Microscopes and methods for microscopy

Abstract

The present invention relates to a system for detecting an emission light beam path comprising at least an illumination control device and an illumination objective lens for illuminating and scanning a sample with excitation light, a detection beam path comprising at least a microscope objective lens for directing emission light emitted by the sample in the direction of the camera, and a camera for recording an image of the sample. The system further comprises a control unit for controlling at least the illumination control device and the camera, the control unit being configured to synchronize in each case a read area of ​​the sensor range of the camera with the position of the excitation light defined by the illumination control device, the detection beam path including an image splitter unit for splitting the emission light into a plurality of partial beam paths each generating a partial image of the sample on the sensor range of the camera, the partial images being adjacent to one another on the sensor range such that linear areas in the partial images corresponding to the position of the excitation light on or within the sample as defined by the illumination control device are present on one or more of the same lines in the sensor range.

Description

[Technical field]

[0001] In a first aspect, the present invention relates to a microscope according to the preamble of claim 1. Furthermore, the present invention relates to a method for microscopy. [Background technology]

[0002] A typical type of microscope is known, for example from DE 10 200 0133 A1, and comprises an illumination beam path with at least an illumination control and an illumination objective for illuminating and scanning a sample, in particular linearly, with excitation light, a detection beam path with at least a microscope objective for directing emission light emitted by the sample towards the camera, a camera for recording an image of the sample, and a control unit for controlling at least the illumination control and the camera, the control unit being configured to synchronize in each case the read area of ​​the sensor range of the camera with the position of the excitation light defined by the illumination control.

[0003] The study of processes in cell aggregates, organs or whole animals is becoming more and more prevalent in biomedical research. Therefore, the objects of investigation are generally no longer flat two-dimensional objects on glass substrates, but three-dimensional objects intended to be observed in their entire volume at high speed and high sensitivity. For three-dimensional microscopic imaging, laser scanning microscopes (LSM) are well established, but cannot meet the demands for high volumetric rates.

[0004] Higher frame rates can be realized by parallelization. A solution in this regard is presented, for example, in US Pat. No. 5,399,633. In that case, the sample is scanned with a linear light distribution that is synchronized with the rolling shutter of the CMOS camera. The rolling shutter then acts like a movable electronic confocal diaphragm and thus allows the optical sectioning known from LSM with corresponding high-contrast imaging. Although this technique already allows indeed higher frame rates, the volume rate is still limited to a few volumes per second. Furthermore, line illumination in a larger axial sample area increases the sample load, since the light intensity does not fall quadratically as in the case of point illumination, but only linearly with the distance from the focal plane. The emission intensity therefore remains high over a large axial range, without any information about the sample being captured. This unavoidable effect in the case of line illumination is disadvantageous, especially with regard to nonlinear bleaching, as exhibited by the relevant fluorescent proteins (Non-Patent Document 1).

[0005] In order to reduce the sample load and at the same time increase the volume rate, an axially parallelized microscope system has further been proposed (Non-Patent Document 2). In that case, reflective slits are arranged in three different axial planes, thereby reflecting the radiation from the planes conjugate thereto towards the camera. The proposed arrangement is complex in the sense that the radiation needs to be rescanned onto the camera. As a result, many successive imaging stages are required, which give rise to losses and imaging aberrations. Furthermore, this arrangement is not very adaptable, since the reflective element performing the function of the confocal line stop is integrated in the beam path in a positionally fixed and invariant manner. However, as in a point scanning system, this stop also needs to be adaptable to the objective lens.

[0006] The size of e.g. 1 Airy Unit (AU) is usually used as the aperture size. The Airy unit is defined here by imaging the point spread function in the sample, determined by the numerical aperture (NA) of the objective, onto the aperture plane with the corresponding magnification as given by the objective and the tube lens. Adaptations are required if adjustments for different resolution levels and screen sizes are required. In the case of the solution proposed by Tsang et al., the aperture is imaged onto the camera. In case of surface defects, disturbing fringes can therefore occur in the image.

[0007] From wide-field microscopy, so-called image division techniques are then known, which allow multiple object planes to be arranged next to each other on the camera sensor (Patent Document 2 or Non-Patent Document 3). In particular the arrangement from Sheng Xiao et al. can be prone to image artifacts, since the optical interface is arranged close to the image plane. Alternative configurations, such as the mechanism described in Patent Document 3, are relatively complex and do not seem suitable for series production. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,335,898 [Patent Document 2] British Patent No. 2442576B [Patent Document 3] International Publication No. 13106731A1 [Non-patent literature]

[0009] [Non-Patent Document 1] Sci.Rep.2015 Oct 20;5:15348.doi:10.1038 / srep15348 [Non-Patent Document 2] Jean-Marc Tsang et al.,Biomed.Opt.Expr.12,1339(2021) [Non-Patent Document 3] Sheng Xiao et al.,Optica 7,1477(2020) Summary of the Invention [Problem to be solved by the invention]

[0010] It may be considered an objective of the present invention to detail a microscope and a microscopy method in which high frame and volume rates are achieved with an acceptably light load on the specimen and acceptable instrumental complexity. [Means for solving the problem]

[0011] This object is achieved by a microscope having the features of claim 1 and by a method having the features of claim 24.

[0012] The above-mentioned type of microscope is developed according to the invention by the fact that the detection beam path includes an image splitter unit for splitting the emission light into a number of partial beam paths, each of which generates a partial image of the sample in the sensor range of the camera, the partial images being adjacent to one another on the sensor range such that linear areas in the partial images corresponding to the position of the excitation light on or in the sample as defined by the illumination control device are on the same line or lines on the sensor range.

[0013] In the microscopy method according to the present invention, the following steps are performed: a sample is illuminated, in particular linearly, with excitation light by an illumination objective and scanned, emission light emitted by the sample is directed towards a camera via a microscope objective, downstream of the microscope objective the emission light is split into a number of partial beam paths, each of which generates a partial image of the sample on the sensor range of the camera, the partial images of the sample are located adjacent to one another on the sensor range such that linear areas in the partial images corresponding to the position of the excitation light on or within the sample are located on one or more same lines in the sensor range, and finally the read area of ​​the sensor range of the camera is synchronized with the position of the excitation light on the sample.

[0014] A microscope according to the invention is suitable for carrying out the method according to the invention. The method according to the invention can in particular be carried out by means of a microscope according to the invention.

[0015] Preferred configurations of the microscope according to the invention and advantageous variants of the method according to the invention are explained below, in particular in conjunction with the dependent claims and the figures.

[0016] The excitation light is electromagnetic radiation, in particular in the visible spectral range and adjacent ranges. The only requirement imposed on the contrast principle according to the invention is that the sample emits emission light as a result of illumination with the excitation light. Typically, the emission light is fluorescence emitted by the sample, in particular the dye molecules present therein, as a result of illumination with the excitation light.

[0017] The term "illumination beam path" refers to all optical beam guiding and beam modifying components, e.g. lenses, mirrors, prisms, gratings, filters, diaphragms, beam splitters, by and through which the excitation light is guided from a light source, e.g. a laser, to a sample to be examined. The illumination beam path includes at least an illumination control device, e.g. a scanner with at least one galvanometer mirror, and an illumination objective. The illumination objective may be a microscope objective of a type known per se. The illumination beam path may be realized by the illumination beam path of a laser scanning microscope.

[0018] The light emitted by the sample under examination as a result of illumination by the excitation light is called emission light and reaches the camera via a detection beam path. The term "detection beam path" refers to all beam-guiding and beam-modifying optical components, such as lenses, mirrors, prisms, diffraction gratings, filters, diaphragms, beam splitters, by and through which the emission light is guided from the sample under examination to the camera. The camera is a sufficiently fast optical detector with a two-dimensional spatially resolved sensor range, the pixels of which can be read at least locally, in particular line-by-line, at high speed.

[0019] The term "control unit" refers to all hardware and software components, which interact with the components of the microscope according to the invention for the intended function of the software components. In particular, the control unit may have a computing device, for example a PC, and a camera control device, which is able to rapidly read out the measurement signals, in particular from the lines of the sensor range. According to the invention, the control unit is in each case configured to synchronize the read area of ​​the sensor range of the camera, in particular the lines of the sensor range, with the position of the excitation light on or in the sample, the position of which is defined by the illumination control device, for example a scanner, in order to position it more precisely by the settings of the scanner. The control unit may also be configured to evaluate the image data provided by the camera.

[0020] The term "image splitter unit" refers to all beam directing and beam modifying optical components, e.g. lenses, mirrors, prisms, diffraction gratings, filters, diaphragms, beam splitters, by and through which the emitted light is directed into at least two partial images onto the sensor range of the camera.

[0021] The camera can be, for example, a CMOS or sCMOS camera with rolling shutter electronics. The rolling shutter is expediently synchronized with the scanning speed of the line illumination. The read-out camera pixels are therefore optically conjugate with the instantaneous position of the line focus on the sample. It is likewise possible to use a SPAD camera whose read-out matrix is ​​dynamically adjustable, thus allowing synchronization with the advancement of the line focus. As an example, a camera of the type "pco.edge 10 bi CLHS" from Excelitas PCO GmbH, 93309 Kelheim, Germany, can be used.

[0022] For fluorescence microscopy using multiple dyes, it may be preferred that the camera is a color resolution camera. As an example, the camera may be equipped with a Bayer filter.

[0023] If the camera is configured to read out two or more areas, in particular two or more lines, in rapid succession, a higher parallelization of image recording is possible, and thus higher frame and volume rates are possible. As an example, the camera may have multiple rolling shutter apertures. For the purposes of the present invention, such cameras may also be used by the method according to the invention, which is adapted to the general situation both in terms of illumination and detection. In particular, a corresponding illumination of the sample is provided for each read area available therein. As an example, using a camera with multiple rolling shutter apertures, the sample may be illuminated simultaneously by multiple illumination lines guided over the sample by an illumination control device, in particular a scanner.

[0024] For receiving and holding a sample, the microscope may be equipped with a sample mount of a type known per se.

[0025] For example, in the case of transmitted light illumination, the illumination objective and the microscope objective can be separate objectives. However, the illumination objective and the microscope objective can be one and the same objective. A main color splitter can then be conveniently present to separate the emission light from the portion of the excitation light in the light totally reflected back from the sample. Optionally, there can be multiple microscope objectives, in particular interchangeable manually or by a controller, for example in an arranged manner on a turret or linear slide.

[0026] At least one light source, in particular a laser, may be present to provide the excitation light. The spectral composition of the excitation light is adjustable, in particular between two or more colors. The excitation light may also be polychromatic at the same time, for example if different dyes are intended to be detected simultaneously. For these or other applications, the main color splitter may be a double bandpass filter or a multi-bandpass filter.

[0027] In a manner known per se, the illumination beam path may include a lens system for imaging of one or more scanners in the back focal plane of the illumination objective.

[0028] One key concept of the invention can be considered as follows: firstly, the light emission emitted by the sample and containing the microscopic information to be extracted is then split into a number of partial beams each generating a partial image of the sample in the plane of the sensor range. A further key concept of the invention is that the partial images are arranged above the sensor range of the camera, so that a fast readout, in particular a fast readout of the pixels of a line, can be used for all partial images simultaneously.

[0029] As an example, the image splitter unit may position three partial images of the sample from different depths adjacently on the sensor range of the camera, so that the position of the line focus for all partial images is incident on the same sensor line. If the propagation direction of the line is parallel or coincident with the optical axis of the microscope objective, images corresponding to, for example, three planes of the sample covering the same area laterally are positioned on the camera. If the propagation direction of the line illumination is inclined, the images are laterally offset with respect to each other. What is important is that the adjustable electronic detection aperture of the camera substantially coincides with the area illuminated by the line in the respective plane.

[0030] The invention provides a microscope and a microscopy method in which the energy input into the sample, in particular based on axial multiple scanning, is used more effectively for imaging purposes. The effective exposure time of the sample is thereby shortened, and higher frame and volume rates can be achieved for the same total radiation load. The complexity of the technical equipment is kept reasonable in this case. In particular, existing microscopes can be retrofitted with the components necessary to realize the invention. Optionally, the detection optical unit can be adapted to the specific sample to be examined and to the specific measurement task.

[0031] In the case of the microscope according to the invention and the method according to the invention, linear regions in the partial images which lie adjacent to one another on the sensor area can in particular be optically conjugate with a linear illuminated region on or in the sample.

[0032] In this context, optically conjugate means in particular that between optically conjugate ranges or range regions there is a point-to-point relationship mediated by the optical unit. In a particular example, therefore, between points on or in a linear illuminated area on or in the sample there is a point-to-point relationship with a point in the linear area of ​​the partial image. The term point-to-point relationship mediated by the optical unit between a point in the object space (of the sample) and a point on the sensor range of the camera means that a beam emanating from a point in the object space is imaged to a point on the sensor range of the camera by the optical unit.

[0033] In principle, the present invention is realized in a configuration in which the distance between the illumination objective and the sample is changed and adjusted manually. In one advantageous configuration of the microscope according to the present invention, there is a controllable displacement device for changing the distance between the illumination objective and the sample, and the control unit is configured to control the displacement device, in particular in a manner coordinated with the control of the illumination control device. With the addition of this device, volume scanning can be performed in an automated manner.

[0034] The sample is preferably illuminated and scanned with a linear distribution of excitation light. For this purpose, it is advantageous for a cylindrical optical unit or an anamorphic optical unit, such as a cylindrical lens, in particular a cylindrical optical unit that can be pivoted into the illumination beam path, to be present in the illumination beam path for the purpose of generating a linear distribution of excitation light in the sample plane. Alternatively or additionally, a Powell lens, in particular a Powell lens that can be pivoted into the illumination beam path, can be present in the illumination beam path for the purpose of generating a linear distribution of excitation light in the sample plane.

[0035] In principle, for many applications, it may be sufficient if the width of the linear distribution of the excitation light remains unchanged. In an advantageous further development of the microscope according to the invention, there is a second cylindrical optical unit, for example an astigmatism lens, which is pivotable in particular in the illumination beam path and is present in the illumination beam path for the purpose of varying the width of the line focus in the sample plane.

[0036] In principle, for many applications, it may be sufficient if the axial distribution of the excitation light, i.e. the intensity distribution of the excitation light in the optical axis direction, has a constant width. However, particularly for volume scanning, it may be advantageous if the illumination beam path includes optical means, in particular lenses and / or diaphragms, for adjusting the numerical aperture and thus the focal depth or focal depth of the linear illumination. As an example, a zoom optical unit may be present for adjusting the focal depth of the linear illumination in the illumination beam path. Alternatively or additionally, a diaphragm device may be present for adjusting the focal depth of the linear illumination in the illumination beam path.

[0037] The illumination controller serves to spatially steer or position the excitation light on or in the sample. The illumination controller may for example comprise a scanner of a type known per se, in particular an amperometric scanner. Alternatively or additionally, the illumination controller may comprise in particular a controllable and / or programmable micromirror array DMD.

[0038] If the extent of the linear distribution of the excitation light in the direction of the line is greater than the extent of the observed screen size of the partial image, then one scanner is sufficient for moving the linear distribution of the excitation light on or through the sample in a direction transverse to the direction of the line. In a further advantageous addition to the microscope according to the invention, the illumination control device comprises a second scanner for scanning in a direction parallel to the line illumination. This scanner may in particular function to scan the sample in the extension direction of the illumination line and / or to adapt the length of the illumination line in the sample to a desired value.

[0039] Alternatively or additionally, in this context, also in the illumination beam path, the excitation filter can be replaced by a cylindrical lens, for example in a filter cube, which is then scanned. This technical solution can be easily retrofitted to any laser scanning microscope.

[0040] The invention can in principle be realised if the image splitter unit is arranged anywhere between the sample and the camera.

[0041] The image splitter unit may comprise components known per se for splitting the emission light. In one configuration, the image splitter unit comprises at least one diffractive device for splitting the emission light, in particular a diffraction grating and / or a spatial light modulator. The diffractive device is preferably arranged in or near a pupil of the detection beam path.

[0042] Pupil plane is understood to mean the back focal plane of the microscope objective, ie the focal plane located opposite the sample, or the plane that is optically conjugate with the back focal plane of the microscope objective.

[0043] In another variant of the microscope according to the invention, the image splitter unit for splitting the emission light alternatively or additionally comprises at least one monolithic component, for example consisting of an adhesively bonded and / or coated prism or plate. Such a component may in particular be non-adjustable.

[0044] In an advantageous variant of the microscope according to the invention, which allows in particular a relatively simple retrofitting to existing microscope systems, the image splitter unit is arranged in the detection beam path between the intermediate image downstream of the tube lens and the camera.

[0045] The image splitter unit can be realized, for example, in such a way that the emission light is split by a beam splitter and then directed by respective separate optical means to diffraction-limited partial images on the camera. In one advantageous configuration of the microscope according to the invention, the image splitter unit has a relay lens system with an entrance lens and an exit lens, between which at least one beam splitter is arranged. In this variant, at least the exit lens of the relay lens system can be used simultaneously by at least two partial beam paths.

[0046] In order to be able to measure the point spread function laterally, for example if individual dye molecules are intended to be localized, it is advantageous for the magnification of the relay lens system of the image splitter unit to be selected such that the diameter of the Airy disk above the sensor range of the camera is at least four times the size of the distance between adjacent pixels of the camera. In this case, the diameter of the Airy disk is understood to mean the diameter of the first diffraction minimum, i.e. the first dark ring.

[0047] Equivalent to this is the requirement that the magnification of the relay lens system of the image splitter unit is selected such that the full width at half maximum of the point spread function belonging to the Airy disk above the sensor range of the camera is at least twice the magnitude of the distance between adjacent pixels of the camera. The full width at half maximum of the point spread function is understood here to mean the diameter of the area where the intensity of the point spread function drops to half its maximum value at its boundary.

[0048] A particular advantage of the invention is that there is a design freedom with respect to the parameter(s) in which the partial images are different. As an example, the partial images may belong to planes axially spaced apart from one another in the sample. To this end, at least one of the beam splitters, in particular two or more of the beam splitters or each of them, may be a neutral splitter, i.e. the partial beam paths may be identical from a spectral standpoint. In another variant, the partial images differ from one another from a spectral standpoint. As an example, the first dye may be essentially visible in the first partial image and the second dye may be essentially visible in the second partial image.

[0049] For this purpose, at least one of the beam splitters, in particular several of the beam splitters or each of them, may be a dichroic beam splitter, i.e. the partial beam paths may differ in terms of spectrum. The use of dichromatic or polychromatic excitation light may be advantageous in this case.

[0050] A mixture of these variants is in principle possible, i.e. the partial images can both belong to axially spaced sample planes and be spectrally distinct, which can be advantageous in concrete situations.

[0051] In a manner known per se, at least one of the beam separators, in particular two or more of the beam separators or each of them, may be arranged to be interchangeable and / or switchable, for example on a linear slide or turret.

[0052] It is preferable that the partial beam paths are provided to the exit lens of the relay lens system such that at least two associated partial images are arranged adjacent to each other on the sensor range of the camera, so that a linear area in the partial image that is in particular optically conjugate with a linear illuminated area on or within the sample is present on one or more lines in the sensor range.

[0053] A further advantageous configuration of the microscope according to the invention is characterized by the fact that a deflection element, in particular a deflection mirror, which is in particular controllable, is present in at least one of the partial beam paths of the image splitter unit, preferably in several or all of the partial beam paths of the image splitter unit. The control unit is then preferably further configured to be able to satisfy the confocal condition for all images of the respective plane as confocal read-out area by controlling the controllable deflection element using the control signal for the camera.

[0054] In connection with the beam splitter used, it has already been explained that in a variant of the invention, at least two partial images can belong to axially offset areas of the sample. By way of example, in a further variant of the microscope according to the invention, additional lenses can be arranged in at least one of the partial beam paths of the image splitter unit, preferably in several or all of the partial beam paths of the image splitter unit. Different axial orientations of the sample planes belonging to the partial images can be achieved by one or more lenses.

[0055] Furthermore, a development of the microscope according to the invention is particularly advantageous, in which the axial distance between the sample planes, to which the partial images belong and which are optically conjugate with the plane of the sensor range, is adjustable. By way of example, the focal length of the at least one additional lens may be adjustable in a variable manner, in particular infinitely. The at least one additional lens may be an electrically adjustable lens (ETL). The control unit may be configured to control the focal length of the at least one additional lens.

[0056] Alternatively, glass blocks of different thicknesses can be introduced into the region between the output lens and the camera's sensor range in a plane in which the partial images no longer overlap, thereby creating the respective desired axial offsets of the partial images relative to each other.

[0057] In one advantageous addition, in the case of a microscope according to the invention, deflection elements and / or glass blocks may be present for the purpose of adjusting the axial orientation of the pupil relative to the partial beam paths in the image splitter unit.

[0058] In a further preferred configuration of the microscope according to the invention, a field stop is present for the purpose of adapting the size of the partial images in an intermediate image plane in the detection beam path. Appropriate adjustment of the field stop makes it possible to prevent the partial images from overlapping on the sensor range of the camera.

[0059] In an important variant of the method according to the invention, images of large volumetric regions are recorded, i.e. a volume scan is performed. In comparison with the prior art, this is possible according to the invention more quickly and by reducing the light burden on the sample, i.e. by treating the sample relatively gently.

[0060] In a manner known per se, regions of interest can be selected, in particular in an automated manner, from the image of the large volumetric region and then examined more closely using a laser scanning microscope.

[0061] Furthermore, in a manner known per se, based on the measurement data of the laser scanning microscope on the sample, a decision can be made, in particular in an automated manner, as to which volume region of the relevant sample is to be selected for recording its image.

[0062] In the case of volume scanning, the sample planes belonging to the partial images are axially offset with respect to each other, the distance between the microscope objective and the sample is changed over a certain scanning movement with a defined step size, and a number of images corresponding to the number of partial images is recorded for each distance between the sample and the microscope objective. The individual images can then be combined by the control unit to form a 3D image of the observed volume.

[0063] In principle, the focal depth or depth of focus of the linear illumination can be unchanged. The focal depth of the linear illumination is preferably adjusted to a value that corresponds to the axial distance between two sample planes that are optically conjugate with the plane of the sensor range of the camera - in particular the distance between two axially outer sample planes, if three or more sample planes are imaged simultaneously. This is particularly advantageous for volume scanning, where an optimal ratio between image quality and light load on the sample is desired.

[0064] Besides simple Gaussian illumination profiles, more complex beam shaping can also be used, for example multiple foci can be generated using diffractive optical elements.

[0065] In order to sample the point spread function axially, the axial distance between the observed sample planes must not exceed half the axial focal depth of the image of the microscope objective in the detection beam path. This is also called axial Nyquist sampling. If in the case of a volume scan the axial distance between the sample planes is assumed to be larger than the axial step size, this gives rise to the requirement that the axial step size, in particular the axial step size of the adjustment of the distance between the sample and the microscope, is selected to be less than half the axial focal depth of the image of the microscope objective in the detection beam path. However, in principle it is also possible for the axial distance between the sample planes belonging to the partial images to be equal to or smaller than the focal depth of the image of the microscope objective in the detection beam path. The axial step size can then be correspondingly larger. Applications of the invention that do not fulfill the axial Nyquist condition, i.e. undersampling, are of course also possible. This may be desirable, for example, when fast volume scans are intended to be performed.

[0066] In a preferred variant of the method according to the invention, in the case of volume scanning, the axial distance between the sample planes belonging to the partial images is an integer multiple of the axial step size. Apart from the planes at the axial limits of the scanning volume, a number of images of the sample planes are then recorded in each case, specifically according to the number of partial images defined by the image splitter unit. The selection of this step size also makes it possible to check the correct linking of the individual subsequent image stacks in each case.

[0067] In another variant of the method according to the invention, in the case of volume scanning, the axial step size is an integer multiple of the axial distance between the sample planes belonging to the sub-images. By way of example, the axial step size may be n times the axial distance between the sample planes belonging to the sub-images, where n is the number of sample planes and sub-images. Fast volume scanning is possible by selecting the axial step size.

[0068] Mixed forms of these variants are possible.In the case of volume scanning, it is advantageous that the axial distance between the sample planes and the axial step size can be freely selected within a wide range.

[0069] A further increase in frame rate and volume rate is possible if the sample is scanned for several linear illumination areas and the areas of the camera sensor range which correspond to the linear illumination areas are read out in each case in a synchronous manner.

[0070] Typically, the excitation light is irradiated onto the sample parallel to the optical axis of the illumination objective. However, for certain samples, it may be preferred that the sample is irradiated with a linear laser illumination at an angle to the optical axis of the illumination objective. The sample areas corresponding to the partial images are then laterally displaced from one another. The sampled volume may substantially correspond to a parallelepiped.

[0071] Further advantages and features of the present invention will be explained in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0072] [Figure 1] 1 is a schematic diagram of an exemplary embodiment of a microscope according to the present invention; [Diagram 2] FIG. 1 is a schematic diagram of a flow diagram of the illumination situation within the sample. [Diagram 3] FIG. 13 is a schematic diagram of the sensor range of a camera with two partial images, each corresponding to a different z-depth within a sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Identical and identically acting components are generally identified by the same reference numbers in the drawings.An exemplary embodiment of a microscope 200 according to the present invention will now be described with reference to FIGS.

[0074] A microscope 200 according to the invention, shown diagrammatically in FIG. 1, comprises as essential components an illumination beam path, a detection beam path and a control unit 100, eg a PC.

[0075] The illumination beam path serves to illuminate and scan the sample S with excitation light 12 and in the illustrated example comprises, among other components, a scanner 20 as illumination control device and an illumination objective 40. Upon illumination with excitation light 12, the sample S emits emission light 14, which is imaged via the detection beam path onto a sensor area 98 of a camera 90. Among other components, the detection beam path comprises a microscope objective 40, which is identical to the illumination objective 40 in the illustrated example, and, as an essential element part of the microscope according to the invention, an image splitter unit 60. The image splitter unit 60 serves to split the emission light 14 coming from the sample S and to image it in a limited manner onto the sensor area 98 of the camera. The camera 90 serves to record an image of the sample S. A control unit 100 serves to control at least the scanner 20 and the camera 90.

[0076] The illumination beam path may be realized by the illumination beam path of a laser scanning microscope.

[0077] Excitation light 12 is provided in the illumination beam path by a laser 10 and is scanned over the sample S by a scanner 20 through a microscope objective 40. Imaging from the scanner 20, e.g. one or more scanning mirrors, into the back focal plane (pupil plane) of the illumination objective 40 is provided via a lens system consisting of a scanning optical unit 22 and a tube lens 24. A cylindrical optical unit, in particular a cylindrical lens 18, preferably generates a linear distribution of the excitation light 12 in the pupil plane and in the sample plane. Due to the Fourier relationship between the pupil plane and the image plane, these planes are perpendicular to each other.

[0078] Additionally or alternatively, a cylindrical lens may be present at the illumination entrance side of the main beam splitter 30 described below to generate a linear distribution of the excitation light 12 .

[0079] In the illustrated exemplary embodiment, a switchable or exchangeable, eg pivotable, second cylindrical optical unit, eg a cylindrical lens 16, is additionally present to adjust the width of the line focus.

[0080] In the illustrated exemplary embodiment, a zoom optical unit 13 is additionally present in the illumination beam path to allow adjusting the focal depth or the depth of focus of the line illumination in the sample S. Additionally or alternatively, an aperture device (not shown in FIG. 1 ) may be present in the illumination beam path to adjust the focal depth of the illumination.

[0081] The line focus is moved by a scanner 20 in a direction transverse to the extension of the line focus in the sample S. Optionally, a second scanner may be present as part of the illumination control device to scan the excitation light 12 in a direction parallel to the line illumination.

[0082] In the illustrated exemplary embodiment, the distance between the microscope objective 40 and the sample S can be adjusted by a displacement device 42, for example a motorized z-drive.

[0083] The emission light 14, in particular the fluorescence light, generated in the sample S due to illumination by the excitation light 12 is collimated by the microscope objective 40 and deflected in the detection beam path towards the camera 90 by the main color splitter 30, a dichroic beam splitter. The emission light 14, i.e. the fluorescence light, does not therefore pass through the scanner 20.

[0084] The intermediate image plane 52 downstream of the tube lens 50 is followed by an image splitter unit 60 which, in the illustrated exemplary embodiment, generates three partial images from different depth regions of the sample S and arranges them adjacent to each other on a sensor area 98 of a camera 90, e.g. a CMOS chip or a SPAD array. The camera 90 can also be a color resolution camera.

[0085] The camera 90 , the scanner 20 and the displacement device 42 are operatively connected to each other via a control unit 100 .

[0086] The control unit 100 is particularly configured to synchronize the position of the line illumination in the sample S - defined by the settings of the scanner 20 - with the respective read area of ​​the sensor range 98 of the camera 90, for example in the manner of a rolling shutter. The control unit 100 may also be configured to control the displacement device 42, particularly in a manner coordinated with the control of the scanner 20.

[0087] In the exemplary embodiment shown in FIG. 1, an image splitter unit 60 is arranged in the detection beam path downstream of the tube lens 50 between the intermediate image 52 and a camera 90 .

[0088] The image splitter unit 60 has an optical relay lens system with an entrance lens 62 and an exit lens 64, which images the intermediate image plane 52 onto a sensor range 98 of the camera 90. The magnification of the relay lens system can now be selected such that a desired number of partial images, i.e. three partial images in the exemplary embodiment of FIG. 1, are imaged onto the sensor surface 98, so that the point spread function can be sampled laterally. This means that the magnification of the relay lens system 62, 64 of the image splitter unit 60 is selected, for example, such that the diameter of an Airy disk at the sensor range 98 of the camera 90 is at least four times as large as the distance between adjacent pixels of the camera 90. Optionally, a field stop can be present in the intermediate image plane 52 to avoid overlapping of the partial images onto the sensor range 98 of the camera 90.

[0089] In the illustrated exemplary embodiment, beam splitters 72, 82 reside between the entrance lens 62 and the exit lens 64 of the relay lens system and split the emitted light 14 into first, second and third partial beam paths.

[0090] In the first partial beam path, the emitted light 14 passes from the beam splitter 72 via an adjustable mirror 74 and a lens 76 to the exit lens 64 and is imaged by the exit lens into a first partial image 91 onto a sensor range 98 of the camera 90.

[0091] In the second partial beam path, the emitted light 14 passes from the beam splitter 82 via an adjustable mirror 84 and a lens 86 to the exit lens 64 and is imaged by the exit lens into a second partial image 92 onto a sensor range 98 of the camera 90.

[0092] In the third partial beam path, the emission light 14 passes through both beam splitters 72 , 82 and passes directly to the exit lens 64 from which it passes to a third partial image onto a sensor range 98 of the camera 90 .

[0093] In the illustrated exemplary embodiment, the neutral splitter 82 can also be a simple mirror. The third partial beam path, and therefore the third partial image, is then omitted.

[0094] The control unit 100 uses the control signal of the camera 90 to control the adjustable mirrors 74, 84 so that the confocal readout region ensures that the confocal condition is met for all partial images in each plane.

[0095] The first partial beam path and the second partial beam path are supplied to the exit lens 64 of the relay lens system 62, 64 such that at least two associated partial images 91, 92 are arranged adjacent to each other on the sensor range 98 of the camera 90, so that linear areas 93, 94 in the partial images 91, 92 that are optically conjugate with linear illuminated areas on or within the sample S lie on one or more same lines 95 in the sensor range 98 (Figure 3).

[0096] Additionally, deflecting elements or glass blocks (not shown in FIG. 1) may be present in the first and / or second partial beam paths to adjust the pupil orientation relative to the partial images.

[0097] The lens 76 in the first partial beam path defines the axial orientation of a plane S1 in the sample S, which is optically conjugate with the plane of the sensor range 98. The lens 86 in the second partial beam path defines the axial orientation of a plane S2 in the sample S, which is optically conjugate with the plane of the sensor range 98. The axial orientation of the sample planes S1 and S2, which are optically conjugate with the plane of the sensor range 98, can therefore be adjusted by suitable selection of the lenses 76 and 86. Preferably, in particular for volume scanning, a certain axial distance is adjusted for the sample planes S1 and S2. Expediently, by means of the zoom optical unit 13, the focal depth of the illumination by the excitation light 12 can then be adjusted in addition thereto, so that the two sample planes are effectively illuminated, but adjacent areas are illuminated as little as possible by the excitation light 12. The adjustment of the focal depth is intended to have the effect that the adjacent areas have a lower illumination density, i.e. a larger area is illuminated. This is achieved when the furthest sample planes observed simultaneously are still just within the focal depth of the illumination, as will be explained in the next paragraph.

[0098] Preferably, the focal depth of the linear illumination is adjusted to a value that corresponds to the axial distance between two sample planes S 1 , S 2 that are optically conjugate with the plane of the sensor range 98 of the camera 90 .

[0099] Advantageously, the focal length of the lenses 76, 86 is adjustable by lenses that are either exchangeable or, for example, electrically adjustable (so-called electrically adjustable lenses, ETL), and their focal length is then also adjustable by the control unit 100. The distance between the sample planes S1, S2 conjugate to the camera image plane and the focal plane of the microscope objective 40 can be adjusted in this way.

[0100] It is also preferred that the adjustable lenses 76, 86 are adjustable in particular so that the sample planes S1 and S2 are not axially spaced apart. As an example, the focal length can be set to infinity for both lenses 76, 86. For the configuration shown in FIG. 1, the first partial image 91 and the second partial image 92 are then ideally identical.

[0101] For convenience, beam splitters 72 and 82 may then be replaced with different dichroic beam splitters consistent with the observation of chromatically distinct dyes. As an example, a first dye may be represented in the centroid term of a first partial image 91 and a second dye may be represented in the centroid term of a second partial image 92. Thus, the sample S is observed simultaneously in two color channels, even though line illumination is directed across the sample S in a manner synchronized with the rolling shutter of camera 90.

[0102] The sample S may be, for example, a cell conglomerate with a thickness of about 15 μm. The microscope objective 40 may be, for example, an objective 40x / 1.2. The focal depth of the imaging is then about 0.5 μm, so that an axial Nyquist sampling with a step size of 0.25 μm is realized. Instead of two partial images as shown, for example three partial images may also be recorded simultaneously. The axial distance between the three sample planes may be adjusted, for example, to 2.5 μm, i.e. the outer sample planes are axially separated by 5 μm. The axial movement of 5 μm may then be traversed uniformly in multiple steps of 0.25 μm without the need for jumps of different magnitudes, which may involve different time delays. The number of images recorded is reduced by a factor of three to only 20 images. With an image repetition rate of the camera of 120 fps (frames per second), six volumes per second will be recorded. The advantage in this case is that there is coverage for each of the first and last planes of the region present in the sample (see FIG. 2), so that the correct association of the image records can be guaranteed.

[0103] It is further preferred to controllably position the partial images 91, 92 on the camera 90. In this case, it is possible to realize a control of the deflection mirrors 74, 84, which are adjustable in particular in a motorized manner, on the basis of a signal on the sensor range 98 of the camera 90 in the control unit 100 according to defined criteria, which control ensures that the positioned partial images 91, 92 do not overlap and furthermore adjoin one another in such a way that the confocal readout area optimally overlaps with the illumination area.

[0104] FIG. 2 shows a schematic diagram of the microscope objective 40 in relation to the sample S. An immersion medium for refractive index matching is located between the microscope objective 40 and the sample S. Two sample planes S1 and S2 are shown. The first sample plane S1 is optically conjugate with a first partial image 91 (FIG. 3) via a first partial beam path running via the elements 62, 72, 74, 76, 64. The second sample plane S2 is optically conjugate with a second partial image 92 (FIG. 1, FIG. 3) via a second partial beam path running via the elements 62, 82, 84, 86, 64. The extent of the axial distribution of the excitation light 12 approximately corresponds to the axial distance between the sample planes S1 and S2.

[0105] 3 shows a schematic diagram of the sensor area 98 of the camera 90. As can be seen, a first partial image belonging to the first partial beam path, which is an image of the sample plane S1, lies adjacent to a second partial image 92 belonging to the second partial beam path, which is an image of the sample plane S2. A linear area 93 in the first partial image 91 corresponds to a region that is optically conjugate with the position of the line illumination in the first sample plane S1. A linear area 94 in the second partial image 92 corresponds to a region that is optically conjugate with the position of the line illumination in the second sample plane S2. As can be seen, the first partial image 91 and the second partial image 92 lie adjacent to each other on the sensor area 98, so that the linear areas 93, 94 in the partial images 91, 92 that are optically conjugate with the position of the line illumination in the second sample plane S2 lie on one or more same lines 95 in the sensor area 98. The control unit 100 is configured to read out and evaluate the measurement signals measured from the pixels lying in the line or lines 95. If with the aid of the scanner 20 the line focus in the sample S is displaced transversely to the extension direction of the line focus, the area respectively read out from the sensor range 98 of the camera 90 is also simultaneously guided in the direction of the arrow 96. [Explanation of symbols]

[0106] 10 Light sources, especially lasers 12 Excitation light 13 Zoom optical unit for adjusting the focal depth of the illumination 14 Emitted light 16 Cylindrical lenses, especially those capable of being pivoted 18 Cylindrical lenses, especially those capable of being pivoted 20 Illumination control device, e.g. galvanometer scanner 22 Scanning optical unit 24 Tube lens in the illumination beam path 30 Primary Color Splitter 40 Illumination objective lenses, microscope objective lenses 42 Controllable Displacement Device 44 Immersion fluid (optional based on objective lens 40) 50 Tube lens in the detection beam path 52 Intermediate Image Plane 60 Image splitter unit 62 Inlet Lens 64 Exit Lens 72 Beam splitters, especially neutral splitters 74 Deflection devices, in particular mirrors, in particular controllable mirrors 76 Lenses, especially lenses with adjustable focal length 82 Beam splitters, especially neutral splitters 84 Deflection devices, in particular mirrors, in particular controllable mirrors 86 Lenses, especially lenses with adjustable focal length 90 Camera 91 First Part Image 92 Second Part Image 93 A linear area coincident with or optically conjugate to a linear illuminated area on or in the sample S 94 A linear area coincident with or optically conjugate to a linear illuminated area on or in the sample S 95 One or more lines read from sensor range 98 96 Direction of movement of the line or lines to be read 98 Sensor range of camera 90 100 Control device, especially PC 200 Microscope according to the present invention S sample S1 First sample plane S2 2nd sample plane

Claims

1. an illumination control device (20) and an illumination beam path including at least an illumination objective (40) for illuminating and scanning a sample (S) in a particularly linear manner with excitation light (12); a detection beam path comprising at least a microscope objective (40) for directing emission light (14) emitted by the sample (S) towards a camera (90); said camera (90) for recording an image of said sample (S); A microscope comprising a control unit (100) for controlling at least the illumination control device (20) and the camera (90), the control unit (100) is configured to synchronize in each case the read area of ​​the sensor range (98) of the camera (90) with the position of the excitation light (12) defined by the illumination control device (20), The detection beam path comprises an image splitter unit (60) for splitting the emission light (14) into a plurality of partial beam paths, each of which generates a partial image (91, 92) of the sample (S) on the sensor range (98) of the camera (90), and the partial images (91, 92) that are adjacent to each other on the sensor range (98) are located on one or more of the same lines (95) on the sensor range (98) so that linear regions (93, 94) in the partial images (91, 92) correspond to the position of the excitation light (12) on or within the sample (S) defined by the illumination control device (20).

2. 2. The microscope of claim 1, wherein the linear areas (93, 94) in the partial images (91, 92) adjacent to each other on the sensor range (98) are optically conjugate with a linear illuminated area on or within the sample (S).

3. the presence of a controllable displacement device (42) for varying the distance between the illumination objective (40) and the sample (S); 2. The microscope according to claim 1, characterized in that the control unit (100) is configured to control the displacement device (42), in particular in a manner coordinated with the control of the illumination control device (20).

4. 2. The microscope according to claim 1, characterized in that a cylindrical optical unit (18), which is particularly rotatable into the illumination beam path, is present in the illumination beam path for generating a linear distribution of the excitation light (12) in the sample plane (S1, S2).

5. 2. The microscope according to claim 1, characterized in that a second cylindrical optical unit (16), which is rotatable in particular into the illumination beam path, is present in the illumination beam path for varying the width of the line focus in the sample plane (S1, S2).

6. 2. The microscope according to claim 1, wherein a lens (13) and / or a diaphragm for adjusting the focal depth of the linear illumination is present in the illumination beam path.

7. 2. The microscope according to claim 1, characterized in that the illumination control device comprises at least one scanner (20) and / or at least one micromirror array.

8. 2. The microscope according to claim 1, characterized in that the image splitter unit (60) for splitting the emitted light comprises at least one diffractive device, in particular a diffraction grating and / or a spatial light modulator.

9. 2. The microscope according to claim 1, characterized in that the image splitter unit (60) for splitting the emitted light comprises at least one monolithic component, for example consisting of an adhesively bonded and / or coated prism or plate.

10. 2. The microscope according to claim 1, wherein the illumination control device has a second scanner for scanning the excitation light in a direction parallel to the line illumination.

11. 2. The microscope according to claim 1, wherein the image splitter unit (60) is arranged in the detection beam path between the intermediate image (52) downstream of the tube lens (50) and the camera (90).

12. 2. The microscope of claim 1, wherein the image splitter unit (60) comprises a relay lens system with an entrance lens (62) and an exit lens (64), between which at least one beam splitter (72, 82) is arranged.

13. 13. The microscope of claim 12, wherein the magnification of the relay lens system (62, 64) of the image splitter unit (60) is selected so that the diameter of an Airy disk over the sensor range (98) of the camera (90) is at least four times the size of the distance between adjacent pixels of the camera (90).

14. 13. The microscope according to claim 12, characterized in that at least one of the beam splitters (72, 82), in particular two or more of the beam splitters (72, 82) or each of them, is a neutral splitter or a dichroic beam splitter.

15. 13. The microscope of claim 12, wherein the partial beam paths are supplied to the exit lens (64) of the relay lens system such that the at least two associated partial images (91, 92) are arranged adjacent to one another on the sensor range (98) of the camera (90), so that linear areas (93, 94) in the partial images (91, 92) that are optically conjugate with linear illuminated areas on or in the sample (S) lie on one or more of the same lines (95) on the sensor range (98).

16. 2. The microscope according to claim 1, characterized in that a controllable deflection element (74, 84), in particular a deflection mirror, is present in at least one of the partial beam paths of the image splitter unit (60), preferably in several or all of the partial beam paths of the image splitter unit (60).

17. 17. The microscope of claim 16, wherein the control unit (100) is configured to satisfy the confocal condition for all images in each plane by the confocal readout region by controlling the controllable deflection elements (74, 84) using control signals from the camera (90).

18. 2. The microscope according to claim 1, wherein the at least two partial images (91, 92) belong to axially offset regions of the sample (S).

19. 2. The microscope of claim 1, wherein additional lenses (76, 86) are arranged in at least one of the partial beam paths of the image splitter unit (60), preferably in several or all of the partial beam paths of the image splitter unit (60).

20. 2. The microscope according to claim 1, characterized in that the axial distance between the sample planes (S1, S2) to which the partial images (91, 92) belong and which are optically conjugate with the plane of the sensor range (98) is adjustable.

21. 2. The microscope according to claim 1, characterized in that the focal length of the at least one additional lens (76, 86) is adjustable in a variable manner, in particular to infinity.

22. 2. The microscope according to claim 1, characterized in that deflection elements and / or glass blocks are present for adjusting the axial orientation of the pupil relative to the partial beam paths in the image splitter unit (60).

23. 2. The microscope according to claim 1, characterized in that the camera (90) is a CMOS or sCMOS camera with rolling shutter electronics or a SPAD camera.

24. 2. The microscope according to claim 1, characterized in that the camera (90) is a color resolution camera.

25. 2. A microscope according to claim 1, characterized in that the camera (90) is configured to read out from two or more areas, in particular two or more lines, in rapid succession.

26. 2. The microscope according to claim 1, wherein the sample areas corresponding to the partial images (91, 92) are laterally displaced with respect to one another.

27. 2. The microscope according to claim 1, characterized in that a field stop is present for adapting the size of the partial images (91, 92) in an intermediate image plane (52) in the detection beam path.

28. The sample (S) is illuminated and scanned, in particular linearly, with excitation light (12) by an illumination objective (40), Emission light (14) emitted by the sample (S) is directed towards a camera (90) via a microscope objective (40); The emitted light (14) downstream of the microscope objective (40) is split into a plurality of partial beam paths, each of the partial beam paths generates a partial image (91, 92) of the sample (S) on a sensor field (98) of the camera (90); the partial images (91, 92) of the sample (S) are adjacent to each other on the sensor area (98), such that linear areas (93, 94) in the partial images (91, 92) corresponding to the position of the excitation light (12) on or within the sample (S) are on one or more same lines (95) of the sensor area (98); 10. A method for microscopy, characterized in that the read area of ​​the sensor range (98) of the camera (90) is synchronized with the position of the excitation light (12) on the sample.

29. 29. The method of claim 28, wherein the focal depth of the linear illumination is adjusted to a value corresponding to the axial distance between two sample planes (S1, S2) that are optically conjugate with the plane of the sensor range (98) of the camera (90).

30. 29. The method of claim 28, characterized in that for volume scanning the axial step size is less than or equal to half the axial focal depth of the imaging of the microscope objective (40).

31. 29. A method according to claim 28, characterized in that in the case of a volume scan, the axial distance between the sample planes (S1, S2) belonging to the sub-images is an integer multiple of the axial step size.

32. 29. The method of claim 28, wherein in the case of a volume scan, the axial step size is an integer multiple of the axial distance between the sample planes belonging to the sub-images.

33. 29. The method of claim 28, characterized in that the sample (S) is scanned using a plurality of linear illumination areas, and the areas of the sensor range (98) of the camera (90) corresponding to the linear illumination areas are read in each case in a synchronous manner.

34. 29. The method of claim 28, characterized in that linear laser illumination is emitted onto the sample (S) at an angle relative to the optical axis of the illumination objective (40).

Images