microscope

Abstract

The present invention relates to a microscope comprising a light source for transmitting excitation light, an illumination beam path for guiding the excitation light onto and / or into a sample, a scanning device for changing the area on and / or within the sample exposed to the excitation light, at least one two-dimensional spatially resolved detector for detecting light emitted by the sample, a detection beam path having a microscope objective lens for guiding at least a portion of the light emitted by the sample to the detector, and a control unit for controlling the scanning device and evaluating measurement data from the detector. The microscope, according to the present invention, is characterized in that it includes a detection unit having a detector and a variable focal length optical unit for imaging a sample onto the detector at variable magnification, and a controllable and optionally operable dispersion device for spectrally separating at least a portion of the light emitted by the sample, and the control unit is configured to control the dispersion device.

Description

Technical Field

[0001] The present invention relates to a microscope as described in the preamble of claim 1.

Background Art

[0002] A general microscope includes a light source that transmits excitation light, an illumination beam path that guides the excitation light onto and / or into a sample, a scanning device that changes a site on and / or in the sample exposed to the excitation light, at least one two-dimensional spatially resolving detector that detects light emitted by the sample, a detection beam path having a microscope objective lens that guides at least a part of the light emitted by the sample to the detector, and a control unit that controls the scanning device and evaluates measurement data from the detector. This type of microscope is known, for example, from Patent Document 1.

[0003] Laser scanning microscopy is an excellent tool for observing three-dimensional samples because it suppresses out-of-focus light by spatial filtering with a confocal aperture to generate a very high-contrast image of an arbitrary sample. In recent years, various measurement methods have been developed for this technology. For example, spectral imaging using a spectrometer-like detector (Patent Document 2), or high-resolution imaging by spatial oversampling of a point spread function (PSF) using a camera-like sensor known as image scanning microscopy (ISM) (Patent Document 1).

[0004] Due to these various different measurement modes, laser scanning microscopes (LSMs) are often complex and expensive. Therefore, it is almost impossible in terms of cost with commercially available technology to combine spectral imaging and spectral multi-channel ISM in one LSM system. Furthermore, the detection efficiency often decreases with the number of measurement modes integrated into the system. Therefore, until now, creating spectral multi-channel image recordings has only been possible with commercially available LSM systems at a lower resolution or only sequentially in what is known as a multi-track mode.

[0005] In 2021, an ISM using a 5x5 pixel SPAD array was reported. Such sensors and similar sensors are already commercially available. This essentially means that sensors are available that provide access to ISM with a significantly smaller structural size than multi-anode PMTs. Currently, the number of pixels available in commercially available SPAD arrays that can be used for scanning microscopy is rather limited. However, this is not a limitation in principle, but rather due to a limited data rate. The number of pixels in these CMOS SPAD camera sensors is expected to increase significantly within a few years. One-million-pixel CMOS SPAD arrays already exist (Non-Patent Literature 1 and Non-Patent Literature 2).

[0006] The objective of the present invention can be considered to be to identify a configuration that enables both spectral imaging and ISM in a simplified setup.

[0007] This objective is achieved by a microscope having the features of claim 1. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] German Patent Application Publication No. 102020120190 Specification [Patent Document 2] West German Patent Application Publication No. 10038528 [Non-patent literature]

[0009] [Non-Patent Document 1] Edoardo Charbon, Kazuhiro Morimoto, “Megapixel SPAD arrays for imaging: a versatile tool for quantum experimentalists and consumers alike”, [online], April 13, 2021, Society of Photo-Optical Instrumentation Engineers (SPIE), Proc. SPIE 11721, Advanced Photon Counting Techniques XV, 1172104, [displayed as of July 25, 2025], Internet <URL:https: / / doi.org / 10.1117 / 12.<2589786>

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0010] The above microscope further developed by the present invention includes a detector, an optical unit with a variable focal length for imaging a sample onto the detector at a variable magnification, and a controllable and optionally operable dispersion device for spectrally separating at least a part of the light emitted by the sample, and a control unit is also configured to control the dispersion device.

[0011] Preferred exemplary embodiments of the microscope according to the present invention are described below, particularly in relation to the dependent claims and drawings.

[0012] This invention is based on the finding that high-speed two-dimensional resolution detectors, particularly SPAD arrays, can be used, on the one hand, as cameras for oversampling detection of PSF, and on the other hand, for detecting the spectrum of emission emitted from a sample.

[0013] The basic concept of the present invention can be considered as enhancing a microscope using a two-dimensional spatially resolved detector by, on the one hand, providing variable magnification in the detection beam path, and on the other hand, providing a variably operable dispersion device in the detection beam path for both image scanning microscopy and spectral imaging.

[0014] An essential advantage of the present invention can be seen as the ability to implement both the image scanning microscopy measurement mode and the spectral imaging measurement mode in the same apparatus, and therefore with a reduced layout compared to the prior art.

[0015] Excitation light is electromagnetic radiation, particularly in the visible spectral range and adjacent ranges. The only requirement imposed on the principle of contrast provision by this invention is that the sample emits light and / or deflects, scatters, or reflects the excitation light as a result of being irradiated with excitation light. Typically, the emitted light is fluorescence emitted by the sample, particularly by the pigment molecules present therein, as a result of being irradiated with excitation light.

[0016] At least one light source, such as a laser, is present to supply the excitation light. The spectral composition of the excitation light may be adjustable, particularly between two or more colors. The excitation light may be multicolored simultaneously, for example, if it is intended that different dyes be detected at the same time.

[0017] The term illumination beam path refers to all optical beam guidance and beam modulation components, such as microscope objective lenses, lenses, mirrors, prisms, diffraction gratings, filters, diaphragms, beam splitters, modulators, such as spatial light modulators (SLMs), through and by these means that excitation light from a light source is guided to the sample to be observed. Beam modulation components also include dispersive elements that cause optical dispersion, particularly diffractive elements, such as structures like diffraction gratings or volume holograms, which may have a partially spatially periodic structure.

[0018] As a result of the sample to be observed being irradiated with excitation light, the light emitted and / or deflected by the sample, for example, scattered light, may be called emitted light and reaches at least one detector via the detection beam path.

[0019] The term "detection beam path" refers to all beam guidance and beam modulation optical components, such as objective lenses, lenses, mirrors, prisms, diffraction gratings, filters, diaphragms, beam splitters, modulators, such as spatial light modulators (SLMs), through and by these means that emitted light is guided from the sample to be observed to the detector. The microscope objective lens is part of the detection beam path.

[0020] Imaging a sample means that at least a portion of the sample is imaged. The visible region is demarcated by the sensor's field of view and / or by a field diaphragm that is optionally present in the detection beam path.

[0021] The term control unit refers to all hardware and software components that interact for the components of the microscope according to the invention and their intended functions. In particular, the control unit can comprise a computing device such as a PC, for example, and a camera controller that can rapidly read out measurement signals. The computer resources of the control unit can be distributed among a plurality of computers and optionally also among computer networks, in particular via the Internet. The control and evaluation unit can in particular comprise normal operating devices and peripheral devices such as a mouse, keyboard, screen, storage medium, joystick, Internet connection, etc. The control unit can in particular read image data from a detector and be configured and operative to control a light source. According to the invention, the control unit is also configured to control a decentralized device. The control unit can also be configured to adjust the magnification using an optical unit with variable focal length.

[0022] In a typical exemplary embodiment, the illumination beam path and / or the detection beam path in the microscope according to the invention comprises at least one of the components, namely a main beam splitter, a scanning optical unit, a tube lens. A part of the illumination beam path and the detection beam path can be provided by the same optical component. A beam splitter that separates the excitation light from the emission light, i.e., the light emitted by the sample, is referred to as the main beam splitter. Typically, the detection beam path thus contains substantially or not at all components of the excitation light downstream of the main beam splitter. Thus, at least one detector in the detection unit cannot be saturated by the excitation light. The main beam splitter can be a dichroic beam splitter.

[0023] The sample can be illuminated using a separate optical unit, in particular via a separate microscope objective. However, generally the sample is illuminated by the same microscope objective that is also a component of the detection beam path.

[0024] The scanning device may comprise a two-dimensional scanner arranged in the illumination beam path. This scanner can also be a component of the detection beam path, i.e., the light emitted by the sample and to be detected is descanned. For example, the two-dimensional scanner can be a galvanometer scanner. Additionally or alternatively, the scanning device may include a sample stage displaceable transversely in an operable manner. The sample stage can advantageously be displaceable transversely in two independent coordinate directions each perpendicular to the direction of the optical axis of the microscope objective. The scanning device can also be realized by a combination of a two-dimensional scanner and a transversely displaceable sample stage.

[0025] In a preferred exemplary embodiment, the detection unit comprises an aperture diaphragm having an adjustable size. The aperture diaphragm can perform two functions. First, out-of-focus emitted light can be narrowed thereby, and second, it acts as the entrance aperture in the spectral dispersion mode, which will be described in detail below. In the spectral decomposition mode or dispersion mode, the size of the entrance aperture affects the spectral resolution and, in the case where there are multiple spectral detection channels, also affects the separation of the detection channels. Advantageously, an incident optical unit may also be present to generate an intermediate image plane at which the aperture diaphragm can be positioned.

[0026] In a particularly preferred exemplary embodiment, the variable focal length optical unit forms an image of the same plane of the sample on the detector regardless of the focal length setting. This achieves the advantage that the detector can remain at the same location in each case regardless of the setting of the variable focal length optical unit.

[0027] The variable focal length optical unit can be formed by a zoom optical unit. The advantage achieved in this context is that the magnification of the imaged sample plane can be adjusted particularly finely. In particular, the magnification can be set particularly accurately to an advantageous value taking into account the pixel pitch parameter of at least one detector used and the spread of the point spread function.

[0028] The term "point image distribution function" refers to the intensity distribution of light transmitted through a lens that satisfies a specific working diameter of the lens. This function is usually abbreviated as PSF (point image distribution function). The working diameter of the lens used can be, but does not have to be, the maximum possible diameter. This means that the PSF does not have to correspond to the full utilization of the maximum available numerical aperture. If the numerical aperture is not fully utilized, the spread of the PSF increases compared to maximum utilization.

[0029] However, a variable focal length optical unit can also be formed by an interchangeable lens system, which allows the magnification to be set to multiple discrete values. This may be sufficient for many applications.

[0030] A particular advantage of the present invention as described herein is that the detection unit can be upgraded to a spectral multichannel variant with relatively little effort. Accordingly, one particularly preferred exemplary embodiment is characterized in that the detection unit has a first detection channel, the first detection channel comprises at least a detector, the detection unit has at least one further detection channel, each further detection channel has a two-dimensional spatially resolved detector, and at least one color splitter is present in the detection beam path to guide the spectral components of light emitted by the sample to be detected to each further detection channel.

[0031] In the simplest variation of spectral multichannel, there may be a color splitter having a filter edge that, based on the filter edge, splits the light to be detected into at least long-wave and short-wave components. The short-wave component enters, for example, a first detection channel, and the long-wave component enters a second detection channel accordingly. In particular, the color splitter may be a dichroic beam splitter.

[0032] To allow the distribution of spectral components across the detection channel to be adapted to the sample, it is advantageous when the spectral positions of the filter edges of the dichroic splitter can be varied discretely, or, particularly advantageously, continuously. However, a color splitter can also be realized by a filter arrangement (interference filter, e.g., bandpass filter) having many filter layers in which the light to be detected is reflected a suitable number of times.

[0033] At least one controllable and optionally operable dispersant is designed for spectrally separating the light to be detected. Spectral separation refers to spectral decomposition such that, depending on the wavelength of each component, the spectral components of the light are emitted in different spatial directions after passing through the device for spectral separation. Separation can be achieved using diffraction and / or refraction. The characteristic of a dispersant to be optionally operable refers to the fact that it either optionally separates the light to be detected in the detection beam path or does not. In a preferred embodiment, the dispersant is inserted into or removed from the detection beam path for this purpose. Alternatively or additionally, the detection beam path is redirected and the dispersant in question becomes part of the redirected detection beam path. The characteristic of a dispersant to be controllable refers to the fact that optional operation can be controlled by a control unit.

[0034] For embodiments of the present invention, it is sufficient to have only a single controllable and optionally operable dispersion device for spectrally separating the light to be detected. In the case of a multi-channel detection unit, the dispersion device may be located upstream of at least one color splitter in the detection beam path. The device then acts together for all spectral channels.

[0035] Advantageously, a controllable and optionally operable dispersant for spectrally separating the light to be detected may be effective in only one of the detection channels, and / or there may be at least one further controllable and optionally operable dispersant for spectrally separating the light to be detected, which is effective in only one of the detection channels. For example, the dispersant present in the present invention may be part of a first detection channel.

[0036] Where this description refers to the fact that at least one component may have a particular characteristic or feature, this also means that multiple or all components of each type may have their respective characteristics or features.

[0037] For example, in an advantageous modification, a controllable and optionally operable dispersive device for spectrally separating the light to be detected can also be present in each individual detection channel. This has the advantage that the spectral dispersion can be specifically set in each individual detection channel. For example, the dispersive devices and color splitters to be used in each individual detection channel can be adapted to each other. This modification is particularly advantageous when prisms are used as dispersive elements, because their spectral dispersion decreases with increasing wavelength.

[0038] A controllable and selectively operable disperser for spectrally separating light to be detected may be formed by, in particular in or for at least one detection channel, a component, namely, at least one of the following: a diffraction grating, particularly a transmission or reflection diffraction grating, that is particularly insertable into the detection beam path by swivel; a prism, particularly a direct-view prism or a Pellin-Broca prism, that is particularly insertable into the detection beam path by swivel; or one of the following components. The disperser may also include holographically fabricated components. Alternatively, a controllable and selectively operable disperser may also be realized when the detection beam path for operating the disperser is redirected, for example by a swiveling mirror, such that the disperser becomes part of the subsequently redirected detection beam path. In this case, the disperser must also be considered insertable. In this case, the disperser itself may or may not be fixed in place.

[0039] In the simplest case, to switch to spectral detection mode, a spectral disperser is inserted into the beam path.

[0040] Advantageously, when switching to spectral imaging, the magnification of the image representation from the sample to the two-dimensional spatially resolved detector is adapted so that the full width at half maximum (FWHM) of the PSF is on the order of a few pixels, ideally only one pixel or less, allowing for simultaneous measurement of the largest possible spectral range. Furthermore, the spectral resolution of the detection system can be improved by two consecutive image recordings and shifting the spectrum on the sensor for subsequent calculations.

[0041] In microscopy, obtaining an appropriate gray value for the image to be recorded is generally always desirable. In other words, the goal is to use the detector capacity appropriately, that is, to avoid operating in the saturation range where the proportional relationship between the measured signal and the amount of light is lost, or at a very low count rate where the signal is small compared to the detector noise.

[0042] In this regard, it should be noted that the count rate of the detector used is typically limited to a few megahertz, and at most a few tens of MHz. In the case of SPAD arrays, the physical reason for the limited count rate lies in the characteristics of the photon count of SPAD pixels, which have an inherent dead time during avalanche quenching. Therefore, in order to obtain a suitable gray value in images generated with pixel residence times of about 1 microsecond or less, which are typical in laser scanning microscopy, it may be advantageous to disperse the emission of the dye to multiple pixels of the detector. To some extent, this has already been achieved by spectral dispersion. In this regard, further improvement is possible in a further particularly preferred exemplary embodiment, in which there is a cylindrical optical unit in the detection unit and / or at least one detection channel that can be optionally inserted into the detection beam path, and the cylindrical optical unit displaces the image plane axially in a direction perpendicular to the direction in which the dispersion device of each detection channel causes spectral separation. The axial displacement of the image plane, i.e., the displacement in the optical axis direction, disperses the light in the wavelength range to be detected to multiple pixels of the detector. In extreme cases, the light in the wavelength range to be detected may be dispersed to all pixels in the relevant row or column of the detector. In particular, cylindrical optical units that can be inserted by rotation can be used to defocus the PSF in an axis oriented perpendicular to the dispersion direction, thereby extending the PSF across many pixels on the detector.

[0043] Only a single cylindrical optical unit, which can be optionally inserted, may exist and may be located in the detection beam path upstream of at least one color splitter. This cylindrical optical unit then acts together for all spectral channels. In another advantageous variation, a separate cylindrical optical unit, which can be optionally introduced, is present in each of the multiple detection channels, or in each individual detection channel. The distribution of the PSF to the detector pixels can then be implemented in the corresponding channels or individually in each channel. Different cylindrical optical units may also be used in various detection channels, as needed. At least one of the cylindrical optical units may comprise, or be realized by, at least one cylindrical lens.

[0044] In a further preferred embodiment of the present invention, at least one of the detection unit and / or detection channels comprises a filter adjustable with respect to its spectral transmission and / or reflectance properties. This filter may, for example, function to set the spectral detection bandwidth for the ISM and to limit the bandwidth to a dye selected in each case.

[0045] In this case, it is particularly preferable that at least one of the filters has a setting that transmits and / or reflects light over a wide bandwidth. This setting can then be advantageously selected for spectral resolution measurement modes. For example, at least one of the filters may have multiple discrete filter segments, each of which can be inserted into the detection beam path. Alternatively or in addition thereto, at least one of the filters may be implemented by a continuously adjustable filter in a different embodiment.

[0046] In a further particularly preferred configuration, an adjustable optical unit for laterally shifting spectrally separated light relative to the detector of each detection channel is present within the detection unit and / or within at least one detection channel. Using the adjustable optical unit for laterally shifting spectrally separated light, the offset of the partial spectrum within the detection channel on the detector of this detection channel can be adjusted particularly advantageously.

[0047] An adjustable optical unit for lateral shift may be implemented within the detection unit and / or within at least one detection channel by comprising, or by, at least one of the following components: an adjustable tiltable mirror, an adjustable tiltable parallel plane transparent plate, and especially a glass plate. This makes it possible to optimally offset the spectral range in each case related to the excited fluorophore on each of the individual detector sensors.

[0048] Mechanical manipulation of components within the beam path, such as rotation in and out of the beam path, pushing into the beam path, pulling out of the beam path, and tilting, can be achieved in commonly known ways by operable motor drives.

[0049] Pixelated detectors, particularly SPAD array detectors, typically have a fill factor of less than 1, meaning the detector has regions between pixels where incident photons are not detected. Therefore, the detector can only detect incident photons on a portion of its surface. To compensate for this characteristic, the detector may be equipped with a microlens array, where each pixel, e.g., each SPAD pixel, may be assigned a microlens. In one preferred modification, a multilens array is placed in front of at least one detector. Placing it in front of the detector means the multilens array is located upstream of the detector in the beam path, i.e., the light to be detected passes through the multilens array before entering the detector. The multilens array may be placed on the associated detector. The multilens array and the associated detector can then form a structural unit.

[0050] In a preferred exemplary embodiment, pixels, particularly adjacent pixels, for example, pixels in the direction of a detector row and / or detector column, can be binned in at least one of the detectors. When each individual pixel of the detector is read out separately, each pixel provides a separate detection signal corresponding to the amount of light to be detected reaching each part of the pixel. When multiple pixels are binned, a single “bin” provides a detection signal equal to the sum of the amounts of light to be detected reaching the pixels forming the “bin.” Thus, the number of detection signals to be evaluated is reduced, and the detector readout can be accelerated. It is advantageous to bin entire pixel rows and / or entire pixel columns. Depending on the measurement task, it may be useful to bin pixels in a direction perpendicular to the direction of spectral dispersion. In this case, spectral resolution is maintained while achieving high dynamism and reduced data rate by using multiple detector elements. For other measurement tasks, it may be useful to bin pixels in the direction of spectral dispersion. This reduces spectral resolution, but can increase readout speed and, consequently, frame rate. Furthermore, both binning methods described can be performed in combination.

[0051] It is also advantageous to define spectral channels by binning on the sensor. For example, two dyes can be detected, each with a bandwidth of 50 nm, and emitting light at, for example, 500 nm and 580 nm. Two bins corresponding to the wavelength ranges of 500 + / - 25 nm and 580 + / - 25 nm can then be unfolded. Each of the two bins then corresponds to one spectral channel, and all other data is discarded or binned and transferred in another way.

[0052] At least one two-dimensional spatially resolved detector is a sufficiently fast optical detector having a two-dimensional spatially resolved sensor surface. In particular, the detector may be a camera, especially a CCD, sCMOS, CMOS, or SPAD camera chip, or a camera having a SPAD array. Particularly preferably, each detector within each detection channel may have a SPAD array. Many properties of fast two-dimensional resolved detectors, especially SPAD arrays, form the basis for their preferred use. For example, these detectors can detect fluorescence signals in time resolution. Since fluorophores often differ very much in their fluorescence lifetime, the inclusion of this parameter in the mathematical separation problem is advantageously possible. Furthermore, the sensor elements can be flexibly switched on and off. Flexible switching patterns of pixel matrices for such applications are described in International Publication No. 2020207571A1.

[0053] Two-dimensional spatially resolved detectors and their corresponding optical setups are particularly well-suited for detecting PSFs of laterally oversampled emitted light, in order to satisfy at least the Nyquist theorem. This is especially important when performing so-called scanning microscopy (brand name Carl Zeiss: Airyscan). However, conventional confocal measurements are also possible with this detector, for example, by adding the values ​​of all pixels, or at least a larger number of pixels. The detector may preferably have a rectangular or hexagonal arrangement of pixels.

[0054] In a particularly preferred configuration of the microscope according to the present invention, a color splitter device is provided together with a rotor on which a plurality of color splitters are arranged, and different color splitters can be inserted into the detection beam path by setting different rotational positions of the rotor, and the spectral components of light emitted by the sample can be supplied to each detection channel through these color splitters. This makes it possible to arrange the detection channels in a very compact manner.

[0055] The color splitters may be configured such that each of the color splitters sequentially reflects each spectral component of the light and transmits one component, and the transmitted spectral component may be supplied to one of the detection channels in each case. The color splitters may also be bandpass filters in particular.

[0056] In one advantageous development of this color splitter device, different groups of color splitters may be inserted into the detection beam path by setting different rotational positions of the rotors. Particularly advantageous, the color splitter may be positioned on a rotor on the outer surface of a cylinder, with the rotor's axis of rotation collinear with the cylinder's main axis and oriented laterally, and especially perpendicularly, to the direction of light incidence to the color splitter device. This achieves the advantage that the beam direction of the resulting detection beam path is maintained after the modified configuration due to the stable rotation of the filter surface around the cylinder's main axis. This is typical, for example, in SPAD array detectors, typically 1 mm in size. 2 This is particularly advantageous due to the significantly smaller sensor surface area.

[0057] Further advantages and features of the present invention are described below in reference to the accompanying drawings. [Brief explanation of the drawing]

[0058] [Figure 1] This is a schematic diagram showing a microscope according to the present invention. [Figure 2]This is a schematic diagram illustrating an exemplary embodiment of a detection unit for a microscope according to the present invention. [Figure 3] This figure illustrates the irradiation of a two-dimensional spatially resolved detector in different operating modes of the microscope according to the present invention. [Figure 4] This figure shows an exemplary embodiment of a filter device for a microscope according to the present invention. [Modes for carrying out the invention]

[0059] An exemplary embodiment of the microscope 100 according to the present invention will be described with reference to Figures 1 to 3. The microscope 100 schematically shown in Figure 1 comprises, as basic components, a light source 1, an illumination beam path having a microscope objective lens 7, a detection beam path, a detection unit 10, and a control unit 11. The light source 1 is typically a laser, in particular having multiple wavelengths, and is used to emit excitation light 2, which is directed onto and / or into the sample 8 to be observed via the illumination beam path. In the schematic example, the illumination beam path comprises a main beam splitter 3, a scanning device 4, a scanning optics unit 5, a tube lens 6, and a microscope objective lens 7. In the microscope 100, a stand for receiving the sample may be present in a known manner in the sample chamber below the microscope objective lens 7. The setup shown in Figure 1 substantially corresponds to a laser scanning microscope (LSM) for sequential scanning and illumination of a sample 8 using an excitation laser as the light source 1.

[0060] Starting from the light source 1, the excitation light 2 first reaches the main beam splitter 3, from where it is guided towards the scanning device 4, which may be, for example, a two-dimensional Garbo scanner. From there, the excitation light 2 reaches the microscope objective lens 7 via the scanning optical unit 5 and the tube lens 6, which focuses the excitation light 2 onto and / or into the sample 8.

[0061] In particular, the light 9 emitted by the sample 8 as a result of irradiation with excitation light 2 may also be called emitted light 9, and is guided to the detection unit 10 via the detection beam path. The emitted light may be fluorescence light from the dye in which the sample 8 was prepared.

[0062] The first component of the detection beam path is a microscope objective lens 7, which receives and collimates the emitted light 9. Through the tube lens 6 and the scanning optical unit 5, the emitted light 9 reaches the scanning device 4, where it is descanned and then reaches the main beam splitter 3, where, in contrast to the excitation light 2, it is transmitted to the detection unit 10, where it is fixedly imaged onto a two-dimensional spatially resolved detector 28 provided according to the present invention.

[0063] In the exemplary embodiment shown, the control unit 11 controls at least the light source 1 and the scanning device 4, and functions to evaluate the measurement data from the detection unit 10, i.e., from at least one detector 28. For example, the control unit 11 can adjust the intensity of the excitation light 2 depending on the location on or within the sample 8, and optionally depending on the intensity of the emitted light 9 emanating from the sample location of interest. Finally, the control unit 11 may, if necessary, generate an image of the sample 8 after appropriate calculations using the measurement data.

[0064] An exemplary embodiment of a detection unit 10 for a microscope 100 according to the present invention will be described with reference to Figure 2. The detection unit 10 shown therein has, as its basic components, a pinhole optical unit 21 having an adjustable size variable aperture diaphragm, a variable focal length optical unit 22 present according to the present invention, and a first spectral detection channel 30a and a second spectral detection channel 30b.

[0065] In the configuration schematically shown in Figure 2, the first detection channel 30a and the second detection channel 30b contain substantially the same optical components, which are optionally optimized for different spectral compositions of the partial beam. Equivalent optical components in the two detection channels 30a and 30b are each denoted by the same reference numeral, with the components of the first detection channel 30a being additionally denoted by the letter 'a' and the components of the second detection channel 30b being additionally denoted by the letter 'b'.

[0066] The first detection channel 30a and the second detection channel 30b each contain two-dimensional spatially resolved detectors 28a and 28b, respectively, which in an exemplary embodiment are SPAD array detectors.

[0067] The pinhole optical unit 21 generates an intermediate image plane in which an adjustable aperture diaphragm, also called a pinhole, is positioned. The aperture diaphragm can be used, on the one hand, to suppress out-of-focus emission, and on the other hand, it can be used as an incident aperture for spectral resolution mode. In the latter case, the size of the aperture diaphragm affects the spectral resolution and therefore also affects the separation of spectral detection channels.

[0068] Downstream of the pinhole optical unit 21 is a variable focal length optical unit 22, which in the illustrated exemplary embodiment is implemented by a schematically illustrated zoom optical unit. The variable focal length optical unit 22 is designed such that its rear focal plane is always in the plane of the SPAD detectors 28a, 28b. In other words, the planes of the SPAD detectors 28a, 28b are optically conjugate to the plane of the adjustable aperture diaphragm. On the one hand, the illumination of detectors 28a, 28b after changing the microscope objective lens can be appropriately adapted by the variable focal length optical unit 22. On the other hand, for spectral resolution measurement modes, the magnification from the plane of the adjustable aperture diaphragm to detectors 28a, 28b can be reduced in such a way that the FWHM of the PSF on detectors 28a, 28b is on the order of the pixel distance d (see Figure 3B) or less in order to improve spectral resolution. The size of the spectral range placed on the sensor depends on the angular dispersion generated within the prism and the distance between the prism and the sensor.

[0069] For example, in a color splitter 23 which may be a discretely or continuously adjustable dichroic beam splitter, the detection beam path is separated into two detection channels 30a and 30b downstream of a variable focal length optical unit 22 in an exemplary embodiment shown in Figure 2, the two detection channels 30a and 30b then transmit different spectral ranges of emitted light 9 downstream of the color splitter 23.

[0070] Directly downstream of the color splitter 23 are controllable and optionally operable dispersers 24a and 24b, respectively, which function to spectrally separate the emitted light 9 in the first detection channel 30a and the second detection channel 30b. In the illustrated exemplary embodiment, the dispersers 24a and 24b are each realized by a displaceable combination of different glass wedges, which can be motorized to move in or out of the beam path. This movement is indicated by double-headed arrows. When the spectral dispersers 24a and 24b are in the beam path, i.e., operational, the emitted light 9 is separated or spectrally spread by the spectral dispersers 24a and 24b downstream of each disperser 24a and 24b in the dispersion plane. In Figure 2, the dispersion plane is identical to the drawing plane.

[0071] The spectrally separated emitted light 9 is then incident on cylindrical optical units 25a and 25b, respectively, which shift the focal position relative to the direction perpendicular to the detection surface so that the emitted light 9 is dispersed across many pixels within the planes of the SPAD array detectors 28a and 28b perpendicular to the dispersion direction (see Figure 3B). The cylindrical optical units 25a and 25b substantially do not change the focal point in the dispersion direction. The image area in the dispersion direction is a function of wavelength. This fact will be explained in more detail below in relation to Figure 3B.

[0072] Using angle-adjustable parallel plane plates 26a and 26b, the spectra generated by the dispersion devices 24a and 24b in each case can be shifted relative to detectors 28a and 28b, depending on the settings of the color splitter 23 in each case, so that the spectral region of interest is located as centrally as possible on detectors 28a and 28b. For example, the spectra may be shifted laterally so that the spectral centroid of the emission is located centrally on one of the detectors 28a and 28b in each case, or so that the entire emission spectral bandwidth is detected by each detector 28a and 28b.

[0073] Finally, downstream of components 26a and 26b, there are adjustable filters 27a and 27b, respectively, which can be used to set the spectral detection bandwidth for the first detection channel 30a and the second detection channel 30b for the scanning microscopy (ISM) measurement mode.

[0074] In ISM operation, the dispersers 24a, 24b and cylindrical optical units 25a, 25b are removed from the beampath in the direction indicated by the arrows, and the adjustable filters 27a, 27b are set to positions that, together with the color splitter 23, allow for spectral discrimination of undesirable signals.

[0075] The movement of the dispersive device 24a in the first detection channel 30a can be performed together with the movement of the dispersive device 24b in the second detection channel 30b, but it can also be performed independently. Therefore, it is possible to operate both detection channels 30a and 30b in ISM mode, both detection channels 30a and 30b in spectral resolution mode, or the first detection channel 30a in ISM mode and the second detection channel 30b in spectral resolution mode, or vice versa. This means that ISM data and spectral image data can be generated simultaneously.

[0076] The emitted light reflected by filters 27a and 27b may also be directed to further detectors not shown in Figure 2. As already mentioned above, the reflection of spectral components of the detected light to further detection channels can also be performed using a further color splitter, which may be located downstream of the color splitter 23 and before one or both of the components 24a and 24b.

[0077] In dispersion mode, the adjustable filters 27a and 27b can be set to positions where, as far as possible, only broadband reflection reduction layers are located in the beam path.

[0078] The order of the optical components within detection channels 30a and 30b may differ without imposing any limitations on the described function. However, due to the focusing effect of the cylindrical optical units 25a and 25b, it is useful to place them downstream of the dispersion devices 24a and 24b in each case.

[0079] A multi-lens array may be present before detectors 28a and 28b to compensate for the finite fill factor of these detectors, which is not shown in Figure 2. Each pixel of detectors 28a and 28b may be assigned a microlens. Preferably, SPAD array detectors with a multi-lens array having a large solid angle of reception on the input side are used, so as completely as possible image the angularly separated spectra generated by the dispersive devices 24a and 24b and the cylindrical optical units 25a and 25b onto the respective active surfaces of detectors 28a and 28b. For this purpose, the focal lengths of the lenses in the multi-lens array should not be selected to be excessively small so that changes in the angle of incidence do not cause excessively large changes in the image offset. Furthermore, care must be taken to ensure that the image representation of the system pupil generated on the active surface region of detectors 28a and 28b is sufficiently smaller than the sensor surface area belonging to a single pixel. The SPAD array detectors 28a and 28b used can have good sensitivity across the entire wavelength spectrum, but can also be optimized for a specific range of the optical spectrum to be detected in each detection channel.

[0080] Figures 3A and 3B schematically show sensor surfaces with typical light distributions for ISM operation (Figure 3A, left) and spectral resolution operation (Figure 3B, right), respectively.

[0081] In the example shown, the detector pixels are arranged at right angles. However, hexagonal or other arrangements are also possible. In Figure 3A, the PSF of the emitted light 9 is positioned approximately on the detector surface such that the maximum intensity of the PSF is located approximately in the center of the detector. This ensures that the intensity of the PSF at the periphery of the detector is reduced as much as possible. Furthermore, the magnification of the image representation is selected by appropriate adjustment of the variable focal length optical unit 22 (see Figure 2) such that the diameter of the PSF, more precisely the diameter of the first Airy ring, is sampled in at least five pixels or five pixel bins. This problem is related by the equation FWHM(psf)≧5d This is expressed as follows, where d is the distance between pixels in the detector (pixel pitch), or the pixel bin. A pixel bin, that is, a group of adjacent pixels coupled to a bin in particular, can be called a pixel bin.

[0082] Figure 3B shows the arrangement of the PSF for a single wavelength λ0 light on the detector in spectral resolution mode. As can be seen, the PSF has a strongly asymmetric shape due to the effect of the cylindrical optical unit 25a or 25b. In Figure 3B, the dispersion direction extends horizontally, i.e., along the x-direction (see coordinate system in Figure 3). This means that PSFs for other wavelengths not shown in Figure 3B will be located to the right or left of the shown PSF, depending on whether the target wavelength is greater or less than wavelength λ0. Using the variable focal length optical unit 22, the magnification is favorably set for this mode, as can be seen in Figure 3B, such that the FWHM of the PSF for a single wavelength is less than the pixel distance d. In this way, improved spectral resolution is obtained. Furthermore, to ensure that as many pixels of the detector as possible contribute to signal generation for individual wavelength PSFs and to provide good dynamics of gray values ​​in the image, the PSFs are defocused perpendicular to the dispersion direction, i.e., in the y-direction, by the cylindrical optical units 25a, 25b. As a result, many pixels are illuminated by light of the same wavelength λ0 in the y-direction.

[0083] Figure 4 shows an exemplary embodiment of a color splitter device 80 in which incident emitted light 9 can be distributed across a total of five spectral detection channels Ch1, ..., Ch5. Reference numerals λ1 to λ5 indicate the wavelength spectra of the emitted light 9 in each of the detection channels Ch1, ..., Ch5. Each of the reference numerals λ1 to λ5 can also represent a finite spectral range, as well as a plurality of discontinuous spectral ranges.

[0084] The color splitter device 80 has a rotor 60 on which color splitters BP1, ..., BP4 are initially arranged, distributed circumferentially along the outer surface of the cylinder, and these color splitters form a first color splitter group. In the situation shown in Figure 4, the color splitters BP1, ..., BP4 of this first group are located within the beam path. The axis of rotation of the rotor 60, which extends perpendicular to the plane of the paper through the point indicated by M, is collinear with the main axis of the cylinder and is oriented perpendicular to the direction in which the emitted light 9 is incident on the color splitter device 80.

[0085] In the situation shown in Figure 4, the color splitter device 80 operates as follows: Incident emitted light 9 enters through window W1 and is incident on the first color splitter BP1. The first spectral component λ1 is transmitted through color splitter BP1 and separated into the first detection channel Ch1. The spectral components of the emitted light 9 that are not transmitted by color splitter BP1 are substantially reflected by color splitter BP1 and then incident on the second color splitter BP2. There, the second spectral component λ2 is transmitted and separated into the second detection channel Ch2. Similarly, the process continues through color splitters BP3 and BP4, where spectral components λ3 and λ4 are separated into the third detection channel Ch3 and the fourth detection channel Ch4, respectively.

[0086] The remaining spectral component λ5 of the emitted light 9 reflected by the fourth color splitter BP4 is incident on the fifth detection channel Ch5 through the exit window W2.

[0087] In the exemplary embodiment shown, the entrance window W1 and the exit window W2 are neutral, meaning that the light passing through them is not spectrally altered. However, it would also be possible to provide filters there.

[0088] Assuming that no light is absorbed by the color splitters BP1 to BP4, and by components W1 and W2, the incident emitted light 9 is completely split between the detection channels Ch1 to Ch5.

[0089] The particularly advantageous characteristics of the color splitter apparatus 80 shown in Figure 4 are derived from the fact that additional color splitters BP5 to BP8 are arranged on the outer surface of the cylinder 60, thereby forming a second group of color splitters. By rotating the cylinder 60 30° clockwise, it can be ensured that the component W2, which previously acted as an exit window, becomes an entry window for the incident emitted light 9. The beam path is the same as described above, provided that the color splitters BP1, ..., BP4 of the first group are replaced by the color splitters BP5, ..., BP8 of the second group, and window W3 is replaced by the exit window W2. The color splitters BP5, ..., BP8 of the second group may have spectrally different characteristics compared to the color splitters BP1, ..., BP4 of the first group.

[0090] Switching from observing a first sample prepared with a first dye selection, particularly using the first spectrum of excitation light 2, to observing a second sample prepared with a second dye selection different from the first selection, particularly using the second spectrum of excitation light 2 different from the first spectrum, is especially convenient when using such a color splitter apparatus. A particular advantage is that the stable rotation of the filter surface around the cylindrical principal axis ensures that the beam direction of the resulting detection beam path is perfectly maintained even after setting up another group of color splitters. This is especially true when typically 1 mm 2 This is a significant advantage when using SPAD array detectors with a smaller sensor surface area.

[0091] In the exemplary embodiments shown, each color splitter group includes four color splitters, but this is not necessarily required. Depending on the available space and experimental requirements, there may be more or fewer color splitters and / or color splitter groups, and correspondingly more or fewer spectral detection channels.

[0092] The light coupled to each detection channel can be further adjusted before detection using filters. This is shown in Figure 4 as an example of a first detection channel Ch1 in which filters F1 and F2 are positioned in the beam path. [Explanation of Symbols]

[0093] 1 light source 2 Excitation light 3. Main beam splitter, dichroic beam splitter 4. Scanning device, 2D scanner 5. Scanning Optical Unit 6 Tube Lenses 7. Microscope objective lens 8 samples 9. Light emitted by sample 8, emitted light 10 detection units 11 Control Unit 21 Adjustable aperture diaphragm 22. Variable focal length optical unit, zoom optical unit 23 Color Splitter 24a Controllable and optionally operable distributed device for the first detection channel 30a 24b Controllable and optionally operable distributed device for the second detection channel 30b 25a A cylindrical optical unit that is optionally inserted into the detection beam path within the first detection channel 30a. 25b A cylindrical optical unit that is optionally inserted into the detection beam path within the second detection channel 30b. 26a Adjustable optical unit for lateral shift of spectrally separated light relative to detector 28a 26b Adjustable optical unit for lateral shift of spectrally separated light relative to detector 28b 27a Filters adjustable with respect to spectral transmission and / or reflection characteristics 27b Filters adjustable with respect to spectral transmission and / or reflection characteristics 28 Two-dimensional spatially resolved detector 28a Two-dimensional spatially resolved detector of the first detection channel 30a 28b 2D spatially resolved detector of the second detection channel 30b 30a First detection channel 30b Further detection channel, second detection channel 60 rotors 80 Color Splitter Device BP1, ..., BP8 Color Splitter BP1, ..., BP4 First color splitter group BP5, ..., BP8 Second color splitter group Ch1, ..., Ch5 Detection Channels F1 filter F2 filter FWHM(psf) Full width at half maximum of PSF PSF (point image distribution function) x Linear coordinate direction y Linear coordinate direction z represents the linear coordinate direction, the direction of the optical axis z, and especially the direction of the optical axis of the microscope objective lens 7. λ wavelength λ0 Wavelength of light in the illustrated intensity distribution λi Wavelength spectrum of emitted light 9 in detection channel i References [1] West German Patent Application Publication No. 10038528 [2] West German Patent Application Publication No. 10201220 [3] M.Buttafava et al., Optica 7,755(2020). [4]https: / / piimaging.com [5] International Publication No. 2020207571A1

Claims

1. The system includes a light source (1) for transmitting excitation light (2), The system includes illumination beam paths (3, 4, 5, 6, 7) for guiding the excitation light (2) onto and / or into the sample (8), The system includes a scanning device (4) for changing the location on and / or within the sample (8) that is exposed to the excitation light (2), The system includes at least one two-dimensional spatially resolved detector (28) for detecting the light (9) emitted by the sample (8), The detection beam path includes a microscope objective lens (7) for guiding at least a portion of the light (9) emitted by the sample (8) to the detector (28), The system includes a control unit (11) for controlling the scanning device (4) and for evaluating the measurement data from the detector (28). In a microscope, A detection unit (10) is provided, which includes the detector (28) and a variable focal length optical unit (22) for imaging the sample (8) onto the detector (28) at a variable magnification, and a controllable and optionally operable dispersion device (24a) for spectrally separating at least a portion of the light (9) emitted by the sample. The control unit (11) is also configured to control the distributed devices (24a, 24b). A microscope characterized by the following features.

2. The detection unit (10) is equipped with an aperture diaphragm having an adjustable size. Characterized by, The microscope according to claim 1.

3. The optical unit (22) with a variable focal length forms an image of the same plane of the sample (8) on the detector (28) or the detectors (28a, 28b), regardless of the focal length setting. Characterized by, The microscope according to claim 1 or 2.

4. The optical unit with a variable focal length is formed by a zoom optical unit (22). Characterized by, A microscope according to any one of claims 1 to 3.

5. The optical unit with a variable focal length is formed by a lens exchange system, thereby allowing the magnification to be set to a plurality of discrete values. Characterized by, A microscope according to any one of claims 1 to 3.

6. At least a first detection channel (30a) comprising the detector (28a) is formed within the detection unit (10), At least one additional detection channel (30b) is present within the detection unit (10), Each further detection channel has a two-dimensional spatially resolved detector (28b), In order to guide the spectral components of the light emitted by the sample (8) to be detected to each of the further detection channels (30b), at least one color splitter (23) is present in the detection beam path, Characterized by, A microscope according to any one of claims 1 to 5.

7. The controllable and optionally operable dispersion devices (24a, 24b) for spectrally separating the light to be detected are effective in only one of the detection channels (30a, 30b), and / or There exists at least one further controllable and optionally operable dispersion device (24a, 24b) for spectrally separating the light to be detected, which is effective only in one of the detection channels. Characterized by, The microscope according to claim 6.

8. The controllable and optionally operable dispersion device (24a, 24b) for spectrally separating light to be detected in at least one detection channel (30a, 30b) comprises, or is formed by, at least one of the following components: a diffraction grating, particularly a transmission or reflection grating, that can be rotated within the detection beam path; or a prism, particularly a direct-view prism or a Pellin-Broca prism, that can be rotated within the detection beam path. Characterized by, A microscope according to any one of claims 1 to 7.

9. Within the detection unit (10), particularly within at least one detection channel (30a, 30b), there is a cylindrical optical unit (25a, 25b) which can be optionally inserted into the detection beam path, and the cylindrical optical unit displaces the image plane axially in a direction perpendicular to the direction in which the dispersion device or one of the dispersion devices (24a, 24b) causes spectral splitting. Characterized by, A microscope according to any one of claims 1 to 8.

10. The detection unit (10) and / or at least one of the detection channels (30a, 30b) has a filter (27a, 27b) that is adjustable with respect to its spectral transmission and / or reflection characteristics. Characterized by, A microscope according to any one of claims 1 to 9.

11. At least one of the filters (27a, 27b) has a setting that allows the light to be transmitted and / or reflected over a broadband. Characterized by, The microscope according to claim 10.

12. At least one of the filters (27a, 27b) has a plurality of discrete filter segments, each of which can be inserted into the detection beam path. Characterized by, The microscope according to claim 10 or 11.

13. At least one of the filters (27a, 27b) is implemented by a continuously adjustable filter. Characterized by, A microscope according to any one of claims 10 to 12.

14. Within the detection unit and / or within at least one detection channel (30a, 30b), there is an adjustable optical unit (26a, 26b) for shifting laterally the spectrally separated light relative to the detector (28a, 28b) of each of the detection channels (30a, 30b). Characterized by, A microscope according to any one of claims 1 to 13.

15. The adjustable optical units (26a, 26b) within the detection unit and / or within at least one detection channel (30a, 30b) have, or are realized by, at least one of the following components: an adjustable tilt mirror, an adjustable tilt parallel plane transparent plate, particularly a glass plate, or an electrically controllable element that generates a phase gradient so that the focus shifts laterally. Characterized by, The microscope according to claim 14.

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

17. In at least one of the detectors (28a, 28b), pixels, in particular adjacent pixels, for example, adjacent pixels in the row and / or column direction of the detector, and in particular pixels perpendicular to the dispersion direction, can be joined by binning. Characterized by, A microscope according to any one of claims 1 to 16.

18. At least one of the detectors (28a, 28b) is equipped with a SPAD array. Characterized by, A microscope according to any one of claims 1 to 17.

19. There exists a color splitter device (80) having a rotor (60) on which multiple color splitters (BP1, ..., BP8) are arranged, By adjusting the different rotational positions of the rotor (60), different color splitters (BP1, ..., BP7) can be inserted into the detection beam path, and the spectral components of the light (9) emitted by the sample (8) can be supplied to each detection channel (30a, 30b) via them. Characterized by, A microscope according to any one of claims 1 to 18.

20. By adjusting the different rotational positions of the rotor (60), different groups of color splitters (BP1, ..., BP4; BP5, ..., BP8) can be inserted into the detection beam path, and / or The color splitter is configured such that each of the color splitters (BP1, ..., BP4; BP5, ..., BP8) sequentially reflects one spectral component of the light and transmits one component. The transmitted spectral components can be supplied to one of the detection channels (Ch1, ..., Ch5). Characterized by, The microscope according to claim 19.

21. The aforementioned color splitters (BP1, ..., BP8) are arranged on the outer surface of the cylinder on the rotor (60), The rotation axis of the rotor (60) is on the same line as the main axis of the cylinder and is oriented laterally, and particularly perpendicularly, with respect to the direction of incidence of the light to the color splitter device (80). Characterized by, The microscope according to claim 19 or 20.

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