Multimodal scanning microscope and methods for its operation

Abstract

The invention relates to a scanning microscope (M) comprising a main color splitter (3) for separating illumination radiation and detection radiation coming from a sample chamber; and at least one detection beam path (10.1, 10.2) for guiding the detection radiation from the main color splitter (3) to an area detector (12) located in the detection beam path (10.1, 10.2), which is configured to detect detection radiation and provide measured values, wherein the area detector (12) comprises a plurality of detector elements (12.1), the measured values ​​of which can each be evaluated individually and / or in groups. A key feature is that either switching elements (20) are present in the detection beam path, by means of which at least a first operating state and a second operating state can be set; or the operating states are generated by means of a correspondingly configured evaluation unit (13). The selection of the detector elements (12.1) to be read out is carried out by means of the control / evaluation unit (13). The invention also relates to a module (Md) and an operating method.

Description

[0001] The invention relates to a scanning or raster microscope that can be operated in various operating states (modes) according to the preamble of the main claim. The invention also relates to a module and a method for operating the scanning microscope according to the preamble of the independent claim.

[0002] Light microscopes, especially laser scanning microscopes, are increasingly being used for a variety of microscopy techniques. For example, there is a desire to be able to perform various applications such as multi-channel confocal experiments, multi-channel high-resolution experiments, or even spectral experiments with a single microscope. At the same time, high requirements regarding noise performance (signal-to-noise ratio, SNR), resolution, and image acquisition speed must be met.

[0003] Microscopes are known from the prior art which, for example, using gradient color splitters, allow a flexible spectral division of a detection radiation, for example an emission light, into several different physical point and / or area sensors (e.g. DE 10 2006 034 908 B4, DE 10 2012 205 722 B4, DE 10 2023 100 926 or DE 10 2020 213 714 A1).

[0004] The disadvantage here is that the required system architecture becomes more complex and costly the higher the number of required channels. Furthermore, the high number of optical surfaces in the detection beam path leads to a loss of sensitivity and thus a poorer SNR.

[0005] It is also known that the spectral information of the detected radiation can be separated by using dispersive elements and a line or area sensor in the detection beam path (also: emission beam path) (DE 100 38 526 B4). Dispersive elements as defined in this description deflect incident rays to varying degrees depending on their respective wavelength due to refraction or diffraction. If necessary, the image data of several colorants and / or dyes present in the sample can be separated from one another using suitable demixing algorithms. It is also described that when using sensitive area sensors and a suitable dispersive element, the spatially and spectrally distorted point spread function (PSF) can be reconstructed and a spectral high-resolution image can be generated (e.g., DE 10 2020 120 190 A1).

[0006] However, imaging systems that allow all or many of the above-mentioned uses are technically complex and cost-intensive.

[0007] The invention is based on the object of proposing possible designs of a microscope that can be used for different imaging methods. At the same time, the achievable quality of the acquired measured values ​​should be high and the costs for the technical implementation of the invention should be comparatively low. The invention should offer the option of a modular design for such a microscope.

[0008] The problem is solved using a multimodal scanning microscope, also known as a scanning microscope, a module, and two variants of an operating method. Advantageous further developments are the subject of the dependent claims.

[0009] The scanning microscope used to solve this problem, hereinafter also referred to as a microscope, has a main color splitter for separating illumination radiation and detection radiation coming from a sample chamber. After the main color splitter, there is at least one detection beam path that guides the detection radiation from the main color splitter to an area detector located in the detection beam path. The area detector is configured to capture detection radiation and provide measurement values. The area detector has a plurality of detector elements, each of which can be evaluated individually and / or in groups. It is therefore possible to freely decide from which detector elements the measurement values ​​are read individually, or which measurement values ​​are combined (“binned”) or ignored.It is also possible to record measured values ​​from individual detector elements and store them for subsequent processing. In addition, the measured values ​​from individual detector elements can be provided in a summarized form.

[0010] A characteristic feature of a microscope according to the invention is that in at least one of the existing detection beam paths there is an advantageously controllable adjustment optics which, depending on the wavelength or a wavelength range of the detection radiation, adjusts an extension (size) of a diffraction pattern in the form of diffraction disks (Airy disk) of the detection radiation on the area detector.

[0011] Using wavelength-dependent adjustment optics, it is possible to adjust the size of a diffraction disk on the area detector. This allows, for example, a diffraction disk to be imaged with the same size on the area detectors of different detection beam paths, for example, on the same number of detector elements or according to the respective detector size, even though the detection radiation in the detection beam paths comprises different wavelengths or wavelength ranges.

[0012] With wavelength-dependent adjustment optics (also abbreviated to "adjustment optics"), the invention can address the fact that, for example, with detected radiation in a range of 400 nm to 900 nm, the size of the diffraction disk changes continuously depending on the wavelength and exhibits a size difference of approximately a factor of 2.25 across the specified range. This disadvantage is eliminated by the wavelength-dependent adjustment optics according to the invention. Furthermore, the optical design of other optical elements in the detection beam path is advantageously simplified.

[0013] In one possible embodiment of the invention, a wavelength-dependent adaptation optics can be formed by a circular optics which, with regard to its optical effect, in particular its focus position, is designed to the expected wavelengths of the detection radiation in the relevant detection beam paths.

[0014] The adaptation optics can be permanently installed in each detection beam path. In other possible embodiments, the adaptation optics can be interchangeable to accommodate different wavelength ranges of the detection radiation and allow for a wide range of applications of the invention.

[0015] The adjustment optics can consist of a single optical element. In other designs, it can also comprise multiple optically active elements that are optionally adjustable, for example, with a controlled adjustment, in order to be able to influence detection radiation of different wavelengths or wavelength ranges as precisely as possible with just one adjustment optics.

[0016] In one embodiment of the microscope, switching elements are present in the detection beam path, which can be brought into at least two switching states, so that in the detection beam path, depending on the switching state, at least a first operating state with a confocal and a second operating state with a spectral detection of measured values ​​of the detection radiation is or can be effected by means of the area detector.

[0017] In each operating state, control commands from an existing controller are used to select the appropriate detector elements to be read individually or in groups. An evaluation unit is also available, configured to evaluate measured values ​​from at least two operating states depending on the currently selected operating state.

[0018] As an alternative to the microscope design described above, at least a first operating state with confocal and a second operating state with spectral acquisition of measured values ​​of the detection radiation can be achieved by using an existing and appropriately configured evaluation unit. The operating states are generated by this unit by evaluating the acquired measured values ​​depending on the currently selected operating state. The configuration of the detection beam path can optionally remain unchanged. For example, the detection beam path can be configured for spectral acquisition. As a result of an appropriate execution of the evaluation by the evaluation unit, a purely confocal acquisition without spectral resolution can be achieved despite a spectral splitting of the detection radiation.

[0019] For the purposes of this description, confocal detection means that portions of the detection radiation that do not originate in the focus or in a confocal volume are eliminated for the detection of the detection radiation. A confocal volume is used, for example, when, in fluorescence correlation spectroscopy (FCS), excitation radiation is directed into a narrow volume of a sample to be observed, which coincides with the initial volume of the detected detection radiation. For simplicity, we will refer to this as a focus below.

[0020] Eliminating unwanted portions of the detection radiation can be achieved in two different ways. In a first possible embodiment, the area detector can be arranged in a plane conjugate to the focus or the confocal volume (the so-called pinhole plane). Each detector element of the area detector can be read out individually and thus only detects the detection radiation originating from the focus. In this sense, a number of detector elements can also be combined (binning), which jointly detect the detection radiation originating from the focus. In this way, in conjunction with optional magnification optics (see below), it is possible to adjust which portions of the diffraction disks (Airy units) are detected together. For example, it is possible for the central diffraction disk to be detected with five detector elements.In such an embodiment of the invention, a physical pinhole in the detection beam path is possible, but not required.

[0021] In a second design variant, a pinhole (usually a pinhole) is arranged in the detection beam path. Its aperture is adjusted to the detection beam path and dimensioned so that only the detection radiation originating from the focus or confocal volume reaches the area detector. Out-of-focus portions of the detection radiation are blocked by the pinhole's confinement.

[0022] From the preceding explanation, it can be seen that spectral detection within the meaning of this description of the invention also involves a confocal configuration of at least one detection beam path. However, in contrast to confocal detection, in spectral detection the detection radiation is split (dispersed) in at least one direction depending on the wavelength. Different wavelengths of the detection radiation then impinge on different areas of the area detector.

[0023] A key advantage of the invention lies in the ability to perform different detection or imaging methods by selecting the respective operating modes while using the same area detector. In contrast to prior art technical implementations, in which a detected detection radiation is directed to a different detector for each imaging method, the invention allows a multimodal microscope in its basic form to be realized with only one detector.

[0024] The term "switching elements" refers to optically active elements that can be introduced into the relevant detection beam path in a controlled manner and influence the detection radiation there. Several switching elements can be present per detection beam path. Switching elements can be, for example, confocal apertures, focusing optics, defocusing optics, as well as spectrally separating or spectrally splitting (dispersive) optical elements such as prisms and gratings.

[0025] The switching elements each comprise a drive and, if necessary, a carrier for holding the optically active element. The latter can be arranged, for example, on a wheel, a turret, or a slider. The optically active elements can also be manually pivoted into or out of the detection beam path by means of a mechanism, but preferably controlled by a motor. It is also possible for switching elements to remain in the detection beam path, and the change in operating state is effected by a corresponding switching state of the switching element. Such switching elements can be, for example, spatial light modulators (SLMs) or acousto-optical filters (AOTFs).

[0026] Additionally, additional optical elements can be reversibly incorporated into the respective detection beam paths to further modify the detection radiation in the respective operating states. Such additional elements can be, for example, emission filters and correction elements. For example, the beam position can be corrected using a plane-parallel plate that is transparent to the detection radiation and can be tilted about at least one axis perpendicular to the detection beam path. Alternatively, pivotable or movable mirrors can be provided to correct the beam position. At least some of the additional optical elements can be adjustable in a controlled manner.

[0027] The detection radiation can, in particular, be fluorescent radiation. For this purpose, an object to be imaged, for example, a biological sample or components thereof, can be provided with fluorophores. Particularly when the microscope is designed as a laser scanning microscope (LSM), the emission of fluorescent radiation as detection radiation (emission radiation) can be achieved by means of laser radiation serving as excitation radiation.

[0028] To increase the flexibility of the microscope according to the invention, in one possible embodiment, the detection beam path can be divided into at least a first and a second detection beam path. In further embodiments of the microscope according to the invention, the detection radiation can be divided into more than two detection beam paths. For the sake of simplicity, the following description focuses on a first and a second detection beam path. An area detector as described above is present in at least one of the detection beam paths. Other types of detectors can be present in the other detection beam paths, for example, secondary electron multipliers (PMTs)—single or as an array—photodiodes, or CCD detectors.

[0029] In further embodiments of the microscope according to the invention, an area detector is present in at least two of the detection beam paths. This advantageously allows, for example, large wavelength ranges to be detected to be addressed by dividing the wavelength range and allocating it proportionally to the detection beam paths (see also below).

[0030] The detection radiation can be divided between the at least one first and one second detection beam path using a beam splitter. Through its action, the incident detection radiation is divided between the detection beam paths, for example, with regard to its intensity, polarization, and / or spectral composition. The section of the detection beam path between the main color splitter and the beam splitter is also referred to below as the common detection beam path.

[0031] One possible design includes a beam splitter with a fixed splitting ratio. In other designs, the beam splitter can be variable. For example, several beam splitters with different properties can be arranged on a turret or slider. Depending on the requirements, the beam splitter that best suits the desired detection method is inserted into the detection beam path. It is also possible to use graduated filters, whose relative position also depends on the desired image acquisition and the wavelengths of the detection radiation to be captured, and is selected and adjusted accordingly.

[0032] As already mentioned, one of the switching elements can be a dispersive optical element, through which incident detection radiation is spectrally split, i.e., depending on its wavelength, in a direction orthogonal to the optical axis of the detection beam path. The split detection radiation is directed onto the area detector, and the associated measured values ​​are recorded by it with spatial resolution. The detection radiation can be spatially split into several wavelengths, in a simple case, spatially into two wavelengths, or wavelength ranges. In another embodiment, it is also possible to extract only one wavelength from a wavelength range of the detection radiation and make this available for detection alongside the remaining wavelength range.

[0033] A dispersive optical element is preferably a prism or grating. An AOM (acousto-optic modulator) or an SLM (spatial light modulator) is also possible.

[0034] In a further possible embodiment of the invention, a beam-shaping optic can be incorporated into the relevant detection beam path together with the dispersive optical element. Through its action, the detection light is distributed orthogonally to the direction of spectral splitting and adapted to a usable detection area of ​​the area sensor. Such a beam-shaping optic can, for example, be a cylindrical optic. This broadens the beam of the detection radiation perpendicular to the direction of spectral splitting and distributes it over a specific area on the sensitive surface (detection area) of the area detector. In this way, for example, the existing width or height of the area detector can be utilized as effectively as possible, thus achieving good dynamics.Advantageously, a spectral resolution (wavelength range per detector element) achieved in the direction of spectral splitting is recorded with a plurality of detector elements. Each wavelength is thus directed to and recorded by multiple detector elements. This allows, for example, the presence of defective detector elements to be compensated for.

[0035] Additionally, a magnification optic can be integrated into the respective detection beam path, allowing adjustment of the diffraction pattern of the detection radiation. The diffraction pattern is defined, in particular, by the intensity distribution of a so-called Airy disc. This magnification optic, which can be designed, for example, as a circular optic, serves the purpose of adjusting the size of the Airy disc to be imaged according to the selected operating state.

[0036] To adjust the diameter of the PSF, for example, when different objectives are used in the microscope, an optical element or device can advantageously be present by means of which such adjustment can be carried out in a controlled manner. A zoom lens (magnification changer, Pankrat) or a correspondingly designed interchangeable lens group can be present in the detection beam path, and usually in the common detection beam path. Control can be achieved using the existing control system. The zoom lens or lens group serves to influence the diameter of a beam of the detection radiation perpendicular to its optical axis.

[0037] To improve the quality of the measured values, especially to largely avoid extra-focal contributions of the detection radiation, a pinhole is advantageously arranged in an intermediate image plane in front of the beam splitter in a common detection beam path. The aperture of the pinhole can be fixed or adjustable. It is also possible to place different pinholes in the common detection beam path depending on the application.

[0038] In other microscope designs, a physical pinhole may be omitted. The pinhole effect is then generated digitally, as explained in more detail below. One or more optical lenses can be arranged downstream of the pinhole, which serve to appropriately shape and guide the detection radiation emerging from the pinhole on the detector side. The pinhole and downstream lenses can be collectively referred to as imaging optics.

[0039] A microscope according to the invention can be further advantageously configured in that, for example, an optical decoupling element is arranged optically upstream of the beam splitter for decoupling a portion of the detection radiation into another beam path. In further embodiments of the invention, the decoupling element can also be moved into and out of the common detection beam path.

[0040] A decoupled portion of the detection beam can be guided along this additional beam path and directed to additional detection modules, such as multi-channel modules with point detectors or single- or multi-channel modules with spatially resolved detectors. The additional beam path can be used to add additional detectors, for example, by splitting (cascading) the additional beam path using additional beam splitters according to the respective requirements.

[0041] The decoupling can be carried out, for example, by means of a beam splitter as the decoupling element, which, for example, has a fixed splitting ratio and / or is designed as a color splitter.

[0042] The invention also encompasses an arrangement or module designed for coupling to a coupling point of a detection beam path. Such a module allows an existing laser scanning microscope (LSM) to be converted very easily into a microscope according to the invention. If the existing LSM offers the option of extracting at least a portion of the captured detection radiation from the original detection beam path and making it available to another beam path, the module according to the invention can be coupled to this additional beam path.

[0043] For this purpose, the module has a beam path in a housing for guiding coupled detection radiation to an area detector. A coupling element is formed for the purpose of coupling the module to the microscope. This serves to mechanically and, if necessary, optically couple the module to a coupling point of the microscope and to admit a portion of the detection radiation guided in the detection beam path along the further beam path. An imaging optics system is provided along the optical axis of the module and comprises optical elements for shaping the coupled detection radiation. These can be, for example, a pinhole, optical lenses and / or additional apertures and filters. A dispersive optical element is provided between the imaging optics and the area detector. This element can be moved into and out of the beam path in a controlled manner.In a further embodiment of the module according to the invention, the dispersive optical element can remain in the beam path. In the latter case, various detection methods (see above) are implemented by means of an evaluation and analysis of the acquired measured values, as explained below.

[0044] The area detector of a module according to the invention is also designed to detect detection radiation and to provide measured values ​​and has a plurality of detector elements, the measured values ​​of which can each be evaluated individually and / or in groups.

[0045] The object underlying the invention is further achieved with an operating method for operating a microscope according to the invention described above. The method comprises controlling the switching elements to place the microscope in one of at least first and second operating states. In the selected operating state, measured values ​​of a detection radiation are acquired. To place the microscope in the other of the first and second operating states, the switching elements are controlled accordingly, and measured values ​​of a detection radiation are acquired in the other operating state.

[0046] A characteristic of the method according to the invention is that, in at least one of the detection beam paths of the microscope, both the measured values ​​in the first operating mode and the measured values ​​in the second operating mode are acquired with the same area detector. The at least two operating modes allow confocal acquisition of measured values ​​in the first operating mode and spectral acquisition in the second operating mode.

[0047] If, for example, the detection beam path is divided into a first detection beam path and a second detection beam path, different operating states can be realized simultaneously in the respective detection beam paths.

[0048] The invention enables different evaluations of the measured values ​​to be carried out depending on the operating state. For example, in a first operating state, confocal detection of the detection radiation can take place. The PSF is imaged centrally on the area sensor. The illumination is advantageously selected so that as many detector elements as possible are used and, for example, up to five Airy units (AU) of the PSF are imaged onto the area detector. In the case of high intensities of the detection radiation, this can be distributed across a plurality of detector elements, while at low intensities the illuminated area on the detector can be reduced, for example to detect less background signal per detector element.

[0049] In the "classic" confocal mode, all detector elements can be read out (binned) together. By changing the size of the pinhole of the imaging optics, the pinhole aperture used for detection can be adjusted between zero and, for example, five AU in confocal mode.

[0050] In a further embodiment, the mechanical pinhole can be omitted. By selecting the readout detector elements, which represent a circular overall area in particular, different virtual pinholes can be simulated. In the case of confocal acquisition, the effect of a pinhole located in the detection beam path can optionally be simulated by selecting the detector elements of the area detector to be read out around an optical axis of a beam of detection radiation incident on the area detector. This further simplifies the technical design and complexity of the microscope.

[0051] In the first operating mode, the microscope can also be operated in a high-resolution mode. In this case, not all detector elements are combined, but the PSF is scanned and detected spatially resolved. A high-resolution image can be calculated using downstream deconvolution and reconstruction algorithms. In this process configuration, a smaller region of the PSF, for example, 1.2 AU, can also be advantageously imaged onto the area detector.

[0052] The modes described above allow for the functionality of a classic two-channel confocal microscope or a two-channel high-resolution system. Furthermore, optimized operating modes for the detection of both high and very low light intensities are possible.

[0053] The second operating mode, with spectral detection, also offers several detection modes. If the dispersive optical element in front of the area detector is retracted into the respective detection beam path, the detection radiation guided in the respective detection beam path is spectrally split in at least one direction orthogonal to the optical axis of the detection beam path.

[0054] The split detection radiation is preferably directed onto the area detector in an approximately rectangular illumination area (useful area), whereby the entire extent of the area sensor in the direction of the split should be used as far as possible. The best possible utilization of the extent helps to cover a large spectral range and / or achieve high spectral resolution. The extent preferably ensures a uniform height—perpendicular to the direction of the split—of the useable area. If the area detector is not rectangular, this results in unused sections of its detection area. Since the individual detector elements can be read out individually, the invention advantageously enables the selection and readout of only those detector elements encompassed by the useable area. In this way, the acquisition of the measured values ​​can be accelerated and the proportion of dark noise and the amount of data to be processed can be reduced.

[0055] For example, in a microscope with an area detector in each of a first and a second detection beam path, it is possible to distribute detection radiation with a spectral emission range of, for example, 400 - 900 nm between the two area detectors. A dispersive element, which is advantageously optimized for the respective wavelength range, is arranged upstream of each of the area detectors. At the beam splitter, the detection radiation can be distributed between the two detection beam paths, for example in the center of the emission range at 650 nm (400 to 650 nm or 651 to 900 nm). In an alternative design of the microscope, a dispersive element can be arranged upstream of the beam splitter and thus upstream of the division into the various detection beam paths in a common beam path.

[0056] This means that approximately 250 nm of spectral width must be imaged onto each area detector. For example, if the area detector has 25 detector elements (pixels) across its width, this results in a theoretical spectral sampling of approximately 10 nm per pixel. The practically achievable spectral resolution per pixel is lower in practice, taking into account some overlap and the edge steepness of the beam splitter.

[0057] To achieve good spectral resolution, the PSF should cover as few pixels as possible in the direction of the splitting. Therefore, compared to the classic confocal mode, a magnification is necessary. In the above example, in non-spectral mode, five AUs correspond to 18 pixels, i.e., 3.6 pixels / AU. If, for example, one AU is to be distributed across two pixels during spectral acquisition (spectral mode), a magnification by a factor of 1 / 1.8 is required.

[0058] Spectral acquisition can also be used to image a sample with a single measurement that is multicolored, for example, labeled with several different fluorophores. Through appropriate post-processing, multiple dyes and / or stains in samples can be separated and combined to create a multi-channel confocal image. In possible embodiments of the method, for example, up to twelve dyes and / or stains can be computationally unmixed. This significantly saves users time in data acquisition and advantageously increases the number of possible experimental applications.

[0059] Due to the highly sensitive and individually readable detector elements, as well as the ability to evaluate the measured values ​​of the detector elements in parallel using different methods if necessary, confocal acquisitions or imaging are possible even when the detection beam is spectrally split. Therefore, the dispersive optical element can be left in the detection beam path, possibly at the expense of reduced performance, while still achieving the different operating states.

[0060] A further method embodiment, which again requires further magnification, allows not only the separation but also the reconstruction of the PSF. This information can also be used to generate a high-resolution image. Based on measured values ​​of a diffraction pattern, in particular its lateral intensity distribution, of a detection radiation captured by the area detector, the position of an origin of the detection radiation in the sample space can be determined. The lateral resolution of the position of the origin is advantageously higher than diffraction-limited optical detection (localization) of the origin. In this operating mode, the size of the PSF can advantageously be adjusted to a compromise value between the optimal values ​​for spectral and confocal detection using the magnification optics.

[0061] Obtained information on the specific formation of the PSF can also be advantageously used for the further operation of the microscope. For example, the characteristics of a point spread transfer function (PSF) of the microscope can be determined based on measured values ​​of the diffraction pattern and the lateral intensity distribution. Knowledge of the PSF can be taken into account when generating future control commands. The control commands can be used to control controllable (optical) elements of the microscope and / or to evaluate the acquired measured values ​​based on the determined PSF and to modify them if necessary.

[0062] The object of the invention is also achieved with a further operating method for operating a microscope. The further method comprises the steps of controlling a controller and an evaluation unit in order to place the microscope in one of first and second operating states. In the respectively selected operating state, measured values ​​of a detection radiation are acquired and evaluated according to the selected operating state. In order to place the microscope in the other of the first and second operating states, at least the evaluation unit is subsequently controlled. This is followed by the acquisition of measured values ​​of a detection radiation in the other operating state.

[0063] What is also characteristic here is that in at least one of the detection beam paths of the microscope, both the measured values ​​in the first operating state and the measured values ​​in the second operating state are recorded with the same area detector.

[0064] In this alternative method, the different operating states are generated by the function of the evaluation unit in conjunction with the advantages of the area detector, in particular its selective readability of its detector elements and its high sensitivity.

[0065] The advantages of the invention lie in the possibility of providing a cost-optimized, comparatively simple, yet highly flexible multimodal microscope, as well as proposing a module and operating method. Advantageous functionalities include, for example, a large detectable emission range of, for example, 400 to 900 nm, the option of simultaneous multi-channel acquisition with confocal resolution even with large pinhole apertures of, for example, five AU and more, as well as simultaneous multi-channel acquisition with optimized imaging ratios. The invention offers the option of conducting investigations using fluorescence correlation spectroscopy (FCS) and for detecting the movement of objects in the sample. Furthermore, a plurality of different dyes can be spectrally separated in a single image acquisition.

[0066] The invention is explained in more detail below using exemplary embodiments and figures. They show: Fig. 1 a schematic representation of a laser scanning microscope according to the prior art; Fig. 2 a schematic representation of a first embodiment of a detection beam path of a microscope according to the invention; Fig. 3 a schematic representation of an embodiment of an imaging optics in a detection beam path of a microscope according to the invention; Fig. 4 a schematic representation of a second embodiment of a detection beam path of a microscope according to the invention; Fig. 5 a schematic representation of an embodiment of a module according to the invention for coupling to a detection beam path of a microscope; Fig. 6 a schematic representation of the detection surface of a surface detector and a first embodiment of a region of the detection surface of a microscope according to the invention exposed to detection radiation; Fig. 7 a schematic representation of the detection surface of a surface detector and a second embodiment of a region of the detection surface of a microscope according to the invention exposed to detection radiation; and Fig. 8 a schematic representation of the detection surface of a surface detector and a third embodiment of a region of the detection surface of a microscope according to the invention exposed to detection radiation.

[0067] In the Fig. Figure 1 shows a schematic representation of a scanning microscope M in the form of a laser scanning microscope (microscope M) according to the prior art. This comprises a light source 1, for example a laser light source, from which a beam of excitation radiation is emitted and guided along an excitation beam path 2. Optional optical elements for shaping and / or collimating the excitation radiation are not shown.

[0068] The excitation radiation is directed onto a main color splitter 3, which is transparent to the excitation radiation and allows it to pass through. After the main color splitter 3, the excitation radiation passes through a section of the beam path of the microscope M, referred to as the combined beam path 210, along which the excitation radiation and a detection radiation (see below) are or can be guided together.

[0069] By means of a subsequently arranged scanner 5, the excitation beam, previously deflected by a mirror 4, can be deflected in a controlled manner and directed into the entrance pupil EP of an objective 6. The mirror 4 allows for a compact design and can be omitted in other versions of the microscope M if no deflection of the excitation beam path 2 is required or provided.

[0070] The excitation radiation deflected by the scanner 5 is focused by the action of the objective 6 into a sample space in which the sample 7 to be imaged can be present on a sample table 8.

[0071] A detection radiation caused in the sample 7 by the excitation radiation is detected by the objective 6 and guided along a detection beam path 10 (shown with broken solid lines), which coincides with the excitation beam path 2 up to the main color splitter 3.

[0072] In further embodiments of the device according to the invention, the detection radiation can be captured by means of an additional lens (not shown). In such a case, the excitation beam path 2 and the detection beam path 10 can be completely separated from one another, or they can be recombined into the common beam path 210, for example, by means of an additional color splitter (not shown).

[0073] In the example shown, the detection radiation is converted ("descanned") into a stationary beam as a result of passing through the scanner 5 and reaches the skin color splitter 3. This splitter is reflective for the wavelength of the detection radiation, which differs from the wavelength of the excitation radiation. The detection radiation reflected by the main color splitter 3 reaches an optics 11 located in the detection beam path 10, by means of which the detection radiation is directed onto a detector 12.

[0074] The scanner 5 and optionally the light source 1 are connected to a controller 13 for the exchange of data and control commands. The controller 13 is, for example, a computer or a suitable control circuit.

[0075] Optionally, the control unit 13 and the detector 12 can be connected to each other, for example, to enable the control unit 13 to generate and / or validate control commands based on the acquired brightness information (measured values) of the detector 12. These control commands serve, for example, to control the light source 1, the scanner 5, and / or an optional drive 9 of the sample stage 8.

[0076] To explain the invention, the Fig. 2, Fig. 4 and Fig. 5 only shows the detection beam paths of microscopes M according to the invention. The microscope M is symbolized by a frame. For the sake of clarity, other optical elements such as optical lenses and the like are only shown when reference is made to them.

[0077] In a first embodiment of a detection beam path 10 of a microscope M according to the invention, a detection radiation passes along the optical axis oA of a common detection beam path 10 into an imaging optics 11, in which the detection radiation is formed by means of optical elements ( Fig. 2, see also Fig. 3). By means of a magnification optics 14 in the form of a controlled adjustable zoom optics 14, the diameter of the PSF can be adapted to the area sensor 12. The diameter can be adjusted, for example, as a result of the use of different lenses 6 (see Fig. 1) be different.

[0078] The magnification optics 14 represent a means for controlled variation of the beam width of the detection radiation, by means of which the beam width of the detected detection radiation can be adapted to the size of a detection area of ​​a spatially resolving detector 12, which is also arranged in an intermediate image ("pinhole plane") in the detection beam path 10. The goal is to illuminate the detection area as completely as possible when using confocal detection. Accordingly, the detection radiation is adjusted manually or in a controlled manner with respect to the beam width using the zoom optics 14 and a zoom drive (not shown).

[0079] The detection radiation reaches a beam splitter 16, through which the detection radiation is split between a first detection beam path 10.1 and a second detection beam path 10.2. Depending on the design of the beam splitter 16, the splitting can occur according to a fixed or controllable ratio, and the detection radiation can be distributed proportionally between the detection beam paths 10.1 and 10.2. In further embodiments, the splitting can occur based on the spectral properties of the detection radiation. For example, detection radiation of a specific wavelength range can be distributed to the first detection beam path 10.1 and detection radiation of a different wavelength range can be distributed to the second detection beam path 10.2.

[0080] Along the first detection beam path 10.1, the detection beam reaches an optional correction element 18, by means of which, for example, the beam position relative to the optical axis oA can be corrected. Such a correction element 18 can be a plane-parallel glass plate whose inclination about y and / or about z relative to the optical axis oA can be adjusted. Furthermore, at least one filter 17 can be present. The correction element 18 and filter 17 are optionally controllable. The filters 17 can be moved into or out of the detection beam path 10.1 as needed (symbolized by a double arrow).

[0081] An adjustment optics 19 for wavelength-dependent adjustment of the beam of the detection radiation is arranged upstream of the correction element 18. This adjustment optics can comprise several optical elements in various configurations. The adjustment optics 19 allows the dimensions of a diffraction disk of the detection radiation to be captured to be adapted to the area detector 12.

[0082] A dispersive optical element 20 is inserted into the first detection beam path 10.1 and splits the detection radiation in a direction orthogonal to the optical axis oA, for example, in the direction of the y-axis of the Cartesian coordinate system shown. The dispersive optical element 20 is connected to a drive 20.1 and can be moved into and out of the detection beam path 10.1 by its action. Depending on the specific design of the dispersive optical element 20, the splitting can also be influenced by the actual, currently implemented feed path of the dispersive optical element 20. Depending on which part of the dispersive optical element 20 the detection radiation passes through, the splitting can occur spatially and / or spectrally differently. The split detection radiation reaches a surface detector 12 and is detected by its detector elements 12.1 (see Fig. 6 to 8).

[0083] In addition, optionally together with the dispersive optical element 20, a beam-shaping optic 24, for example a cylindrical lens 24, can be moved into the first detection beam path 10.1. Due to its effect, during spectral detection, the detection radiation can be stretched in the direction of the splitting and into a range that can be used to record measured values ​​(useful range NB, see Fig. 6 to 8).

[0084] The previously discussed correction elements 18 and filters 17 are present in the second detection beam path 10.2, which can be used for data acquisition by reflecting a portion of the detection radiation at the beam splitter 16. An adjustment optics 19 for wavelength-dependent adjustment is also present. The dispersive optical element 20 is moved out of the second detection beam path 10.2 in the illustrated operating state. The detection radiation is confocally detected in the second detection beam path 10.2 by the detector 12. The second detection beam path 10.2 also contains an optional optical system for post-magnification 26, with the aid of which the detection radiation to be imaged or a diffraction pattern can be finally adjusted in its dimensions.

[0085] An output coupling 15 can optionally be arranged upstream of the beam splitter 16. This can also be designed as a beam splitter and serves to direct a portion of the detection radiation into another beam path 10.3 (shown with a dashed solid line). Additional detectors and / or other technical elements can be variably connected to this beam path. In further embodiments of the invention, the output coupling 15 can also be arranged upstream of the zoom optics 14.

[0086] The controller 13 can also perform the functions of an evaluation unit 13. In the following, reference is made to a combined control and evaluation unit 13. This selects the detector elements 12.1 to be read out, the method of reading them (e.g., individually and / or collectively), temporarily stores measured values ​​if necessary, and applies different imaging methods to the measured values. Furthermore, a pinhole (see Fig. 3) can be simulated. In further embodiments, the controller and evaluation unit 13 can also be separate technical units that are merely connected to each other in a manner suitable for data transmission.

[0087] The Fig. 2, Fig. 4 and Fig. 5 each shown imaging optics 11 is in the Fig. 3 shows a possible embodiment in more detail. The imaging optics 11 can comprise a pinhole 21 arranged in an intermediate image plane. The pinhole 21, which functions as a confocal pinhole, can optionally be adjustable in terms of its aperture width by means of a drive 21.1. The imaging optics 11 typically comprises at least one optical lens, through the action of which a detection radiation coming from the pinhole 21 is directed along the common detection beam path 10.

[0088] Instead of the pinhole diaphragm 21, further designs may include a slit diaphragm, which can optionally be adjusted in terms of its opening width, rotational position, and / or the shape of its opening by means of a drive 21.1. The drive 21.1 can be controlled by the controller 13.

[0089] A technically simplified embodiment of a microscope M according to the invention is shown in Fig. 4. Currently, the dispersive optical elements 20 are inserted into the respective detection beam paths 10.1 and 10.2. The detection radiation is captured accordingly by the area detectors 12 (illustrated by hatched areas). An adaptation optics 19 is present in each of the detection beam paths 10.1 and 10.2.

[0090] In such a simplified design, a fixed adjustment of the beam path and optimization of the tolerance budget can eliminate the need for correction elements 18. Furthermore, the susceptibility to errors in operation is reduced because fewer elements need to be correctly adjusted.

[0091] In the example, filter 17 and correction elements 18 are Fig. 4 have been omitted. However, these could be retrofitted optionally.

[0092] The beam splitter 16 can be designed such that it spectrally distributes the detection radiation guided in the common detection beam path 10 to the first and second detection beam paths 10.1, 10.2, respectively. For example, a first portion of a wavelength range encompassed by the detection radiation can be directed into the first detection beam path 10.1 and detected there. A second portion of the wavelength range can be reflected into the second detection beam path 10.2 and detected there. The adaptation optics 19 are designed for the respective wavelength ranges. Such spectral division by means of the beam splitter 16 can, of course, also be implemented in the first embodiment (see Fig. 2). The beam splitter 16 can be fixed to simplify the design.

[0093] In a further embodiment, the dispersive optical elements 20 can also be arranged fixedly in the respective detection beam path 10.1, 10.2, whereby the drives 20.1 can be dispensed with and less installation space is required.

[0094] In order to adjust the different operating states or apply different detection methods for permanently installed dispersive optical elements 20, the measured values ​​to be evaluated are selected and analyzed accordingly. Adaptation to different lenses and / or magnifications is achieved in this embodiment by an adapted selection of the detector elements 12.1 to be read out and, if necessary, combined.

[0095] A module Md for coupling to a detection beam path of a microscope M shows Fig. 5. The module Md has a housing 23 in which an imaging optics 11, an adjustment optics 19, and a dispersive optical element 20 are arranged along an optical axis oA. The latter can optionally be moved into and out of the further beam path 10.3. Furthermore, an area detector 20 is provided for capturing the detection radiation.

[0096] To connect the module Md to a microscope M, a coupling element 22 is formed on the housing 23. This can be brought into contact with a correspondingly shaped counterpart (coupling point 25) on another beam path 23 of the microscope M, establishing a preferably detachable connection. At least the area detector 12, and optionally also drives 20.1, 21.1 of the dispersive element 20 and / or the pinhole 21, can be connected to the controller 13 of the microscope M. Optionally, the module Md has its own controller 13 and a data memory, for example, to provide a program (detection and control software) required to operate the module Md, to allow necessary communication with the microscope M, and, if necessary, to store operating data and measured values.

[0097] In all embodiments of the invention, the controller 13 serves to generate and provide control commands, the execution of which enables the invention, in particular the operating method. It is connected to the controllable elements and drives, and optionally to the area detectors 12, in a manner suitable for data exchange.

[0098] With the Fig. The utilization of an exemplary area detector 12 under different operating conditions is shown as an example.

[0099] Using the example of an area detector 12 with a rectangular detection surface on which a plurality of detector elements 12.1 are arranged, the useful area NB available for the acquisition of measured values ​​is outlined with a broken solid line and highlighted with an oblique hatching ( Fig. 6).

[0100] In the case of confocal detection (first operating state), the useful area NB is circular ( Fig. 7). With a rectangular area detector 12, detector elements 12.1 that cannot be used to acquire measured values ​​therefore remain unused. To avoid unnecessary capacity or to avoid reading large numbers of detector elements 12.1 unnecessarily, the detector elements 12.1 to be read can be selected based on the expected or actual useful range NB.

[0101] If the detection radiation is spectrally split, for example, along the y-axis (illustrated by an arrow), it can also be shaped using, for example, a cylindrical lens 24. The useful range NB is limited in the z-axis direction. In this case, non-illuminated detector elements 12.1 can also be selectively excluded from the readout process. Reference symbol 1 light source 2 Illumination beam path 210 common beam path 3 main color dividers 4 mirrors 5 Scanning setup 6 Lens 7 Sample 8 Sample table 9 Drive sample table 10 Detection beam path 10.1 first detection beam path 10.2 second detection beam path 10.3 further beam path 11 Imaging optics, optical lens 12 detector, area detector 12.1 Detector elements 13 Evaluation unit, control 14 Magnifying optics, zoom optics 15 Extract 16 beam splitters 17 filters 18 Correction element 19 wavelength-dependent adjustment optics 20 dispersive elements 20.1 Drive (of the dispersive element 17) 21 aperture, pinhole 21.1 Drive (of pinhole 20) 210 combined beam path 22 Coupling element 23 housings 24 cylindrical lens 25 coupling point 26 Re-enlargement EP entrance pupil M microscope Md module NB Usable area oA optical axis QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited patent literature

[0000] DE 10 2006 034 908 B4

[0003] DE 10 2012 205 722 B4

[0003] DE 10 2023 100 926

[0003] DE 10 2020 213 714 A1

[0003] DE 100 38 526 B4

[0005] DE 10 2020 120 190 A1

[0005]

Claims

[1] Microscope (M), in particular scanning microscope, comprising - a main color splitter (3) for separating an illumination radiation and a detection radiation coming from a sample chamber, - at least one detection beam path (10.1, 10.2) for guiding the detection radiation from the main color splitter (3) to a surface detector (12) located in a detection plane in the detection beam path (10.1, 10.2), which is configured to capture detection radiation and to provide measured values, wherein the surface detector (12) has a plurality of detector elements (12.1), the measured values ​​of which can each be evaluated individually and / or in groups; characterized by , that - in at least one of the existing detection beam paths (10.1, 10.2) there is an adjustment optics (19) which adjusts an extension of a diffraction disk of the detection radiation on the area detector (12) as a function of the wavelength or a wavelength range of the detection radiation; and either switching elements (20) are present in the detection beam path, which can be brought into at least two switching states, so that in the detection beam path (10.1, 10.2), depending on the switching state, at least a first operating state with a confocal and a second operating state with a spectral detection of measured values ​​of the detection radiation by means of the area detector (12) is effected, and an evaluation unit (13) is present which is configured to evaluate measured values ​​of the at least two operating states depending on the currently selected operating state, or at least a first operating state with a confocal and a second operating state with a spectral detection of measured values ​​of the detection radiation is effected by providing a correspondingly configured evaluation unit (13) and evaluating the detected measured values ​​by means of the evaluation unit (13) depending on the currently selected operating state; and In each of the operating states, control commands from an existing control system (13) are used to select the detector elements to be read out individually or in groups. [2] Microscope (M) according to claim 1, characterized by that the detection beam path (10.1, 10.2) is divided into at least a first detection beam path (10.1) and a second detection beam path (10.2), wherein an area detector (12) is present in at least one of the detection beam paths (10.1, 10.2). [3] Microscope (M) according to claim 2, characterized bythat a surface detector (12) is present in at least two of the detection beam paths (10.1, 10.2). [4] Microscope (M) according to one of claims 2 and 3, characterized by in that a beam splitter (16) is provided for dividing the detection radiation between the at least first and second detection beam paths (10.1, 10.2), by the effect of which incident detection radiation is divided between the detection beam paths (10.1, 10.2) with regard to its intensity and / or its spectral composition and / or its polarization. [5] Microscope (M) according to one of the preceding claims, characterized bythat at least one of the switching elements (20) present is a dispersive optical element, by the action of which incident detection radiation is spectrally split in a direction orthogonal to the optical axis (oA) of the detection beam path (10.1, 10.2), the split detection radiation being directed onto the area detector (12). [6] Microscope (M) according to claim 5, characterized by that, together with the dispersive optical element (20), a beam-forming optic (24) can be introduced into the relevant detection beam path, by the action of which detection light is distributed transversely to the direction of a spectral splitting and adapted to a usable detection area (NB) of the area sensor (12). [7] Microscope (M) according to one of the preceding claims, characterized bythat a magnifying optic (14) for influencing a diameter of a beam of the detection radiation is present in the detection beam path. [8] Microscope (M) according to one of the preceding claims, characterized by that a pinhole diaphragm (21) is arranged in an intermediate image plane in a common detection beam path (10) in front of the beam splitter (16). [9] Microscope (M) according to one of the preceding claims, characterized by that an optical decoupling element (15) for decoupling a portion of the detection radiation into a further beam path (10.3) is provided optically upstream of the beam splitter (16). [10] Module (Md) for coupling to a coupling point (25) of a detection beam path (10), comprising a beam path (10.3) for guiding a coupled detection radiation to a surface detector (12); a coupling element (22) for coupling to the coupling point (25) and for admitting a portion of a detection radiation guided in the detection beam path (10) along the beam path (10.3), an imaging optics (11) comprising optical elements for shaping the coupled detection radiation, an adjustment optics (19) which adjusts an extension of a diffraction disk of the detection radiation on the area detector (12) as a function of the wavelength or a wavelength range of the detection radiation; and a dispersive optical element (20) between the adaptation optics (19) and the area detector (12), wherein the area detector (12) is designed to detect detection radiation and to provide measured values, and the area detector (12) has a plurality of detector elements (12.1), the measured values ​​of which can each be evaluated optionally individually and / or in groups. [11] Operating method for operating a microscope according to one of claims 1 to 9 or a module (Md) coupled to the microscope (M) according to claim 10, comprehensively the steps - controlling switching elements (20) to place the microscope (M) into one of first and second operating states, - recording measured values ​​of a detection radiation in the selected operating state, - controlling switching elements (20) in order to place the microscope (M) into the other of the first and second operating states; - recording measured values ​​of a detection radiation in the other operating state, characterized by , that at least in one of the detection beam paths (10.1, 10.2) of the microscope (M) both the measured values ​​in the first operating state and the measured values ​​in the second operating state are recorded with the same area detector (12). [12] Operating method for operating a microscope (M) according to one of claims 1 to 9 or a module (Md) coupled to the microscope (M) according to claim 10, comprehensively the steps - controlling a controller (13) and an evaluation unit (13) to place the microscope (M) into one of first and second operating states, - recording measured values ​​of a detection radiation and evaluating the measured values ​​according to the selected operating mode, - controlling at least the evaluation unit (13) in order to place the microscope (M) into the other of the first and second operating states; - recording measured values ​​of a detection radiation in the other operating state, characterized by , that at least in one of the detection beam paths (10.1, 10.2) of the microscope (M) both the measured values ​​in the first operating state and the measured values ​​in the second operating state are recorded with the same area detector (12). [13] Operating method according to claim 11 or 12, characterized by that the effect of a pinhole diaphragm (21) located in the detection beam path (10) is simulated by selecting the detector elements (12.1) of the area detector (12) to be read out around an optical axis (oA) of a beam of detection radiation incident on the area detector (12). [14] Operating method according to one of claims 10 to 13, characterized bythat a position of an origin of the detection radiation in the sample space is determined on the basis of measured values ​​of a diffraction pattern of a detection radiation detected by means of the area detector (12), wherein the lateral resolution of the position of the origin is higher than a diffraction-limited optical detection of the origin. [15] Operating method according to one of claims 10 to 13, characterized by that the characteristic of a point image transfer function of the microscope (M) is determined on the basis of measured values ​​of a detection radiation detected by means of the area detector (12), and future control commands to controllable elements of the microscope (M) and / or evaluations of the detected measured values ​​are generated or carried out depending on the determined point image transfer function.

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