Super-resolution microscope having rapid quasi-confocal detection

Abstract

In order to increase the resolving power of quasi-confocal line scanning microscopes comprising a main beam splitter for coupling the illumination beam path and the detection beam path, after descanning by the illuminating beam deflection unit, in a "re-scan" method the sample light is scanned once more in the detection beam path by said sample light being moved over the sensor, downstream of the confocal diaphragm, through a second beam deflection unit, in the transverse direction, to result in linear focus. The illumination line can be structured in order to increase the resolution in the longitudinal direction. The optical arrangement has the disadvantage of considerable light losses on account of a large number of optical interfaces to be passed. A microscope in which the illuminating beam deflection unit is optically arranged between the light source and the main beam splitter such that the sample light reaches the sensor remotely from the illuminating beam deflection unit (non-descanned), and a control unit moves the sample light beam over the sensor only by means of the second beam deflection unit in an operating mode, is more light sensitive and more flexible. Depending on the operating mode, synchronisation with the first beam deflection unit can be provided. Due to the high light sensitivity, the microscope is particularly suitable for fluorescence microscopy.

Description

[0001] Super-resolution microscope with fast quasi-confocal detection

[0002] The invention relates to a microscope with an illumination beam path, a detection beam path and a beam splitter, wherein the detection beam path has a sample chamber, a microscope objective, a tube lens, an intermediate image generated by the tube lens and a two-dimensional spatially resolving optoelectronic sensor with detection optics for imaging the intermediate image onto the sensor, and the illumination beam path has a light source and a first adjustable beam deflection unit for moving an illumination light beam (“scanning”, “rasterizing”) through the sample chamber, and the detection beam path has a second adjustable beam deflection unit for moving a sample light beam over the sensor, wherein the illumination beam path and the detection beam path are optically coupled to a common beam path by means of the beam splitter in such a way thatIllumination light from the light source passes through the beam splitter through the microscope objective into the sample chamber, and sample light from the sample chamber passes through the microscope objective, through the beam splitter, and then through the second beam deflection unit to the sensor. The beam deflection units are arranged in or at least near a respective plane that is optically conjugated with a back focal plane of the microscope objective, which, in the case of telecentric beam paths, corresponds to a pupil plane of the microscope objective.

[0003] Confocal fluorescence imaging delivers high-contrast, high-resolution images of biological samples. As described in DE 197 02 753 A1, the sample is scanned point-by-point using laser illumination via a beam deflection unit. The generated sample light is descanned using the same beam deflection unit, so that a stationary beam of light is present on the optical axis from each sample location. The light is detected through a confocal pinhole. This pinhole largely shields the sensor from out-of-focus light, resulting in high contrast. A disadvantage, however, is the comparatively long acquisition time due to point-by-point scanning. This inevitably also results in a high phototoxic exposure to the sample from the excitation light. These disadvantages of point-by-point imaging can be reduced through parallelization.Examples of this are confocal microscopes with Nipkow disks and light-sheet microscopes, the latter requiring additional illumination optics. Parallelization can alternatively be achieved using linear illumination, as in EP 1 617 258 A1. To achieve confocality in at least one dimension (perpendicular to the illumination line), linear detection with a slit aperture is necessary to suppress out-of-focus light. Along the line, the resolution is comparable to that of a wide-field microscope.

[0004] To further increase the resolution across the line (super-resolution beyond the diffraction limit), the sample light in the detection beam path can be re-scanned after being de-scanned by the illuminating beam deflection unit. For this purpose, it is moved across the sensor behind the confocal aperture by a beam deflection unit (De Luca et al.: "Re-scan confocal microscopy: scanning twice for better resolution" in Biomedical Optics Express 2013, Vol. 4, No. 11, p. 2644). The pixels of the super-resolved image of the sample must be calculated by linking intensities of different pixels at different times. This optical arrangement has the disadvantage of considerable light losses due to the large number of optical interfaces that must be passed through.

[0005] To increase the resolution along the illumination line, the intensity can be additionally structured (modulated) in its longitudinal direction using the "re-scan" method (Shen et al.: "Confocal rescan structured illumination microscopy for real-time deep tissue imaging with superresolution" in Advanced Photonics Nexus 2023, Vol. 2 (1 ), p. 016009-1). Each location on the sample must be illuminated in at least three different phases (different positions of the line structure) and imaged in a corresponding number of phase images. As in structured illumination microscopy (SIM) in the widefield, the overall image must be calculated by linking all phase images (demodulation).

[0006] The low light sensitivity of the optical arrangements used in the "re-scan" method remains even with structured illumination. Furthermore, the arrangements are limited to the specific scanning microscopy technique and are therefore inflexible. The confocal aperture, in particular, renders the system inflexible or at least complex, as it must be mechanically adjustable with high precision to allow a minimum degree of flexibility. Furthermore, such mechanical adjustments tend to be slow.

[0007] The invention is based on the object of improving a microscope of the type mentioned above so that the detection beam path is simplified, thereby achieving greater light transmission efficiency and thus light sensitivity. At least in certain embodiments, greater flexibility of the microscope should also be enabled.

[0008] The object is achieved by a microscope having the features specified in claim 1.

[0009] Advantageous embodiments of the invention are specified in the subclaims.

[0010] According to the invention, the first beam deflection unit is optically arranged between the light source and the beam splitter, so that the sample light reaches the sensor away from the first beam deflection unit. The microscope also comprises a control unit configured to move the sample light beam across the sensor by means of the second beam deflection unit in one operating mode. This movement is, in principle, initially independent of any movement of the first beam deflection unit. Depending on the operating mode, synchronization with the first beam deflection unit can be provided.

[0011] The sample light reaches the sensor away from the first beam deflection unit, which can be described as non-descanned detection. In contrast to conventional "re-scan" microscopes, the movement of the first beam deflection unit is not reversed, not even by the second beam deflection unit. The beams (sample light beams) emanating successively from different locations in the sample chamber due to the scanning illumination thus fall at correspondingly different angles to the optical axis into each plane conjugate to the rear focal plane of the microscope (and thus at different angles to the optical axis onto the second beam deflection unit). According to the invention, there is no stationary beam in front of the second beam deflection unit.Surprisingly, it was discovered that even with just one beam deflection unit in the detection beam path, scanning a beam bundle is possible, which corresponds to "re-scanning." Accordingly, even in the first operating mode, there is no stationary beam bundle behind the second beam deflection unit; instead, the angle of the beam bundle to the optical axis after passing the second beam deflection unit varies over time.

[0012] To advantageously increase resolution, the angle of the propagation direction of the beams emerging from the sample chamber relative to the optical axis can be increased by the second beam deflection unit. This process can be referred to as "add scanning." For this purpose, the control unit can be configured to move the sample light beam across the sensor in the first operating mode using the second beam deflection unit in such a way that the intermediate image is projected onto the sensor in an enlarged form.In particular, the movement can be carried out in such a way that an amount of a quotient between a first angle, which a propagation direction of a beam of sample light emanating from a location in the sample space immediately behind the second beam deflection unit encloses with an optical axis of the detection optics, and a second angle, which the propagation direction of the beam encloses with an optical axis of the microscope objective immediately in front of the second beam deflection unit, is greater than one.

[0013] The computational evaluation of the sensor's pixel intensities from different points in time to generate a super-resolution image of the sample transverse to the longitudinal direction of the illumination line corresponds to the known evaluation of pixel intensities recorded after desampling and resampling (and thus an intermediate image projected onto the sensor, preferably magnified by a factor of two, one-dimensionally transverse to the longitudinal direction of the illumination line). With a magnification factor of two, which is only effective in one dimension due to the additional scanning, the maximum increase in resolution transverse to the illumination line is achieved. The magnification is achieved with little effort by ensuring that, at least in the first operating mode, the movement of the second beam deflection unit can be synchronized or synchronized with the movement of the first beam deflection unit, for example, via the control unit or another electrical connection.In particular, the control unit can move the first beam deflection unit and the second beam deflection unit with identical or approximately identical angular amplitude in order to achieve the maximum resolution increase by a factor of 2 through the additional scanning with the only one-dimensional mechanical magnification.

[0014] The mechanical and technical requirements for the second beam deflection unit are lower than for conventional "re-scan" arrangements. It only has to meet the same requirements as the first beam deflection unit because it does not have to scan an angular amplitude twice as large as the first, as is the case with the prior art, which could cause problems with linearization and would also limit the overall acquisition speed, for example, in the case of a galvanometer mirror.

[0015] Embodiments in which the illumination beam path has a beam former (e.g., a cylindrical lens) for generating a linear distribution of the illumination light ("illumination line") are advantageous. The linear distribution can be intensity-modulated along its longitudinal direction, in particular periodically intensity-modulated. Such a distribution ("light pattern") is preferably created in the sample space by the beam former, which for this purpose comprises, for example, a spatial light modulator, simultaneously generating at least two linear, mutually interfering light distributions that are present in a plane optically conjugate to a rear focal plane of the microscope objective (BFP). By structuring along the illumination line, it is possible to increase the resolution along the illumination line by recording and evaluating raw images in multiple phase positions of the modulated light distribution.Preferably, the beam former generates the distribution of the illumination light in such a way that a (modulated or unmodulated) illumination line is created in the sample space, which is imaged onto these pixel lines by the detection beam path geometrically parallel to the pixel lines of the sensor.

[0016] Preferably, the beam shaper can be repeatedly removed from the illumination beam path, in particular by a motor and controlled by the control unit. This allows the microscope to be used with wide-field illumination in alternative operating modes. The repeated motorized removability allows for great flexibility. Alternatively or in addition to removability, the beam shaper can comprise a spatial light modulator (SLM), in particular a phase-modulating SLM in a conjugate pupil plane (rear focal plane of the microscope objective). This can be controlled, in particular, by the control unit in such a way that different illumination light distributions are created in the sample space in temporal alternation. This makes it possible to generate either unmodulated linear illumination or, by means of at least two interference-capable light distributions in the pupil plane / BFP, modulated linear illumination in the sample space.The switching can be achieved very quickly, for example, at a frequency of 50 Hz to 100 Hz by rewriting the phase pattern on the SLM. Using an SLM, rapid switching between point illumination and linear illumination would also be possible, for example, for optical manipulation alternating with observation of the sample. The first beam deflection unit can be conveniently adjustable in two dimensions for this purpose.

[0017] In particularly advantageous embodiments, the first beam deflection unit is designed for two-dimensional scanning of the sample space in a first dimension transverse to the longitudinal direction of the linear illumination light distribution and in a second dimension parallel to the longitudinal direction. By moving in the second dimension, the illumination light distribution can be brought into different phase positions with little effort and in a short time in order to acquire the number of differently illuminated raw images required to increase resolution.This purpose can preferably be served by a control unit which places the first beam deflection unit in at least three different positions along the second dimension, wherein two of the resulting positions of the illumination light distribution in the sample space at the positions along the second dimension are spaced from one another by less than a length of the illumination light distribution in the longitudinal direction, in particular by a period length of the intensity-modulated light distribution or less than a period length, and wherein in particular a separate raw image is created for each of the positions.

[0018] As an alternative to shifting the light pattern along the (intensity-modulated) illumination line by the first beam deflection unit, such a shift can be achieved by means of a pivotable, transparent, plane-parallel plate or by means of an electro-optical phase shifter, each in the illumination beam path. As an alternative to a shift, the illumination beam path can comprise optics for illuminating the sample space at different phase positions of the intensity-modulated illumination light distribution, in particular a spatial phase modulator (phase-influencing spatial light modulator) and / or an electro-optical modulator in conjunction with a quarter-wave plate. In this alternative, the first beam deflection unit can advantageously be designed for only one-dimensional scanning of the sample space transverse to the longitudinal direction of the linear illumination light distribution.A beam deflection unit that can be adjusted only in one dimension is more cost-effective and, above all, more stable.

[0019] Compared to conventional SIM image acquisition, which requires 13 or even 15 raw images, a resulting image can be calculated using only three raw images. A region of interest (ROI) of the sample space can be selected at least in the longitudinal direction of the illumination line using the second dimension of the first beam deflection unit 21. A small region of interest can be scanned and recorded extremely quickly with the illumination line.

[0020] In all embodiments, the sensor, in particular a CMOS sensor, an EMCCD sensor, an iCCD sensor, or a SPAD array sensor, can have a deactivatable line-shaped electronic shutter arranged confocally to the intermediate image. The electronic shutter can preferably be implemented in the form of a correspondingly operated "rolling shutter," for example as in WO 2006 / 008637 A1, in particular with the ability to synchronize or synchronize an adjustment of the first beam deflection unit with a dynamic position (movement) of the shutter, at least in the first operating mode (so that the position of the electronic shutter opening corresponds to the position of the image of the illumination line from the sample space on the sensor). The electronic shutter allows for the suppression of out-of-focus sample light and thus, like a mechanical confocal shutter, both an axial and lateral resolution improvement.An increase in lateral resolution (perpendicular to the illumination line) is possible due to the magnified image generated by the second beam deflection unit, but also without a confocal aperture. Out-of-focus light is also at least partially suppressed by calculating from multiple raw images at different illumination phase positions. The improvement in axial resolution is not affected by the aperture size. However, the aperture size can be used to balance the signal-to-noise ratio and the depth of field.

[0021] The use of a sensor with an electronic aperture has the advantage of allowing rapid switching between confocal and wide-field detection, for example between consecutive images.

[0022] For synchronization purposes, the sensor can preferably have a lightsheet readout mode (referred to by Hamamatsu as "Lightsheet Readout Mode") and an output to which information about the position and / or movement of the aperture is present, in particular a signal indicating the beginning of an image (frame) on the sensor. Sensors with such a mode and a corresponding output are commercially available from Hamamatsu: https: / / www.hamamatsu.com / eu / en / product / cameras / cmos- cameras / lightsheet-readout-mode.html. Hamamatsu refers to the output as an "external trigger output." The control unit is expediently electrically connected to the sensor and the beam deflection unit.

[0023] A microscope is particularly versatile in which the control unit is electrically connected to the sensor and the beam deflection units and, in the first selectable operating mode, activates the electronic aperture and synchronizes the movements of the first beam deflection unit, the second beam deflection unit and the movement of the electronic aperture and / or, in a second selectable operating mode, activates the electronic aperture, synchronizes the movement of the first beam deflection unit and the movement of the electronic aperture and operates the second beam deflection unit in a constant position, in particular in a neutral position, and / or, in a third selectable operating mode, deactivates the electronic aperture and operates the second beam deflection unit in a constant position and / or, in a fourth selectable operating mode, deactivates the electronic aperture and operates the first and second beam deflection units as in the first operating mode.

[0024] Such a system can operate in a classic wide-field mode (e.g., with incoherent illumination via a lamp as the light source, whose illumination light is guided into the sample chamber via the microscope objective, away from the first beam deflection unit, for example, through another output of the microscope) or in laser wide-field mode (third operating mode), as well as in a confocal mode (second operating mode), and in an additional scanning mode (first and fourth operating modes) without requiring any mechanical modification to the system. Furthermore, quasi-simultaneous phase measurements (e.g., phase contrast, differential interference contrast, intensity transport equation, differential phase contrast) are also possible with the sensor in other operating modes.All that is necessary is to leave one or both beam deflection units in their rest position (neutral position) and activate the electronic aperture and, if necessary, adjust its size accordingly or deactivate it. This can be done quickly and easily from the control unit, for example via software. This enables the implementation of measurement tasks that would not be possible with a conventional "re-scan" system. Especially for multimodal imaging of the same sample, the multiple use of the same sensor is generally advantageous, as the data from the various measurements are then already aligned with pixel precision. Furthermore, the cost is low because only one sensor is required for the different methods.

[0025] An advantageous option may therefore be that the control unit removes the beam shaper from the illumination beam path in the third operating mode and / or that intensity modulation of the illumination light can be deactivated or is deactivated in the second operating mode (so that an unmodulated illumination line is created in the sample space) and / or that the control unit comprises a selection unit for selecting one of several operating modes and that the control unit operates the microscope in the selected operating mode after the selection, in particular with an additional selection option in the selection unit between several sub-modes, each with different light sources, in particular for the third operating mode. This enables flexible, cost-effective use with low operating effort.

[0026] A microscope is advantageously compact and light-efficient if the detection beam path between the intermediate image generated by the tube lens and the detection optics is free of confocal field diaphragms, in particular slit diaphragms, and in particular is free of image planes conjugated with the intermediate image and / or if the detection beam path between the intermediate image generated by the tube lens and the detection optics has exactly one plane conjugated to the rear focal plane of the microscope objective.

[0027] A microscope can be constructed to be particularly compact and light-efficient if the common beam path has an optical system, preferably a scanning lens, between the intermediate image and the beam splitter for generating a plane conjugate to the rear focal plane of the microscope objective on or near the first beam deflection unit and on or near the second beam deflection unit. This means that, on the one hand, the beam splitter is arranged in collimated light, so that any contamination has only a minimal impact on its transmission quality. On the other hand, this allows a compact coupling of the illumination and detection beam paths without the need for additional collimation, as is the case in the prior art. In particular, this eliminates the need for additional relay optics.

[0028] Particularly advantageously, the conjugate plane (pupil plane) can be created by reflection at the beam splitter, so that the first beam deflection unit lies in or near the reflected conjugate plane, in particular with transmission of the conjugate pupil plane into the detection beam path on or near the second beam deflection unit, so that the second beam deflection unit lies in or near the transmitted conjugate plane. In this way, no additional optics are required in the detection beam path, which keeps the number of optical interfaces to a minimum and thereby maximizes the light sensitivity of the detection. The transmission into the detection beam path leads (compared to a reflected arrangement) to better extinction of the excitation light, so that an improved contrast between the measurement signal and the excitation signal is achieved at the detector.

[0029] Preferably, both the illumination light on its path to the microscope objective and the sample light on its path to the sensor pass through the same intermediate image generated by the tube lens. The beam splitter can be arranged as a main beam splitter or main color splitter, optically positioned between the first beam deflection unit and the intermediate image generated by the tube lens.

[0030] By sharing the same intermediate image for illumination and detection, only a single optical port of the microscope is advantageously occupied. Additional outputs are available for other uses, thus providing even greater flexibility. In particular, fast quasi-confocal microscopy can be combined in a multimodal manner with other methods that require their own output. The inventive arrangement of illumination and detection beam paths, each extending to or from the respective output,The light source from the intermediate image, or only from the detection beam path from the intermediate image to the sensor, can thus be advantageously integrated into a single module, reducing manufacturing and installation costs and enabling a more compact and stable design. In the case of a combined module with illumination and beam path, the actual light source can be located outside the module and optically connected to it, for example, via optical fiber(s) or free beam coupling into the module. Furthermore, compared to light sheet microscopes, only a single illumination optic in the form of the microscope objective is required.

[0031] Alternatively, the microscope can have a second tube lens that creates a second intermediate image, with the illumination light passing through the second intermediate image (e.g., at a second optical port of the microscope) on its way to the microscope objective. For example, an existing laser scanning module can be used for illumination. Only the detection beam path, for example, is then connected to the first intermediate image at the first port of the microscope as a separate module.

[0032] In one possible embodiment, the detection beam path between the beam splitter and the detection optics can have a secondary color splitter, which splits the sample light into two spectrally disjoint components and directs them to disjoint regions of the sensor or to a respective sensor (for example, as in DE 10 2021 134 427 A1 or in DE 10 2023 005 252 A1). The illumination beam path can have a second light source with a different emission wavelength than the first light source. In this way, two spectral ranges belonging to the emission wavelengths, in particular from two different fluorescent dyes, can be recorded simultaneously. The first and second light sources can be the same light source, for example in the form of a multi-line laser or a broadband light source, in particular a white light source.For precise structuring, the two colors can advantageously be superimposed on the first steel deflection unit, whereby the grating constants of the illumination pattern in the sample are identical.

[0033] Advantageously, the first beam deflection unit comprises MEMS micromirrors, in particular MEMS micromirrors that can be continuously adjusted around two orthogonal spatial directions. This allows a module (with illumination and detection beam paths, each up to the intermediate image or only the detection beam path from the intermediate image) to be provided in a compact, quiet, and cost-effective manner. Two-dimensionally adjustable MEMS scanners also have the advantage that the deflection mirror is always located in the optical pupil / BFP, which optimizes the accuracy of the illumination focus volume placement. The second axis can be used, in particular, to shift the illumination line along its longitudinal direction to generate the various illumination phase positions of structured illumination. Furthermore, it can be used to define the illumination area and / or generate a longer (modulated or unmodulated) or more homogeneous (unmodulated) line.In addition, the second axis can be used to generate very small movements along the illumination line, which homogenize the line illumination (without SIM mode). Finally, this also ensures good image field illumination when the sample is optically manipulated via the first beam deflection unit.

[0034] As an alternative to a two-dimensionally adjustable MEMS micromirror, the first beam deflection unit can be formed in a conventional manner from two one-dimensionally adjustable mirrors, in particular galvanometer mirrors, preferably with an optical relay that images one mirror onto the other.

[0035] Particularly preferred are embodiments in which the microscope has a stand on which the microscope objective is arranged, in particular in a lens turret, wherein the stand has a first connection, in the region of which the tube lens and the intermediate image are arranged, and at least one further connection with a further tube lens and a further intermediate image, wherein the sample light can be guided to both outputs simultaneously or sequentially by means of at least one second beam splitter, in particular a repeatedly removable beam splitter, or by means of a mirror, wherein the first beam splitter, the detection optics, the sensor, the first and second beam deflection units, and a scanning optics are arranged within a module that is detachably mechanically and optically connected to one of the connections. In this way, a flexibly usable microscope system with high light sensitivity and a short acquisition time can be provided.

[0036] In a further embodiment, the invention also comprises a microscope with an evaluation unit that acquires multiple raw images from at least two, preferably three, different phase positions of the illumination light distribution in the sample space and calculates a resulting image from the raw images with increased resolution in at least two spatial directions, in particular by solving a system of equations that describes the convolution of an unknown sample with point spread functions for illumination and detection for the various phase positions in the spatial or frequency domain. The invention is explained in more detail below using exemplary embodiments.

[0037] The drawings show:

[0038] Fig. 1 a microscope,

[0039] Fig. 2 the optical conditions in the microscope,

[0040] Fig. 3 an alternative microscope and

[0041] Fig. 4 Principles of the control and synchronization regime.

[0042] In all drawings, corresponding parts have the same reference symbols.

[0043] Fig. 1 shows a schematic representation of a microscope 1. It consists of a stand 2, an "add-scan" module 3, and a laser module 4. The stand 2 has a, for example, telecentric microscope objective 5 with tube lenses 6, which each generate an intermediate image ZB in the area of ​​two outputs 7 / 7'. The microscope objective 5 is mounted on a lens turret (not shown separately). The stand also has pivotable beam splitters 9, for example neutral splitters or color splitters, in order to selectively and configurably direct light proportionally to the outputs and / or to the eyepiece 10 and / or light from a lamp 11 to the microscope objective 5 and from there into the sample chamber P. In the sample chamber, the sample rests on a motor-driven, three-dimensionally movable stage (not shown for the sake of clarity).

[0044] The laser module 4 comprises, purely by way of example, four lasers 12 with different emission wavelengths, the respective intensities of which can be adjusted using a respective AOTF 13. Alternatively (not shown), one or more of the lasers can be directly modulated. Their illumination light is coupled into optical fibers 15 via coupling optics 14 and guided to the scanning module 3, where it is collimated, for example, using longitudinally movable collimators 16. The collimators 16 can be used to compensate for longitudinal chromatic aberrations and / or to focus the illumination light into different depths of the sample space P. Alternatively (not shown), the different emission wavelengths can be combined, for example, in the laser module, so that only a single optical fiber 15 is required and collimators 16 can be dispensed with.Via a mirror 17 and a beam combiner 18, the illumination light combined in this way reaches a deflection mirror 19, which deflects the illumination light such that, after it has passed a beam former 20, which comprises, for example, a phase-modifying spatial light modulator (SLM) and a cylindrical lens, it falls onto the first beam deflection unit 21, for example a continuously two-dimensionally adjustable microelectromechanical system (MEMS) with micromirrors. From the beam splitter 22, the illumination light passes via the scanning optics 23, the intermediate image ZB and one of the tube lenses 6 to the microscope objective 5 and from there into the sample space P. Since the beam former 20 focuses the illumination light in one dimension into the plane lying on the beam deflection unit 21 and conjugated with the pupil of the microscope objective 5, a basically linear illumination focus volume (illumination line) results in the sample space.Using the SLM, the illumination light can be modified to form two separate points or truly parallel lines on the first beam deflection unit, which is arranged in the conjugate pupil plane PE', which interfere in the sample space in such a way that the illumination focus volume is illuminated with a periodic intensity pattern along its longer extension (longitudinal direction of the illumination line).

[0045] Sample light, in particular fluorescence essentially excited in a line pattern in the sample by the illumination light, travels in the opposite direction via the intermediate image ZB to the beam splitter 22. The portion of the illumination light reflected in the sample and en route there is reflected back to the first beam deflection unit 21 by the beam splitter 22, which is configured, for example, as a dichroic notch filter and thus acts as the main color splitter. Fluorescence contained in the sample light is transmitted, particularly due to the Stokes shift, through the beam splitter 22 to the second beam deflection unit 24, for example, a one-dimensionally continuously adjustable MEMS mirror or a galvanometer mirror.The first scanning optics 23, which collimates the sample light, are designed such that a plane PE" conjugate to the pupil PE of the microscope objective 5 lies on the one hand on the first beam deflection unit 21 and on the other hand on the second beam deflection unit 24. Behind the second beam deflection unit 24, the sample light is focused by a detection optics 25 onto the two-dimensional spatially resolving sensor 28, for example, a CMOS chip or a matrix of single-photon counting avalanche photodiodes (SPAD array). In the neutral position (zero position) of the second beam deflection unit 24, the optical axes of the microscope objective 5 and the detection optics 25 coincide. The sensor 28 has an electronic slit diaphragm in the form of a "rolling shutter," the slit width of which is adjustable and which can be completely deactivated.The illumination line in the sample chamber is aligned so that it is imaged parallel to the pixel rows of the sensor 28 (and thus parallel to the electronic slit diaphragm).

[0046] A control unit 29 with a selection unit 30 for, for example, four different operating modes (with further sub-modes) is electrically connected to the stand 2, the "add-scan" module 3, and the laser module 4. It controls the mechanical and optical components contained therein, and also receives measured values ​​from the sensors contained therein, in particular from sensor 28.

[0047] If the user selects the first operating mode ("Add-Scan Mode"), the control unit 29 activates the electronic diaphragm of the sensor 28 and moves the sample light beam coming from the sample chamber over the sensor 28 by means of the second beam deflection unit 24 such that the intermediate image ZB is projected onto the sensor 28, enlarged in the direction of movement. The movement of the second beam deflection unit 24 is synchronized with the movement of the first beam deflection unit 21, and both are moved with, for example, identical angular amplitudes. The movement of the beam deflection units 21, 24 is also synchronized by the control unit 29 with the movement of the electronic slit diaphragm of the sensor 28, so that the latter acts as a confocal diaphragm and the illumination focus volume is imaged into the slit diaphragm opening.The sample light arriving at the second beam deflection unit 24 already has an angle of incidence (relative to the optical axis of the microscope objective 5) that depends on its origin in the sample chamber. The synchronization of the first beam deflection unit 21 and the second beam deflection unit 24 enhances this effect. Due to the identical amplitude and the selected in-phase oscillation direction, the sample light reflected by the second beam deflection unit 24 has an angle twice as large relative to the optical axis of the detection optics 25. In a conventional system, this angle would be zero due to the de-sampling there, and the sample light beam would be at rest.

[0048] The increase in the angle results in a one-dimensional magnification across the illumination line, in the case of doubling by identical amplitudes of the beam deflection units 21 / 24 a “non-optical” anisotropic magnification by a factor of M mech=2, which may result in a purely optical (typically isotropic) magnification M opt is added by the detection optics 25 in conjunction with the scanning optics 23. On the one hand, this leads to a corresponding broadening of the point spread function (PSF), but on the other hand, it also means that the distance between two points of the sample space imaged onto the sensor 28 is increased more than the width of the PSF increases, which corresponds to a "re-assignment" or "re-scanning". The image recorded at the sensor 28 must be compressed (back-projected) one-dimensionally by the factor Mmech in the direction transverse to the illumination line in order to correctly reproduce the proportions of the sample space. Since the imaged points were spread more than the PSF was widened, despite the compression in the transverse direction, a better resolution remains than in a comparable diffraction-limited wide-field image - directly optically and mechanically without complex reconstruction calculations.However, the resolution in the longitudinal direction of the illumination line is not affected.

[0049] The second beam deflection unit 24 acts in the direction transverse to the illumination line as follows. The image B of a fluorescent object O in the sample space in the object-side focal plane of the microscope objective onto the sensor 28 can be described with a convolution, where the optical magnification is defined without loss of generality as M op t=1 can be assumed:

[0050] Here, H em x D - x0,y D - y o the intensity point mapping function for mapping objects in the object plane into the detection plane on sensor 28. The coordinates with index 0 describe the sample space, the coordinates with index D the image space on sensor 28.

[0051] Scanning the object Ö with the fluorescence-exciting illumination line at the location xo / yo then leads to:

[0052] Inserting into equation (1) gives:

[0053] By integrating over the sampling vector x s perpendicular to the longitudinal direction of the illumination line, a wide-field image results. By adding the one-dimensional confocal electronic aperture D(x), also at the position x s , surrendered:

[0054] - *s)

[0055] From these equations, it can be deduced that the PSF is characterized by the fact that in the longitudinal direction of the illumination line, imaging occurs as in the wide-field case, while in the direction of the effect of the confocal electronic shutter (i.e., transverse to the illumination line), the point image corresponds to a convolution of the excitation PSF with the slit function D:

[0056] For a confocal aperture with D(x) = <5(x), the fully confocal PSF again follows, whereas for an open aperture with D(x) = 1, the convolution results in a constant and the image of the point emitter corresponds to the emission PSF. To keep the equations compact, the imaging for the case of the second beam deflection unit 24 is presented below only in the affected variable.

[0057] Along the direction y D Along the illumination line (and along the electronic aperture formed by the respective sensor row), the point image can further be described by wide-field imaging.

[0058] The second beam deflection unit 24 results in a shift of the detection PSF by a second scanning vector x s ' .

[0059] The second beam deflection unit 24 is connected in the first (and fourth) operating mode to the first beam deflection unit 21 according to x s ' = (M mech - l~)xs synchronized. The case Mmech = 1 describes a stationary second beam deflection unit 24, while Mmech = 2 means that the second beam deflection unit 24 oscillates with the same angular amplitude and optically in the same direction as the first beam deflection unit 21, which moves the illumination line through the sample space. For Mmech = 0, the second beam deflection unit 24 compensates for the effect of the first beam deflection unit 21.

[0060] The effective intensity PSF is given by:

[0061] This finally gives for M mech = 2: which is in particular consistent for r|=2 with Equation S9 in Shen et al., in Advanced Photonics Nexus 2023, Vol. 2 (1), p. 016009-1 , Supplementary Information.

[0062] In addition, this result agrees with formula 3 in CJR Sheppard: “Superresolution in Confocal Imaging” in Optik 1988, vol. 80, p. 53. This gives an indication of the mathematical relationship to so-called image scanning, which can also be explicitly demonstrated.

[0063] Generalized to the two-dimensional case, the following applies:

[0064] Additional structured illumination (through intensity modulation along the line) can be used to increase resolution in the longitudinal direction of the illumination line. For this purpose, the control unit 29 places the first beam deflection unit 21 in at least two, preferably at least three different positions along the longitudinal direction of the illumination line, such that adjacent resulting positions (phase positions) in the sample space are spaced from each other by less than one period length of the intensity-modulated light distribution, and creates a separate raw image of the sample space for each of the positions. Conveniently, the region of interest in the sample space is first completely scanned for each of these phase positions of the illumination pattern, and a respective raw image with one-dimensional (perpendicular to the line) increased resolution is created.This process is repeated with the second phase position and, for example, with the third phase position and, if necessary, further phase positions. From the (in particular three) raw images, a result image with increased resolution is then calculated according to the SIM method, for example by solving the known system of equations. The compression by Mmech perpendicular to the illumination line for backprojection described above can be carried out before or after the SIM reconstruction (for example, comprising transforming the raw images into the frequency domain, separating order spatial frequency spectra and shifting the order spatial frequency spectra in the frequency domain, as well as backtransforming them into the spatial domain, see, for example, Shen et al., in Advanced Photonics Nexus 2023, Vol. 2 (1), p. 016009-1, Sections 2.1 and 2.2) because the structuring along the illumination line and the reconstruction of the increased resolution in this direction are independent of the additional scanning perpendicular to the illumination line. Before the super-resolution SIM reconstruction, the raw images can be high-pass filtered, preferably in the frequency domain after Fourier transformation, in order to reduce out-of-focus background by optical sectioning in the sense of an OS-SIM treatment (Neil et al.: “Method of obtaining optical sectioning by using structured light in a conventional microscope” in Optics Letters 1997, Vol. 22, No. 24, p. 1905). The control unit can be configured to calculate the resulting image with increased resolution, in particular according to the aforementioned steps. By combining the structuring of the illumination line for a 2D-SIM illumination along the longitudinal direction of the line (and the electronic pixel line aperture) and the additional scanning with M. m ech=2 results in the effective PSF:

[0065] This PSF can be used in particular for deconvolution that further improves resolution.

[0066] If the user selects the second operating mode, the control unit 29 activates the electronic shutter, synchronizes the movement of the first beam deflection unit 21 and the movement of the electronic shutter, but keeps the second beam deflection unit 24 constantly in its neutral position and deactivates intensity modulation. This mode allows image acquisition as with a conventional line-scanning confocal microscope. In the third operating mode, the control unit 29 deactivates the electronic shutter and also keeps the second beam deflection unit 24 in its neutral position. This enables conventional wide-field image acquisition. The user can use the lamp 11 as a wide-field light source as an alternative to the lasers 12.

[0067] In a fourth selectable operating mode, the control unit 29 deactivates the electronic shutter and operates the first and second beam deflection units 21, 24 as in the first operating mode. This mode allows the use of the "add-scan" method with less suppression of out-of-focus light. However, the calculation of the resulting image from the structured raw images also suppresses out-of-focus light to a certain degree. Since a shutter is not absolutely necessary, a sensor 28 can also be used without one.

[0068] In Fig. 2, exemplary optical relationships are shown by the focal length fi of the scanning optics 23 and the focal length f2 of the detection optics 25. Both the first beam deflection unit 21 and the second beam deflection unit 24 are arranged at an identical distance d from the beam splitter 22, so that both lie in a respective conjugate pupil plane PE' and PE". The ratio of the focal lengths M opt=f2 / fi describes the optical magnification of the intermediate image ZB on the sensor 28. In addition, there is the one-dimensional mechanical magnification Mmech due to the movement of the second beam deflection unit 24, which, in the case of a synchronous, in-phase movement with the first beam deflection unit 21, results from the ratio of the angular amplitudes oci of the first beam deflection unit 21 and oc2 of the second beam deflection unit 24 as M m ech= (oci+a2) / ai. Also shown as section AA is the effect of the beam former 20 on the illumination pattern with the intensity distribution l PE(x,y) in the illumination-side conjugate pupil plane PE' in the first operating mode. It consists of two separate, parallel lines that interfere in the intermediate image ZB due to the Fourier transformation effected by the scanning optics 23 to form a line grating with the intensity distribution lzß(x,y), which is transmitted through the tube lens 6 and the microscope objective 5 into the sample space. A double arrow indicates the movement of the line grating in the xzB direction for scanning the sample by means of the first beam deflection unit 21. The different illumination phases are achieved by adjusting the first beam deflection unit 21 such that the line grating is offset in the yzß direction, preferably by less than one period of the line grating.

[0069] Fig. 3 shows an alternative embodiment in which the illumination beam path is provided by a separate laser scanning module 31. In addition to the beam former 20 and the first beam deflection unit 21, this module has its own scanning optics 32, a main beam splitter 33, a slit diaphragm 34, and a line detector 35. The module 31 provides a linear light distribution in the sample space as in Fig. 1. The "add-scan" module 3 here only comprises parts of the detection beam path (from the intermediate image ZB to the sensors 28). It has a secondary color splitter 26 between the beam splitter 22 and the detection optics 25, which splits the sample light into two spectrally disjoint components and directs them to a respective sensor 28, 28'. The control unit 29, for example, switches on two lasers 12 with different emission wavelengths in order to simultaneously record two fluorescent dyes. Since the beam splitter 22 is expediently arranged as shown in Fig.1 is designed as a color splitter, the line detector 35 cannot be used in this operating mode because no sample light reaches it (but is directed into the “Add-Scan” module).

[0070] Fig. 4 schematically illustrates the connections and functions of the control unit 29 and the synchronization in the first operating mode. For example, the beam deflection units 21, 24 are synchronized with the sensor 28. The control unit 29 can adjust the magnification factor M by changing the ratio of the amplitudes of the beam deflection units 21, 24. The control unit 29 can also move the sample stage or select one of several microscope objectives 5 from the objective turret.

[0071] List of reference symbols

[0072] 1 microscope

[0073] 2 tripod

[0074] 3 “Add-Scan” module

[0075] 4 laser module

[0076] 5 microscope objective

[0077] 6 tube lenses

[0078] 7 Exit

[0079] 8

[0080] 9 beam splitters

[0081] 10 eyepiece

[0082] 11 Lamp

[0083] 12 lasers

[0084] 13 AOTF

[0085] 14 Coupling optics

[0086] 15 optical fibers

[0087] 16 Collimator

[0088] 17 mirrors

[0089] 18 beam combiners

[0090] 19 Deflecting mirrors

[0091] 20 beam formers

[0092] 21 First beam deflection unit

[0093] 22 main beam splitters

[0094] 23 Scanning optics

[0095] 24 Second beam deflection unit

[0096] 25 Detection optics

[0097] 26 secondary color splitters

[0098] 27

[0099] 28 Sensor

[0100] 29 Control unit

[0101] 30 selection unit

[0102] 31 Laser scanning module

[0103] 32 scanning optics

[0104] 33 Main beam splitter 34 Slit diaphragm

[0105] 35 line detector

[0106] P rehearsal room

[0107] E.g. intermediate image

[0108] PE pupil plane

[0109] PE'(') Conjugate pupil plane

Claims

Patent claims 1 . Microscope (1) with an illumination beam path, a detection beam path and a beam splitter (22), wherein the detection beam path has a sample chamber (P), a microscope objective (5), a tube lens (6), an intermediate image (ZB) generated by the tube lens (6), and a two-dimensional spatially resolving optoelectronic sensor (28) with detection optics (25) for imaging the intermediate image (ZB) onto the sensor (28), and the illumination beam path has a light source (12) and a first adjustable beam deflection unit (21) for moving an illumination light beam through the sample chamber (P), and the detection beam path has a second adjustable beam deflection unit (24) for moving a sample light beam over the sensor, wherein the illumination beam path and the detection beam path are optically coupled to a common beam path by means of the beam splitter (22),that illumination light from the light source (12) passes through the microscope objective (5) into the sample chamber (P) via the beam splitter (22) and the sample light from the sample chamber (P) passes through the microscope objective (5) via the beam splitter (22) and then via the second beam deflection unit (24) to the sensor (28), characterized in that the first beam deflection unit (21) is arranged optically between the light source (12) and the beam splitter (22) so that the sample light reaches the sensor (28) away from the first beam deflection unit (21), and characterized by a control unit (29) which is configured to move the sample light beam over the sensor (28) by means of the second beam deflection unit (24) in an operating mode.

2. Microscope according to the preceding claim, wherein the control unit (29) is configured to move the sample light beam over the sensor (28) in the first operating mode by means of the second beam deflection unit (24) in such a way that the intermediate image (ZB) is projected onto the sensor (28) in an enlarged manner, in particular by a movement such that an amount of a quotient between a first angle, which a propagation direction of a beam of the sample light emanating from a location in the sample space directly behind the second beam deflection unit with an optical axis of the detection optics, and a second angle which the propagation direction of the beam immediately in front of the second beam deflection unit includes with an optical axis of the microscope objective (5) is greater than one.

3. Microscope according to the preceding claim, wherein at least in the first operating mode the movement of the second beam deflection unit (24) is synchronizable or synchronized with the movement of the first beam deflection unit (21), in particular with identical or approximately identical angular amplitude of the first beam deflection unit (21) and the second beam deflection unit (24).

4. Microscope according to one of the preceding claims, wherein the illumination beam path has a beam former (20) for generating a linear distribution of the illumination light, in particular a distribution intensity-modulated along its longitudinal direction, in particular a periodically intensity-modulated distribution, in particular such a distribution in the sample space by simultaneously generating at least two linear light distributions capable of interference with one another, which are present in a plane (PE') optically conjugate to a rear focal plane (PE) of the microscope objective (5).

5. Microscope according to the preceding claim, wherein the beam former (20) is repeatedly removable from the illumination beam path, in particular motorized and controlled by the control unit (29), and / or comprises a spatial light modulator, in particular controlled by the control unit (29) in such a way that different illumination light distributions are created in the sample space in temporal alternation.

6. Microscope according to claim 4 or 5, wherein the first beam deflection unit (21) is designed for two-dimensional scanning of the sample space in a first dimension transverse to a longitudinal direction of the line-shaped illumination light distribution and in a second dimension parallel to the longitudinal direction, in particular with a control unit which places the first beam deflection unit (21) in at least three different positions along the second dimension, wherein two of the resulting positions of the illumination light distribution in the sample space at the positions along the second dimension are spaced from one another by less than a length of the illumination light distribution in the longitudinal direction, in particular by a period length of the intensity-modulated light distribution or less than a period length, and in particular creates a separate raw image for each of the positions.

7. Microscope according to claim 4 or 5, wherein the illumination beam path comprises an optic (20) for illuminating the sample space in different phase positions of the intensity-modulated illumination light distribution, in particular a spatial phase modulator and / or an electro-optical modulator in conjunction with a quarter-wave plate, in particular with the first beam deflection unit (21) designed for only one-dimensional scanning of the sample space transversely to a longitudinal direction of the linear illumination light distribution.

8. Microscope according to one of claims 4 to 7, wherein the sensor (28), in particular a CMOS sensor or a SPAD sensor, has a deactivatable line-shaped electronic diaphragm which is arranged confocally to the intermediate image, in particular in the form of a rolling shutter, in particular with the ability to synchronize an adjustment of the first beam deflection unit with a dynamic position of the diaphragm.

9. Microscope according to the preceding claim, wherein the control unit (29) is electrically connected to the sensor (28) and the beam deflection units (21, 24) and in the first selectable operating mode activates the electronic diaphragm and synchronizes the movements of the first beam deflection unit (21), the second beam deflection unit (24) and the movement of the electronic diaphragm and / or in a second selectable operating mode activates the electronic diaphragm, synchronizes the movement of the first beam deflection unit (21) and the movement of the electronic diaphragm and operates the second beam deflection unit (24) in a constant position, in particular in a neutral position, and / or in a third selectable operating mode electronic shutter is deactivated and the second beam deflection unit (24) is operated in a constant position and / or in a fourth selectable operating mode the electronic shutter is deactivated and the first and second beam deflection units (21, 24) are operated as in the first operating mode.

10. Microscope according to the preceding claim, wherein the control unit (29) removes the beam former (20) from the illumination beam path in the third operating mode and / or an intensity modulation of the illumination light can be deactivated or is deactivated in the second operating mode and / or the control unit comprises a selection unit (30) for selecting one of a plurality of operating modes and the control unit (29) operates the microscope after the selection in the selected operating mode, in particular with an additional selection option in the selection unit between a plurality of sub-modes, each with different light sources (12), in particular for the third operating mode.

11. Microscope according to one of the preceding claims, wherein the detection beam path between the intermediate image (ZB) generated by the tube lens (6) and the detection optics (25) is free of confocal field diaphragms, in particular slit diaphragms, and in particular is free of image planes conjugated with the intermediate image and / or wherein the detection beam path between the intermediate image (ZB) generated by the tube lens (6) and the detection optics (25) has exactly one plane (PE') conjugated to the rear focal plane (PE) of the microscope objective (5).

12. Microscope according to one of the preceding claims, wherein the common beam path optically comprises an optical system (23), preferably a scanning optical system, between the intermediate image (ZB) and the beam splitter (22) for generating a conjugated to the rear focal plane (PE) of the microscope objective (5). plane (PE') on or near the first beam deflection unit (21) and on or near the second beam deflection unit (24).

13. Microscope according to one of the preceding claims, wherein both the illuminating light on its way to the microscope objective (5) and the Sample light on its way to the sensor (28) passes through the same intermediate image (ZB) generated by the tube lens (6), in particular with the beam splitter (22) arranged as a main beam splitter or main color splitter optically between the first beam deflection unit and the intermediate image (ZB) generated by the tube lens (6), or wherein the microscope has a second tube lens (6') which generates a second intermediate image (ZB), and the illuminating light passes through the second intermediate image (ZB) on its way to the microscope objective (5).

14. Microscope according to one of the preceding claims, wherein the detection beam path between the beam splitter (22) and the detection optics (25) has a secondary color splitter (26) which splits the sample light into two spectrally disjoint components and directs these to disjoint regions of the sensor (28) or to a respective sensor (28, 28'), in particular with a second light source (12) with an emission wavelength different from the first light source (12) in the illumination beam path.

15. Microscope according to one of the preceding claims, wherein the first beam deflection unit (21) comprises MEMS micromirrors, in particular MEMS micromirrors continuously adjustable about two orthogonal spatial directions.

16. Microscope (1) according to one of the preceding claims, wherein the microscope has a stand (2) on which the microscope objective (5) is arranged, wherein the stand (2) has a first connection (7), in the region of which the tube lens (6) and the intermediate image (ZB) are arranged, and at least one further connection (7') with a further tube lens (6') and a further intermediate image (ZB), wherein the sample light can be guided simultaneously or sequentially to both outputs (7, 7') by means of at least one second beam splitter (9), in particular a repeatedly removable beam splitter, or by means of a mirror, wherein the first beam splitter (22), the detection optics (25), the sensor (28), the first and second beam deflection units (21, 24) and a scanning optics (23) are arranged within a module (3) which is detachably mechanically and optically connected to one of the connections (7).

Images