Microscope

Abstract

The invention relates to a microscope having: a light source for emitting excitation light; an illumination light path for guiding the excitation light onto and / or into the sample; a scanning device for changing the position on and / or in the sample at which the excitation light is applied; at least one two-dimensional spatially resolved detector for detecting light emitted from the sample; a probe light path having a microscope objective for guiding at least a portion of the light emitted from the sample onto a probe; and a control unit for actuating the scanning device and for evaluating measurement data of the detector. According to the invention, the microscope is characterized in that a detection unit is provided, the detection unit comprises a detector and has a variable-focal-length optical device for imaging the sample onto the detector at a variable magnification, and a controllable and selectively activatable dispersion device for spectrally splitting at least a part of the light emitted from the sample, the control unit is also configured to actuate the dispersion device.

Description

Field of the Invention

[0001] The present invention relates to a microscope according to the preamble of claim 1. Background Art

[0002] A microscope of the generic type has: a light source for emitting excitation light; an illumination optical path for guiding the excitation light onto and / or into a specimen; a scanning device for changing the position of the excitation light incident on and / or in the specimen; at least one two-dimensional spatially resolving detector for detecting light emitted from the specimen; a detection optical path having a microscope objective for guiding at least a part of the light emitted from the specimen to the detector; and a control unit for driving the scanning device and for evaluating the measurement data of the detector. Such a microscope is known, for example, from DE 10 2020 120 190 A1.

[0003] Laser scanning microscopes are excellent tools for studying three-dimensional specimens because they suppress light outside the focus based on the spatial filtering of a confocal aperture and thus produce images with very high contrast of any specimen. In recent years, various different measurement methods have been developed for this technology, such as spectral imaging using a spectroscopic detector (DE 10 038528 A1) or high-resolution imaging using a camera-like sensor with spatial oversampling of the point spread function (PSF), i.e., so-called image scanning microscopy (ISM) (DE 10 2020 120 190 A1).

[0004] Due to the diversity of such different measurement modes, laser scanning microscopes (LSMs) tend to be complex and costly. Therefore, integrating spectral imaging and spectral multi-channel ISM into an LSM system is hardly comparable in price to common technologies on the market. In addition, as the number of measurement modes integrated in the system increases, the detection efficiency often also decreases. Therefore, using common LSM systems on the market has so far only been able to create spectral multi-channel image acquisitions either with a lower resolution or in a so-called multi-track mode sequentially.

[0005] In 2021, it was reported that ISM adopted a SPAD array containing 5×5 pixels. Such sensors and similar sensors are already commercially available. Therefore, compared with multi-anode PMTs, sensors with significantly smaller structural dimensions can in principle be provided for ISM. Currently, the number of available pixels in commercially available SPAD arrays that can be used in scanning microscopes is still quite limited. However, this is not a limitation in principle, but rather due to limited data transfer rates. It is foreseeable that the number of pixels in these CMOS-SPAD camera sensors will increase significantly within a few years. Currently, there are already CMOS-SPAD arrays with 1 million pixels (https: / / doi.org / 10.1117 / 12.2589786 and https: / / doi.org / 10.1364 / OPTICA.386574). Summary of the Invention

[0006] It is regarded as the task of the present invention to describe a component that can achieve both spectral imaging and ISM with a simplified structure.

[0007] This task is solved by a microscope having the features of claim 1.

[0008] The above microscope is improved according to the present invention in such a way that there is a detection unit, which includes a detector and has an optical device with a variable focal length for imaging a specimen onto the detector with a variable magnification and a controllable and selectively activatable dispersion device for spectral splitting at least a part of the light emitted from the specimen, wherein the control unit is also configured to control the dispersion device.

[0009] Preferred embodiments of the microscope according to the present invention will be explained hereinafter, in particular in conjunction with the dependent claims and the drawings.

[0010] The present invention is based on the following recognition, that is, a fast two-dimensional resolution detector, especially a SPAD array, can on the one hand be used as a camera for oversampling detection of the PSF, and on the other hand can also be used as a detector for detecting the spectrum of the radiation emitted from the specimen.

[0011] What can be regarded as the basic concept of the present invention is that by providing a variable magnification in the detection optical path on the one hand and a variably activatable dispersion device in the detection optical path on the other hand, a microscope with a two-dimensional spatially resolved detector is functionally upgraded to be used for both image scanning microscopy and spectral imaging.

[0012] It can be regarded as a main advantage of the present invention that the measurement modes of, on the one hand, image scanning microscopy and, on the other hand, spectral imaging can be implemented using one and the same set of components, and thus can be implemented in a manner that is less costly in terms of the set of components compared to the prior art.

[0013] The excitation light is electromagnetic radiation, in particular electromagnetic radiation in the visible spectral range and adjacent ranges. For the present invention, the requirement for the principle of contrast formation is only that the sample emits emitted light and / or deflects, scatters or reflects the excitation light when irradiated with the excitation light. Typically, the emitted light is fluorescence emitted by the sample, in particular dye molecules present therein, when irradiated with the excitation light.

[0014] To provide the excitation light, there is at least one light source, such as a laser. The spectral composition of the excitation light can be adjustable, in particular adjustable between two or more colors. For example, when different dyes need to be detected simultaneously, the excitation light can also be simultaneously multi-color.

[0015] The term "illumination optical path" refers to all optical components that guide and change the light beam, such as microscope objectives, lenses, mirrors, prisms, gratings, filters, apertures, beam splitters, modulators, such as spatial light modulators (SLMs), using and via which the excitation light from the light source is guided onto the sample to be examined. The components that change the light beam also include dispersive and in particular diffractive elements that cause optical dispersion, such as structures such as gratings or volume holograms, which may in particular partially have a spatially periodic structure.

[0016] The light emitted and / or deflected (e.g., scattered) from the sample to be examined when irradiated with the excitation light can be referred to as emitted light and reaches at least one detector via the detection optical path.

[0017] The term "detection optical path" refers to all optical components that guide and change the light beam, such as objectives, lenses, mirrors, prisms, gratings, filters, apertures, beam splitters, modulators, such as spatial light modulators (SLMs), using and via which the emitted light is guided from the sample to be examined to the detector. The microscope objective is part of the detection optical path.

[0018] Imaging the sample means imaging at least a part of the sample. The visible range is limited by the field of view of the sensor and / or by a possible field aperture in the detection optical path.

[0019] The term "control unit" refers to all hardware and software components that cooperate with the components of the microscope according to the invention to make these components work as specified. In particular, the control unit can have computing devices, such as a PC, and a camera control unit for being able to quickly read measurement signals. The computing resources of the control unit can be distributed to multiple computers and, if necessary, to a computer network, especially also via the Internet. The control and evaluation unit can in particular have common operating devices and peripherals, such as a mouse, a keyboard, a screen, a storage medium, a joystick and an Internet connection. The control unit can in particular read in image data from the detector and can also be used to drive the light source. According to the invention, the control unit can also be configured to drive a dispersion device. The control unit can also be configured to adjust the magnification using an optical device with variable focal length.

[0020] In a typical embodiment, in the microscope according to the invention, the illumination optical path and / or the detection optical path has at least one of the following components, namely, a main beam splitter, scanning optics, and a tube lens. The illumination optical path and the detection optical path can be partially provided by the same optical components. The main beam splitter refers to a beam splitter that separates the excitation light from the emitted light (i.e., the emitted light from the specimen). Typically, therefore, the detection optical path downstream of the beam of the main beam splitter contains almost or completely no component of the excitation light. Thus, at least one detector in the detection unit is not over-irradiated by the excitation light. The main beam splitter can be a dichroic beam splitter.

[0021] Illumination of the specimen can be achieved using separate optics, especially via a separate microscope objective. However, generally, the specimen is illuminated via the same microscope objective that is also part of the detection optical path.

[0022] The scanning device can include a two-dimensional scanner arranged in the illumination optical path. The scanner can also be part of the detection optical path, that is, it scans the light emitted from the specimen and to be detected. The two-dimensional scanner can be, for example, a galvo scanner. As a supplement or alternative, the scanning device can also include a controllable specimen stage that can move laterally. The specimen stage can advantageously move laterally in two independent coordinate directions respectively perpendicular to the optical axis of the microscope objective. The scanning device can also be realized by a combination of a two-dimensional scanner and a laterally movable specimen stage.

[0023] In a preferred embodiment, the detection unit has an aperture diaphragm with an adjustable size. The aperture diaphragm can perform two functions. On the one hand, it can block out-of-focus emitted light, and on the other hand it acts as an entrance opening for a spectral dispersion mode, which will be described in more detail below. In the spectral resolution or dispersion mode, the size of the entrance opening will affect the spectral resolution and, when there are multiple spectral detection channels, also the separation of the detection channels. Advantageously, there may also be an entrance optics in order to produce an intermediate image plane in which the aperture diaphragm can be positioned.

[0024] In a particularly preferred embodiment, the variable focal length optics image one and the same plane of the specimen onto one detector or multiple detectors regardless of the focal length adjustment. This achieves the advantage that one detector or multiple detectors can each be kept in the same position regardless of how the variable focal length optics are adjusted.

[0025] The variable focal length optics can be formed by a zoom optics. The advantage achieved here is that the magnification of the imaged specimen plane can be adjusted particularly finely. In particular, the magnification can be adjusted very precisely to an advantageous value in view of the pixel pitch parameter of at least one of the detectors used and the extent of the point spread function.

[0026] The term "point spread function" refers to, for example, the conversion of an incident parallel beam (which fills a specific effective diameter of the lens) into the resulting light intensity distribution by a lens. Also commonly used terms for this function are point distribution function, point imaging function or the English term Point-Spread-Function (PSF). The effective diameter of the lens can be, but does not have to be, the maximum possible diameter. That is to say, the PSF does not necessarily correspond to making full use of the maximum available numerical aperture. In the case where the numerical aperture is not fully utilized, the extent of the PSF will be increased compared to the case of maximum utilization.

[0027] However, it is also possible that the variable focal length optics are formed by a lens replacement system with which the magnification can be adjusted to a plurality of discrete values. For many applications, this may already be sufficient.

[0028] A particular advantage of the present invention shown here is that the detection unit can be upgraded to a spectral multi-channel variant relatively easily. Thus, a particularly preferred embodiment is characterized in that a first detection channel is formed in the detection unit, which first detection channel has at least a detector, there is at least one further detection channel in the detection unit, each further detection channel having a two-dimensional spatially resolving detector, and there is at least one dichroic beam splitter in the detection optical path in order to guide a spectral component of the light emitted by the specimen and to be detected into the respective further detection channels.

[0029] In the simplest variant of a spectral multi-channel system, there can be a dichroic beam splitter with a filter edge that divides the light to be detected (relative to the filter edge) into at least a long-wave component and a short-wave component. Then, the short-wave component enters the first detection channel, and the long-wave component enters the second detection channel accordingly. The dichroic beam splitter can in particular be a dichroic beam splitter.

[0030] In order to be able to match the division of the spectral components into these detection channels to the sample, it is advantageous if the spectral position of the filter edge of the dichroic beam splitter can be varied discretely or, particularly advantageously, continuously. However, the dichroic beam splitter can also be implemented by a filter assembly with a plurality of filter layers on which the light to be detected is reflected accordingly multiple times (interference filter, for example band-pass filter).

[0031] At least one controllable and selectively activatable dispersive device for spectral splitting of the light to be detected is designed to perform spectral splitting of the light to be detected. Spectral splitting means spectral decomposition, i.e., that the spectral components of the light are emitted in different spatial directions according to the wavelength of each component after passing through the device for spectral splitting. The splitting can be carried out using diffraction and / or refraction. The selectively activatable characteristic of the dispersive device involves that the dispersive device selectively either splits the light to be detected in the detection optical path or does not split it. In a preferred design, the dispersive device is introduced into or removed from the detection optical path for this purpose. Alternatively or additionally, the detection optical path is redirected such that the mentioned dispersive device becomes part of the redirected detection optical path. The controllable characteristic of the dispersive device involves that the selective activation can be controlled by a control unit.

[0032] To implement the present invention, it is sufficient to have a controllable and selectively activatable dispersive device for spectral splitting of the light to be detected. In the case of a multi-channel detection unit, the dispersive device can be arranged upstream of the beam of at least one or a plurality of dichroic beam splitters in the detection optical path. Then, the dispersive device acts on all spectral channels jointly.

[0033] Advantageously, the controllable and selectively activatable dispersive device for spectral splitting of the light to be detected acts only in one of the detection channels, and / or there can be at least one additional controllable and selectively activatable dispersive device for spectral splitting of the light to be detected that acts only in one of the detection channels. For example, the dispersive device present according to the invention can be part of the first detection channel.

[0034] If it is stated in this specification that at least one component can have a specific property or a specific feature, this also means that several or all components of the respective type can have the respective property or the respective feature.

[0035] For example, in an advantageous variant, a drivable and selectively activatable dispersion device for spectral splitting of the light to be detected can also be present in each individual detection channel. The advantage achieved thereby is that the spectral dispersion can be adjusted in a targeted manner in each individual detection channel. For example, the dispersion device and the dichroic beam splitter to be used in the respective detection channels can be coordinated with each other. This variant is particularly advantageous when using a prism as the dispersion element, since the spectral dispersion of the prism decreases with increasing wavelength.

[0036] In particular, a drivable and selectively activatable dispersion device for spectral splitting of the light to be detected in at least one detection channel or for at least one detection channel can have at least one of the following components or be formed by at least one of the following components: a grating that can be introduced into the detection light path, in particular a swing-in grating, in particular a transmissive grating or a reflective grating; a prism that can be introduced into the detection light path, in particular a swing-in prism, in particular a direct vision prism or a Perrin - Broca prism. The dispersion device can also have holographically produced components. Alternatively, when, for example, a swingable mirror is used to redirect the detection light path for activating the dispersion device so that the dispersion device then becomes part of the redirected detection light path, a drivable and selectively activatable dispersion device can also be realized. In this case, the dispersion device itself can but does not have to be arranged with a fixed relative orientation.

[0037] In the simplest case, to switch to the spectral detection mode, the spectral dispersion device is introduced into the light path.

[0038] Advantageously, when switching to spectral imaging, the magnification of the image of the sample onto the two - dimensional spatially resolving detector is adapted such that the full width at half maximum (FWHM) of the PSF reaches the order of several pixels, ideally the order of only one pixel or less, so that as large a spectral range as possible can be measured simultaneously. In addition, the spectral resolution of the detection system can be improved by shifting the spectrum on the sensor for taking two successive images and then calculating them.

[0039] In microscopy, it is generally always desired that the image to be captured has reasonable gray values. In other words, one strives to reasonably utilize the maximum capacity of the detector used, that is, neither operating in the saturation region (where the proportional relationship between the measured signal and the light quantity is lost), nor on the other hand operating at a very low count rate (where the signal is very small compared to the detector noise).

[0040] Furthermore, it should be noted in this regard that the count rate of the detector used is typically limited to a few megahertz, and in the best case up to several tens of MHz. In the case of a SPAD array, the physical reason for the limited count rate is the nature of photon counting of the SPAD pixels and their inherent dead time during avalanche quenching. Therefore, in order to obtain reasonable gray values in the generated image in the case of a pixel dwell time typical for a laser scanning microscope of about 1 microsecond or shorter, it is advantageous to distribute the emission of the dye over multiple pixels of the detector. To some extent, this has already been achieved through spectral dispersion. Further improvement is possible in a further particularly preferred embodiment, in which in the detection unit and / or in at least one detection channel, there is a cylindrical optical device that can be selectively introduced into the detection optical path, and the cylindrical optical device axially moves the image plane in a direction perpendicular to the direction in which the dispersion device in the associated detection channel causes spectral decomposition. The axial movement of the image plane, that is, the movement in the optical axis direction, causes the light in the wavelength range to be detected to be distributed over multiple pixels of the detector. In the extreme case, the light in the wavelength range to be detected can be distributed over all pixels in the relevant row or column of the detector. By the cylindrical optical device that can be introduced, especially swung in, the PSF can be defocused on the axis perpendicular to the dispersion direction and thus expanded to multiple pixels on the detector.

[0041] There can be only a single selectively introducible cylindrical optical device, which can be arranged upstream of the beam of at least one dichroic mirror or a plurality of dichroic mirrors in the detection optical path. The cylindrical optical device then acts on all spectral channels jointly. In another advantageous variant, there are separate selectively introducible cylindrical optical devices in multiple detection channels or in each individual detection channel. The distribution of the PSF to the detector pixels can then be carried out uniquely in the corresponding channel or in each channel. If necessary, different cylindrical optical devices can also be used in various detection channels. At least one of the cylindrical optical devices can have at least one cylindrical lens, or can be realized by at least one cylindrical lens.

[0042] In a further preferred embodiment of the invention, the detection unit and / or at least one of the detection channels has a filter whose spectral transmission and / or reflection characteristics can be adjusted. The filter can be used, for example, to adjust the spectral detection bandwidth for ISM and to limit it to the dyes selected respectively.

[0043] Particularly preferably here, at least one of the filters has an adjustment mode for broadband transmission and / or reflection of light. This adjustment mode can then advantageously be selected for the spectrally resolved measurement mode. For example, at least one of the filters can have a plurality of discrete filter segments which can each be introduced into the detection optical path. Alternatively or additionally, in another embodiment, at least one of the filters can be realized by a continuously adjustable filter.

[0044] In a further particularly preferred design, in the detection unit and / or in at least one of the detection channels, there is an adjustable optical device for laterally displacing the spectrally split light relative to the detector of the respective detection channel. By means of the adjustable optical device for laterally displacing the spectrally split light, it is possible to particularly advantageously adjust the projection of the partial spectrum in the detection channel onto the detection in the detection channel.

[0045] The adjustable optical device for lateral displacement in the detection unit and / or in at least one of the detection channels can have at least one of the following components or be realized by one of the following components: an adjustable tiltable mirror, an adjustable tiltable plane-parallel transparent plate, in particular a glass plate. Thereby, the spectral ranges relevant for the respective excited fluorophores can be optimally projected onto each individual detector sensor.

[0046] The mechanical operation of the components in the optical path, such as swinging into and out of the optical path, moving into, out of the optical path and tilting, can in principle be known manner by a drivable motor-driven device.

[0047] Pixelated detectors, in particular SPAD array detectors, usually have a fill factor of less than 1, which means that the detector has areas between the pixels where photons incident there cannot be detected. Therefore, the detector can only detect incident photons on a part of the detector surface. To compensate for this property, the detector can be provided with a microlens array, where a microlens can be assigned to each pixel (for example to each SPAD pixel). In a preferred variant, a multi-lens array is arranged at least in front of the detector. Arranging in front of the detector means that the multi-lens array is in the optical path upstream of the detector's light beam, i.e. the light to be detected passes through the multi-lens array before hitting the detector. The multi-lens array can be arranged on the associated detector. The multi-lens array and the associated detector can form a structural unit.

[0048] In a preferred embodiment, in at least one detector, pixels, especially adjacent pixels, such as pixels adjacent in the direction of a detector row and / or a detector column, can be combined by pixel binning. If each individual pixel of the detector is read separately, each pixel will provide a separate detection signal according to the amount of light to be detected received at its respective pixel position. If multiple pixels are combined by pixel binning, a single "binned region" will provide a detection signal such that the detection signal corresponds to the sum of the amounts of light to be detected received on the pixels forming the "binned region". Thereby, the number of detection signals to be evaluated is reduced and the readout speed of the detector is accelerated. Advantageously, an entire pixel row and / or an entire pixel column can be combined by pixel binning. Depending on the measurement task, it may be appropriate to combine pixels in a direction perpendicular to the spectral dispersion direction by pixel binning. In this case, the spectral resolution is maintained, but at the same time a high dynamic range is achieved by using multiple detector elements and reducing the data rate. In other measurement task scenarios, it may be appropriate to bin the pixels in the spectral dispersion direction. Thereby, the spectral resolution is reduced, but the readout speed and the image frame rate can be increased. In addition, the above two pixel binning measures can also be combined.

[0049] Advantageously, the spectral channels on the sensor are also defined by pixel binning. For example, two dyes each emitting with a bandwidth of 50 nm at, for example, 500 nm or 580 nm can be detected. Then two binned regions can be opened up, which correspond to wavelength ranges of 500 + / - 25 nm and 580 + / - 25 nm. Each of these binned regions corresponds to a spectral channel, and all the remaining data is then discarded or otherwise pixel-binned and transmitted.

[0050] At least one two-dimensional spatially resolved detector is a sufficiently fast optical detector having a two-dimensional spatially resolved sensor surface. The detector can in particular be a camera, especially a camera having a CCD, sCMOS, CMOS or SPAD camera chip or a SPAD array. Particularly preferably, in each detection channel, the respective detector can have a SPAD array. A series of properties of the fast two-dimensional resolved detectors, especially SPAD arrays, form the basis for their preferred use. Thus, these detectors can detect fluorescence signals in a time-resolved manner. Since fluorophores often differ in their fluorescence lifetimes, it may be advantageous to incorporate this parameter into the mathematical analysis problem. In addition, the sensor elements can be switched on and off flexibly. For such applications, a flexible switching pattern for the pixel matrix is described in WO2020207571A1.

[0051] Two-dimensional spatially resolved detectors and corresponding optical structures are particularly suitable for detecting the PSF of emitted light in a way of lateral oversampling (at least meeting the Nyquist theorem). This is particularly important when the so-called image scanning microscopy (Carl Zeiss brand name: Airyscan) method should be performed. However, it is also possible to perform traditional confocal measurements using this detector, for example, by adding the values of all or at least most of the pixels. The detector can preferably have a rectangular or hexagonal pixel arrangement.

[0052] In a particularly preferred design of the microscope according to the invention, there is a dichroic device with a rotor on which a plurality of dichroics are arranged. By adjusting different rotational positions of the rotor, different dichroics can be introduced into the detection optical path. Via the dichroics, a spectral component of the light emitted by the specimen can be delivered to each detection channel. Thereby, the detection channels can be arranged very compactly.

[0053] The dichroics can each be configured to reflect one spectral component of the light and transmit one component in turn on each dichroic, and the transmitted spectral component can be delivered to one of the detection channels respectively. The dichroics can be band-pass filters.

[0054] In an advantageous refinement of such a dichroic device, different groups of dichroics can be introduced into the detection optical path by adjusting different rotational positions of the rotor.

[0055] Particularly advantageously, the dichroics can be arranged on the circumferential side surface of a cylinder on the rotor, where the rotation axis of the rotor is collinear with the main axis of the cylinder and is oriented transversely, especially perpendicularly, to the incident direction of the light onto the dichroic device. The advantage achieved thereby is that due to the stable rotation of the filter surface around the main axis of the cylinder, the beam direction of the generated detection optical path remains unchanged after the configuration changes. Since the sensor surface is typically small for, for example, a SPAD array detector (usually significantly less than 1 mm 2 )), this is particularly advantageous in this regard. Description of the Drawings

[0056] Further advantages and features of the present invention will be described below in conjunction with the drawings. Among them:

[0057] Figure 1 : shows a schematic diagram of a microscope according to the present invention;

[0058] Figure 2 : shows a schematic diagram of an embodiment of the detection unit of a microscope according to the present invention;

[0059] Figure 3: shows a diagram of the illumination of a two-dimensional spatially resolved detector in different operating modes of a microscope according to the present invention; and

[0060] Figure 4 : shows an embodiment of a filter device for a microscope according to the present invention. Detailed Description

[0061] An embodiment of a microscope 100 according to the present invention is explained with reference to Figures 1 to 3 . The microscope 100 schematically shown in Figure 1 has a light source 1, an illumination optical path with a microscope objective 7, a detection optical path, a detection unit 10, and a control unit 11 as main components. The light source 1, typically a laser, is especially a laser with multiple wavelengths, which is used to emit excitation light 2, and the excitation light is guided through the illumination optical path onto and / or into the specimen 8 to be examined. In the schematically shown example, the illumination optical path includes a main beam splitter 3, a scanning device 4, scanning optics 5, a tube lens 6, and a microscope objective 7. In the microscope 100, in a known manner, a holder for accommodating the specimen can be present in the specimen space below the microscope objective 7. Figure 1 The structure shown in

[0062] is basically equivalent to a laser scanning microscope (LSM) for sequentially scanning and illuminating the specimen 8 using an excitation laser as the light source 1.

[0063] Starting from the light source 1, the excitation light 2 first reaches the main beam splitter 3, and then is guided from the main beam splitter in the direction of the scanning device 4, which can be a two-dimensional galvanometer scanner. From there, the excitation light 2 reaches the microscope objective 7 via the scanning optics 5 and the tube lens 6, and the microscope objective focuses the excitation light 2 onto and / or into the specimen 8.

[0064] The first component of the detection optical path is the microscope objective 7, which receives the emission light 9 and collimates it. Via the tube lens 6 and the scanning optics 5, the emission light 9 reaches the scanning device 4, where it is scanned, and then is transmitted through the main beam splitter 3 (differently from the excitation light 2 there) to the detection unit 10, and is imaged onto the two-dimensional spatially resolved detector 28 present according to the present invention in a relatively fixed orientation in the detection unit.

[0065] In the illustrated embodiment, the control unit 11 is at least used to drive and control the light source 1, the scanning device 4, and is used to evaluate the measurement data of the detection unit 10 (i.e., at least one detector 28). For example, the control unit 11 can adjust the intensity of the excitation light 2 depending on the position on or within the specimen 8 and, if necessary, depending on the intensity of the emitted light 9 from the relevant specimen position. Finally, the control unit 11 can, if necessary, generate an image of the specimen 8 based on an appropriate calculation of the measurement data.

[0066] An embodiment of the detection unit 10 for the microscope 100 according to the invention is explained with reference to Figure 2 which. The detection unit 10 shown therein has a pinhole optics 21 with an adjustable, variable-sized aperture stop, an optical device 22 with a variable focal length according to the invention, and a first spectral detection channel 30a and a second spectral detection channel 30b as main components.

[0067] In Figure 2 the components schematically shown, the first detection channel 30a and the second detection channel 30b basically comprise the same optical components, which are optimized, if necessary, according to the different spectral compositions of the split light beams. The equivalent optical components in the two detection channels 30a and 30b are respectively provided with the same reference numerals and, incidentally, the components of the first detection channel 30a are additionally provided with the letter a, while the components of the second detection channel 30b are additionally provided with the letter b.

[0068] In the first detection channel 30a and in the second detection channel 30b, two-dimensional spatially resolved detectors 28a or 28b are respectively provided, where, in the embodiment, they are both SPAD array detectors.

[0069] The pinhole optics 21 generates an intermediate image plane in which an adjustable aperture stop (which can also be referred to as a pinhole) is arranged. The aperture stop can, on the one hand, be used to block out-of-focus radiation and, on the other hand, the aperture stop can be used as an incident opening for the spectral resolution mode. In the latter case, the size of the opening of the aperture stop affects the spectral resolution and thus also affects the separation of the spectral detection channels.

[0070] Downstream of the light beam of the pinhole optical device 21, there is an optical device 22 with variable focal length, which is realized by a zoom optical device schematically shown in the illustrated embodiment. The optical device 22 with variable focal length is designed such that its rear focal plane is always located in the plane of the SPAD detectors 28a, 28b. In other words, the plane of the SPAD detectors 28a, 28b is optically conjugate to the plane of the adjustable aperture diaphragm. By using the optical device 22 with variable focal length, on the one hand, the illumination of the detectors 28a, 28b can be appropriately adapted after replacing the microscope objective. On the other hand, for the spectral resolution measurement mode, the magnification from the plane of the adjustable aperture diaphragm to the detectors 28a, 28b can be reduced so that the FWHM of the PSF on the detectors 28a, 28b reaches the pitch d of the pixels of the detectors 28a, 28b (see Figure 3 B) or less, in order to improve the spectral resolution. The size of the spectral range projected onto the sensor is related to the angular dispersion generated in the prism and the distance between the prism and the sensor.

[0071] On a dichroic beam splitter 23, which can be, for example, a dichroic beam splitter that can be adjusted in a discrete or continuous manner, in the illustrated embodiment Figure 2 In the illustrated embodiment, the detection optical path is split into two detection channels 30a and 30b downstream of the light beam of the optical device 22 with variable focal length, which guide different spectral ranges of the emitted light 9 downstream of the beam of the dichroic beam splitter 23.

[0072] Downstream of the beam of the dichroic beam splitter 23, dispersive devices 24a, 24b that can be driven and selectively activated are respectively provided, which are used for spectral splitting of the emitted light 9 in the first detection channel 30a or the second detection channel 30b. In the illustrated embodiment, the dispersive devices 24a and 24b are respectively realized by a combination of movable glass wedges made of different materials, and these different glass wedges can be moved into or out of the optical path in a motor-driven manner. This movement is respectively indicated by double arrows. When the spectral dispersive devices 24a, 24b are in the optical path and in the activated state, the emitted light 9 is split or spectrally fan-shaped expanded in the dispersion plane downstream of the respective dispersive devices 24a, 24b. In Figure 2 In it, the dispersion plane is the same as the drawing plane.

[0073] The spectrally split emitted light 9 then respectively impinges on cylindrical optical devices 25a and 25b, which shift the focusing position in a direction perpendicular to the detection plane, so that the emitted light 9 is distributed over many pixels perpendicular to the dispersion direction in the plane of the SPAD array detectors 28a, 28b (see Figure 3B). In the dispersion direction, the cylindrical optical devices 25a, 25b basically do not change the focusing. The image position in the dispersion direction is a function of the wavelength. This factual situation will be explained in more detail below in conjunction with Figure 3 B.

[0074] By using the plane-parallel plates 26a, 26b whose angles can be adjusted, according to the adjustment of the beam splitter 23, the spectra respectively generated by the dispersion devices 24a, 24b can be shifted relative to the detectors 28a, 28b, so that the spectral range of interest is as much as possible located at the centers of the detectors 28a, 28b. For example, the spectra can be shifted laterally so that the emission spectral peaks are respectively located at the centers of the detectors 28a, 28b, or as much as possible the entire spectral emission bandwidth is captured by the relevant detectors 28a, 28b.

[0075] Finally, downstream of the light beams of the components 26a, 26b, there are respectively adjustable filters 27a, 27b. By using these adjustable filters, the spectral detection bandwidths for the first detection channel 30a and the second detection channel 30b can be adjusted for the image scanning (ISM) measurement mode.

[0076] During ISM operation, the dispersion devices 24a, 24b and the cylindrical optical devices 25a, 25b are removed from the optical path in the direction indicated by the arrow, and the adjustable filters 27a, 27b are advantageously adjusted to positions that can, together with the beam splitter 23, achieve spectral discrimination of unwanted signals.

[0077] The movement of the dispersion device 24a in the first detection channel 30a can be carried out jointly with the movement of the dispersion device 24b in the second detection channel 30b, but can also be carried out independently. Therefore, it is possible to operate the two detection channels 30a, 30b in the ISM mode, operate the two detection channels 30a, 30b in the spectral resolution mode, or operate the first detection channel 30a in the ISM mode while operating the second detection channel 30b in the spectral resolution mode, or vice versa. Therefore, ISM data and spectral image data can be generated simultaneously.

[0078] The emitted light reflected on the filters 27a, 27b can also be redirected to Figure 2 other detectors not shown. As described above, the spectral components of the detected light being reflected into other detection channels can also be achieved by means of additional beam splitters, which can be arranged downstream of the beam splitter 23 and before one or both of the components 24a, 24b.

[0079] In the dispersion mode, the adjustable optical filters 27a, 27b can advantageously be adjusted to a position such that, as far as possible, only the broadband antireflection layer is present in the optical path.

[0080] The order of the optical components in the detection channels 30a, 30b can be different without restricting the function. However, due to the focusing action of the cylindrical optics 25a, 25b, it is also expedient to arrange these cylindrical optics downstream of the beams of the dispersion devices 24a, 24b, respectively.

[0081] To compensate for the limited fill factor of these detectors, a multi-lens array can be present in front of the detectors 28a, 28b, but this is not shown in Figure 2 Here, a microlens can be assigned to each pixel of the detectors 28a, 28b. Preferably, SPAD array detectors with a multi-lens array are used, which have a large acceptance solid angle on their input side in order to image the angle-split spectra generated by the dispersion devices 24a, 24b and the cylindrical optics 25a, 25b as completely as possible onto the respective active areas of the detectors 28a, 28b. For this purpose, the focal lengths of the lenses of the multi-lens array should not be chosen too small, so that a change in the angle of incidence does not cause too large a change in the image projection. In addition, it should also be noted that the image of the system pupil generated on the active areas of the detectors 28a, 28b should be significantly smaller than the sensor surface belonging to a single pixel. The SPAD array detectors 28a, 28b used can have good sensitivity over the entire wavelength spectrum; but they can also be optimized according to the specific range of the spectrum to be detected in the respective detection channels.

[0082] Figure 3 In, the sensor surfaces with typical light distributions for ISM operation ( Figure 3 A, left) and spectrally resolved operation ( Figure 3 B, right) are schematically shown in FIGS. A and B, respectively.

[0083] In the example shown, the pixels of the detector are arranged in a rectangular pattern. However, a hexagonal or other arrangement is also possible. In Figure 3 A, the PSF of the emitted light 9 is positioned on the detector surface approximately such that the intensity maximum of the PSF is approximately at the center of the detector. Thus, it is ensured that the intensity of the PSF decays as much as possible at the edges of the detector. In addition, by appropriately adjusting the variable focal length optics 22 (see Figure 2 ), the magnification of the image is selected such that the PSF (more precisely, the diameter of the first Airy ring) is scanned by at least five pixels or a five-pixel merging region. This situation is represented by the following relationship:

[0084] FW HM (psf) ≥ 5d

[0085] Wherein, d is the pitch of the pixels of the detector (pixel pitch) or the pitch of the pixel binning region. The pixel binning region, i.e., a group of especially adjacent pixels (which are combined into a binning region) can be referred to as the pixel binning region.

[0086] Figure 3 B shows the arrangement of the PSF of light of the only wavelength λ0 on the detector in the spectral resolution mode. As can be seen, due to the action of the cylindrical optics 25a or 25b, the PSF has a strongly asymmetric shape. In Figure 3 B, the dispersion direction extends horizontally, i.e., in the x direction (see Figure 3 the coordinate system in). This means that Figure 3 the PSFs of other wavelengths not shown in B will be located to the right or left of the shown PSF depending on whether the relevant wavelength is greater than or less than the wavelength λ0. For this mode, the variable focal length optics 22 can advantageously adjust the magnification so that the FWHM of the PSF for a single wavelength (as Figure 3 visible in B) is less than the pixel pitch d. Thus, an improved spectral resolution is achieved. In addition, in order to enable the PSF for each wavelength to contribute as many pixels of the detector to signal generation and thus provide a good gray scale dynamic in the image, the cylindrical optics 25a, 25b are used to defocus the PSF perpendicular to the dispersion direction, i.e., in the y direction. This results in light of the same wavelength λ0 illuminating multiple pixels in the y direction.

[0087] Figure 4 An embodiment of the dichroic device 80 is shown, by which the received radiant light 9 can be distributed into a total of five spectral detection channels Ch1,..., Ch5. The reference signs λl to λ5 refer to the wavelength spectra of the radiant light 9 in the respective detection channels Ch1,..., Ch5. Each of the reference signs λl to λ5 can represent a limited spectral range and can also represent a plurality of discontinuous spectral ranges.

[0088] The dichroic device 80 has a rotor 60, on which first the dichroic elements BP1,..., BP4 are arranged distributed in the circumferential direction along the circumferential side surface of the cylinder, and these dichroic elements form the first group of dichroic elements. In Figure 4 the case shown, the dichroic elements BP1,..., BP4 of the first group of dichroic elements are in the optical path. The axis of rotation of the rotor 60, which extends perpendicular to the plane of the paper and passes through the point marked with M, is collinear with the main axis of the cylinder and is especially oriented perpendicular to the incident direction of the radiant light 9 onto the dichroic device 80.

[0089] InFigure 4 In the case shown, the beam splitter device 80 operates as follows: The received radiation light 9 enters through the window W1 and impinges on the first beam splitter BP1. The first spectral component λl is transmitted through the beam splitter BP1 and coupled out into the first detection channel Ch1. The spectral component of the radiation light 9 that is not transmitted through the beam splitter BP1 is basically reflected by the beam splitter BP1 and then impinges on the second beam splitter BP2. There, the second spectral component λ2 is transmitted and coupled out into the second detection channel Ch2. Similarly, the beam splitters BP3 and BP4 also continue the process, where the spectral components λ3 and λ4 are respectively coupled out into the third detection channel Ch3 or the fourth detection channel Ch4 on these beam splitters.

[0090] The remaining spectral component λ5 of the radiation light 9 is reflected on the fourth beam splitter BP4 and reaches the fifth detection channel Ch5 via the exit window W2.

[0091] In the illustrated embodiment, the entrance window W1 and the exit window W2 are neutral, and the light passing through there does not change spectrally. However, it is also possible that a filter is provided there.

[0092] Assuming that no light is absorbed in the beam splitters BP1 to BP4 and the components W1 and W2, the received radiation light 9 will be completely divided into the detection channels Ch1 to Ch5.

[0093] Then, in Figure 4 The particularly advantageous property of the beam splitter device 80 shown in is that additional beam splitters BP5 to BP8 are additionally arranged on the circumferential side surface of the cylinder 60, and a second set of beam splitters is formed by these additional beam splitters. By twisting the cylinder 60 clockwise by 30°, it is possible to make the component W2, which has hitherto served as the exit window, become the entrance window for the incident radiation light 9. Then, the optical path is the same as described before, except that the positions of the first set of beam splitters BP1,..., BP4 are replaced by the positions of the second set of beam splitters BP5,..., BP8, and the position of the exit window W2 is replaced by the window W3. The second set of beam splitters BP5,..., BP8 may have spectrally different properties compared to the first set of beam splitters BP1,..., BP4.

[0094] Switching from the inspection of a first specimen prepared with a first selected dye, especially in the case of using the first spectrum of excitation light 2, to the inspection of a second specimen prepared with a second dye different from the first selection, especially in the case of using a second spectrum different from the first spectrum of excitation light 2, is particularly convenient in the case of using such a dichroic device. A particular advantage is that, due to the stable rotation of the filter surface around the main axis of the cylinder, the beam direction of the resulting detection optical path remains completely unchanged after adjusting another component of the dichroic device. Especially when using a SPAD array detector with a typically sensor surface smaller than 1 mm 2 this is an important advantage.

[0095] In the illustrated embodiment, each component of the dichroic device includes four dichroic devices respectively, but this is of course not mandatory. Depending on the available structural space and according to the experimental requirements, there may also be more or fewer dichroic devices and / or groups of dichroic devices, and correspondingly more or fewer spectral detection channels.

[0096] Before detection, the light coupled out into the respective detection channels can be further processed in the case of using filters. This is Figure 4 exemplarily shown for the first detection channel Ch1, where filters F1 and F2 are positioned in the optical path.

[0097] List of reference numerals

[0098] 1 Light source

[0099] 2 Excitation light

[0100] 3 Main beam splitter, dichroic beam splitter

[0101] 4 Scanning device, two-dimensional scanner

[0102] 5 Scanning optics

[0103] 6 Tube lens

[0104] 7 Microscope objective

[0105] 8 Specimen

[0106] 9 Light emitted from specimen 8, emitted light

[0107] 10 Detection unit

[0108] 11 Control unit

[0109] 21 Adjustable aperture diaphragm

[0110] 22 Variable focal length optics, zoom optics

[0111] 23 Dichroic device

[0112] Dispersion device of the first detection channel 30a that can be driven and selectively activated

[0113] Dispersion device of the second detection channel 30b that can be driven and selectively activated

[0114] Cylindrical optical device 25a that can be selectively introduced into the detection optical path in the first detection channel 30a

[0115] Cylindrical optical device 25b that can be selectively introduced into the detection optical path in the second detection channel 30b

[0116] Adjustable optical device 26a for laterally displacing the spectrally split light relative to the detector 28a

[0117] Adjustable optical device 26b for laterally displacing the spectrally split light relative to the detector 28b

[0118] Filter 27a that can be adjusted in terms of spectral transmission and / or reflection characteristics

[0119] Filter 27b that can be adjusted in terms of spectral transmission and / or reflection characteristics

[0120] Two-dimensional spatially resolved detector

[0121] Two-dimensional spatially resolved detector 28a of the first detection channel 30a

[0122] Two-dimensional spatially resolved detector 28b of the second detection channel 30b

[0123] First detection channel 30a

[0124] Another detection channel, second detection channel 30b

[0125] Rotor 60

[0126] Beam splitter device 80

[0127] Beam splitters BP1, ..., BP8

[0128] First group of beam splitters BP1, ..., BP4

[0129] Second group of beam splitters BP5, ..., BP8

[0130] Detection channels Ch1, ..., Ch5

[0131] Filter F1

[0132] Filter F2

[0133] FWHM (psf) Full Width Half Maximum of the PSF

[0134] PSF, psf Point Spread Function

[0135] x linear coordinate direction

[0136] y linear coordinate direction

[0137] z linear coordinate direction, direction of the optical axis z, in particular the direction of the microscope objective 7

[0138] λ wavelength

[0139] Wavelength of the light of the intensity distribution shown by λ0

[0140] λi Wavelength spectrum of the emitted light 9 in detection channel i

[0141] References

[0142] [1] DE10038528A1

[0143] [2] DE10201220A1

[0144] [3] M. Buttafava er al., Optica 7, 755 (2020) (Authored by M. Buttafava et al., Published in the journal Optica 7, 755, 2020)

[0145] [4] https: / / piimaging.com

[0146] [5] WO2020207571A1.

Claims

1. A microscope, the microscope having: a light source (1) for emitting excitation light (2), an illumination optical path (3, 4, 5, 6, 7) for guiding the excitation light (2) onto and / or into a specimen (8), a scanning device (4) for changing the position on and / or in the specimen (8) onto which the excitation light (2) is incident, at least one two - dimensionally spatially resolving detector (28) for detecting light (9) emitted from the specimen (8), a detection optical path having a microscope objective (7) for guiding at least a part of the light (9) emitted from the specimen (8) to the detector (28), a control unit (11) for driving the scanning device (4) and for evaluating the measurement data of the detector (28), characterized in that there is a detection unit (10) which comprises the detector (28) and has a variable - focal - length optical device (22) for imaging the specimen (8) onto the detector (28) with a variable magnification and a controllable and selectively activatable dispersion device (24a) for spectrally splitting at least a part of the light (9) emitted from the specimen, wherein the control unit (11) is also configured to drive the dispersion device (24a, 24b).

2. The microscope according to claim 1, characterized in that the detection unit (10) has an aperture diaphragm of adjustable size.

3. The microscope according to claim 1 or 2, characterized in that the variable - focal - length optical device (22) images one and the same plane of the specimen (8) onto the detector (28) or multiple detectors (28a, 28b) independently of the focal - length adjustment.

4. The microscope according to any one of claims 1 to 3, characterized in that the variable - focal - length optical device is formed by a zoom optical device (22).

5. The microscope according to any one of claims 1 to 3, characterized in that the variable - focal - length optical device is formed by a lens replacement system with which the magnification can be adjusted to a plurality of discrete values.

6. The microscope according to any one of claims 1 to 5, characterized in that a first detection channel (30a) is formed in the detection unit (10), the first detection channel having at least the detector (28a), at least one additional detection channel (30b) is present in the detection unit (10), each additional detection channel having a two - dimensionally spatially resolving detector (28b), and at least one dichroic beam splitter (23) is present in the detection optical path for guiding a spectral component of the light (9) emitted from the specimen (9) to be detected into the respective additional detection channels (30b).

7. The microscope according to claim 6, characterized in that The energy-driven and selectively activatable dispersion devices (24a, 24b) for spectrally splitting the light to be detected act only in one of the detection channels (30a, 30b), and / or there is at least one additional energy-driven and selectively activatable dispersion device (24a, 24b) for spectrally splitting the light to be detected that acts only in one of the detection channels.

8. The microscope according to any one of claims 1 to 7, characterized in that the energy-driven and selectively activatable dispersion devices (24a, 24b) for spectrally splitting the light to be detected in at least one detection channel (30a, 30b) have at least one of the following components or are formed by at least one of the following components: a grating that can be swung into the detection optical path, in particular a transmission grating or a reflection grating; a prism that can be swung into the detection optical path, in particular a direct vision prism or a Perrin - Broca prism.

9. The microscope according to any one of claims 1 to 8, characterized in that in the detection unit (10), in particular in at least one detection channel (30a, 30b), there are cylindrical optical devices (25a, 25b) that can be selectively introduced into the detection optical path, and the cylindrical optical devices axially shift the image plane in a direction perpendicular to the direction in which the dispersion device or one of the dispersion devices (24a, 24b) causes spectral decomposition.

10. The microscope according to any one of claims 1 to 9, characterized in that the detection unit (10) and / or in at least one detection channel (30a, 30b) thereof has filters (27a, 27b) that can be adjusted in terms of their spectral transmission and / or reflection characteristics.

11. The microscope according to claim 10, characterized in that at least one of the filters (27a, 27b) has an adjustment mode of broadband transmission and / or reflection of light.

12. The microscope according to claim 10 or 11, characterized in that at least one of the filters (27a, 27b) has a plurality of discrete filter segments, and the filter segments can be respectively introduced into the detection optical path.

13. The microscope according to any one of claims 10 to 12, characterized in that at least one of the filters (27a, 27b) is realized by a continuously adjustable filter.

14. The microscope according to any one of claims 1 to 13, characterized in that in the detection unit and / or in at least one detection channel (30a, 30b), there are adjustable optical devices (26a, 26b) for laterally moving the spectrally split light relative to the detectors (28a, 28b) of the respective detection channels (30a, 30b).

15. The microscope according to claim 14, characterized in that The adjustable optical devices (26a, 26b) in the detection unit and / or in at least one detection channel (30a, 30b) have at least one of the following components or are implemented by at least one of the following components: an adjustable tiltable mirror, an adjustable tiltable plane-parallel transparent plate, in particular a glass plate, an electrically controllable element which generates a phase gradient such that the focus is laterally displaced.

16. The microscope according to any one of claims 1 to 15, characterized in that a multi-lens array is arranged in front of at least one of the detectors (28a, 28b).

17. The microscope according to any one of claims 1 to 16, characterized in that in at least one of the detectors (28a, 28b), pixels, in particular adjacent pixels, such as pixels adjacent in the direction of a detector row and / or a detector column and in particular adjacent perpendicular to the dispersion direction, can be combined by pixel binning.

18. The microscope according to any one of claims 1 to 17, characterized in that at least one of the detectors (28a, 28b) has a SPAD array.

19. The microscope according to any one of claims 1 to 18, characterized in that there is a dichroic device (80) having a rotor (60) on which a plurality of dichroics (BP1, ..., BP8) are arranged, by adjusting different rotational positions of the rotor (60), different dichroics (BP1, ..., BP7) can be introduced into the detection optical path, via which a spectral component of the light (9) emitted from the specimen (8) can be conveyed to each detection channel (30a, 30b).

20. The microscope according to claim 19, characterized in that by adjusting different rotational positions of the rotor (60), different dichroic groups (BP1, ..., BP4; BP5, ..., BP8) can be introduced into the detection optical path, and / or the dichroics are each configured to, in turn, reflect one spectral component of the light and transmit one component on each of the dichroics (BP1, ..., BP4; BP5, ..., BP8), and wherein the transmitted spectral components can each be conveyed to one of the detection channels (Ch1, ..., Ch5).

21. The microscope according to claim 19 or 20, characterized in that the dichroics (BP1, ..., BP8) are arranged on the circumferential side surface of a cylinder on the rotor (60), and the axis of rotation of the rotor (60) is collinear with the main axis of the cylinder and is oriented transversely, in particular perpendicularly, with respect to the direction of incidence of the light on the dichroic device (80).

Images