into a stationary strip A' by means of an intermediate image in the erecting optics 34. The strip A' is inclined with respect to a plane 43 which is optically conjugate to the plane shown in the Figures 1 and 2focal plane of the optics 22, designated 44.

[0047] As in Figure 1 As shown, the erection optics 34 comprises a first partial optic in the form of the objective 36 and a second partial optic, which is inclined relative thereto and is formed from the objective 38 and the tube lens 40. These two partial optics 36 and 38, 40, respectively, which are inclined relative to one another, ensure that the strip A' is imaged in the correct position onto the line detector 42. The descanning effect of the scanning mirror 20 also ensures that the illuminated sample area A is imaged in a fixed position onto the line detector 42 via the stationary intermediate image A'.

[0048] At this point it should be noted that the representation of the detection beam path in Figure 2 is greatly simplified. In fact, the part of the detection beam path lying between the scanning mirror 20 and the lens 36 runs perpendicular to the plane of the Figure 2, while the part of the detection beam path following it in the direction of the line detector 42 is inclined from the plane of the Figure 2 runs out.

[0049] In the Figures 3 and 4 a microscope 50 is shown which is an inventive further development of the SCAPE microscope 10 according to the Figures 1 and 2 The microscope 50 according to the invention is modified from the conventional SCAPE microscope 10 in that it scans the sample at a given time not with a single illuminating light beam, but with several spatially separated illuminating light beams. Those functional components of the microscope 50 that correspond to those of the conventional SCAPE microscope 10 are provided with the Figures 1 and 2 used reference symbols and are not explained again below.

[0050] In the embodiment according to the Figures 3 and 4the light source 14 emits three illumination light beams 52, 54 and 56. For simplification, the illumination light beams 52, 54 and 56 are Figures 3 and 4 only represented in the form of their main rays.

[0051] The illumination light beams 52, 54, 56 are reflected by a lens 46 from the mirror 18 and from there focused onto the scanning mirror 20. As in Figure 3 As shown, the illumination light beams 52, 54, 56 are tilted relative to one another onto the scanning mirror 20 such that, after passing through the scanning lens 24 and the tube lens 26, they enter the entrance pupil of the objective 28 tilted relative to one another along the y-axis. This tilt along the y-axis results in the illumination light beams 52, 54, 56 having a first parallel offset along the x-axis within the sample (cf. Figures 5 and 6 ). Furthermore, the illumination light beams, as in Figure 4shown, are reflected at the scanning mirror 20 in such a way that, after passing through the scanning lens 24 and the tube lens 26, they fall into the entrance pupil of the objective 28 tilted to one another along the x-axis.

[0052] This tilting causes the illumination light beams 52, 54, 56 to have a second parallel offset along the y-axis within the sample (cf. Figures 5 and 6 ).

[0053] By tilting the scanning mirror 20 about the x-axis, the sample is scanned along the y-axis with the illumination light beams 52, 54, 56. Accordingly, when the scanning mirror 20 is tilted about the y-axis, the sample is scanned along the x-axis with the illumination light beams 52, 54, 56.

[0054] The illumination light beam 52 illuminates a strip-shaped sample region B within the sample. Correspondingly, the illumination light beam 54 illuminates a strip-shaped sample region C and the illumination light beam 56 illuminates a strip-shaped sample region D. The fluorescence radiation originating from the illuminated sample regions B, C and D returns to the scanning mirror 20 in the form of separate detection light beams 58, 60 and 62 after passing through the objective 28, the tube lens 26 and the scanning lens 24. The scanning mirror 20 reflects the detection light beams 58, 60 and 62 into the erection optics 34. In this, intermediate images of the sample regions illuminated by the illumination light beams B, C and D are generated, which are inclined relative to the plane 43, which is conjugate to the focal plane 44 of the optics 22. In the simplified representation according to Figure 3 These intermediate images are labeled B', C' and D' respectively.

[0055] The microscope 50 has a detector 71 formed from three line sensors 66, 68, 70, which detects the light beams 58, 60 and 62, respectively. The intermediate image B' is imaged in the correct position on the line sensor 66, the intermediate image C' is imaged in the correct position on the line sensor 68, and the intermediate image D' is imaged in the correct position on the line sensor 70. As shown in the Figures 3 and 4 As shown, the line sensors 66, 68 and 70 are offset from one another according to the offset of the illuminated sample areas B, C and D within the sample and, accordingly, the intermediate images B', C', D'.

[0056] In the Figures 5 and 6 It is again illustrated how the strip-shaped sample areas B, C and D illuminated by the illumination light beams 52, 54, 56 are imaged in the form of the detection light beams 58, 60 and 62 onto the line sensors 66, 68 and 70, respectively. Figure 5 with Figure 3 and Figure 6 with Figure 4 . The Figures 5 and 6, in which the erection optics 34 is only indicated, show in particular that the line sensors 66, 68 and 70 are arranged both axially, ie along the z-axis or optical axis O of the lens 28, and laterally, ie offset from one another along the x-axis. Figures 5 and 6 The scanning directions in which the illumination light beams 52, 54, 56 are moved by tilting the scanning mirror 20 are also indicated. If the scanning mirror 20 is tilted about the x-axis, the illumination light beams 52, 54, and 56 are moved along the y-axis. If, however, the scanning mirror 20 is tilted about the y-axis, the illumination light beams 52, 54, and 56 are moved along the x-axis.

[0057] In the Figures 7 and 8A modified embodiment of the microscope 50 is shown as a second exemplary embodiment. In this exemplary embodiment, glass blocks 72 and 74, respectively, are arranged in front of the line sensors 66 and 68. The glass blocks 72, 74 serve to compensate for the offset of the illuminating light beams along the optical axis O of the objective 28. The line sensors 66, 68, 70 can thus be arranged in one plane. In particular, it is also possible to provide a one-piece area sensor with separate sensor areas corresponding to the line sensors instead of the three line sensors 66, 68, 70.

[0058] The Figures 9 and 10 show a third non-claimed example in which the microscope operates in the manner of an SPIM microscope. Accordingly, in the third example, two separate objectives are provided on the sample side, of which an objective 80 serves as a detection objective and an objective 82 (cf. Figure 9) acts as an illumination lens. The two lenses 80 and 82 are arranged with their optical axes perpendicular to each other.

[0059] In the Figures 11 and 12 The third example is shown with a geometric representation of the respective light beams.

[0060] Figure 13 shows, using a block diagram, an example of the implementation of the microscope according to the invention in the manner of a SCAPE microscope. In the illustration according to Figure 13 The arrows between individual functional components indicate the path of the illumination light or the detection light within the microscope.

[0061] The realization according to Figure 13As exemplary functional components, it includes a laser 90 as a light source, which may have a beam expander, a beam splitter 92 in the form of a grating, an acousto-optical beam splitter (AOBS), a spatial light modulator (SLM), or a digital micromirror array, a 2D scanner 94 in the form of one or more galvanometer mirrors or MEMS mirrors, a scan lens 96, a tube lens 98, and an objective 100 that serves both for illumination and detection. The 2D scanner 94, the scan lens 96, the tube lens 98, and the objective 100 form a telecentric system. In the part of the detection light path adjoining the 2D scanner 94, the microscope further contains an erection optics 102, which has, for example, two objectives tilted relative to one another, as well as several line or strip sensors 104 or, in an alternative embodiment, one or more glass blocks arranged upstream of an area sensor.

[0062] Finally, Figure 14 a block diagram with functional components for implementing the microscope according to the invention as a SPIM microscope. Again, the arrows between the functional components indicate the light path of the illumination light and the detection light, respectively. Figure 14 As can be seen, the SPIM implementation differs from the SCAPE implementation only by the use of a 1D scanner 94' (such as an acousto-optical deflector (AOD), a galvanometer mirror, or a MEMS mirror) instead of the 2D scanner 94, and the use of two separate sample-side objectives 100', 100" as illumination and detection objectives, respectively, instead of the single sample-side objective 100.

[0063] The embodiments described above are merely exemplary. A number of modifications are conceivable for implementing the teachings of the invention. For example, in the embodiment according to the Figures 3 and 4 The mirror 18 is provided for coupling the illumination light. However, instead of the mirror 18, a dichroic mirror element can also be provided, which couples the illumination light in transmission and directs the detection light in reflection onto the detector (or vice versa).

[0064] The embodiments described above relate to the implementation of the microscope according to the invention in the manner of a SCAPE microscope or an SPIM microscope. As already mentioned above, the invention is not limited to these implementations. For example, an implementation as an OPM microscope is also conceivable. In particular, it is with reference to the SCAPE version according to the Figures 3 and 4It is also possible, instead of the tilting of the 2D scanning mirror 20 provided there, to move the objective 36 in the sense of an OPM application (cf. the publication by Kumar et al. mentioned above) in order to effect the synchronous displacement of the illumination light bundles and the plane to be imaged. In this case, a beam coupling via the objective 36 is then required. Thus, while the scanning unit according to the invention has a 2D scanning mirror in the SCAPE application, it comprises a 2D scanning mirror in the OPM application (with reference to the Figures 3 and 4 ) an actuator which moves the lens 36 along the optical axis to offset the illumination along the y-axis, and a scanner (or alternatively a cylindrical lens) to offset the illumination along the x-axis. List of reference symbols

[0065] 10 Conventional SCAPE microscope 12 Illumination unit 14 Light source 16 Illumination light beam 18 Mirror 20 Scanning mirror 22 Optics 24 Scanning lens 26 Tube lens 28 Objective 30 Sample 32 Detection light beam 34 Erecting optics 36 Objective 38 Objective 40 Tube lens 42 Line sensor 43 Plane 44 Focus plane 46 Lens 50 Microscope 52, 54, 56 Illumination light beam 58, 60, 62 Detection light beam 66, 68, 70 Line sensors 71 Detector 72, 74 Glass blocks 80 Detection objective 82 Illumination objective 90 Laser 92 Beam splitter 94 2D scanner 96 Scanning lens 98 Tube lens 100Lens 100', 100"Lenses 104Strip Sensors 106Glass Blocks

Claims

1. A microscope (50) for imaging a sample (30), comprising: an illumination unit (12) for emitting illumination light onto the sample (30), a detector (71) for detecting detection light originating from the sample (30), optics (22) for focusing the illumination light, emitted by the illumination unit (12), into the sample (30) and for focusing the detection light, originating from the sample (30), onto the detector (71), and a scanning unit (20) for scanning the sample (30) with the illumination light, wherein the illumination unit (12) is configured to emit the illumination light in the form of separate illumination light beams (52, 54, 56) onto the scanning unit (20) such that the illumination light beams (52, 54, 56) are simultaneously focused on spatially separated, strip-shaped sample areas (A, B, C) when scanning the sample (30), the detector (71) is configured to detect the detection light in the form of separate detection light beams (58, 60, 62), originating from the spatially separated, strip-shaped sample areas (A, B, C) simultaneously and spatially separated from one another, the sample areas (A, B, C), illuminated by the illuminating light beams (52, 54, 56), are located in several sample planes, the detector (71) comprises several partial detectors (66, 68, 70), each of which is assigned to one of the sample planes, wherein the respective partial detector (66, 68, 70) detects the detection light beam (58, 60, 62), originating from the sample plane assigned to this partial detector (66, 68, 70), and the partial detectors are arranged in a depth of field of detection optics, arranged upstream of the detector, characterized in that the optics (22) simultaneously contains a single objective (28) which forms an illumination objective for focusing the illumination light beams (52, 54, 56), emitted by the illumination unit (12) into the sample (30), and a detection objective for focusing the detection light beam (58, 60, 62), originating from the sample (30), onto the detector (71).

2. The microscope (50) according to claim 1, characterized in that the sample planes are offset parallel to one another.

3. The microscope (50) according to claim 1 or 2, characterized in that the illumination unit (12), the scanning unit (20) and the optics (22) interact in such a way that the illumination light beams (52, 54, 56) in the sample (30) have a mutual parallel offset.

4. The microscope (50) according to claim 3, characterized in that the parallel offset includes a first parallel offset along a first axis (x) which is perpendicular to the optical axis of a detection objective (28), contained in the optics (22), and / or a second parallel offset along a second axis (y) which is perpendicular to the optical axis (O) of the detection objective (28), wherein the first axis (x) and the second axis (y) are aligned perpendicular to one another.

5. The microscope (50) according to any one of the preceding claims, characterized in that the scanning unit (20) is configured to scan the sample (30) with the illuminating light beams (52, 54, 56) along a first axis (x) which is perpendicular to the optical axis (O) of a detection objective (28), contained in the optics (22), and / or along a second axis (y) which is perpendicular to the optical axis (y) of the detection objective (28), wherein the first axis (x) and the second axis (y) are perpendicular to each other.

6. The microscope (50) according to claim 5, characterized in that the scanning unit comprises a scanning mirror (20), wherein the scanning mirror (20) is tiltable about the second axis (y) in order to scan the sample (30) with the illumination light beams (52, 54, 56) along the first axis (x), and / or the scanning mirror (20) is tiltable about the first axis (x) in order to scan the sample (30) with the illumination light beams (52, 54, 56) along the second axis (y).

7. The microscope (50) according to any one of the preceding claims, characterized in that the scanning unit (20) is additionally configured to descan the detection light beams (58, 60, 62), focused on the detector (71).

8. The microscope (50) according to any one of the preceding claims, characterized in the illumination unit (12) and the scanning unit (20) cooperate in such a way that the illumination light beams (52, 54, 56), tilted relative to one another about the second axis (y), are incident into an entrance pupil of the objective (28) in order to effect the first parallel offset along the first axis (x) in the sample (30), and / or the illumination unit (12) and the scanning unit (20) cooperate in such a way that the illuminating light beams (52, 54, 56), tilted relative to one another about the second axis (x), are incident into the entrance pupil of the objective (28) in order to cause the second parallel offset along the second axis (y) in the sample (30).

9. The microscope (50) according to any one of the preceding claims, characterized in that the illumination unit (12) and the scanning unit (20) interact in such a way that the illumination light beams (52, 54, 56) each illuminate only an off-centre partial area of an entrance pupil of the objective (28).

10. The microscope (50) according to any one of the preceding claims, characterized by an erecting optics (34), arranged downstream of the scanning unit (20) in the propagation direction of the detection light, with a first partial optics (36) for generating an intermediate image and a second partial optics (38, 40), inclined relative to the first partial optics for imaging the intermediate image in the correct position on the detector (71).

11. The microscope (50) according to any one of the preceding claims, characterized by at least one element (72, 74) arranged upstream of the detector (71) for influencing the optical path length, which compensates for the offset of the sample planes.

12. The microscope according to any one of the preceding claims, characterized in that the detector (71) is a surface detector with several detector portions which form the partial detectors.

13. The microscope (50) according to any one of the preceding claims, characterized in that the detector (71) comprises several line detectors (66, 68, 70) which form the partial detectors.

14. The microscope (50) according to claim 13, characterized in that the line detectors (66, 68, 70) are arranged along the optical axis of a detection optics, arranged upstream of the detector, with an offset from one another which corresponds to the offset of the associated sample planes.

15. The microscope (50) according to any one of the preceding claims, characterized in that the illumination unit (12) has a single light source (14) which emits the separate illumination light beams (52, 54, 56).

16. The microscope (50) according to any one of the preceding claims, characterized in that the illumination unit (12) has several light sources, each emitting one of the illumination light beams (52, 54, 56).

17. The microscope (50) according to any one of the preceding claims, characterized in that the illumination unit (12) is configured to emit the illumination light beams (52, 54, 56) with different wavelengths.

18. The microscope (50) according to any one of the preceding claims, characterized in that the detector comprises a TDI line camera.

19. The microscope (50) according to any one of the preceding claims, characterized in that the scanning unit (20) and the optics (22) form a telecentric system.

20. A method for microscopically imaging a sample (30), in which illuminating light is emitted onto the sample (30) by means of an illumination unit (12), in which detection light, originating from the sample (30), is detected by means of a detector (71), in which, by means of an optics (22), the illumination light, emitted by the illumination unit (12), is focussed into the sample (30) and in which the detection light, originating from the sample (30), is focussed onto the detector (71), wherein the sample (30) is scanned with the illumination light by means of a scanning unit (20) and the detection light, focused on the detector (71), is descanned, wherein the illuminating light is emitted onto the scanning unit (20) in the form of separate illuminating light beams such that the illuminating light beams (52, 54, 56) are simultaneously focused onto spatially separated, strip-shaped sample areas (A, B, C) when scanning the sample (30), the detection light is detected by the detector (71) in the form of separate detection light beams (58, 60, 62) originating from the spatially separated, strip-shaped sample areas (A, B, C) simultaneously and spatially separated from one another, the sample areas (A, B, C), illuminated by the illuminating light beams (52, 54, 56), are located in several sample planes, the detector (71) comprises several partial detectors (66, 68, 70), each of which is assigned to one of the sample planes, wherein the respective partial detector (66, 68, 70) detects the detection light beam (58, 60, 62), which originates from the sample plane, assigned to this partial detector (66, 68, 70), and which are arranged in a depth of field of a detection optics, arranged upstream of the detector, characterized in that the optics (22) contains a single objective which forms an illumination objective for focusing the illumination light beams (52, 54, 56), emitted by the illumination unit (12) into the sample (30), and simultaneously a detection objective for focusing the detection light beam (58, 60, 62), originating from the sample (30), onto the detector (71).