Super-resolution microscope with fast quasi-confocal detection

Abstract

In order to improve the resolving power of a quasi-confocal line scanning microscope comprising a main beam splitter for coupling an illumination light path and a detection light path, after scanning by an illumination beam deflection unit, in a "rescanning" method, the sample light is moved on the sensor downstream of the confocal diaphragm in a direction transverse to the line-shaped focal point by a second beam deflection unit, the sample light being scanned again in the detection light path. The illumination line can be structured in order to improve the resolution in the longitudinal direction. The disadvantage of this optical arrangement is a considerable light loss due to the large number of optical interfaces. A microscope, in which the illumination beam deflection unit is optically arranged between the light source and the main beam splitter, such that the sample light bypasses the illumination beam deflection unit to the sensor (non-rescanning), and in which a control unit moves the sample light beam on the sensor only by means of the second beam deflection unit in one operating mode, is more sensitive to light and more flexible. Depending on the operating mode, a synchronization with the first beam deflection unit can be provided. Due to the high light sensitivity, this microscope is particularly suitable for fluorescence microscopy.

Description

Technical Field

[0001] This invention relates to a microscope having an illumination optical path, a detection optical path, and a beam splitter. The detection optical path includes a sample space, a microscope objective, a tube mirror, an intermediate image generated by the tube mirror, and a two-dimensional spatially resolved photoelectric sensor with a detection optics unit. The detection optics unit images the intermediate image onto the sensor. The illumination optical path includes a light source and a first settable beam deflection unit for moving ("scanning", "grating scan") the illumination beam through the sample space. The detection optical path includes a second settable beam deflection unit for moving the sample beam onto the sensor. The illumination optical path and the detection optical path are optically coupled by the beam splitter to form a common optical path, such that illumination light from the light source passes through the microscope objective via the beam splitter and enters the sample space, and sample light from the sample space passes through the microscope objective, through the beam splitter, and subsequently through the second beam deflection unit to reach the sensor. In this case, the beam deflection units are arranged in or at least near the back focal plane of the microscope objective, which, in the case of a telecentric optical path, corresponds to the pupil plane of the microscope objective. Background Technology

[0002] Confocal fluorescence imaging provides high-contrast, high-resolution images of biological samples. Similar to DE 197 02 753 A1, the sample is scanned point-by-point via a beam deflection unit using laser illumination, and the generated sample light is descanned using the same beam deflection unit, ensuring that the stationary beam from each sample location is on the optical axis and detected via a confocal pinhole. Due to this aperture, the sensor is essentially shielded from defocused light, resulting in high contrast. However, the relatively long acquisition time due to point-by-point scanning is a disadvantage. This also inevitably leads to a high phototoxicity load on the sample caused by the excitation light.

[0003] These drawbacks of point-by-point imaging can be mitigated through parallelization. For example, confocal microscopes with Nipkow disks and light-sheet microscopes, the latter requiring additional illumination optics. In an alternative, parallelization can be achieved using linear illumination, as described in EP 1 617 258 A1. To achieve confocality in at least one dimension (transverse to the illumination line), a slit aperture is used for linear probing to suppress defocusing. Along this line, the resolution is comparable to that of a wide-field microscope.

[0004] To further improve the resolution laterally to the line (super-resolution beyond the diffraction limit), the sample light can be rescanned in the probe path after descanning by the illumination beam deflection unit. For this purpose, a beam deflection unit is used laterally downstream of the confocal stop to move the sample light across the sensor (De Luca et al., “Re-scanconfocal microscopy: scanning twice for better resolution,” Biomedical OpticsExpress 2013, Vol. 4, No. 11, p. 2644). The pixels of the super-resolution image of the sample must be calculated by correlating the intensities of different pixels at different times. This optical arrangement suffers from significant light loss due to the need to traverse numerous optical interfaces.

[0005] To improve resolution along the illumination line, during the rescan method, the illumination line can also be structured (modulated) in terms of intensity in its longitudinal direction (Shen et al., “Confocal rescan structuredillumination microscopy for real-time deep tissue imaging with superresolution”, Advanced Photonics Nexus 2023, Vol. 2 (1), p. 016009-1). In this case, each location on the sample must be illuminated at least three different phases (different relative positions of the line structure) and imaged in a corresponding number of phase images. As with Structured Illumination Microscopy (SIM) in wide field, the overall image (demodulation) must be calculated by correlating all phase images.

[0006] The low light sensitivity of the optical arrangements used in rescanning methods persists even under structured illumination. Furthermore, these arrangements are limited by specific scanning microscopy methods, thus lacking flexibility. In particular, confocal stops make the system inflexible or at least complex, because to achieve a degree of flexibility, the stop must be designed to be mechanically highly precise. Moreover, such mechanical setting is often slow. Summary of the Invention

[0007] The problem this invention aims to solve is to improve the type of microscope described at the beginning, thereby simplifying the detection optical path and achieving higher light transmission efficiency and thus higher light sensitivity. At least in certain embodiments, greater flexibility of the microscope is also intended.

[0008] This problem is solved by a microscope having the features described in claim 1.

[0009] Advantageous configurations of the invention are given in the dependent claims.

[0010] This invention specifies that a first beam deflection unit is optically arranged between the light source and the beam splitter, such that the sample light bypasses the first beam deflection unit to reach the sensor. The microscope includes a control unit configured to move the sample beam on the sensor using a second beam deflection unit in an operating mode. In principle, this movement is initially independent of the 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 by bypassing the first beam deflection unit; this can be called non-scan detection. Unlike known rescanning microscopes, the movement of the first beam deflection unit is not canceled out, nor is it canceled out by the second beam deflection unit. The beams (sample beams) emitted sequentially from different positions in the sample space due to scanning illumination are thus incident at correspondingly different angles relative to the optical axis onto each plane conjugate to the back focal plane of the microscope (and therefore incident at different angles relative to the optical axis onto the second beam deflection unit). According to the invention, there is no stationary beam upstream of the second beam deflection unit. Surprisingly, it has been found that beam scanning corresponding to rescanning can be achieved even when there is exactly one beam deflection unit in the detection optical path. Therefore, there is also no stationary beam downstream of the second beam deflection unit (in the first operating mode); instead, the angle of the beam relative to the optical axis changes over time after passing through the second beam deflection unit.

[0012] To advantageously improve resolution, the angle of the propagation direction of the beam from the sample space relative to the optical axis can even be increased by the second beam deflection unit. This method can be referred to as additional scanning. For this purpose, the control unit can be configured to move the sample beam on the sensor in a first operating mode by means of the second beam deflection unit in such a way that the intermediate image is imaged on the sensor in a magnified manner. Specifically, this movement can make the absolute value of the following quotient greater than 1: the quotient of a first angle and a second angle, where the first angle is the angle formed by the propagation direction of the sample light beam emitted from a position in the sample space with the optical axis of the probe optics unit immediately downstream of the second beam deflection unit, and the second angle is the angle formed by the propagation direction of the beam with the optical axis of the microscope objective immediately upstream of the second beam deflection unit.

[0013] In this case, to generate a super-resolution image of the sample in the transverse direction to the longitudinal direction of the illumination line, the sensor pixel intensity at different times is calculated and evaluated, corresponding to a known evaluation of the pixel intensity acquired by descanning and rescanning (and thereby image the intermediate image onto the sensor in a magnified manner in one dimension transverse to the longitudinal direction of the illumination line, preferably magnified by 2x). The maximum improvement in resolution transverse to the illumination line is achieved by adding a 2x magnification factor that is effective only in one dimension through additional scanning.

[0014] The amplification can be achieved with minimal overhead by synchronizing the movement of the second beam deflection unit with, or already has, the movement of the first beam deflection unit, at least in the first operating mode, for example, through a control unit or any other electrical connection. Specifically, the control unit can move the first and second beam deflection units with the same or approximately the same angular amplitude to achieve maximum resolution improvement through additional scanning and by utilizing a 2x mechanical amplification effective only in one dimension.

[0015] The mechanical requirements for the second beam deflection unit are lower than those in conventional rescanning arrangements. It only needs to meet the same requirements as the first beam deflection unit because—unlike existing technologies—it does not need to scan with an angular amplitude twice that of the latter, which could otherwise lead to linearization problems, for example, for galvanometer mirrors, and would also limit the overall acquisition speed.

[0016] An embodiment in which the illumination path includes a beam shaper (e.g., a cylindrical lens) for generating a linear distribution (“illumination line”) of illumination light is advantageous. In this case, the linear distribution can be intensity modulated along its longitudinal direction, particularly periodically. Preferably, at least two linear light distributions that can interfere with each other and exist in a plane optically conjugate with the back focal plane (BFP) of the microscope objective are simultaneously generated by the beam shaper (e.g., a spatial light modulator for this purpose), such that such a distribution (“light pattern”) appears in the sample space. By structuring along the illumination line, the resolution along the illumination line can be improved by acquiring and evaluating the original image at multiple phase angles of the modulated light distribution. Preferably, the beam shaper generates the illumination light distribution in such a way that it produces (modulated or unmodulated) an illumination line in the sample space, which is imaged onto the pixel row by the probe path in a manner geometrically parallel to the sensor pixel row.

[0017] Preferably, the beam shaper can be repeatedly removed from the illumination path, particularly by a motor and controlled by a control unit. Therefore, the microscope can also use wide-field illumination in alternative operating modes. Repeatable motor-driven removal allows for great flexibility. As an alternative or supplement to removability, the beam shaper can include a spatial light modulator (SLM), particularly a phase-modulated SLM located in the conjugate pupil plane (the back focal plane of the microscope objective). Specifically, the latter can be controlled by a control unit so that different illumination light distributions alternate over time in the sample space. Thus, unmodulated linear illumination can be selectively generated, or modulated linear illumination can be generated in the sample space by means of at least two interfering light distributions in the pupil plane / BFP. Switching can be very rapid, for example, at frequencies from 50 Hz to 100 Hz, by rewriting the phase pattern on the SLM. The SLM also allows for rapid switching between linear and point illumination, for example, for alternating between sample observation and optical manipulation. For this purpose, advantageously, the first beam deflection unit can be set in two dimensions.

[0018] In a particularly advantageous embodiment, the first beam deflection unit is designed to perform a two-dimensional scan of the sample space in a first and a second dimension, the first dimension being transverse to the longitudinal direction of the linear illumination distribution and the second dimension being parallel to that longitudinal direction. By moving along the second dimension, the illumination distribution can be positioned at different phase angles with minimal overhead and within a short time, enabling the acquisition of a number of original images of different illuminations required to improve resolution. Preferably, this can be achieved by a control unit that positions the first beam deflection unit in at least three different poses along the second dimension, wherein at these poses, two of the resulting positions of the illumination distribution in the sample space are spaced apart from each other by a length less than the longitudinal direction of the illumination distribution, particularly by a period length of the intensity modulation light distribution or less than a period length, and specifically, a separate original image is created for each pose.

[0019] As an alternative to shifting the light pattern along the (intensity-modulated) illumination line via a first beam deflection unit, this shift can be achieved, respectively, by means of a pivotable transparent parallel plate or an electro-optic phase shifter located in the illumination optical path. Alternatively, the illumination optical path can include optical units for illuminating the sample space at different phase angles to modulate the illumination light distribution with intensity, particularly spatial phase modulators (spatial light modulators that affect phase) and / or electro-optic modulators combined with quarter-wave plates. In this alternative, the first beam deflection unit can advantageously be designed to scan the sample space only in one dimension transverse to the longitudinal direction of the linear illumination light distribution. A beam deflection unit that can be set in only one dimension is less costly and, in particular, more stable.

[0020] Compared to conventional SIM image acquisition, which requires 13 or even 15 raw images, the resulting image can be calculated using only three raw images. In this case, the region of interest (ROI) in 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. Small ROIs can be scanned and acquired extremely quickly using the illumination line.

[0021] In all embodiments, the sensor, particularly a CMOS sensor, EMCCD sensor, iCCD sensor, or SPAD array sensor, may include a deactivated linear electronic stop arranged confocally with the intermediate image. Preferably, the electronic stop can be implemented as a "rolling shutter" that operates in a suitable manner, such as in WO 2006 / 008637 A1, especially in at least a first operating mode, where the setting of the first beam deflection unit is synchronized with or has been synchronized with the dynamic position (movement) of the stop (such that the relative position of the opening of the electronic stop coincides with the position of the illumination line from the sample space on the sensor). The electronic stop allows suppression of defocused sample light, thus allowing improved resolution in both the axial and lateral directions, similar to a mechanical confocal stop. However, due to magnified imaging by means of a second beam deflection unit, lateral resolution (lateral to the illumination line) can be improved even without a confocal stop (light control mechanism). Defocused light is also suppressed at least partially by calculation based on multiple raw images at different illumination phase angles. The improvement in axial resolution is unaffected by the size of the stop. However, a trade-off between signal-to-noise ratio and imaging depth can be struck by adjusting the size of the aperture.

[0022] The advantage of using a sensor with an electronic aperture is that it allows for rapid switching between confocal and wide-field detection, such as switching between consecutive images.

[0023] For synchronization purposes, the sensor preferably has a lightsheet readout mode (Hamamatsu's designation) and an output that provides information about the position and / or movement of the aperture, particularly a signal indicating the start of an image ("frame") on the sensor. Commercially available sensors with this mode and corresponding output are available from Hamamatsu: https: / / www.hamamatsu.com / eu / en / product / cameras / cmos-cameras / lightsheet-readout-mode.html. Hamamatsu refers to this output as an "external trigger output." Advantageously, the control unit is electrically connected to both the sensor and the beam deflection unit.

[0024] The following microscopes are particularly suitable for the following configuration: A control unit is electrically connected to the sensor and the beam deflection unit, and in a first selectable operating mode, the control unit activates the electronic aperture and synchronizes the movement of the first beam deflection unit, the second beam deflection unit, and the electronic aperture; and / or, in a second selectable operating mode, the control unit activates the electronic aperture, synchronizes the movement of the first beam deflection unit with the movement of the electronic aperture, and operates the second beam deflection unit in a constant pose, particularly a neutral pose; and / or, in a third selectable operating mode, the control unit deactivates the electronic aperture and operates the second beam deflection unit in a constant pose; and / or, in a fourth selectable operating mode, the control unit deactivates the electronic aperture and operates the first and second beam deflection units as in the first operating mode.

[0025] Such a system can operate in conventional wide-field modes (e.g., using a lamp as a light source for incoherent illumination, the illumination light of which, for example, bypasses a first beam deflection unit and is guided into the sample space via a microscope objective, for example, through different outputs of the microscope), or in laser wide-field modes (third operating mode), confocal modes (second operating mode), and additional scanning modes (first and fourth operating modes), without requiring mechanical modifications to the system. Furthermore, the sensor can perform phase measurements (e.g., phase contrast, differential interference contrast, intensity transmission equation, differential phase contrast) almost simultaneously in other operating modes. All that is required is to keep one or two beam deflection units in their stationary position (neutral pose) and to enable the electronic aperture and optionally set its size accordingly, or to deactivate it. This can be easily and quickly implemented by the control unit, for example, through software. This enables measurement tasks that are impossible using conventional rescanning systems. Especially in the case of multimodal acquisition of the same sample, sharing the same sensor is often advantageous because the data from various measurements are thus aligned with each other at pixel precision. Furthermore, since each method requires only one sensor, the cost is lower.

[0026] Therefore, an advantageous option is that the control unit removes the beam shaper from the illumination path in the third operating mode; and / or, the intensity modulation of the illumination light can be disabled or has been disabled in the second operating mode (resulting in an unmodulated illumination line in the sample space); and / or, the control unit includes a selection unit for selecting one operating mode from multiple operating modes, and the control unit operates the microscope in the selected operating mode after selection, particularly with additional selection options in the selection unit between multiple sub-modes with different light sources, especially for the third operating mode. This enables flexible and cost-effective use with minimal operating overhead.

[0027] The following microscopes are advantageously compact and have high optical efficiency: the probe optical path between the intermediate image generated by the tube lens and the probe optics does not have a confocal field stop, in particular not a slit stop, and especially not an image plane conjugate to the intermediate image; and / or the probe optical path between the intermediate image generated by the tube lens and the probe optics contains exactly a plane conjugate to the back focal plane of the microscope objective.

[0028] By positioning the common optical path optically between the intermediate image and the beam splitter, including optical units, preferably scanning lenses, for generating a plane conjugate to the back focal plane of the microscope objective on or near the first beam deflection unit and on or near the second beam deflection unit, a particularly compact and optically efficient microscope can be constructed. Thus, on the one hand, the beam splitter is arranged in the collimated light, so any potential contamination has only a minor impact on its transmission quality. On the other hand, this allows for compact coupling of the illumination and probe optical paths without the need for additional collimation as in the prior art. In particular, this eliminates the need for additional relay optical units.

[0029] Advantageously, in this configuration, the conjugate plane (pupil plane) can be generated due to reflection at the beam splitter, thus the first beam deflection unit is located in or near the reflected conjugate plane, and specifically, the conjugate pupil plane, transmitted through the beam, is located on or near the second beam deflection unit in the probe optical path, such that the second beam deflection unit is located in or near the transmitted conjugate plane. In this way, no additional optical units are required in the probe optical path; this keeps the number of optical interfaces to a minimum, thereby maximizing the light sensitivity of the probe. Transmission into the probe optical path (compared to a reflective arrangement) improves the extinction of the excitation light, thus achieving a higher contrast of the measurement signal relative to the excitation signal at the detector.

[0030] Preferably, both the illumination light and the sample light pass through the same intermediate image generated by the tube mirror on their paths to the microscope objective and on their paths to the sensor. In this case, the beam splitter, which serves as the main beam splitter or main dichroic beam splitter, can be optically positioned between the first beam deflection unit and the intermediate image generated by the tube mirror.

[0031] Advantageously, by sharing the same intermediate image for illumination and detection, only a single optical port of the microscope is occupied. Other possible outputs can be used for other purposes, thus providing greater flexibility to the microscope. In particular, rapid quasi-confocal microscopy can be combined in a multimodal manner with other methods that require their own outputs. Therefore, the arrangement of the illumination and detection optical paths according to the invention (leading to and exiting the intermediate image, respectively), or the arrangement of the detection optical path only from the intermediate image to the sensor, can be advantageously integrated into a single module, thereby reducing manufacturing and installation costs and achieving a more compact and stable construction. In the case of a combined module with illumination and detection optical paths, the actual light source can be arranged outside the module and can be optically connected to or has already been connected to the module, for example, via optical fiber or through free beam input coupling into the module. Compared to light sheet microscopes, only a single illumination optical unit in the form of a microscope objective is required.

[0032] Alternatively, the microscope may include a second tube for generating the second intermediate image, wherein the illumination light passes through the second intermediate image (e.g., at the second optical port of the microscope) in its path to the microscope objective. Therefore, for example, an existing laser scanning module can be used for illumination. In this case, for example, only the probe light path, as a dedicated module, is connected to the first intermediate image at the first port of the microscope.

[0033] In one possible embodiment, the detection optical path between the beam splitter and the detection optics unit may include a secondary dichroic beam splitter that splits the sample light into two spectrally non-intersecting components and directs them to non-intersecting regions on the sensor or to respective sensors (e.g., as in DE 10 2021 134 427A1 or DE 10 2023 005 252 A1), and the illumination optical path may include a second light source having an emission wavelength different from the first light source. This allows for the simultaneous acquisition of two spectral ranges associated with the emission wavelength, particularly two spectral ranges of two different fluorescent dyes. The first and second light sources may be the same light source, such as a multi-line laser or a broadband light source, particularly a white light source. For precise structuring, the two colors can advantageously be superimposed on each other at the first beam deflection unit, where the grating constants of the illumination patterns in the sample are the same.

[0034] Advantageously, the first beam deflection unit comprises MEMS micromirrors, particularly MEMS micromirrors capable of continuous adjustment around two orthogonal spatial directions. Therefore, modules (with illumination and probe paths respectively leading to the intermediate image, or only a probe path starting from the intermediate image) can be provided in a compact, quiet, and cost-effective manner. The two-dimensional tunable MEMS scanner also offers the advantage that the deflection mirror is always located within the optical pupil / BFP, which optimizes the positioning accuracy of the illumination focal volume. In particular, the second axis can also be used to move the illumination line along its longitudinal direction to generate different illumination phase angles for structured illumination. Furthermore, this allows for defining the illumination area and / or generating longer (modulated or unmodulated) lines or more uniform (unmodulated) lines. Additionally, the second axis can be used to generate very small movements along the illumination line, and these movements homogenize the line illumination (without SIM mode). Finally, good image field illumination is also guaranteed if the sample is optically manipulated using the first beam deflection unit.

[0035] As an alternative to two-dimensional tunable MEMS micromirrors, the first beam deflection unit can conventionally be formed by two mirrors, each tunable in one dimension, particularly a galvanometer mirror, preferably having an optical relay that images one mirror onto the other.

[0036] A particularly preferred embodiment is the microscope comprising a frame housing microscope objectives, particularly those arranged in a turret. The frame includes a first port with a tube lens and intermediate image arranged within its region, and at least one other port with another tube lens and another intermediate image. Sample light can be simultaneously or sequentially directed to both outputs by means of at least one second beam splitter, particularly a removable beam splitter, or by means of a mirror. The first beam splitter, a probe optics unit, a sensor, first and second beam deflection units, and a scanning lens are arranged within a module optically and mechanically detachably connected to one of the ports. This allows for a flexible microscope system with high light sensitivity and short acquisition time.

[0037] In another embodiment, the invention also includes a microscope having an evaluation unit that receives multiple raw images from at least two, preferably three, different phase angles of illumination light distribution in the sample space, and calculates a resulting image with improved resolution in at least two spatial directions based on the raw images, in particular by solving a set of equations that describe the convolution of the unknown sample with the point spread function of illumination and detection for each phase angle in the spatial or frequency domain.

[0038] The present invention will now be described in detail based on exemplary embodiments. Attached Figure Description

[0039] In the attached diagram: Figure 1 A microscope is shown; Figure 2 This shows the optical conditions under the microscope; Figure 3 An alternative microscope is shown; and Figure 4 The principle of the control and synchronization scheme is shown. Detailed Implementation

[0040] In all the accompanying drawings, the corresponding parts use the same reference numerals.

[0041] Figure 1 Microscope 1 is schematically shown. It consists of a frame 2, an additional scanning module 3, and a laser module 4. The frame 2 includes microscope objectives 5, such as telecentric microscope objectives, and tubes 6 that generate intermediate images ZB in the regions of the two output ends 7 / 7', respectively. The microscope objectives 5 are arranged on an objective turret (not shown separately). The frame also includes a pivotally accessible beam splitter 9, such as a neutral intensity beam splitter or a dichroic beam splitter, to selectively direct light proportionally to the output ends and / or eyepieces 10, and / or to direct light from lamp 11 to microscope objectives 5 and from there into the sample space P. In the sample space, the sample is located on a stage that can be moved three-dimensionally by a motor (not shown for clarity).

[0042] By way of example only, laser module 4 includes four lasers 12 with different emission wavelengths, the intensity of which can be set by means of a corresponding AOTF 13. Alternatively (not shown), one or more lasers can be directly modulated. Their illumination light is coupled to an optical fiber 15 via an input coupling optics unit 14 and guided to a scanning module 3, where the illumination light is collimated, for example, by means of a longitudinally movable collimator 16. The collimator 16 can be used to compensate for longitudinal chromatic aberration and / or to focus the illumination light to different depths in the sample space P. Alternatively (not shown), the different emission wavelengths can, for example, be combined in the laser module, so that only a single optical fiber 15 is needed, and the collimator 16 can be eliminated. The illumination light, thus combined, reaches a deflector 19 via a reflector 17 and a beam combiner 18. The deflector 19 deflects the illumination light in such a way that, after passing through a beam shaper 20, which includes, for example, a phase-modulated spatial light modulator (SLM) and a cylindrical lens, the illumination light is incident on a first beam deflection unit 21, which is, for example, a microelectromechanical system (MEMS) with micromirrors that can be continuously set in two dimensions. Starting from a beam splitter 22, the illumination light reaches the microscope objective 5 via a scanning lens 23, an intermediate image ZB, and one of the tube mirrors 6, and from there enters the sample space P. Because the beam shaper 20 focuses the illumination light in one dimension onto a plane conjugate to the pupil of the microscope objective 5 and located on the beam deflection unit 21, a substantially linear illumination focal volume (illumination line) is produced in the sample space. With the help of SLM, the illumination light can be modified in such a way that the illumination light forms two separation points or truly parallel lines on the first beam deflection unit arranged in the conjugate pupil plane PE', and these separation points or parallel lines interfere with each other in the sample space in such a way that the illumination focal volume is illuminated by a periodic intensity pattern along its longer extension direction (the longitudinal direction of the illumination line).

[0043] The sample light, particularly including the fluorescence in the sample excited essentially linearly by the illumination light, reaches beam splitter 22 via the intermediate image ZB in the opposite direction. The illumination light components reflected in the sample and on their way to the sample are reflected back to the first beam deflection unit 21 by beam splitter 22, which is designed, for example, as a dichroic trap filter and thus serves as the primary dichroic beam splitter. The fluorescence contained in the sample light, particularly due to Stokes shift, is transmitted through beam splitter 22 to the second beam deflection unit 24, which is, for example, a MEMS mirror or galvanometer mirror that can only be continuously set in one dimension. The first scanning lens 23 for collimating the sample light is designed such that the plane PE, conjugate with the pupil PE of the microscope objective 5, is located on one side of the first beam deflection unit 21 and on the other side of the second beam deflection unit 24. Downstream of the second beam deflection unit 24, the sample light is focused by the probe optics unit 25 onto a two-dimensional spatially resolved sensor 28, such as a CMOS chip or a single-photon counting avalanche photodiode array (SPAD array). In the neutral pose (zero position) of the second beam deflection unit 24, the optical axes of the microscope objective 5 and the probe optics unit 25 coincide. The sensor 28 includes an electronic slit aperture in the form of a rolling shutter, the slit width of which is settable and can be completely deactivated. Illumination lines in the sample space are aligned to image onto the sensor 28 in a manner parallel to the pixel rows of the sensor 28 (and therefore also parallel to the electronic slit aperture).

[0044] The control unit 29, which has a selection unit 30 for, for example, four different operating modes (with additional sub-modes), is electrically connected to the frame 2, the additional scanning module 3, and the laser module 4. It controls the mechanical and optical components contained therein, and receives measurements from the sensors contained therein, particularly sensor 28.

[0045] If the user selects the first operating mode (“Additional Scan Mode”), the control unit 29 activates the electronic aperture of the sensor 28 and moves the sample beam from the sample space onto the sensor 28 by means of the second beam deflection unit 24, so that the intermediate image ZB is magnified and imaged onto the sensor 28 in the direction of movement. The control unit synchronizes the movement of the second beam deflection unit 24 with the movement of the first beam deflection unit 21, and makes both move, for example, with the same angular amplitude. Furthermore, the control unit 29 synchronizes the movement of the beam deflection units 21 and 24 with the movement of the electronic slit aperture of the sensor 28. As a result, the slit aperture acts as a confocal aperture, and the illumination focal volume is imaged into the opening of the slit aperture.

[0046] In this configuration, the sample light incident on 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 source in the sample space. The synchronization of the first beam deflection unit 21 with the second beam deflection unit 24 amplifies this effect. Due to the same amplitude and the chosen in-phase oscillation direction, the sample light reflected from the second beam deflection unit 24 has an angle twice the size relative to the optical axis of the probe optics unit 25. In a conventional system, this angle would be zero due to the descanning performed there; the sample beam would be stationary.

[0047] The increase in angle results in a one-dimensional magnification transverse to the illumination line; when the angle is doubled due to the beam deflection units 21 / 24 having the same amplitude, the generation factor is M. mech = 2, a “non-optical” anisotropic amplification, and this amplification is superimposed on the potential purely optical (typically isotropic) amplification M produced by the combination of the probe optics unit 25 and the scanning lens 23. opt This results in a corresponding broadening of the point spread function (PSF), and also causes the increase in the distance between two points on sensor 28 to the image of the sample space to be greater than the increase in the PSF width, which corresponds to redistribution or rescanning. The image acquired on sensor 28 must be processed according to a factor M in the direction transverse to the illumination line. mech One-dimensional compression (backprojection) is performed to accurately reproduce the scale of the sample space. Because the imaging points are more expanded than the PSF, better resolution is retained compared to comparable diffraction-limited wide-field imaging, despite the compression in the lateral direction—directly through an optical-mechanical means without complex reconstruction calculations. In contrast, the resolution in the longitudinal direction of the illumination line is unaffected in this respect.

[0048] The second beam deflection unit 24 operates in the direction transverse to the illumination line as follows: A fluorescent object located within the object-side focal plane of the microscope objective in the sample space. The image B on sensor 28 can be described using convolution, where, without loss of generality, the optical magnification M can be assumed. opt = 1:

[0049] here, It is the intensity point spread function used to image an object in the object plane onto the detection plane of sensor 28. The coordinates of subscript O describe the sample space; the coordinates of subscript D describe the image space on sensor 28.

[0050] Subsequently, using the location at position x O / y O The fluorescence excitation illumination line at the object The scan yielded the following results:

[0051] Substituting into equation (1), we get:

[0052] Scan vector transverse to the longitudinal direction of the illumination line Integrating yields a wide-field image. Then, adding another image located at the same point... The one-dimensional confocal electron aperture D(x) at the location is obtained as follows:

[0053] Based on these equations, it can be deduced that the PSF is characterized by the existence of imaging similar to the wide-field case in the longitudinal direction of the illumination line, while in the direction of the confocal electronic stop (i.e., transverse to the illumination line), the point image corresponds to the convolution of the excitation PSF and the slit function D:

[0054] For satisfying The confocal stop, in this case, again achieves a fully confocal PSF, while for satisfying An open aperture is used, convolution yields a constant, and the image of the point emitter corresponds to the emitted PSF.

[0055] To keep the equations compact, the imaging in the case of the second beam deflection unit 24 is given below only for the affected variables. This applies to the direction along the illumination line (and along the electronic aperture formed by the corresponding sensor rows). In this case, point images can still be described using wide-field imaging.

[0056] The second beam deflection unit 24 causes the probe PSF to move according to the second scan vector. Shift:

[0057] In the first (and fourth) operating modes, the second beam deflection unit 24 according to Synchronized with the first beam deflection unit 21. M mech The case of = 1 describes the stationary second beam deflection unit 24, while M mech = 2 indicates 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 that moves the illumination line through the sample space. For M mech = 0, the second beam deflection unit 24 compensates for the effect of the first beam deflection unit 21.

[0058] The effective strength PSF is:

[0059] for The final result is:

[0060] This is particularly true for η=2, which corresponds to Equation S9 given by Shen et al. in Advanced Photonics Nexus 2023, vol. 2(1), p. 016009-1, Supplementary Information. Furthermore, this result corresponds to Equation 3 in CJR Sheppard's "Super-Resolution in Confocal Imaging" in Optik 1988, vol. 80, p. 53. This demonstrates a mathematical relationship with so-called image scanning, which can also be explicitly shown.

[0061] Extending to the two-dimensional case, the following holds true:

[0062] Additional structured illumination (through intensity modulation along the line) can be used to improve resolution in the longitudinal direction of the illumination line. For this purpose, control unit 29 positions the first beam deflection unit 21 in at least two, preferably at least three, different poses along the longitudinal direction of the illumination line, such that adjacent resulting positions (phase angles) in the sample space are spaced apart from each other by less than one period length of the intensity-modulated light distribution, and a separate original image of the sample space is created for each pose. Appropriately, the region of interest in the sample space is first fully scanned for each phase angle of the illumination pattern, and original images with improved resolution in one dimension (transverse to the line) are created respectively. This sequence is then repeated for a second phase angle and, for example, a third phase angle, and optionally for other phase angles. The resulting image with improved resolution is then calculated from the (particularly three) original images according to the SIM method, for example, by solving a system of equations known per se. In this case, the aforementioned transverse to the illumination line for backprojection is M... mechCompression can be performed before or after SIM reconstruction (e.g., including transforming the original image to the frequency domain, separating the sub-spatial frequency spectrum and shifting the sub-spatial frequency spectrum in the frequency domain, and inverse transforming it to the spatial domain; see, for example, Shen et al., Advanced Photonics Nexus 2023, vol. 2(1), p. 016009-1, sections 2.1 and 2.2), because structuring along the illumination line and the resolution-enhanced reconstruction in that direction are independent of additional scanning transverse to the illumination line. Prior to super-resolution SIM reconstruction, the original image can be high-pass filtered, preferably in the frequency domain after the Fourier transform, to reduce the defocus background in the sense of optical sectioning in OS-SIM processing (Neil et al., “Method of obtaining optical sectioning by using structured lightin a conventional microscope”, Optics Letters 1997, vol. 22, no. 24, p. 1905). The control unit can be configured, in particular, to calculate the resolution-enhanced resulting image according to the steps described above.

[0063] By structuring the illumination line used for 2D SIM illumination along the longitudinal direction of the line (and the electronic pixel row stop) and M mech The additional scan with a value of 2 is combined to obtain the effective PSF:

[0064] In particular, this PSF can be used for deconvolution to further improve resolution.

[0065] If the user selects the second operating mode, the control unit 29 activates the electronic aperture, synchronizing the movement of the first beam deflection unit 21 with the movement of the electronic aperture, while keeping the second beam deflection unit 24 in its neutral pose and disabling intensity modulation. This mode allows image acquisition like a conventional line-scan confocal microscope. In the third operating mode, the control unit 29 disables the electronic aperture and also keeps the second beam deflection unit 24 in its neutral pose. This allows for conventional wide-field image acquisition. In this case, the user can use lamp 11 as a wide-field light source instead of laser 12.

[0066] In the fourth selectable operating mode, the control unit 29 disables the electronic diaphragm and operates the first and second beam deflection units 21, 24 as in the first operating mode. This mode allows for the use of additional scanning methods, but offers less suppression of defocused light. However, the image calculated from the structured original image also suppresses defocused light to some extent. Since the diaphragm is not mandatory, a sensor 28 without the diaphragm can also be used.

[0067] Figure 2 An exemplary optical condition is shown, formed by the focal length f1 of the scanning lens 23 and the focal length f2 of the probe optics unit 25. In this case, both the first beam deflection unit 21 and the second beam deflection unit 24 are equidistant from the beam splitter 22, and therefore the two units are located in their respective conjugate pupil planes PE' and PE" respectively. The focal length ratio M opt =f2 / f1 describes the optical magnification of the intermediate image ZB on sensor 28. In addition, there is a one-dimensional mechanical magnification M generated by the movement of the second beam deflection unit 24. mech When moving in phase with the first beam deflection unit 21, the mechanical amplification is determined by the ratio of the angular amplitude α1 of the first beam deflection unit 21 to the angular amplitude α2 of the second beam deflection unit 24 according to M. mech = (α1+α2) / α1. Section AA also shows the effect of the beam shaper 20 on the illumination pattern with intensity distribution IPE(x,y) in the illumination-side conjugate pupil plane PE' in the first operating mode. This illumination pattern consists of two separate and parallel lines that interfere in the intermediate image ZB due to the Fourier transform achieved by the scanning lens 23, forming a line grating pattern with intensity distribution IZB(x,y), which is transmitted to the sample space by the tube lens 6 and the microscope objective 5. The double-headed arrows indicate the movement of the line grating pattern in the xZB direction to scan the sample using the first beam deflection unit 21. Different illumination phases are achieved by adjusting the first beam deflection unit 21 to offset the line grating pattern in the yZB direction, preferably by an offset less than one period of the line grating pattern.

[0068] Figure 3 An alternative embodiment is shown in which the illumination light route is provided by a separate laser scanning module 31. In addition to the beam shaper 20 and the first beam deflection unit 21, the latter also includes a dedicated scanning lens 32, a main beam splitter 33, a slit stop 34, and a line detector 35. Module 31 is as follows: Figure 1This provides a linear light distribution in the sample space, as in the example. In this case, the additional scanning module 3 only includes a portion of the probe optical path (from the intermediate image ZB to the sensor 28). Between the beam splitter 22 and the probe optics unit 25, the module includes a secondary dichroic beam splitter 26, which splits the sample light into two spectrally non-overlapping components and directs these components to the corresponding sensors 28, 28', respectively. For example, the control unit 29 activates two lasers 12 with different emission wavelengths to enable the simultaneous detection of two fluorescent dyes. Because the beam splitter 22 appropriately employs... Figure 1 The beam splitter is in the form of a dichroic beam splitter, so the line detector 35 cannot be used in this operating mode because no sample light reaches the line detector (instead, the sample light is directed to the additional scanning module).

[0069] Figure 4 The diagram schematically illustrates the connections and tasks of the control unit 29, as well as synchronization in the first operating mode. Thus, for example, beam deflection units 21 and 24 are synchronized with sensor 28. The control unit 29 can set the magnification factor M by changing the amplitude ratio of beam deflection units 21 and 24. The control unit 29 can also be used to move the sample stage or select one of multiple microscope objectives 5 from the objective turret.

[0070] List of reference numerals

[0071] 1. Microscope

[0072] 2 frames

[0073] 3. Additional scanning module

[0074] 4 Laser Module

[0075] 5. Microscope Objectives

[0076] 6-tube endoscope

[0077] 7 Output terminal 8

[0079] 9 beam splitters

[0080] 10 eyepieces

[0081] 11 lights

[0082] 12 Lasers

[0083] 13 AOTF

[0084] 14 Input Coupled Optical Unit

[0085] 15 optical fibers

[0086] 16 Collimators

[0087] 17. Reflector

[0088] 18 Bundles

[0089] 19 Deflecting Mirror

[0090] 20 Beam Shaper

[0091] 21 First beam deflection unit

[0092] 22 Main beam splitter

[0093] 23 Scanning Lens

[0094] 24 Second beam deflection unit

[0095] 25 Detection Optical Units

[0096] 26 Secondary dichroic beam splitters 27

[0098] 28 sensors

[0099] 29 Control Unit

[0100] 30 Selection Unit

[0101] 31 Laser scanning module

[0102] 32 Scanning Lens

[0103] 33 Main beam splitter

[0104] 34 Slit Aperture

[0105] 35-line detector

[0106] P Sample space

[0107] ZB Intermediate Image

[0108] PE pupil plane

[0109] PE'() conjugate pupil plane

Claims

1. A microscope (1) having an illumination optical path, a detection optical path, and a beam splitter (22), wherein, The detection optical path includes a sample space (P), a microscope objective (5), a microscope tube (6), an intermediate image (ZB) generated by the microscope tube (6), and a two-dimensional spatially resolved photoelectric sensor (28) with a detection optical unit (25). The detection optical unit (25) is used to image the intermediate image (ZB) onto the sensor (28). The illumination optical path includes a light source (12) and a first settable beam deflection unit (21). The first settable beam deflection unit (21) is used to move the illumination beam through the sample space (P). The detection optical path includes a second settable beam deflection unit (24). The second settable beam deflection unit (24) is used to move the sample beam onto the sensor. The illumination optical path and the detection optical path are optically coupled by the beam splitter (22) to form a common light source. The path is such that illumination light from the light source (12) passes through the microscope objective (5) via the beam splitter (22) and enters the sample space (P), and sample light from the sample space (P) passes through the microscope objective (5), via the beam splitter (22) and then via the second beam deflection unit (24) to reach the sensor (28), characterized in that the first beam deflection unit (21) is optically arranged between the light source (12) and the beam splitter (22) such that the sample light bypasses the first beam deflection unit (21) to reach the sensor (28), and is further characterized in that it includes a control unit (29) configured to move the sample beam on the sensor (28) by means of the second beam deflection unit (24) in an operating mode.

2. The microscope according to the preceding claim, wherein, The control unit (29) is configured in a first operating mode to move the sample beam on the sensor (28) by means of the second beam deflection unit (24) in such a way that the intermediate image (ZB) is imaged on the sensor (28) in a magnified manner, in particular by moving it such that the absolute value of the quotient of a first angle and a second angle is greater than 1, wherein the first angle is the angle formed by the propagation direction of the sample light beam emitted from a position in the sample space with the optical axis of the detection optics unit immediately downstream of the second beam deflection unit, and the second angle is the angle formed by the propagation direction of the beam with the optical axis of the microscope objective (5) immediately upstream of the second beam deflection unit.

3. The microscope according to the preceding claim, wherein, At least in the first operating mode, the movement of the second beam deflection unit (24) can be synchronized with or has been synchronized with the movement of the first beam deflection unit (21), especially since the first beam deflection unit (21) and the second beam deflection unit (24) have the same or approximately the same angular amplitude.

4. The microscope according to any one of the preceding claims, wherein, The illumination path includes a beam shaper (20) for generating a linear distribution of the illumination light, particularly a distribution with intensity modulation along its longitudinal direction, particularly a distribution with periodic intensity modulation, particularly such a distribution generated in the sample space by simultaneously generating at least two linear light distributions that can interfere with each other and exist in a plane (PE') optically conjugate with the back focal plane (PE) of the microscope objective (5).

5. The microscope according to the preceding claim, wherein, The beam shaper (20) can be repeatedly removed from the illumination path, particularly in a motor-driven manner and controlled by the control unit (29), and / or includes a spatial light modulator, particularly controlled by the control unit (29), such that different illumination light distributions alternate over time in the sample space.

6. The microscope according to claim 4 or 5, wherein, The first beam deflection unit (21) is designed to perform a two-dimensional scan of the sample space in a first dimension and a second dimension, the first dimension being transverse to the longitudinal direction of the linear illumination light distribution and the second dimension being parallel to the longitudinal direction. In particular, it has a control unit that positions the first beam deflection unit (21) in at least three different poses along the second dimension, wherein, at the poses along the second dimension, two of the resulting positions of the illumination light distribution in the sample space are spaced apart from each other by a length less than the length of the illumination light distribution in the longitudinal direction, in particular by a period length of the intensity modulation light distribution or less than a period length, and in particular, a separate original image is created for each pose.

7. The microscope according to claim 4 or 5, wherein, The illumination optical path includes an optical unit (20) for illuminating the sample space at different phase angles of the illumination light distribution modulated by the intensity, in particular a spatial phase modulator and / or an electro-optic modulator combined with a quarter-wave plate, wherein the first beam deflection unit (21) is configured to scan the sample space only in one dimension transverse to the longitudinal direction of the linear illumination light distribution.

8. The microscope according to any one of claims 4 to 7, wherein, The sensor (28), particularly a CMOS sensor or SPAD sensor, includes a deactivated linear electronic aperture arranged confocally with the intermediate image, particularly in the form of a rolling shutter, and particularly has the ability to synchronize the setting of the first beam deflection unit with the dynamic position of the aperture.

9. The 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 the control unit enables the electronic aperture in the first selectable operating mode and synchronizes the movement of the first beam deflection unit (21), the movement of the second beam deflection unit (24) and the movement of the electronic aperture. And / or, the control unit enables the electronic aperture in a second selectable operating mode, synchronizes the movement of the first beam deflection unit (21) with the movement of the electronic aperture, and operates the second beam deflection unit (24) in a constant pose, particularly in a neutral pose; and / or, the control unit deactivates the electronic aperture in a third selectable operating mode and operates the second beam deflection unit (24) in a constant pose; and / or, the control unit deactivates the electronic aperture in a fourth selectable operating mode and operates the first and second beam deflection units (21, 24) as in the first operating mode.

10. The microscope according to the preceding claim, wherein, The control unit (29) removes the beam shaper (20) from the illumination path in the third operating mode, and / or the intensity modulation of the illumination light can be disabled or has been disabled in the second operating mode, and / or the control unit includes a selection unit (30) for selecting one operating mode from a plurality of operating modes, and the control unit (29) operates the microscope in the selected operating mode after selection, particularly having additional selection options in the selection unit between a plurality of sub-modes with different light sources (12), especially for the third operating mode.

11. The microscope according to any one of the preceding claims, wherein, The probe optical path between the intermediate image (ZB) generated by the tube lens (6) and the probe optical unit (25) does not have a confocal field stop, in particular, it does not have a slit stop, and in particular, it does not have an image plane conjugate to the intermediate image, and / or wherein the probe optical path between the intermediate image (ZB) generated by the tube lens (6) and the probe optical unit (25) contains exactly a plane (PE') conjugate to the back focal plane (PE) of the microscope objective (5).

12. The microscope according to any one of the preceding claims, wherein, The common optical path located between the intermediate image (ZB) and the beam splitter (22) includes an optical unit (23), preferably a scanning lens, for generating a plane (PE') conjugate to the back focal plane (PE) of the microscope objective (5) on or near the first beam deflection unit (21) and on or near the second beam deflection unit (24).

13. The microscope according to any one of the preceding claims, wherein, The illumination light passes through the same intermediate image (ZB) generated by the tube (6) in its path to the microscope objective (5) and the sample light passes through the same intermediate image (ZB) in its path to the sensor (28). In particular, the beam splitter (22) is optically arranged as a main beam splitter or a main dichroic beam splitter between the first beam deflection unit and the intermediate image (ZB) generated by the tube (6), or wherein the microscope includes a second tube (6') that generates a second intermediate image (ZB), and the illumination light passes through the second intermediate image (ZB) in its path to the microscope objective (5).

14. The microscope according to any one of the preceding claims, wherein, The detection optical path between the beam splitter (22) and the detection optical unit (25) includes a secondary dichroic beam splitter (26), which splits the sample light into two non-intersecting components in the spectrum and guides these components to non-intersecting regions on the sensor (28) or to the respective sensors (28, 28'). In particular, the illumination optical path has a second light source (12) whose emission wavelength is different from that of the first light source (12).

15. The microscope according to any one of the preceding claims, wherein, The first beam deflection unit (21) includes a MEMS micromirror, particularly a MEMS micromirror that can be continuously adjusted around two orthogonal spatial directions.

16. The microscope (1) according to any one of the preceding claims, wherein, The microscope includes a frame (2) on which the microscope objective (5) is arranged, wherein the frame (2) includes a first port (7) in which the tube mirror (6) and the intermediate image (ZB) are arranged, and at least one other port (7') having another tube mirror (6') and another intermediate image (ZB), wherein the sample light can be simultaneously or sequentially directed to two outputs (7, 7') by means of at least one second beam splitter (9), particularly a removable beam splitter, or by means of a mirror, wherein the first beam splitter (22), the probe optics unit (25), the sensor (28), the first and second beam deflection units (21, 24) and the scanning lens (23) are arranged in a module (3), the module (3) being optically and mechanically detachably connected to one of the ports (7).

Images