Microscope

Abstract

To quickly record a three-dimensional image of a sample.SOLUTION: A microscope includes: forming a first detection channel having at least one first detector and at least further one detection channel, in a detection unit, in which each of the further detection channel includes at least one detector; making at least one beam splitter for guiding a part of light emitted from a sample to the further detection channel exist in each case, in the detection unit; and making an operation device for axial displacement of a focal region exist in at least the one detection channel, so as to image sample planes separated in an axial direction in at least the two detection channels.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a microscope having the features of the preamble of claim 1.

Background Art

[0002] A general microscope is known from German Patent Application Publication No. 102020120190 and comprises the following components: a light source for transmitting excitation light, an illumination beam 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 exposed to the excitation light, a detection unit for detecting the light emitted from the sample, a detection beam path having a microscope objective for guiding at least a part of the light emitted from the sample to the detection unit, and a control unit for controlling the scanning device and the detection unit and evaluating the measurement data from the detection unit.

[0003] Confocal microscopy is a method commonly used for examining small biological structures by imaging. It is possible to image from single cells in living tissue to living animals. The ability to achieve background suppression and thus three-dimensional image structures is particularly important here.

[0004] A central problem of confocal microscopy is the slowness of its image construction, which is due to the fact that all points of the sample have to be scanned by the laser spot and the associated detection. This is done sequentially and thus slowly. In particular, there is a significant time difference between the imaging of different measurement planes, because the two-dimensional images are usually recorded for each plane and there are steps in the axial direction following each image. The image stack can then be represented, for example, pseudo-three-dimensionally or for each section and projection.

[0005] The ability to measure not only in one plane but simultaneously in multiple planes is interesting for various measurement tasks.

[0006] Another problem associated with basic recording methods is that the high intensities required to focus on small areas of the sample can partially cause significant fading of the sample and optical damage to the sample.

[0007] Higher speeds can be achieved by using a resonant scanner, which can rapidly scan a relatively large image field. The drawback of this method is due to the fact that a sufficiently good signal must be generated within a very short pixel time. For example, when intending to achieve a signal-to-noise ratio of 3, approximately 10 detected photons are required per measurement point. Assuming both a total detection efficiency of about 33% and an average pixel dwell time of 50 nanoseconds, 30 emitted photons are required in 50 nanoseconds, which corresponds to a photon emission rate of 600 MHz. This is only possible with very bright and stable samples.

[0008] Furthermore, it is possible to achieve higher speeds by using various techniques for parallelizing the recording of an image by scanning more than one confocal volume. At this time, various parallelization techniques are possible. The lateral multi-spot method (for example, German Patent Application Publication No. 102016102286.1), the axial multi-spot method [1], [2], and the multi-spot method within the point spread function [3] are known. The lateral decomposition method based on the lateral multiplication of the point spread function (PSF), the so-called multi-spot method, requires a relatively high level of device complexity.

[0009] Axial parallel methods are equally complex and / or inflexible. The distance between planes is often fixed with these techniques [2]. In [1], here the size of the confocal slit given by the reflection structure is also fixed. This is disadvantageous because the pinhole / slit size depends strongly on the wavelength and the selected objective lens, and this parameter is also used to optimize the axial resolution, background suppression, and signal intensity. In [2], the pinholes seem to be more flexible. However, they are in fixed positions, given by physical pinholes, and can be expensive. In this case, displacing the pinhole positions seems to be complex. Furthermore, they must be matched with the illumination by diffractive elements, which makes their use in multicolor experiments doubtful.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0012] One problem addressed by the present invention can be considered to be identifying a microscope that can be used to record three-dimensional images of a sample particularly quickly.

[0013] This problem is solved by a microscope having the features of claim 1.

[0014] According to the present invention, in the following respects, namely, in the detection unit, a first detection channel having at least one first detector and at least one further detection channel are formed, each further detection channel comprising at least one detector, and in the detection unit, in each case at least one beam splitter for guiding a part of the light emitted from the sample to the respective further detection channels is present, and in at least one of the detection channels, there is an operating device for axial displacement of the focal region for imaging sample planes axially spaced in at least two detection channels, a microscope of the above type is developed.

[0015] Advantageous exemplary embodiments of the microscope according to the present invention are explained below, in particular in connection with the dependent claims and the drawings.

[0016] The essential concept underlying the microscope described herein is the concept of axial parallelization of detection, in particular confocal detection.

[0017] One starting point of the present invention is that in the detection configuration of FIG. 2 of the German Patent Application Publication No. 102023100926.5 of the previous application, a gradient color splitter 23 that functions to direct light of different wavelengths to different sensors in each case can advantageously be replaced by a neutral intensity splitter that distributes a ratio such as, for example, 50% / 50% to each sensor.

[0018] It can be considered a further essential concept to achieve an axial offset in various detection channels by arranging an operating device for axially displacing the focal region in at least one of the detection channels.

[0019] The excitation light is electromagnetic radiation, especially in the visible spectral range and adjacent ranges. The only requirement placed on the contrast-providing principle by the present invention is that the sample emits emission light as a result of irradiation with the excitation light and / or deflects, scatters or reflects the excitation light. Typically, the emission light is fluorescence emitted by the sample, especially the dye molecules present therein, as a result of irradiation with the excitation light.

[0020] There is at least one light source, for example a laser, for providing the excitation light. The spectral composition of the excitation light can be adjustable, especially between two or more colors. For example, if different dyes are intended to be detected simultaneously, the excitation light may be polychromatic simultaneously.

[0021] The term "illumination beam path" refers to all optical beam guiding components and beam modifying components, such as microscope objectives, lenses, mirrors, prisms, gratings, filters, diaphragms, beam splitters, modulators, such as spatial light modulators (SLMs), by means of which and through which the excitation light from the light source is guided to and / or into the sample to be examined. The beam modifying components also include dispersive elements that cause optical dispersion, especially diffractive elements, such as structures including gratings or volume holograms that can especially have a spatially periodic structure in part.

[0022] As a result of irradiation with the excitation light, light transmitted and / or deflected, for example scattered, by the sample to be examined can be referred to as emission light and reaches the detection unit via the detection beam path.

[0023] The term "detection beam path" refers to all beam-guiding and beam-changing optical components, such as objective lenses, lenses, mirrors, prisms, gratings, filters, diaphragms, beam splitters, modulators, such as spatial light modulators (SLMs), by means of which and through which the emission light is guided from the sample to be examined to the detector. The microscope objective lens is part of the detection beam path.

[0024] Imaging the sample means that at least a part of the sample is imaged. The visible region is delimited by the field of view of the sensor and / or by an optionally present field stop within the detection beam path.

[0025] The term "control unit" refers to all hardware and software components that interact with the components of the microscope according to the invention for the intended function of the microscope according to the invention. In particular, the control unit can comprise a computing device such as a PC and a camera controller that can rapidly read out measurement signals. The computer resources of the control unit can in particular be further distributed over a plurality of computers and optionally a computer network, in particular via the Internet. The control and evaluation unit can in particular be equipped with customary operating devices and peripherals such as a mouse, keyboard, screen, storage medium, joystick, Internet connection, etc. The control unit can in particular read image data from the detector and can also be configured and function to control the light source.

[0026] In a typical exemplary embodiment, the illumination beam path and / or the detection beam path in the microscope according to the present invention comprises at least one of the following components, namely, a main beam splitter, a scanning optical unit, an objective 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 emitted light, i.e., the light emitted from the sample, is called a main beam splitter. Thus, typically, the detection beam path contains little or no component of the excitation light downstream of the main beam splitter. Thus, the detector in the detection unit cannot be flooded by the excitation light. The main beam splitter can be a dichroic beam splitter.

[0027] The sample can be illuminated using a separate optical unit, in particular via a separate microscope objective. However, in many configurations, the sample is illuminated by one and the same microscope objective that is also a component of the detection beam path.

[0028] The scanning device can 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 to be detected emitted from the sample is descanned, and the detection unit can be arranged in the descanned beam path. For example, the two-dimensional scanner can be a quasi-static scanner. If it is intended to obtain a higher scanning speed, a resonant scanner can also be used. Instead of, or preferably in addition to, this, the scanning device may comprise a controllable sample stage displaceable in the lateral direction. The sample stage can advantageously be displaceable in the lateral direction 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 laterally displaceable sample stage. The resonant scanner and the quasi-static scanner can each be designed as a galvanometric scanner, a MEMS scanner, or any other type of scanner.

[0029] In a preferred exemplary embodiment, the detection unit comprises an adjustable-size aperture stop. The aperture stop can perform two functions. First, it can narrow the unfocused emitted light there, and second, it functions as an entrance aperture for the spectral dispersion mode described in detail below. In the spectral decomposition mode or spectral dispersion mode, the size of the entrance aperture affects the spectral resolution and, if there are multiple spectral detection channels, the separation of the detection channels. Advantageously, there may be an entrance optical unit to create an intermediate image plane where the aperture stop can be placed.

[0030] In a particularly preferred exemplary embodiment, the detection unit has an optical unit with a variable focal length for imaging the sample on the detector at a variable magnification. First, the optical unit with a variable focal length can function to set one or more distances between the sample planes imaged in various channels, which will be described in detail below. Further, the optical unit with a variable focal length can function to adapt the imaging to the pixel grid of the two-dimensional spatial resolution detector.

[0031] In a particularly preferred exemplary embodiment, the optical unit with a variable focal length, in each case, images the same plane of the sample onto one or more detectors regardless of the focal length setting. This achieves the advantage that one or more detectors can remain in the same position in each case regardless of the setting of the optical unit with a variable focal length.

[0032] The optical unit with a variable focal length can be formed by a zoom optical unit. The advantage achieved in this regard is that the magnification of the imaged sample plane can be adjusted particularly finely. In particular, the magnification can be set to particularly accurate and advantageous values taking into account the pixel pitch parameter of the detector used and the range of the point spread function.

[0033] The control unit can also be configured to adjust the magnification using an optical unit with a variable focal length.

[0034] The term "point spread function" refers to, for example, the intensity distribution of light created by a lens from an incoming parallel beam that fills a specific use diameter of the lens. This function is usually abbreviated as PSF (point spread function). The use diameter of the lens used may be the maximum possible diameter, but it does not have to be. This means that the PSF does not have to correspond to the full use of the maximum available numerical aperture. If the numerical aperture is not fully utilized, the range of the PSF, especially in the axial direction, also increases compared to the maximum utilization number.

[0035] However, it is also possible that the optical unit with a variable focal length is formed by a lens exchange system, whereby the magnification can be set to a plurality of discrete values. This may be sufficient for many applications.

[0036] The possibility of high-quality simultaneous recording of two or more planes of a sample should be considered as a first important advantage of the microscope according to the present invention.

[0037] As a result, in the microscope according to the present invention, the distances between various scanning planes can be set very easily but still very accurately.

[0038] Furthermore, in the microscope according to the present invention, a complete confocal operation is possible. In particular, the size of the effective confocal pinhole, which does not necessarily have to be a physical aperture, can be set individually for each detection channel and thus, for example, individually for different observation sample planes.

[0039] In the configuration described herein, image scanning microscopy can also be performed, and a change in binning is equivalent to adjusting the size of the confocal pinhole. This will be described in detail below.

[0040] Recording on one and the same plane with pinholes of different sizes and consequently different binning is possible with a suitable readout using a suitable spatially resolving detector and can be advantageous, for example, for increasing the contrast or improving the axial resolution. This is possible by using neutral density filters if the spatially resolving detector is subjected to the same spectral content in both channels. Furthermore, since the cross-section of the point spread function depends linearly on the central wavelength, it is expedient to select pinholes of different sizes for very different emission light wavelengths.

[0041] The microscope according to the invention also enables a simple reconfiguration from a multicolor system (multi-channel system) that records data from a sample in only one plane to a multi-plane system.

[0042] All imaging and measurement modes that can be performed in one plane using this system can be extended to two or more planes without further modification (confocal imaging, airy scan / image scanning, MPLX, single-shot 3D, Dynamics Profiler).

[0043] A particularly simple exemplary embodiment of the microscope according to the invention is characterized in that, in at least one of the detection channels, the operating device comprises a glass plate arranged in the converging part of the detection beam path.

[0044] The distance between the planes of the sample imaged on the detector depends on both the angular spectrum of the light incident on the glass plate and the thickness of this plate. As an approximation, for a small non-zero convergence angle of the beam, the axial offset Dz is described as follows by the thickness d of the plate and the refractive index n of the plate material. Dz = d×(n - 1) / n

[0045] This axial displacement Dz in the image area is converted into the sample space in the form of an axial offset dz in the sample space according to the following, by means of the lateral magnification V from the sample space to the image area of the detection. dz = Dz / (V×V)

[0046] When adjustable components are used, higher flexibility is possible from the perspective of possible different axial offsets. For example, in at least one of the detection channels, the operating device may comprise a stepped glass plate arranged in the converging part of the detection beam path, and the stepped glass plate can be variably arranged in the detection beam path in order to set different optically effective thicknesses transverse to the optical axis.

[0047] Alternatively, or in addition, in at least one of the detection channels, the operating device can comprise an exchanger having different glass plates arranged in the converging part of the detection beam path, the glass plates having different thicknesses and / or the glass material of the glass plates having different refractive indices.

[0048] If the operating device comprises two glass wedges arranged in the converging part of the detection beam path and the glass wedges are displaceable relative to each other and relative to the optical axis in order to continuously set different optically effective thicknesses, a finer adjustment of the axial position of the sample plane imaged in a particular detection channel is also possible.

[0049] Alternatively, or in addition, finally, in at least one of the detection channels, it is also possible for the operating device to comprise a small telescope, in particular an adjustable small telescope.

[0050] The components of the type described in this specification can each be present in just one detection channel, a plurality of detection channels, or all detection channels. Each operating device can be controlled by a control unit, and thus the user can, for example, select the spacing of the axial planes. The spacing between the planes in the sample imaged in each detection channel can be obtained by changing the thickness d and / or the magnification V, in particular using an optical unit with a variable focal length, such as a zoom optical unit.

[0051] Defocusing is always carried out in only one direction by the above-described operating device for axially displacing the focal region, in particular by adding a path in the glass. Therefore, it may also be advantageous for a device for setting the axial depth of illumination to be configured to defocus the excitation light such that the focus of the excitation beam is located between two detection planes, in particular between the two detection planes that are furthest apart from each other, especially in the center.

[0052] However, in addition to or instead of this, additional optical components may also be present, and the latter provide defocusing in a direction opposite to the direction of defocusing caused by the addition of the path in the glass.

[0053] From a practical perspective, when using an optical unit with a variable focal length, such as a zoom optical unit, it is important that the magnification from the sample to the two-dimensional decomposition detector can vary significantly.

[0054] For example, the total magnification can be 100. To set the interval between planes of 100 nm with a total magnification of V = 100, which is approximately equal to the axial Nyquist scan of a high NA objective lens, an optical path difference of 3 mm of glass, which is a realizable size, is required.

[0055] However, in a microscope mode where the point spread function is greatly enlarged, for example, the cross-section of the point spread function is imaged on the two-dimensional decomposition detector to cover several pixels, for example, 5 pixels or more, a significantly larger magnification up to V = 1000 is possible (Zeiss: "Airyscan" mode). In that case, an optical path difference of 300 mm of glass is required, which is not practical. Especially in such a mode, it may be advantageous for an additional component for setting the focal length, such as an adjustable lens, in particular an electro-optically adjustable lens, or a variable focus lens, to be present in the detection channel between the beam splitter and at least one two-dimensional decomposition detector.

[0056] A combination of a glass plate and an adjustable lens can also be advantageous because the adjustable lens often cannot set a particularly desired accuracy and a low refractive power well.

[0057] To achieve illumination of a sample in an axially extending region, a unit for setting the axial depth of illumination is present in the illumination beam path in a further preferred configuration of the microscope according to the invention.

[0058] In a simple variant, the numerical aperture of the illumination can advantageously be reduced somewhat in order to lengthen the axial distribution of the excitation light and extend it at least across the detection plane. For example, this can be achieved by reducing the diameter of a diaphragm located in the illumination beam path. Thus, the unit for setting the axial depth of illumination preferably comprises an adjustable diaphragm for setting the beam diameter of the excitation light.

[0059] A similar effect, i.e., lengthening the axial distribution of the excitation light and its extent across the detection plane, is possible in a configuration in which the unit for setting the axial depth of illumination comprises at least one diffractive component.

[0060] From the perspective of obtaining illumination of different axial planes with as similar intensity as possible, in embodiments in which the unit for setting the axial depth of illumination is configured to generate a plurality of axially spaced foci within the sample, it involves more effort but can achieve even better characteristics. For example, this is possible using a diffractive optical element (DOE).

[0061] Even more particularly preferred exemplary embodiments are distinguished in that a further optical beam shaping unit is present in front of the main beam splitter in the illumination beam path in a direction opposite to the propagation direction and / or in front of the main beam splitter in the detection beam path in the direction of the propagation direction, and the further optical beam shaping unit functions to generate a special form of the point spread function, in particular an axially encoded point spread function, such as a spiral point spread function, an astigmatic point spread function, or a "twisted" point spread function.

[0062] Axially encoded means that a beam cross-section perpendicular to the optical axis, i.e., perpendicular to the z-axis, has a shape and / or orientation that depends on the z-coordinate and thus the depth coordinate.

[0063] Axially encoded beams can be used to obtain depth information, i.e., for 3D techniques. "Single shot" techniques are examples of such 3D techniques, which are so called because they enable the acquisition of microscopic information about a sample at a position (x,y) for a plurality of values of the z-coordinate by illuminating a single point in the xy plane of the sample. An example is described in German Patent Application Publication No. 102017119531.

[0064] The beam shaping unit may preferably comprise one or more of the following components: a diffractive optical element, a spatial light modulator (SLM), an adjustable lens, a phase plate.

[0065] For example, the beam shaping unit may be arranged in the detection beam path between a pinhole optical unit and an optical unit with variable focal length, or between an optical unit with variable focal length and a beam splitter. In the illumination beam path, the beam shaping unit may be arranged, for example, between a unit for setting the axial depth of illumination and the main beam splitter.

[0066] It is also possible for a unit for setting the axial depth of illumination to provide the function of the beam shaping unit discussed here.

[0067] There is a degree of freedom in design from the perspective of a specific configuration of the beam splitter.

[0068] In an important group of exemplary embodiments of the microscope according to the present invention, at least one of the beam splitters is a neutral intensity splitter. It is also possible that a plurality of beam splitters or all beam splitters are neutral intensity splitters.

[0069] For the two detection channels formed from the incoming beam by the neutral intensity splitter, when there is an operating device for axially displacing the focal region in at least one of the two detection channels and the axial positions in the sample plane imaged by the two detection channels are different, they can be used to image two different axial planes. Under the condition that the neutral intensity splitter is ideally neutral, when the two detection channels do not include different components such as filters and / or no means having different effects on the spectrum of the light to be detected are taken, light of the same wavelength spectrum is detected in the two detection channels.

[0070] A particularly advantageous use of the microscope according to the present invention is possible when at least one of the detectors is a two-dimensional spatial resolution detector, particularly a camera chip, particularly preferably a SPAD array. It is also possible that a plurality of detectors or all detectors are two-dimensional spatial resolution detectors, particularly SPAD arrays. In that case, it is also possible that a plurality of detectors are formed by one and the same camera chip, for example by one and the same SPAD array. In particular, the SPAD array can operate in single photon counting mode (Geiger mode), which is advantageous from the perspective of the signal-to-noise ratio. Spatial resolution means that each pixel of the two-dimensional spatial resolution detector can be read out separately.

[0071] A variable pinhole can be realized using such a detector or array sensor, i.e., the detector can represent an electronically adjustable pinhole. This is particularly advantageous in connection with an optical unit of variable focal length that can roughly adjust the size of the pinhole, such as a zoom system or another adjustable optical unit. Again, the optical unit of variable focal length can function to match the PSF to each detector so that the dynamic range of the detector can be optimally utilized. By electronically setting the detector, it is possible to further assign its own pinhole with an individualized size to each axial plane, i.e., the degree of confocalness can be set individually for each detection channel. This enables the entire system to operate in confocal mode, and background suppression is as good as known from confocal systems.

[0072] In at least one of the two-dimensional spatially resolving detectors, it is particularly advantageous if pixels, especially adjacent pixels, such as pixels adjacent in the direction of a row and / or a column of the detector, especially pixels adjacent perpendicular to the direction of dispersion, can be combined by binning. This enables important advantages from the perspective of data rate and measurement speed.

[0073] A further improvement in this regard can be achieved if, in at least one of the two-dimensional spatially resolving detectors, the pixels can be set individually actively or passively. Passive means that no generation and / or reading of measurement information takes place from the passively set pixels.

[0074] Pixelated detectors, particularly SPAD array detectors, typically have a fill factor of less than 1, which means that the detector has regions between pixels where incident photons are not detected. As a result, the detector can only detect incident photons on a portion of the detector surface. To compensate for this characteristic, a microlens array can be provided for the detector, and a microlens can be assigned to each pixel, for example each SPAD pixel. Therefore, when a multi-lens array is placed in front of the associated detector, a more effective entrance for the two-dimensional spatial resolution detection of the light to be detected into the detector becomes possible.

[0075] In an alternative configuration, at least one of the detectors comprises one or more photomultiplier tubes, particularly with an adjustable entrance pinhole. It is also possible for a plurality of detectors or all detectors to each comprise one or more photomultiplier tubes, particularly each with an adjustable entrance pinhole.

[0076] For example, a hybrid form is also possible where an SPAD array is arranged in some detection channels and photomultiplier tubes are arranged in some detection channels.

[0077] A particular advantage of the microscope according to the invention is that light having different wavelength spectra can also be detected in different detection channels without incurring high costs.

[0078] Another important group of exemplary embodiments of the microscope according to the invention is characterized in that at least one of the beam splitters is a color splitter that forms at least two spectrally different detection channels. It is also possible for a plurality of beam splitters or all beam splitters to be color splitters.

[0079] In that case, a dispersive device is particularly preferably present in at least one of the detection channels in order to spectrally separate at least a portion of the light emitted from the sample.

[0080] The control unit can also be configured to control one or more distributed devices.

[0081] The two detection channels formed by the color beam splitter from the incoming beam can function to image two portions of the wavelength spectrum of the light emitted from the same axial sample plane when the axial positions of the sample planes imaged within the two detection channels are the same.

[0082] However, it may also be preferably possible to use two detection channels formed by the color beam splitter from the incoming beam in order to image the first portion of the wavelength spectrum from the first sample plane and the second portion of the wavelength spectrum from the second axial sample plane axially spaced from the first sample plane.

[0083] As in the situation described above for the neutral intensity splitter, this requires that at least one of the two detection channels has an operating device for axially displacing the focal region and that the axial positions of the sample planes imaged within the two detection channels are different.

[0084] Advantageously, there can be a dispersive device for spectrally separating the light to be detected, which is effective in a plurality of detection channels.

[0085] A microscope can be obtained that can be used particularly flexibly when at least one dispersive device for spectrally separating at least a part of the light emitted from the sample, or a plurality or all of the dispersive devices, is controllable and optionally operable, and the control unit is configured to control at least one dispersive device in each case.

[0086] For example, at least one dispersive device for spectrally separating the light to be detected may comprise or may be formed by at least one of the following components, namely a grating rotatable within the detection beam path, in particular a transmission grating or a reflection grating, a prism rotatable within the detection beam path, in particular at least one of an Amici prism or a Pellin-Broca prism.

[0087] In microscopy, it is generally desirable to obtain reasonable gray values of the recorded images. In other words, the aim is to use the capacity of the detector reasonably, i.e., not to operate them within the saturation range where the proportionality to the light amount of the measurement signal is lost, or at very low counting rates where the signal is small compared to the detector noise and the signal-to-noise ratio is not good.

[0088] In this regard, it should also be noted that the counting rate of the detector elements (pixels) used is typically limited to a few megahertz and at most a few tens of MHz. In the case of a SPAD array, the physical reason for the limited counting rate lies in the photon counting nature of the SPAD pixels with an inherent dead time during the detection process, and further in the dependence on the type of avalanche quenching. Therefore, in order to obtain reasonable gray values of an image with a pixel dwell time of typically less than about 1 microsecond, which is typical for a laser scanning microscope, it may be advantageous to distribute the emission of the dye among a plurality of pixels of the detector. To some extent, this is already achieved by spectral dispersion. In this regard, in a further particularly preferred exemplary embodiment, there is a cylindrical optical unit that can be optionally introduced into the detection beam path within the detection unit and / or at least one detection channel, and the cylindrical optical unit axially displaces the image plane in a direction perpendicular to the direction in which the dispersion device of each detection channel causes spectral splitting, and further improvement is possible. The axial displacement of the image plane, and thus the displacement in the direction of the optical axis, distributes the light in the wavelength range to be detected among a plurality of pixels of the detector. In an extreme case, the light in the wavelength range to be detected can be distributed among all the pixels of the relevant rows or columns of the detector. By using a cylindrical optical unit that can be introduced, in particular rotated, the PSF can be defocused about an axis in a direction perpendicular to the dispersion direction, and thus the PSF can be extended over many pixels on the detector.

[0089] There may only be a single cylindrical optical unit that can be optionally introduced, and it can be arranged in the detection beam path in front of one or more color splitters in a direction opposite to the propagation direction. Then, this cylindrical optical unit acts on all spectral channels together. In another advantageous variant, separate cylindrical optical units that can be optionally introduced are present for each of the plurality of detection channels or for each individual detection channel. Then, the distribution of the PSF between the pixels of the detector can be carried out individually for the corresponding channel or for each channel. If necessary, different cylindrical optical units can also be used for the various detection channels. At least one of the cylindrical optical units may comprise at least one cylindrical lens or may be realized by at least one cylindrical lens.

[0090] Alternatively, or in addition, at least one of the detection channels has a cylindrical optical unit that can be optionally introduced into the detection beam path, and the cylindrical optical unit axially displaces the image plane in a direction perpendicular to the direction in which one of the dispersion devices or the dispersion device causes spectral splitting.

[0091] In a further preferred embodiment of the invention, the detection unit and / or at least one of the detection channels comprises a filter that is adjustable with respect to its spectral transmission characteristics and / or spectral reflection characteristics. This filter can function, for example, to set the spectral detection bandwidths of the ISMs and limit them to the dyes selected in each case.

[0092] In this case, it is particularly preferable that at least one of the filters has a setting in which light is transmitted and / or reflected over a wide bandwidth. This setting can then advantageously be selected for the spectral decomposition measurement mode. For example, at least one of the filters may have a plurality of separate filter segments, each of which can be introduced into the detection beam path. Alternatively or in addition, at least one of the filters may, in different embodiments, be realized by a continuously adjustable filter.

[0093] In an even more particularly preferred configuration, an adjustable optical unit for laterally displacing spectrally separated light with respect to the detector of each detection channel is present in the detection unit and / or at least one detection channel. The adjustable optical unit for laterally displacing spectrally separated light can particularly advantageously adjust the offset of the partial spectrum within the detection channel on the detector of this detection channel.

[0094] The adjustable optical unit for lateral displacement may comprise or be realized by at least one of the following components within the detection unit and / or at least one detection channel, namely an adjustable tiltable mirror, an adjustable tiltable plane-parallel transparent plate, in particular at least one glass plate. This enables the optimal offset of the spectral range associated with the excitation phosphor on each individual detector sensor in each case.

[0095] The mechanical operation of the components in the beam path, for example, pivoting into and out of the beam path, pushing into the beam path, pulling out of the beam path, and tilting, can be realized by a controllable electric drive device.

[0096] Further features and advantages of the microscope according to the invention will be explained below in connection with the drawings.

Brief Description of the Drawings

[0097]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0098] One exemplary embodiment of a microscope 100 according to the present invention will be described with reference to FIG. 1.

[0099] According to the present invention, the microscope 100 includes a light source 10 for transmitting the excitation light 11, an illumination beam path for guiding the excitation light 11 onto and / or into the sample 20, a scanning device 14 for changing the position on and / or in the sample 20 exposed to the excitation light 11, a detection unit 80 for detecting the light 25 emitted from the sample 20, and a detection beam path having a microscope objective lens 17 for guiding at least a part of the light 25 emitted from the sample 20 to the detection unit 80. According to the present invention, there is a control unit 90 for controlling the scanning device 14 and the detection unit 80 and evaluating the measurement data from the detection unit 80.

[0100] The light source 10 can typically be a laser or a laser module having laser light with specifications suitable for a fluorescence microscope, particularly with regard to intensity, spectral characteristics, and temporal structure. In particular, the excitation light 11 may be electromagnetic radiation suitable for exciting the phosphor prepared in the sample 20.

[0101] In the illustrated exemplary embodiment, the illumination beam path through which the excitation light 11 reaches onto and / or into the sample 20 is formed by a unit 12 for setting the axial depth of illumination and the following further components, namely, a main color splitter 13, a scanner 14, a scanning optical unit 15, a tube lens 16, and a microscope objective lens 17.

[0102] The unit 12 for setting the axial depth of illumination may be a variably adjustable aperture, whereby the numerical aperture of the illumination is reduced such that the axial range of the focal volume within the sample 20 increases. In the illustrated example, the focal volume within the sample 20 extends axially in any case such that at least the regions within the planes 21 and 22 within the sample 20 are sufficiently exposed to the excitation light. Through the main color splitter 13, the excitation light 11 reaches the scanner, which can scan the excitation light 11, for example two-dimensionally, and can comprise a static and / or resonant galvanometric scanner. Through the scanning optical unit 15 and the tube lens 16, the excitation light 11 enters the microscope objective lens 17, which focuses it onto the focal volume within the sample 20 and, in the illustrated example, passes through at least the axial planes 21 and 22 of the sample 20.

[0103] As a result of the exposure to the excitation light 11, the sample 20 or the phosphor with which the sample 20 was prepared emits light 25, in particular red-shifted fluorescence, which is guided via the detection beam path to the detection unit 80.

[0104] In the illustrated example, the light 25 to be detected returns to the main color splitter 13 via the microscope objective lens 17, the tube lens 16, the scanning optical unit 15, and the scanner 14. The main color splitter 13 can be configured such that the light 25 to be detected passes through the main color splitter 13 and subsequently impinges on the detection unit 80, which functions to detect the light 25 emitted from the sample 20.

[0105] In the illustrated example, the detection unit 80 is formed with a first detection channel a having a first detector 70a and a second detection channel b having a second detector 70b. Further, according to the present invention, in the illustrated example, there is a beam splitter 40 for guiding a part of the light 25 to be detected to the second detection channel b. The beam splitter 40 can be a neutral intensity splitter, for example, a 50 / 50 neutral intensity splitter. Of course, other splitting ratios are also possible.

[0106] In the illustrated example, an optical unit 35 with a variable focal length that can be a zoom optical unit is present in the detection unit 80 and functions to image the sample 20 at a variable magnification on the detectors 70a, 70b.

[0107] Finally, in the second detection channel b in the example of FIG. 1, there is an operation device 60b schematically shown for axially displacing the focal region. In the illustrated example, this can be a glass plate with a thickness d arranged transversely to the optical axis of the beam path. The beam path converges in this region, i.e., in front of the detector 70b. The glass plate 60b causes a refocusing to slightly different planes. As a result, the glass plate 60b functions to displace the focal region in the sample 20 imaged on the detector 70b in the direction of the optical axis.

[0108] What approximately holds is that the axial offset Dz is described as follows by the thickness d of the plate and the refractive index n of the plate material. Dz = d×(n - 1) / n

[0109] This axial displacement Dz in the image region is converted into an axial offset dz between the planes 21 and 22 in the sample 20 space according to the following, by the lateral magnification V from the sample space 20 to the detection image region. dz = Dz / (V×V)

[0110] Therefore, using the microscope 100 shown in FIG. 1, it is possible to image the sample planes 21, 22 spaced apart in the axial direction by dz into the detection channels a, b. The interval dz between the imaged sample planes can be varied by the parameters of the operation device 60b and the magnification V. In the example of FIG. 1, the magnification V can be easily, accurately, and continuously changed using the zoom optical unit 35.

[0111] The detection unit 80 of the microscope 100 in FIG. 1 also includes an adjustable entrance pinhole 31 disposed in the intermediate image plane, formed by the pinhole optical unit 30, in the detection beam path. The pinhole 31 can be opened to such an extent that light to be detected from both planes can pass through the pinhole 31 and reach the detection channel for the measurement mode in which a plurality of axially spaced planes of the sample 20 are imaged into the detection channels a, b.

[0112] The detectors 70a, 70b are preferably SPAD arrays. From the perspective of the signal-to-noise ratio, it is advantageous to operate the SPAD array in the single-photon counting mode (Geiger mode).

[0113] The pixels of the detectors 70a, 70b can preferably be combined by binning, for example, as described in FIGS. 2 to 4 of WO 2020 / 20757, from the second last paragraph on page 20 to the second paragraph on page 21, especially for forming superpixels. This enables important advantages from the perspective of the data rate and the measurement speed. The disclosure from FIGS. 2 to 4 of WO 2020 / 20757, from the second last paragraph on page 20 to the second paragraph on page 21 is also incorporated herein. FIGS. 3 and 4 of WO 2020 / 20757 show the binning of SPAD pixels advantageous for image scanning, and the active area in FIG. 3 is smaller than that in FIG. 4. The active area can correspond to a specific pinhole size. In principle, the pinhole sizes for detection in different planes, and thus in the detection channels a and b, can be selected to be the same or different. It also depends on the selected objective lens and the system magnification.

[0114] Finally, as is also described in International Publication No. 2020 / 20757, it is also preferable that the pixels in the SPAD arrays 70a, 70b can be individually set actively or passively.

[0115] As a result, variable pinholes can be realized by the SPAD arrays 70a, 70b, which is particularly advantageous in relation to the zoom system 35 that can roughly adjust the size of the pinhole.

[0116] Then, again, the zoom system 35 can function to match the PSF to each detector 70a, 70b so as to optimally use the dynamic range of the associated detectors 70a, 70b. Further, in each axial plane, its own pinhole with an individualized size can be assigned, that is, the degree of confocalness can be individually set for each detection channel a, b.

[0117] Finally, it is also preferable that each microlens array is arranged in front of the SPAD arrays 70a, 70b to compensate for a fill factor of less than 1.

[0118] As described, the electronic pinhole can be created using a two-dimensional spatial resolution detector, particularly an SPAD array. Alternatively, an integrated sensor such as a photomultiplier tube (PMT) can be used, in which case a physical pinhole needs to be attached upstream in each detection channel. This also works, but it is more complex and less flexible. A PMT with a fiber bundle can also be used. However, in this case, the size of the pinhole can only be set relatively roughly. Therefore, confocal sectioning can only be substantially set by an optical unit with a variable focal length, for example, the zoom 35 in FIG. 1. In contrast, in the case of an SPAD array, the electronic pinhole created thereby can be used in combination with the zoom 35. Both can be controlled by the control unit 90 according to the microscope objective lens 17 and the respective desired resolution and measurement method used.

[0119] By using methods such as convolution, the data quality can be significantly improved. New methods based on machine learning or deep learning can also be used directly and advantageously. So-called multiplexing methods, such as the method established for the airy scan sensor [3], can also be used in axially spaced planes. Therefore, the speed of recording the three-dimensional image of the sample can be further increased, and the flexibility in the use of sensor pixels can be very advantageously utilized, as in the case of the confocal mode.

[0120] Advantageously, a method for increasing the depth of field, for example, as described in European Patent Application Publication No. 3650905, can also be used in combination with the microscope according to the present invention. The measurement data in two planes can be acquired simultaneously using the microscope according to the present invention.

[0121] Then, the microscope according to the present invention can also be advantageously used to perform a method for FLIM or FLI(S)M evaluation. In particular, simultaneous FLI(S)M imaging can be performed in a plurality of planes, for example, n planes.

[0122] Finally, the microscope according to the invention advantageously also provides a possible single-shot 3D method with a double capture range.

[0123] In the microscope shown in FIG. 1, the axial spacing dz between the planes 21 and 22 in the sample 20 imaged in the detection channels a and b depends in this example on the parameters of the operating device 60b, the thickness d and the refractive index n of the glass plate, and the magnification V. The magnification V can be changed using the zoom system 35. If the parameters of the operating device can also be changed, a higher flexibility with respect to the change in the axial spacing dz is achieved. An example of how this is possible will be explained in connection with FIGS. 2 and 3.

[0124] FIG. 2 is in the form of a stepped glass plate 61 and schematically shows an operating device that can be variably arranged, for example, with respect to the optical axis 71b of the detection channel b. In the situation shown in FIG. 2, the region of the stepped glass plate 61 with thickness d2 is in the beam path and is effective there. As an example, other areas with thicknesses d1 and d3 are shown, which can be arranged in the beam path respectively by displacing the stepped glass plate 61 transversely to the optical axis 71b in the direction indicated by the double arrow.

[0125] Properties similar to those of the stepped glass plate 61 in FIG. 2 can be achieved by an exchanger that can be used to arrange different glass plates, for example glass plates with different thicknesses and / or different refractive indices, in the beam path 71b.

[0126] If the effective thickness of the glass component within the beam path can be adjusted continuously, even higher flexibility for setting the axial spacing is possible. An example for this purpose is shown in FIG. 3. The operating device in FIG. 3 is formed by two glass wedges 62 and 63 that can be displaced relative to the optical axis 71b (mainly perpendicular to the optical axis 71b and slightly in the direction of the optical axis 71b) so as to achieve different effective thicknesses d4 (FIG. 3a), d5 (FIG. 3b), d6 (FIG. 3c) and d7 (FIG. 3d). The two glass wedges 62 and 63 can be actuated via motor components that can be controlled by a control unit 90.

[0127] The detection channels of the detection unit 80 of the microscope according to the invention can be configured in a number of different deformation forms. In particular, the detection of axially spaced planes can be combined with spectral resolution detection. Two examples thereof will be described in connection with FIGS. 4 and 5.

[0128] FIG. 4 shows a deformation form in which a total of four axially spaced planes z1, z2, z3 and z4 within the sample 20 are observed in the detection channels c, d, e and f respectively. For this purpose, a first beam splitter 40, a second beam splitter 41, and a third beam splitter 42 are present, and advantageously, each can be a 50 / 50 beam splitter.

[0129] Each of the detectors 70c, 70d, 70e and 70f is present in the detection channels c, d, e and f as schematically shown in FIG. 4. Advantageously, each of these detectors can be a SPAD array.

[0130] The component beam reflected from the first beam splitter 40 in FIG. 4 is incident on the second beam splitter 41, whereby the component beam passes through the effective operation device 60cd for detection channels c and d and is further split between detection channels c and d. In detection channel c, there is a further operation device 60c that functions to appropriately set the axial plane z1 detected by the detector 70c. The detector 70d images the axial plane z2. Thus, the operation devices 60cd and 60c are effective for channel c. For detection channel d, only the operation device 60cd is effective.

[0131] The component beam that passes through the first beam splitter 40 and is incident on the third beam splitter 42 is split by the third beam splitter 42 between detection channels e and f. Further, in detection channel e, there is an operation device 60e that functions to appropriately set the axial position of the axial plane z3 detected by the detector 70e. The detector 70f images the axial plane z4.

[0132] The operation devices 60cd, 60c, and 60e can each be a glass plate. However, they may also be formed by any other embodiment described in this specification of the operation device for axially displacing the focal region.

[0133] The operation devices 60e and 60cd are dimensioned such that the focal regions are displaced differently. Otherwise, z2 and z3 would coincide, which is not desirable here. For example, if the operation devices 60e and 60cd are formed from glass plates of the same material, they have different thicknesses.

[0134] Another example of the configuration of the detection channels is shown in FIG. 5, where two axially spaced planes z5 and z6 are each spectrally resolved in each case. The sample plane z5 is imaged in a spectrally resolved manner in the detection channels p and q, and the sample plane z6 is imaged in a spectrally resolved manner in the detection channels r and s. For this purpose, there is also a beam splitter 40, which can be a 50 / 50 neutral intensity splitter as shown in FIG. 4, as well as a first color splitter 43 and a second color splitter 44. Each of the detectors 70p, 70q, 70r and 70s is present in the detection channels p, q, r and s, as schematically shown in FIG. 5. Advantageously, each of these detectors can be a SPAD array.

[0135] The component beam reflected from the beam splitter 40 passes through the effective operating device 60pq for the detection channels p and q and then impinges on the first color splitter 43, whereby this component beam is spectrally split between the detection channels p and q. The component beam passing through the beam splitter 40 impinges on the second color splitter 44, whereby this component beam is spectrally split between the detection channels r and s.

[0136] The sample plane z5 is imaged in the detection channels p and q, which is also caused by the axial displacement of the operating device 60pq. The sample plane z6 is imaged in the detection channels r and s.

[0137] The color splitters 43 and 44 may be the same in the illustrated exemplary embodiment. The color splitters 43 and 44 can also be color gradient splitters in which the change in the limiting wavelength is achieved by mechanical displacement.

[0138] In the illustrated example, the first portion λ1 to λ2 of the spectrum of the light 25 to be detected can be detected in the detection channels p and r, and the second portion λ3 to λ4 of the spectrum of the light 25 to be detected can be detected in the detection channels q and s.

[0139] Finally, in the illustrated example, dispersion units 50p, 50q, 50r, and 50s, each having at least one dispersion device 55p, 55q, 55r, and 55s, are present in each of the spectral detection channels p, q, r, and s. The dispersion devices 55p, 55q, 55r, 55s each function to spectrally separate the light 25 to be detected and can typically be formed by diffractive or refractive components. The dispersion units 50p, 50q, 50r, and 50s can each include additional components such as filters, apertures, lenses, particularly cylindrical lenses, or other optical components.

[0140] In an advantageous modification of the configuration of FIG. 4 in which the beam splitters 41, 42 are neutral intensity beam splitters, the beam splitters 41, 42 can be extended by a dispersion prism such that all detectors 70c to 70f receive, for example, the same spectral range, but here in a spectrally resolved manner.

[0141] If a sufficiently large two-dimensional resolving detector, i.e., a detector having a sufficiently large number of pixels and / or an appropriate geometric arrangement, is available, it may no longer be necessary to distribute the entire spectrum among a plurality of detectors as shown in FIG. 5.

[0142] The modifications of the configuration of FIG. 4 described here can be advantageous in this case. References [1] Tsang et al., <https: / / doi.org / 10.1364 / BOE.417286> [2] Zhao et al., <https: / / doi.org / 10.1364 / BOE.491538> [3] <https: / / www.zeiss.com / microscopy / de / produkte / lichtmikroskope / konfokale-mikroskope / lsm-980-mit-airyscan-2.html> [4] LSM 5 LIVE by ZEISS Explanation of Reference Signs

[0143] 10 Light source, laser module 12 Unit for setting the axial depth of illumination 11 Excitation light 13 Main color splitter 14 Scanner 15 Scanning optical unit 16 Tube lens 17 Microscope objective lens 20 Sample 21 First plane within sample 20 22 Second plane within sample 20 25 Light emitted from sample 20 as a result of exposure to excitation light 11 30 Pinhole optical unit 31 Adjustable pinhole 35 Optical unit with variable focal length, zoom optical unit 40 Beam splitter, neutral intensity splitter 41 Beam splitter, neutral intensity splitter 42 Beam splitter, neutral intensity splitter 43 Beam splitter, color splitter 50p Dispersion unit within detection channel p 50q Dispersion unit within detection channel q 50r Dispersion unit within detection channel r 50s Dispersion unit within detection channel s 55p Dispersion device within detection channel p 55q Dispersion device within detection channel q 55r Dispersion device within detection channel r 55s Dispersion device within detection channel s 60b Operating device for axially displacing the focal region 60cd Operating device for axially displacing the focal region, effective for detection channels c and d 60c Operating device for axially displacing the focal region, effective for detection channel c An operating device for axially displacing the focal region, effective for detection channel e An operating device for axially displacing the focal region, effective for detection channels p and q Step glass plate First glass wedge Second glass wedge Detector in detection channel a (first), SPAD array Detector in detection channel b (second), SPAD array Detector in detection channel c, SPAD array Detector in detection channel d, SPAD array Detector in detection channel e, SPAD array Detector in detection channel f, SPAD array Detector in detection channel p, SPAD array Detector in detection channel q, SPAD array Detector in detection channel r, SPAD array Detector in detection channel s, SPAD array Optical axis Detection unit Detection channel, first detection channel Detection channel, second detection channel Detection channel Detection channel Detection channel Detection channel Detection channel Detection channel Detection channel Detection channel Axial offset in the sample space Axial offset in the image space First thickness of the step glass plate 61 Second thickness of the step glass plate 61 Third thickness of the step glass plate 61 d4 The first effective thickness of the pair of glass wedges 62, 63 d5 The second effective thickness of the pair of glass wedges 62, 63 d6 The third effective thickness of the pair of glass wedges 62, 63 d7 The fourth effective thickness of the pair of glass wedges 62, 63 d8 The fifth effective thickness of the pair of glass wedges 62, 63 z1 The z - coordinate of the plane within the sample 20 imaged on the detector 70c z2 The z - coordinate of the plane within the sample 20 imaged on the detector 70d z3 The z - coordinate of the plane within the sample 20 imaged on the detector 70e z4 The z - coordinate of the plane within the sample 20 imaged on the detector 70f z5 The z - coordinate of the plane within the sample 20 imaged on the detectors 70p and 70q z6 The z - coordinate of the plane within the sample 20 imaged on the detectors 70r and 70s λ1~λ2 The wavelength range detected in the detection channels p and r λ3~λ4 The wavelength range detected in the detection channels q and s

Claims

1. A light source (10) for transmitting excitation light (11), an illumination beam (12, 13, 14, 15, 16, 17) for directing said excitation light (11) onto and / or into a sample (20); a scanning device (14) for varying the position on and / or within the sample (20) exposed to the excitation light (11); a detection unit (80) for detecting light (25) emitted from the sample (20); a detection beam path (17, 16, 15, 14, 13, 30, 35, 40) having a microscope objective (17) for directing at least a portion of the light (25) emitted from the sample (20) to the detection unit (80); A control unit (90) for controlling the scanning device (14) and the detection unit (80) and for evaluating measurement data from the detection unit (80). Under the microscope, In said detection unit (80), a first detection channel (a) with at least one first detector (70a) and at least one further detection channel (b) are formed, each further detection channel (b) comprising at least one detector (70b), in the detection unit (80) there is in each case at least one beam splitter (40) for directing a portion of the light (25) emitted from the sample (20) into a respective further detection channel (b), In at least one of said detection channels (a, b) there is a manipulation device (60) for axial displacement (Dz) of the focal region in order to image axially spaced (dz) sample planes (21, 22) in at least two detection channels (a, b). A microscope characterized by:

2. the presence in said detection unit (80) of a variable focal length optical unit (35) for imaging said sample (20) on said detectors (70a, 70b) with variable magnification; Characterized by:

2. The microscope according to claim 1.

3. in at least one of the detection channels (a, b), the manipulation device comprises a glass plate (60b) arranged in a converging portion of the detection beam path; Characterized by:

3. A microscope according to claim 1 or 2.

4. in at least one of the detection channels, the manipulation device comprises an exchanger having different glass plates arranged in a convergent portion of the detection beam path, each glass plate having a different thickness and / or a glass material of each glass plate having a different refractive index. Characterized by: A microscope according to any one of claims 1 to 3.

5. In at least one of the detection channels, the manipulation device comprises a stepped glass plate (61) arranged in a converging portion of the detection beam path, the stepped glass plate being variably positionable within the detection beam path in order to set different optically effective thicknesses (d1, d2, d3) transverse to an optical axis (71b). Characterized by: A microscope according to any one of claims 1 to 4.

6. In at least one of the detection channels, the manipulation device comprises two glass wedges (62, 63) arranged in a converging part of the detection beam path, the glass wedges being displaceable relative to each other and to the optical axis (71b) in order to continuously set different optically effective thicknesses (d4, d5, d6, d7). Characterized by: A microscope according to any one of claims 1 to 5.

7. In at least one of the detection channels, the manipulation device comprises a telescope, in particular an adjustable telescope. Characterized by: A microscope according to any one of claims 1 to 6.

8. an optical beam shaping unit is present before the main beam splitter (13) in the illumination beam path in the direction opposite to the propagation direction and / or before the main beam splitter (13) in the detection beam path in the direction of the propagation direction, said optical beam shaping unit serving to generate a special form of point spread function, in particular an axially encoded point spread function, e.g. a helical point spread function, an astigmatic point spread function or a "twisted" point spread function; Characterized by: A microscope according to any one of claims 1 to 7.

9. In the illumination path, there is a unit (12) for setting the axial depth of illumination. Characterized by: A microscope according to any one of claims 1 to 8.

10. the unit (12) for setting the axial depth of illumination comprises an adjustable diaphragm for setting the beam diameter of the excitation light (11); Characterized by: The microscope according to claim 9.

11. The unit (12) for setting the axial depth of illumination is configured to generate a plurality of axially spaced focal points within the sample (20). Characterized by: A microscope according to claim 9 or 10.

12. said unit (12) for setting the axial depth of illumination comprises at least one diffractive element, A microscope according to any one of claims 9 to 11.

13. the device for setting the axial depth of illumination is configured to defocus the excitation light such that the focal point of the excitation beam is located, in particular in the center, between two detection planes, in particular between the two detection planes that are furthest from each other; Characterized by: A microscope according to any one of claims 9 to 12.

14. the optical unit with variable focal length images in each case one and the same plane (21, 22) of the sample (20) onto the detectors (70a, 70b), independently of the focal length setting; Characterized by: A microscope according to any one of claims 1 to 13.

15. The variable focal length optical unit comprises a zoom optical unit (35) and / or a lens exchange system that allows the magnification to be set to a number of discrete values. Characterized by: A microscope according to any one of claims 1 to 14.

16. The detection unit (80) comprises an aperture stop (11) of adjustable size. Characterized by: A microscope according to any one of claims 1 to 15.

17. At least one of the beam splitters is a neutral intensity splitter. Characterized by: A microscope according to any one of claims 1 to 16.

18. At least one of the detectors is a two-dimensional spatially resolved detector, in particular a camera chip, particularly preferably a SPAD array. Characterized by: A microscope according to any one of claims 1 to 17.

19. The detectors are formed by one and the same camera chip. Characterized by: A microscope according to any one of claims 1 to 18.

20. In at least one of the two-dimensional spatially resolved detectors (28a, 28b), pixels, in particular adjacent pixels, for example adjacent pixels in the direction of the detector rows and / or the detector columns, in particular adjacent pixels perpendicular to the direction of dispersion, can be combined by binning. Characterized by:

20. A microscope according to claim 18 or 19.

21. In at least one of said two-dimensional spatially resolved detectors (28a, 28b), the pixels can be individually set to be active or passive. Characterized by: A microscope according to any one of claims 18 to 20.

22. a multi-lens array is arranged in front of at least one of said detectors (70a, . . . , 70s); Characterized by: A microscope according to any one of claims 18 to 21.

23. At least one of the detectors comprises one or more photomultiplier tubes, in particular with an adjustable entrance pinhole. Characterized by: A microscope according to any one of claims 1 to 22.

24. At least one of said beam splitters is a color splitter forming at least two spectrally distinct detection channels (p, . . . , s). Characterized by: A microscope according to any one of claims 1 to 23.

25. the presence of a dispersive device (55p, . . . , 55s) in at least one of the detection channels (p, . . . , s) for spectrally separating at least a portion of the light (25) emitted from the sample (20); Characterized by:

25. The microscope of claim 24.

26. The presence of a dispersive device for spectrally separating the light to be detected that is effective in multiple detection channels. Characterized by:

26. A microscope according to claim 24 or 25.

27. at least one of the dispersion devices (55p, . . . , 55s) for spectrally separating at least a portion of the light (25) emitted from the sample is controllable and optionally operable; The control unit (90) is configured to control the distributed device. Characterized by:

27. The microscope of claim 26.

Images