SCANNING DEVICE, LIGHT MICROSCOPE AND SCANNING PROCEDURE

Abstract

The invention relates to a scanning device (1) for a light microscope (100) with an acousto-optic deflector (10) for deflecting a first illumination light beam (L1) of a first wavelength and a second illumination light beam (L2) of a second wavelength, which differs from the first wavelength, and a control unit (13) for generating sound waves of a first frequency range and a second frequency range separate from the first frequency range in the acousto-optic deflector (10), wherein the first frequency range and the second frequency range are selected such that the first illumination light beam (L1) is deflected by the sound waves of the first frequency range and the second illumination light beam (L2) is deflected independently of the first illumination light beam (L2) by the sound waves of the second frequency range.as well as a light microscope (100) with the scanning device (1) and a method for deflecting a first and a second illumination light beam (L1, L2).

Description

Technical field of the invention

[0001] The invention relates to a scanning device for a light microscope, a light microscope with the scanning device, and a method for deflecting two illumination light beams with different wavelengths (scanning method). State of the art

[0002] In contrast to wide-field light microscopy, scanning light microscopy involves illuminating the sample at different points with focused light to obtain images or positional information of structures in the sample.

[0003] For example, in confocal laser scanning microscopy, focused laser light is moved in a grid over or through the sample, and a grid image of the sample is created from the detection light obtained for different illuminated positions, in particular fluorescence light.

[0004] However, the scanning light microscopy methods within the meaning of the invention also include certain localization microscopy methods in which no raster image of the sample is created, but the positions of individual emitters in the sample are determined, for example MINFLUX and MINSTED.

[0005] In the context of the present application, the term MINFLUX method refers to a process in which a sample with an intensity distribution of illumination light containing a local minimum, in particular a zero point, is illuminated, wherein the local minimum is located in the immediate vicinity of a presumed position of a single emitter in the sample, wherein emission light emanating from the emitter (e.g., scattered light, reflected light, or luminescence light, in particular fluorescence light) is detected, and wherein, based on the emission light detected for different positions of the local minimum or based on the emission light detected for different shapes and / or orientations of the intensity distribution of the illumination light, the position of the single emitter is determined using an estimation algorithm. This process can, for example,MINFLUX imaging can be performed sequentially for different emitters in the sample to create a localization map of the emitters, resembling a high-resolution light microscopy image. Alternatively, the position of an emitter moving within the sample can be determined at short intervals, for example, to obtain a trajectory of the emitter with high spatial and temporal resolution (MINFLUX tracking). MINFLUX methods have achieved spatial resolutions in the single-digit nanometer range and temporal resolutions in the microsecond range with very high photon efficiency, especially compared to other localization methods such as PALM / STORM. The illumination light used in MINFLUX methods is usually excitation light, which is scattered or reflected by emitters in the sample or excites them to luminescence, particularly fluorescence.

[0006] A MINFLUX method is known, for example, from the publication “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes” by F. Balzarotti, Y. Eilers, KC Gwosch, AH Gynnå, V. Westphal, FD Stefani, J. Elf and SW Hell, Science 355, 606-612 (2017).

[0007] From the prior art (see, e.g., US 2018 / 0259458 A1 and US 2020 / 0393378 A1), specific MINFLUX methods are also known in which the illumination light is suppression light, in particular STED (stimulated emission depletion) light, combined with focused fluorescence excitation light. Such methods, sometimes referred to as STED-MINFLUX methods, also fall under the term "MINFLUX methods" in the context of this disclosure.

[0008] MINSTED is a specific localization technique for single emitters, in which a sample is scanned along a continuous path around the presumed position of an emitter using a combination of focused excitation light and an intensity distribution of STED light with a central local minimum. The path is fitted based on individual fluorescence photons detected by the emitter. A MINSTED method is described, for example, in the publication "MINSTED fluorescence localization and nanoscopy" by M. Weber, M. Leutenegger, S. Stoldt, S. Jakobs, TS Mihaila, AN Butkevich and SW Hell, Nature Photonics 15, 361-366 (2021).

[0009] Scanning the illumination light over or through the sample can be achieved by moving the sample while the light beam remains stationary (stage scanning) or by moving the light beam while the sample remains stationary (beam scanning). Stage scanning has the disadvantage that relatively large masses often need to be moved, which limits the achievable scanning speed due to inertia. Therefore, in scanning light microscopy, beam scanners are primarily used, at least for lateral scanning perpendicular to the optical axis of the light beam.

[0010] Confocal laser scanning microscopes are often operated with mechanical beam scanners such as galvanometer scanners and resonant scanners. However, mechanical beam scanners are limited in speed. Furthermore, special scanning techniques such as random access scanning are difficult to implement with mechanical scanners.

[0011] The term Random Access Scanning refers here to the rapid scanning of individual points in the sample in an essentially arbitrary sequence (unlike raster scanning). In many implementations of MINFLUX methods, the minimum illumination light must also be positioned as quickly as possible at illumination points around the presumed position of the emitter, which can also be dynamically adjusted. Due to their very high speed, electro-optical and acousto-optic scanning devices are particularly well-suited for such tasks.

[0012] Electro-optical deflectors are technically complex and relatively expensive devices, and have the disadvantage of a small field of view. Furthermore, the deflection of the light beam is necessarily dependent on polarization, which poses particular problems regarding the unscanning of the detection light.

[0013] Acousto-optic deflectors are characterized by lower complexity and a larger field of view compared to electro-optic deflectors and are therefore extremely advantageous, especially for random access scanning and for MINFLUX methods.

[0014] A random-access scanning method based on acousto-optic deflectors is described, for example, in patent disclosure US 2006 / 0140462 A1.

[0015] Basic principles of acousto-optic elements are described, for example, in the review articles “Acousto-Optic Devices - Optical Elements for Spectroscopy” by CD Tran, Analytical Chemistry 64, 971-981 (1992) and “Acousto-optic systems for advanced microscopy” by M. Duocastella, S. Surdo, A. Zunino, A. Diaspro and P. Saggau, J. Phys. Photonics 3, 01 2004 (2021).

[0016] Acousto-optic devices utilize the interactions between light rays and sound waves within a solid (acousto-optic crystal). Sound waves coupled into the crystal via a transducer create periodic differences in the crystal's refractive index. A light ray passing through the crystal is diffracted by these refractive index distributions.

[0017] Depending on the crystal's thickness along the direction of light propagation, the wavelength of the light, the acoustic frequency, and the crystal's static refractive index, two parameter ranges are distinguished: the Raman-Nath regime and the Bragg regime, which lead to different acousto-optic diffraction effects. In the Raman-Nath regime, which occurs with relatively thin crystals, the emerging light beam is dispersed according to its diffraction orders. In the Bragg regime, with relatively thick crystals, all diffraction orders except the first interfere destructively. When the Bragg condition is met... sin θ=λf2cv is satisfied, where θ denotes the angle of incidence of the light ray, λ denotes the wavelength of the light ray, f denotes the frequency of the sound wave, and c v When the speed of sound is specified, the incident light ray is deflected in the first order by the angle θ relative to the optical axis.

[0018] Various acousto-optic devices are known from the prior art, in which the acousto-optic effect is used technically in different ways, in particular by utilizing different geometries, frequency ranges, the Raman-Nath or Bragg regime, as well as with standing (with reflector) or passing (with absorber) acoustic waves. For example, acousto-optic modulators (AOM) are used to control light intensity, acousto-optic filters (AOTF) to select wavelengths, and so-called TAG lenses to focus light beams.

[0019] Patent application US 2023 / 0288715 A1 describes, for example, a beam splitter for splitting illumination light with different wavelengths and detection light based on an AOTF.

[0020] Acousto-optic deflectors (AODs) utilize the acousto-optic effect to deflect the incident light beam, particularly for the rapid scanning of laser beams. AODs operate in the Bragg regime and typically employ a traveling acoustic wave that is absorbed by an absorber at the end of the crystal opposite the transducer. The acoustic frequencies used in AODs are in the MHz range (ultrasound).

[0021] To ensure a variable deflection angle of the light beam, birefringent crystals are frequently used for AODs. This allows the Bragg condition to be fulfilled by so-called tangential phase matching over a relatively wide acoustic frequency range (see, for example, "A review of physical principles and applications of acousto-optic deflectors on the basis paratellurite" by SN Antonov and VA Kotelnikov, Phys Astron Int. J. 3(6), 235-249 (2019)). The deflection angle can then be controlled by adjusting the acoustic frequency over frequency bandwidths of typically around 50-70 MHz, enabling relatively large scan ranges to be achieved when using suitable optics in the sample.

[0022] The review article “Acousto-optic systems for advanced microscopy” by M. Duocastella, S. Surdo, A. Zunino, A. Diaspro and P. Saggau, J. Phy. Photonics 3 (2021), 01 2004, mentions that it is possible to split an incident light beam into different outgoing beams by simultaneously subjecting an acousto-optic crystal of an AOD to several different acoustic frequencies.

[0023] As mentioned above, beam deflection in acousto-optic deflectors is wavelength-dependent. For many applications of light microscopy, the simultaneous use of multiple illumination beams with different wavelengths is extremely advantageous, particularly for observing spatial arrangements of different sample components, e.g., proteins of a protein complex in biological cells labeled with different fluorescent dyes.

[0024] If, for example, random access scanning or a MINFLUX method with two illumination beams of different wavelengths is to be implemented in light microscopes with AODs, according to the state of the art at least two separate AODs in parallel beam paths of the illumination beams are required.

[0025] This increases the required installation space, complexity, energy consumption, and cost of the light microscope. Object of the invention

[0026] Therefore, the object of the present invention is to enable rapid beam scanning of two illumination beams with different wavelengths in an efficient manner with regard to the installation space, complexity, energy requirements and costs. Solution

[0027] This problem is solved by the subject matter of the independent claims (scanning device, light microscope, and scanning method). Advantageous embodiments are specified in the dependent claims and are described below. Description of the invention

[0028] A first aspect of the invention relates to a scanning device for a light microscope, comprising at least one acousto-optic deflector configured to deflect a first illumination beam of a first wavelength and a second illumination beam of a second wavelength that differs from the first wavelength, wherein the scanning device includes a control unit configured to generate sound waves of a first frequency range and a second frequency range separate from the first frequency range in the acousto-optic deflector, wherein the first frequency range and the second frequency range are selected such that the first illumination beam is deflected by the sound waves of the first frequency range and the second illumination beam is deflected by the sound waves of the second frequency range independently of the first illumination beam.

[0029] The invention is not limited to the independent deflection of two illumination light beams of different wavelengths, but optionally more than two illumination light beams of different wavelengths can also be deflected, in particular independently of each other, by the at least one acousto-optic deflector, in particular by more than two separate frequency ranges.

[0030] A scanning device within the meaning of this specification is a device designed to shift illumination light rays focused into a sample within a predetermined image field relative to the sample by means of optics comprising an objective lens.

[0031] The scanning device has at least one acousto-optic deflector. If the scanning device has at least two acousto-optic deflectors, these are arranged one behind the other in the beam path of the first and second illumination beams and are both designed to deflect both the first and the second illumination beams independently of each other, particularly in different spatial directions (see below).

[0032] The acousto-optic deflector can, in particular, comprise an acousto-optic crystal transparent to the illumination light used, a frequency generator for generating sound waves in the ultrasonic range (furthermore, in particular, from 1 MHz to 999 MHz), and a transducer for coupling the sound waves into the acousto-optic crystal. The control unit can, in particular, be connected to or integrated into the frequency generator. The control unit can, in particular, be configured to control the frequency generator such that it generates sound waves of the first frequency range and the second frequency range, which can be coupled into the acousto-optic crystal via the transducer. Furthermore, the acousto-optic deflector can have an absorber opposite the transducer in one direction of sound wave propagation, so that sound waves propagating within the acousto-optic crystal are formed.The acousto-optic crystal can be configured such that the acousto-optic deflector can be operated in the Bragg regime. Optionally, the acousto-optic crystal can be birefringent, i.e., it can have different refractive indices with respect to different propagation directions and polarization directions of the light. If the acousto-optic crystal is birefringent, the acousto-optic deflector can be, in particular, a non-collinear acousto-optic deflector. In this case, the first and second illumination light rays can each be linearly polarized so that only one first-order diffracted ray is produced. The polarization direction can be adjusted such that the illumination light rays incident on the acousto-optic crystal correspond to the extraordinary ray.

[0033] The first illumination beam has a first wavelength, and the second illumination beam has a second wavelength that differs from the first. At least one of the illumination beams can be essentially monochromatic, and in particular, both illumination beams can be monochromatic. This is advantageous because the deflection by the acousto-optic deflector is wavelength-dependent, but it is not strictly necessary. The illumination beams can also have wavelength ranges in which the first wavelength and the second wavelength lie, respectively, with the spectrum of the first illumination beam differing from the spectrum of the second. The different wavelengths can be used, for example, to excite two different dyes (e.g., fluorophores) in the sample to luminescence (especially fluorescence).Different sample structures, e.g., different biomolecules, can be labeled with different dyes to investigate the spatial arrangement of these sample structures.

[0034] The first (acoustic) frequency range and the second (acoustic) frequency range are separated from each other; that is, the first and second frequency ranges do not overlap. The first frequency range can be directly adjacent to the second frequency range or separated from the first frequency range by a cutoff frequency (see below).

[0035] The first illumination beam is deflected by the sound waves selected from the first frequency range independently of the second illumination beam, and the second illumination beam is deflected by the sound waves selected from the second frequency range independently of the first illumination beam. This can mean, in particular, that while the sound waves selected from the first frequency range cause a deflection of both the first and second illumination beams, only the deflected first illumination beam lies within the relevant image field, and vice versa. The other deflected illumination beam (i.e., the second illumination beam deflected by sound waves from the first frequency range and the first illumination beam deflected by sound waves from the second frequency range) can be blocked, for example, by a suitable aperture.

[0036] According to one embodiment, the scanning device has at least one aperture designed to block out the second illumination beam deflected by sound waves of the first frequency range and / or the first illumination beam deflected by sound waves of the second frequency range from an image field.

[0037] During the development of the present invention, it was surprisingly discovered that it is possible to deflect two light beams of different wavelengths independently of each other using a single acousto-optic deflector by selecting suitable acoustic frequencies. In this way, multicolor microscopy with fast random access scanning or a multicolor MINFLUX localization method can be implemented, saving at least one AOD (or even two AODs if deflection is in two directions, such as x and y). This significantly reduces the required installation space, complexity, energy consumption, and cost of the microscope.

[0038] In addition to the undesired effect of the first frequency on the second light beam and vice versa, harmonic mixing vibrations can occur in the acousto-optic crystal due to frequency addition. It has been discovered in the course of the present invention that, in particular, the mixing vibrations 2f1 - f2 and 2f2 - f1 lie within the relevant frequency range and, due to undesired phase agreement with the incident light beam, lead to additional deflected beams. The other relevant mixing vibrations (in particular 2f1, 2f2, f1 + f2, 2f1 + f2, 2f2 - f1) are regularly outside the relevant operating range at typical frequency values.

[0039] Both problems described (undesirable effect of the frequencies on the respective other light beam as well as frequency mixing) can surprisingly be solved by a suitable choice of separate frequency ranges and suitable angles of incidence on the acousto-optic crystal for the first and the second illumination light beam.

[0040] According to one embodiment, the scanning device is designed to direct the first illumination light beam at a first angle of incidence onto the acousto-optic deflector and to direct the second illumination light beam at a second angle of incidence onto the acousto-optic deflector.

[0041] Ideal deflection by an acousto-optic deflector with an optically isotropic crystal can be achieved in particular when the angle of incidence of the light beam to be deflected corresponds to the Bragg angle. θ=λ⋅Fv corresponds to where θ iThe angle of incidence, λ the wavelength of the light, and F the acoustic frequency are denoted. Small deviations in the angle of incidence or the acoustic frequency are tolerable, but reduce the intensity of the deflected beam. The tolerance range that results in sufficient diffraction efficiency is significantly larger for acousto-optic deflectors with anisotropic, especially birefringent, crystals.

[0042] By varying the angle of incidence, the direction of the diffracted beam can be adjusted over a certain range. According to the invention, this can advantageously be used to align the first and second diffracted illumination beams, which diverge due to the separate frequency ranges used for independent manipulation of the beams, relative to each other, in order to direct them onto the same image field in the sample.

[0043] The intensity reduction resulting from deviations from ideal phase alignment can be tolerated for many applications (especially for MINFLUX methods, where relatively low light intensities are sufficient) and / or can be compensated for, in particular, by adjusting the light source or, for example, an AOM (Automatic Optical Mass). Any greater dependence of the intensity on the acoustic frequency caused by the suboptimal angle of incidence can be determined experimentally in an adjustment step and automatically compensated for in subsequent measurements based on this adjustment.

[0044] According to a further embodiment, the first angle of incidence is tuned to a first zero position frequency within the first frequency range, which results in a zero position of the first illumination light beam within an image field and / or the second angle of incidence is tuned to a second zero position frequency within the second frequency range, which results in a zero position of the second illumination light beam within the image field.

[0045] Particularly when the zero position is to lie in the center of the image field achievable by the deflection of the illumination light rays, it is advantageous for the first zero-position frequency to be a center frequency of the first frequency range and / or for the second zero-position frequency to be a center frequency of the second frequency range. The center frequency is the frequency that is equidistant from the lower and upper limits of the respective frequency ranges.

[0046] According to a further embodiment, the first illumination light beam and the second illumination light beam have a relative angle to each other when they strike the acousto-optic deflector, wherein the relative angle θr=λ1v⋅FN,1−λ2v⋅FN,2 is, where θ r the relative angle, λ1 the first wavelength of the first illumination beam, λ2 the second wavelength of the second illumination beam, F N,1 the first zero position frequency and F N,2 The second zero-position frequency is designated. The first zero-position frequency can correspond to the center frequency of the first frequency range, and / or the second zero-position frequency can correspond to the center frequency of the second frequency range. The formula for the relative angle is derived from Bragg's formula. θ=λ⋅Fv.

[0047] In this way, the deflected first illumination beam and the deflected second illumination beam can be aligned with each other in such a way that the available image field for both beams is optimally utilized by aligning the zero positions of the first illumination beam and the second illumination beam.

[0048] According to a further embodiment, the scanning device has a first optical system which is configured to align the first illumination light beam and the second illumination light beam relative to each other such that the first illumination light beam and the second illumination light beam have the relative angle to each other.

[0049] According to another embodiment, the scanning device has a second optical system designed to align the first illumination light beam and the second illumination light beam relative to each other after deflection by the acousto-optic deflector.

[0050] The first optical system and the second optical system can each have, for example, at least one reflective element (e.g., a mirror), at least one dispersive element (e.g., a prism), and / or at least one focusing element (e.g., a lens).

[0051] According to another embodiment, there is a separation zone between the first frequency range and the second frequency range. That is, the first frequency range does not directly border the second frequency range.

[0052] In the course of the invention, it has been recognized that in particular the mixing frequency 2f1 - f2 can lie in the first frequency range and thus influence the deflection of the first illumination light beam, and the mixing frequency 2f2 - f1 can lie in the second frequency range and thus influence the deflection of the second illumination light beam.

[0053] By appropriately selecting the separation range, it is possible, in particular, to ensure that harmonic mixing frequencies, especially 2f1 - f2 and 2f2 - f1, lie outside the relevant frequency range. This makes it possible to deflect the first and second illumination beams independently of each other over a wide image field. An overlap of the deflection ranges of the first and second illumination beams, despite the separation range, can be achieved, in particular, by adjusting the angles of incidence on the acousto-optic deflector, especially the acousto-optic crystal (see above).

[0054] According to one embodiment, the separation range is selected such that for all frequency pairs f1,f2 the conditions 2f1 - f2 < f u , 2f2 - f1 < f u , 2f1 - f2 > f0 and 2f2 - f1 > f0 are satisfied, where f1 is the frequency from the first frequency range, f2 is the frequency from the second frequency range, f ua lower cutoff frequency of a working range of the acousto-optic deflector and f o denotes an upper cutoff frequency of the operating range of the acousto-optic deflector.

[0055] If the acoustic frequency lies within the working range, a phase match occurs with light rays of suitable wavelength, so that the light rays can be deflected by these frequencies.

[0056] According to a further embodiment, the separation range extends from a first threshold frequency to a second threshold frequency, wherein the first threshold frequency f s,1 = fs,1=2fu+fo3 is and the second threshold frequency fs,2=fu+2fo3 is, where f s,1 the first threshold frequency, f s,2 the second threshold frequency, f u a (especially the) lower cutoff frequency of a working range of the acousto-optic deflector and f odenotes an (in particular the) upper cutoff frequency of the operating range of the acousto-optic deflector.

[0057] The first and second threshold frequencies given above can be determined, for example, on the basis of the inequalities 2f1 - f2 < f u , 2f2 - f1 < f u , 2f1 - f2 > f o and 2f z - f1 > f o to determine. In other words, the range of frequency pairs (f1, f2) is sought for which the mixing frequencies 2f1 - f2 and 2f2 - f1 lie outside the interval [f u , f o ], i.e., outside the operating range of the AOD. The first threshold frequency is the intersection of the lines 2f1 - f2 = f u and 2f2 - f1 = f u in the f1 / f2 diagram, and the second threshold frequency is the intersection point of the lines 2f1 - f2 = f oand 2f2 - f1 = f0 in the f1 / f2 diagram. The first and second threshold frequencies do not represent absolute limits. Since the limits of the permissible parameter ranges in the f1 / f2 diagram are not parallel to the axes, there are permissible parameter ranges that lie beyond these threshold frequencies. Furthermore, the separation range can be reduced by making the positions of the first and second illumination beams in the image field within the sample dependent on each other, for example, by shifting both illumination beams together across the entire image field but maintaining a relatively small separation (see below).

[0058] According to a further embodiment, the first frequency range has lower frequencies than the second frequency range, with the first illumination beam having a longer wavelength than the second illumination beam. Light of a longer wavelength is deflected more by the acousto-optic deflector at the same frequency than light of a shorter wavelength. Therefore, in order to optimally utilize the dynamic range of the acousto-optic deflector and to allow both illumination beams to be shifted essentially over the same image field, it is advantageous to assign the lower acoustic frequency range to the light of the longer wavelength.

[0059] According to a further embodiment, the scanning device has a first acousto-optic deflector and a second acousto-optic deflector, wherein the first acousto-optic deflector is configured to deflect the first illumination light beam and the second illumination light beam independently of each other in a first direction, and wherein the second acousto-optic deflector is configured to deflect the first illumination light beam and the second illumination light beam independently of each other in a second direction, in particular wherein the first direction is perpendicular to the second direction.

[0060] In this way, the first illumination light beam and the second illumination light beam can be deflected in two spatial directions (e.g., two lateral directions x and y perpendicular to each other, perpendicular to an optical axis of an objective of a light microscope) by taking advantage of the benefits of the present invention.

[0061] According to a further embodiment, the control unit is designed to adjust the frequencies of the sound waves for deflecting the first illumination beam and the second illumination beam in a range by a predetermined ratio, so that the first illumination beam deflected by the acousto-optic deflector and the second illumination beam deflected by the acousto-optic deflector maintain a relative distance in a focal plane within a predetermined range.

[0062] A range around a predetermined ratio means that pairs of values ​​(f1, f2) are selected such that a quotient f1 / f2 or f2 / f1 lies within a predetermined range of values ​​(e.g., between 1.2 and 1.3), where f1 denotes frequencies from the first frequency range and f2 denotes frequencies from the second frequency range. This corresponds to a specific maximum distance between the deflected illumination light rays in the image field. In the described embodiment, the completely independent deflection of two illumination light rays with an AOD, which is possible in principle with the present invention, is omitted in order to increase the usable frequency range and thus the dynamic range of the AOD during deflection. This can, for example,This can be achieved by ensuring that both illumination beams are always positioned together within the image field and, in particular, that they are positioned at the same location within the sample, or by ensuring that the two illumination beams maintain a maximum distance from each other within the sample. In this way, the separation range between the first and second frequency ranges can be reduced, or even eliminated entirely (i.e., the first and second frequency ranges can be directly adjacent), since certain frequency combinations that lead to undesirable mixing frequencies in the relevant frequency range do not occur at all.Furthermore, the described embodiment has high practical relevance, especially in random-access scanning microscopy and MINFLUX methods, since these methods often involve examining two closely spaced sample structures, for example, a protein complex in a biological cell labeled with different fluorescent dyes at specific positions.

[0063] A second aspect of the invention relates to a light microscope comprising a scanning device according to the first aspect.

[0064] According to one embodiment, the light microscope can be a confocal laser scanning microscope capable of performing random access scanning.

[0065] According to another embodiment, the light microscope can be a MINFLUX microscope.

[0066] The first and second illumination beams can, in particular, be first and second excitation beams, respectively, which excite emitters (especially different emitters for the first illumination beam and the second illumination beam) in the sample to luminescence, especially fluorescence, and / or are reflected and / or scattered by emitters in the sample.

[0067] In this application, emitters are understood to be objects which, when illuminated with excitation light, can be considered point light sources with regard to the measurements according to the invention. The light emitted by the object acting as a point light source can, for example, be scattered light resulting from elastic scattering such as Rayleigh scattering or inelastic scattering such as Raman scattering, or it can be luminescent light, in particular fluorescent light. An emitter can therefore be, for example, a light-reflecting nanoparticle, a quantum dot, a fluorescent dye molecule (fluorophore), or a molecule or nanoparticle labeled with one or more fluorescent dye molecules.Depending on the size of the molecule and the distance between the fluorophores, a molecule labeled with several fluorophores or a nanoparticle labeled with several fluorophores can of course also have several emitters in the sense of the definition used here.

[0068] According to one embodiment, the light microscope has at least one light source configured to generate the first and second illumination beams. That is, the light microscope can have a first light source (e.g., a first laser) for generating the first illumination beam and a second light source (e.g., a second laser) for generating the second illumination beam. Alternatively, the light microscope can have a single light source (e.g., a laser) configured to generate both the first and second illumination beams. This light source can, for example, generate light of multiple wavelengths, from which the first and second illumination beams are then extracted, for example, by means of optical filters. Such a light source could, for example, be a white laser.

[0069] According to a further embodiment, the light microscope has an objective lens configured to focus the first illumination beam and the second illumination beam into or onto a sample. Optionally, the objective lens can also be configured to collect detection light emanating from the sample, in particular reflected light, scattered light, or luminescence light, and further, in particular, fluorescence light.

[0070] According to a further embodiment, the light microscope has a detector designed to detect detection light emanating from the sample, in particular reflected light, scattered light or luminescence light, and further in particular fluorescence light.

[0071] If the light microscope is a confocal microscope, the detector can be positioned in an image plane to the focal plane in or on the sample in which the foci of the first and second illumination light beams are arranged.

[0072] According to one embodiment, the light microscope has a confocal aperture in front of the detector in the direction of propagation of the detection light.

[0073] The detector can be, for example, a photomultiplier, an avalanche photodiode (especially a single photon avalanche diode, SPAD), a hybrid detector or an area detector with multiple detector elements, especially of the types mentioned above.

[0074] According to one embodiment, the light microscope can have a beam splitter designed to separate the first and second illumination light from the detection light.

[0075] According to one embodiment, the scanning device according to the invention can be arranged, in particular, between the at least one light source and the beam splitter, so that the first and second illumination light beams are scanned by the scanning device over or through the sample, but the detection light is not descanned by the scanning device. This non-descanned arrangement in the beam path is particularly important if the at least one acousto-optic deflector of the scanning device is polarization-dependent, as can be the case, for example, with AODs based on birefringent acousto-optic crystals.

[0076] According to another embodiment, a further scanning device (e.g. a galvanometer scanning device) can be arranged between the beam splitter and the lens, in particular for coarse positioning of the image field and / or for partially descanning the detection light.

[0077] According to a further embodiment, the light microscope has at least one light modulator configured to spatially modulate the phase and / or amplitude of the first illumination beam and / or the second illumination beam, such that an intensity distribution of the respective illumination light with a local minimum, in particular a central zero at the geometric focus, is formed at the focus in or on the sample. The light modulator can be, for example, a phase filter or a controllable spatial light modulator (SLM). The intensity distributions can be, for example, donut-shaped, bottle-beam-shaped, or crescent-shaped.

[0078] The formation of an intensity distribution with a local minimum is particularly necessary in a STED microscope or a MINFLUX microscope. Specifically, both the first and second illumination beams are phase- or amplitude-modulated so that they form such an intensity distribution at the focus. At suitable wavelengths, this can be achieved with a single light modulator or with two separate light modulators, each adapted to its respective wavelength, in separate beam paths or in a common beam path.

[0079] In particular, light microscopy can be a multicolor MINFLUX microscope. In such a microscope, intensity distributions of the first and second illumination lights, with their respective local minima, can be positioned at illumination points of an illumination pattern in the sample using the scanning device according to the invention, which has at least one AOD. The detector can then detect the respective amounts of light emitted at each illumination point. A spectral detection system (e.g., with optical filters) can be provided upstream of the detector to separate the detection light from different emitters, each excited by the first and second illumination beams. Subsequently, a processing unit can calculate the positions of the different emitters in the sample from the detected light amounts and the corresponding illumination positions.

[0080] According to a further embodiment, the light microscope has a first optical system which is configured to align the first illumination light beam and the second illumination light beam relative to each other in such a way that the first illumination light beam and the second illumination light beam have a non-zero relative angle to each other when they strike the acousto-optic deflector, in particular the acousto-optic crystal.

[0081] According to a further embodiment, the first light source and the second light source are arranged and aligned such that the first illumination light beam and the second illumination light beam have a non-zero relative angle to each other when they strike the acousto-optic deflector, in particular the acousto-optic crystal.

[0082] According to another embodiment, the light microscope has a second optical system designed to align the first illumination beam and the second illumination beam relative to each other after deflection by the acousto-optic deflector.

[0083] A third aspect of the invention relates to a method for deflecting a first illumination light beam with a first wavelength and a second illumination light beam with a second wavelength that differs from the first wavelength, in particular wherein the method can be carried out with the scanning device according to the first aspect and / or with the light microscope according to the second aspect, wherein sound waves of a first frequency range and a second frequency range separate from the first frequency range are generated in an acousto-optic deflector, wherein the first frequency range and the second frequency range are selected such that the first illumination light beam is deflected by the sound waves of the first frequency range and the second illumination light beam is deflected independently of the first illumination light beam by the sound waves of the second frequency range.

[0084] Further embodiments and advantages of the method according to the third aspect will become apparent from the description of the scanning device according to the first aspect and the light microscope according to the second aspect.

[0085] Advantageous embodiments of the invention are described in the claims, the description, the drawings, and the accompanying explanations. The described advantages of features and / or combinations of features of the invention are merely examples and can have an effect alternatively or cumulatively.

[0086] Regarding the disclosure content (but not the scope of protection) of the original application documents and the patent, the following applies: Further features can be seen in the drawings – in particular the depicted relative arrangements and functional connections. The combination of features from different embodiments of the invention or from features of different claims is also possible, deviating from the chosen cross-references in the claims, and is hereby encouraged. This also applies to features that are illustrated in separate drawings or mentioned in their description. These features can also be combined with features from different claims. Likewise, features listed in the claims can be omitted for further embodiments of the invention, but this does not apply to the independent claims of the granted patent.

[0087] The reference numerals contained in the patent claims do not constitute a limitation of the scope of the subject matter protected by the patent claims. They merely serve the purpose of making the patent claims easier to understand.

[0088] Exemplary embodiments of the invention are described below with reference to figures. These do not limit the subject matter of this disclosure or the scope of protection. Brief description of the characters Fig. Figure 1 shows an embodiment of a scanning device according to the invention; Fig. Figure 2 shows possible deflection areas of the first and second illumination light beams; Fig. Figure 3 shows the deflection areas of the first and second illumination light beams according to an embodiment of the invention; Fig. Figure 4 shows a frequency diagram with frequency ranges that can be used for the operation of the scanning device according to the invention; Fig. Figure 5 shows an embodiment of a light microscope with a scanning device according to the invention; Fig. Figure 6 shows another embodiment of a light microscope, namely a MINFLUX microscope, with a scanning device according to the invention. Description of the characters

[0089] Fig. Figure 1 schematically shows an embodiment of a scanning device 1 according to the invention comprising an acousto-optic deflector 10, a control unit 13 and a first optical system 12. The acousto-optic deflector 10 has an acousto-optic crystal 15, a transducer 16, an absorber 17 and a frequency generator 11.

[0090] The frequency generator 11 is designed to generate ultrasonic waves in the MHz range, which are coupled into the acousto-optic crystal 15 via the transducer 16. The sound waves pass through the acousto-optic crystal 15 in a sound propagation direction S and are absorbed by the absorber 17, which is located opposite the transducer 16 in the sound propagation direction S.

[0091] The control unit 13 is connected to the frequency generator 11 and is designed to control the frequency generator 11 in such a way that it generates sound waves of a first frequency f1 from a first frequency range and sound waves of a second frequency f2 from a second frequency range separate from the first frequency range.

[0092] At an angle to the direction of sound propagation S, a first illumination beam L1 with a first wavelength and a second illumination beam L2 with a second wavelength, which differs from the first wavelength, pass through the acousto-optic crystal 15. The first illumination beam L1 and the second illumination beam L2 initially travel parallel to each other. The second illumination beam L2 is deflected by a first optical system 12, in particular a mirror, such that the first illumination beam L1 and the second illumination beam L2 form an acute relative angle θ with each other upon entering the acousto-optic crystal 15. r exhibit.

[0093] The first illumination beam L1 is deflected by the sound waves of the first frequency range from the acousto-optic deflector 10 by diffraction, and the deflected first illumination beam L1' emerges from the acousto-optic deflector 10. The second illumination beam L2 is deflected independently of the first illumination beam L1 by the sound waves of the second frequency range from the acousto-optic deflector 10, and the deflected second illumination beam L2' emerges from the acousto-optic deflector 10. The deflected illumination beams L1' and L2' can, in particular, be beams of diffraction order 1 or -1. The undiffracted beams (0th order) are in Fig. 1 not shown. These can be hidden, in particular, in the further beam path of the scanning device 1 or the light microscope 100 (su).

[0094] The following will be based on the Fig. 2 an example of the selection of frequency ranges for the independent deflection of the first illumination light beam L1 and the second illumination light beam L2 is explained.

[0095] In the example shown, the operating range, i.e., the range in which phase matching is possible with light beams of suitable wavelengths due to the properties of the acousto-optic crystal 15, extends from 70 MHz to 140 MHz. This range is initially divided into two sub-ranges, each assigned to the first illumination beam L1 and the second illumination beam L2, respectively. For example, the operating range can be halved, i.e., the ranges from 70 MHz to 105 MHz and 105 MHz to 140 MHz are assigned to the respective light beams L1 and L2.It is advantageous to assign the lower acoustic frequency range (here 70 MHz to 105 MHz) to the illumination beam with the longer wavelength (in this embodiment, the monochromatic first illumination beam L1 with a wavelength of 638 nm) and the higher acoustic frequency range (here 105 MHz to 140 MHz) to the illumination beam with the shorter wavelength (in this embodiment, the monochromatic second illumination beam L2 with a wavelength of 561 nm). As an alternative to an even distribution of frequencies, a distribution is also possible that, taking into account the wavelengths of the illumination beams L1 and L2, results in an identical deflection range for both illumination beams L1 and L2.

[0096] The corresponding deflection angles can be determined in particular by the formula θe=λ⋅Fv are calculated, where θ eThe deflection angle, λ the wavelength of the light, F the acoustic frequency and v the speed of sound in the acousto-optic crystal 15.

[0097] As in Fig. As shown in Figure 2, with a speed of sound of 650 m / s (with the same angles of incidence of the illumination light rays L1,L2 on the acousto-optic crystal 15), the deflection angles for the first illumination light ray L1 by frequencies f1 of the first frequency range are between 68.7 mrad and 103.1 mrad, and for the deflection of the second illumination light ray L2 by frequencies f2 of the second frequency range, the deflection angles are between 90.6 mrad and 120.8 mrad.

[0098] In contrast, the frequencies f1 of the first frequency range lead to a deflection of the second illumination light beam L2 in the range of 60.4 mrad to 90.6 mrad, and the frequencies f2 of the second frequency range lead to a deflection of the first illumination light beam L1 in the range of 103.1 mrad to 137.4 mrad.

[0099] The deflection ranges due to the frequencies f1 of the first frequency range are in Fig. 2 are shown as solid lines and the deflection ranges due to the frequencies f2 of the second frequency range are shown as dashed lines.

[0100] For example, by means of suitable apertures, the deflection range of the second illumination light beam L2, deflected by the frequencies f1 of the first frequency range, can be ( Fig. 2, L2 / f1) and the deflection range of the first illumination light beam L1 deflected by the frequencies f2 of the second frequency range ( Fig. 2, L1 / f2) are hidden from the image field.

[0101] This results in an overlap area 20 (hatched area in Fig. 2) of an approximate size of 13 mrad. In this angular range, the illumination light rays L1,L2 can in principle be deflected independently of each other by an AOD, if harmonic mixing shrinkages are neglected (see below).

[0102] By irradiating the AOD 10 with the first illumination light beam L1 and the second illumination light beam L2 at different angles of incidence, i.e. setting a relative angle θ r If the overlap area is not equal to zero, it can be enlarged (see Fig. 3).

[0103] It is convenient to use the centers of the two image areas L1 / f1 and L2 / f2 (see Fig. 2) by adjusting the relative angle θ r be placed in the same position in the room.

[0104] The corresponding relative angle θ r can be achieved, for example, by the formula derived from the Bragg formula θr=λ1v⋅FN,1−λ2v⋅FN,2 are calculated, where θ r the relative angle, λ1 the wavelength of the first illumination light ray L1, λ2 the wavelength of the second illumination light ray, F N,1 a first zero-position frequency within the first frequency range and F N,2 denotes a second zero-position frequency within the second frequency range.

[0105] The center frequencies of the frequency ranges (i.e., 87.5 MHz as the first center frequency and 122.5 MHz as the second center frequency) can be chosen as zero-position frequencies, each corresponding to the center of the respective image area. Fig. 2 are assigned. With the values ​​given above (λ1=638 nm, λ2=561 nm, v=650 m / s) a relative angle θ results. r of 19.8 mrad.

[0106] The adjustment of the relative angle θ r The superimposed image areas (L1 / f1 and L2 / f2) are in Fig. 3 shown. This results in a comparison to Fig. 2 significantly increased overlap area 20 of approximately 30 mrad.

[0107] However, the mixing frequencies 2f1 - f2 and 2f2 - f1, in particular, prevent a completely independent displacement of the illumination light rays L1, L2 without additional deflected rays in the relevant image field. With an operating range of 70 MHz to 140 MHz and a separation of the first and second frequency ranges at 105 MHz according to the embodiment shown here, the mixing frequencies 2f1 - f2 lie in the frequency interval from 0 MHz to 105 MHz, and the mixing frequencies 2f2 - f1 lie in the frequency interval from 105 MHz to 210 MHz. Mixing frequencies can therefore occur throughout the entire operating range from 70 MHz to 140 MHz. The mixing frequencies that lie in the first and second frequency ranges are responsible for the occurrence of unwanted deflected light rays in the relevant image field.

[0108] Fig. Figure 4 shows a diagram in which the second frequency f2 for deflecting the second illumination beam L2 is plotted against the first frequency f1 for deflecting the first illumination beam L1. The parameter ranges in which the mixing frequencies 2f1 - f2 and 2f2 - f1 are below the lower limit f u of the operating range (here 70 MHz) or above the upper limit f o The parameters within the operating range (here 140 MHz) are shown hatched. These parameter ranges are defined by the lines 2f1 - f2 = f u and 2f2 - f1 = f u (top left) or the lines 2f1 - f2 = f o and 2f2 - f1 = f o (bottom right) bounded. The lines 2f1 - f2 = f u and 2f2 - f1 = f u they intersect at that point 2fu+fo3 (also known as first threshold frequency f) s,1 (designated) and the lines 2f1 - f2 = f0 and 2f2 - f1 = f o they intersect at that point fu+2fo3 (also known as second threshold frequency f) s,2 designated).

[0109] With the values ​​f u =70 MHz and f o At a value of 140 MHz, a first threshold frequency f is obtained. s,1 of approximately 93.333 MHz and a second threshold frequency f s,2 of approximately 116.666 MHz.

[0110] Therefore, if in the present embodiment the first frequency range is selected from 70 MHz to 93.333 MHz, the cutoff range from 93.333 MHz to 116.666 MHz and the second frequency range from 116.666 MHz to 140 MHz, the mixing frequencies 2f1 - f2 and 2f2 - f1 are advantageously outside the operating range of the AOD 10 and the corresponding unwanted additional deflected rays can be blocked out of the image area, e.g. with an aperture.

[0111] Since the in Fig. The overlap area 20 shown is in the “forbidden” parameter range under these circumstances (in 20). Fig. 4 (outside the hatched areas) should be the relative angle θ r The incident illumination light rays L1,L2 are set to a non-zero value as described above, in order to function as described in Fig. Figure 3 illustrates how to deflect both illumination light rays L1,L2 in the same image area.

[0112] It is therefore useful to define the zero-position frequencies F N,1 , F N,2 to be selected as the center frequencies of the first and second frequency ranges. Center frequency F N,1 The first frequency range [70 MHz; 93.333 MHz] yields approximately 81.666 MHz as the center frequency F. N,2 The second frequency range [116.666 MHz; 140 MHz] yields approximately 128.333 MHz.

[0113] Based on the formula θr=λ1v⋅FN,1−λ2v⋅FN,2 With λ1=638 nm, λ2=561 nm, v=650 m / s, a relative angle θ is obtained. rof approximately 30.6 mrad between the incident first illumination ray L1 and the incident second illumination ray L2.

[0114] As a result, the illumination light rays L1,L2 can be seen throughout the entire area. Fig. The overlap area 20 shown in section 3 can be deflected without any disturbing influence from mixing frequencies.

[0115] Out of Fig. 4. It is further evident that for certain frequency combinations, frequencies above the first threshold frequency f are also possible. s,1 and below the second threshold frequency f s,2 are "allowed", i.e., lead to mixing frequencies that lie outside the working range (triangular sub-areas 21 of the hatched area in Fig. 4) In this area, however, the permitted frequencies for one illumination beam depend on the frequency used for the other illumination beam.

[0116] A displacement of the illumination light rays L1,L2 over a relatively large image field without disturbing deflected rays due to mixing frequencies can also be achieved if the two deflected illumination light rays L1,L2 have a maximum angle Δθ max adhere to the guidelines, meaning they are shifted together to a certain extent. This is tolerable for many applications, e.g., in multi-color MINFLUX processes.

[0117] For example, for a maximum angle Δθ max = 0 (i.e., the same position in sample 2 is controlled by the deflected first illumination beam L1 and the deflected second illumination beam L2) and an AOD operating range of 70 MHz to 140 MHz, with a first frequency range of 70 MHz to 105 MHz and a second frequency range of 105 MHz to 140 MHz, the entire operating range of the AOD can be used without any interfering influence of the mixing frequencies 2f1 - f2 and 2f1 - f1.

[0118] In this case, for the extreme values ​​f1 = 70 MHz, f2 = 105 MHz, the resulting mixing frequencies are 2f1 - f2 = 35 MHz and 2f2 - f1 = 140 MHz, and for the extreme values ​​f1 = 105 MHz, f2 = 140 MHz, the resulting mixing frequencies are 2f1 - f2 = 70 MHz and 2f2 - f1 = 175 MHz.

[0119] These lie outside or at the edge of the working area, and the corresponding additional deflected light rays can therefore be excluded from the image area.

[0120] For Δθ mm For values ​​> 0, correspondingly smaller usable frequency ranges result without any influence of the mixing frequencies, but these depend on the choice of the parameter Δθ. max , i.e., the distance between the foci of the illumination light rays L1L2 in sample 2 can be greater than that in Fig. 4. Allowed parameter range outlined (hatched area).

[0121] In Fig. Figure 5 shows an embodiment of a light microscope 100 with a scanning device 1 according to the invention, comprising an acousto-optic deflector 10 and a frequency generator 11. The light microscope 100 has a first light source 3 for generating a first illumination beam L1 and a second light source 4 for generating a second illumination beam L2. Both illumination beams L1 and L2 are deflected by the acousto-optic deflector 10. A first optical system 12 in the form of a deflecting mirror is arranged between the second light source 4 and the acousto-optic deflector 10. This system deflects the second illumination beam L2 so that it strikes the acousto-optic crystal 15 at a desired angle of incidence (see Figure 5). Fig. 1) of the acousto-optic deflector 10.

[0122] The first illumination beam L1 and the second illumination beam L2, deflected by the acousto-optic deflector 10, pass together through a beam splitter 14 and a galvanometer scanner 6 with a deflection mirror 60. They are then focused by a lens 7 along an optical axis O into a sample 2. Detection light D emanating from the sample 2, e.g., fluorescent light or reflected / scattered light, is collected by the lens 7, scanned by the galvanometer scanner 6, and reflected by the beam splitter 14 to a detector 8. The detector 8 is coupled to a processing unit 9, which calculates a raster image from the signals of the detector 8. This image can then be displayed by a display unit (not shown).

[0123] The optional galvanometer scanning device 6 can be used, for example, for coarse adjustment of the image field and for scanning the detection light D, and in particular has at least one deflection mirror 60 for each spatial direction of deflection, wherein in Fig. 5 For the sake of simplicity, only one deflection mirror 60 is shown.

[0124] The light microscope 100 can be a confocal laser scanning microscope. In this case, the light sources 3, 4 are lasers and a confocal aperture (not shown) is arranged between the beam splitter 14 and the detector 8, which is located in an image plane to the focal plane of the first illumination light beam L1 and / or the second illumination light beam L2 in the sample 2.

[0125] The 100 light microscope can still be used in Fig. 5 have an optical relay not shown, which has a plane in which the acousto-optic crystal 15 (see Fig. 1) of the acousto-optic deflector 10, is imaged into a back aperture of the objective 7. This results in a tilting of the illumination light rays L1,L2 by the AOD 10, leading to a lateral displacement of the foci of the illumination light rays L1,L2 in the sample 2 perpendicular to the optical axis O.

[0126] The scanning device 1 can be used, for example, for fast random access scanning of the first illumination light beam L1 and the second illumination light beam L2.

[0127] In addition to the illustrated scanning device 1, which deflects the illumination light beams L1, L2 in a first direction x perpendicular to the optical axis O, the light microscope 10 can, in particular between the scanning device 1 and the beam splitter 14, have a further scanning device according to the invention, the AOD 10 of which is arranged such that it deflects the first illumination light beam L1 and / or the second illumination light beam L2 in a second direction y, which is perpendicular to the first direction x and perpendicular to the optical axis O. In this way, for example, fast two-dimensional random access scanning can be implemented. If required, the scanning device can be switched between the two directions shown in the illustration. Fig. In the scanning device 1 shown in Figure 5 and in the further scanning device, a further optical system for deflecting the first illumination light beam L1 and / or the second illumination light beam L2 may be arranged in order to direct the respective illumination light beam L1 / L2 at a desired angle of incidence onto the acousto-optic deflector 10 of the further scanning device. The further optical system may, for example, comprise at least one element for spatially separating the illumination light beams L1, L2 (e.g., a dichroic beam splitter or a dispersive element such as a prism) and optionally at least one element for deflecting the first and / or second illumination light beam L1 / L2 (e.g., a deflecting mirror).

[0128] The light microscope 100 has a control unit 13 configured to control the frequency generator 11 (and optionally the frequency generator of a further scanning device for lateral deflection in a second direction) such that it generates acoustic frequencies in the first frequency range and the second frequency range to illuminate the first illumination beam L1 and the second illumination beam L2, in particular as described above and in Fig. 1 to Fig. Figure 4 illustrates how to control them independently. Furthermore, the light microscope 100 can optionally have at least one optical aperture designed to block out unwanted additional deflected light rays from the image field (not shown). These rays can occur, for example, due to the effect of the first frequency range on the second illumination light beam L2 and the second frequency range on the first illumination light beam L1, as well as due to harmonic mixing frequencies.

[0129] In Fig. Figure 6 shows a light microscope 100, in particular a MINFLUX microscope, with a scanning device 1 according to the invention. The in Fig. The setup of the light microscope 100 shown in Figure 6 is largely identical to the one in Figure 6. Fig. 5 light microscope 100 shown, where identical components are marked with the same reference numerals. With regard to these components, particular reference is made to the description of Fig. 5 referred.

[0130] The in Fig. The light microscope 100 shown in Figure 6 has two additional components: light modulators 5 for spatial phase and / or amplitude modulation of the first illumination beam L1 and the second illumination beam L2. The light modulators 5 can be, for example, controllable phase-modulating liquid crystal SLMs. The light modulators 5 are specifically tuned to the wavelength and, optionally, the polarization direction of the first illumination beam L1 and the second illumination beam L2, respectively. In particular, the acousto-optic crystal 15 (see Figure 6) is used for this purpose. Fig. 1) of the AOD 10 of the scanning device 1 is set up and arranged in such a way that it does not affect, or does not significantly affect, the modulated phase and / or amplitude distribution of the first illumination light beam L1 and the second illumination light beam L2.

[0131] As an alternative to the setup shown, the illumination light beams L1, L2 can also be modulated in phase and / or amplitude by a light modulator 5 after their deflection by the scanning device 1 according to the invention. This is particularly advantageous if the illumination light beams L1, L2 have suitable wavelengths and optionally polarization directions so that they can be phase- and / or amplitude-modulated together by a light modulator 5. In this case, beam splitting and merging after light modulation can be omitted.

[0132] The light modulators 5 are specifically designed to modulate the phase and / or amplitude of the first illumination beam L1 and the second illumination beam L2 such that focusing the illumination beams L1 and L2 at the focus in the sample results in an intensity distribution with a local minimum. The local minimum is, in particular, a zero point and is located at the geometric focus. The intensity distributions can be, for example, donuts, bottle beams, or crescent-shaped light distributions.

[0133] The scanning device 1 according to the invention can be used in a MINFLUX process to arrange the foci of the illumination light beams L1, L2 in rapid succession at defined illumination positions in the sample 2, forming, for example, an illumination pattern (such as a polygon) around a previously estimated position of a single emitter in the sample 2. A light intensity or a photon count can be determined for each illumination position. These photon counts or light intensities, together with their associated coordinates of the illumination positions, can be evaluated by the processing unit 9 to determine an estimated position of a single emitter using a position estimation algorithm. Subsequently, an image of structures in the sample 2 or a trajectory of an emitter moving in the sample 2 can be calculated from the determined emitter positions.

[0134] In particular, the light microscope 100 can, in addition to the illustrated scanning device 1, which deflects the illumination light beams L1, L2 in a first direction x perpendicular to the optical axis O, especially between the scanning device 10 and the beam splitter 14, have a further scanning device 1 according to the invention, the AOD 10 of which is arranged such that it deflects the first illumination light beam L1 and / or the second illumination light beam L2 in a second direction y, which is perpendicular to the first direction x and perpendicular to the optical axis O. A 2D-MINFLUX method can then be carried out with this. For further details of this embodiment, please refer to the description in [reference to relevant section]. Fig. 5 referred.

[0135] Position determination can be carried out, in particular, with different emitters in the sample 2, wherein, in particular, the first illumination beam L1 excites emitters of a first species and the second illumination beam L2 excites emitters of a second species. The first illumination beam L1 and the second illumination beam L2 can be moved independently of one another to respective illumination positions in the sample 2 using the scanning device 1 according to the invention. The areas of the illumination positions of the first illumination beam L1 and the second illumination beam L2 can overlap or be spatially separated from one another.

[0136] The light microscope 100 has a further beam splitter 19 between the beam splitter 14 and the detector 8. This beam splitter spectrally splits the detection light D and directs a portion of the detection light D to a further detector 18, which is also connected to the processing unit 9. If the different emitter species in the sample 1 emit, for example, luminescence light of different wavelengths, the signals from detector 8 can be used to determine the position of a first emitter species excited by the first illumination beam L1, and the signals from the further detector 18 can be used to determine the position of a second emitter species excited by the second illumination beam L2. In this way, a two-color MINFLUX method can be implemented. Reference symbol list 1 scanning device 2 Sample 3 First light source 4 Second light source 5 Light modulator 6 Galvanometer scanning device 7 Lens 8 Detector 9 Calculation unit 10 Acousto-optic deflector 11 Frequency generator 12 First optical system 13 Control unit 14 beam splitters 15 Acousto-optic crystal 16 Transducers 17 absorbers 18 Additional Detector 19. Additional beam splitter 20 Overlap area 21 sub-area 60 deflection mirrors D detection light L1 First illumination beam L2 Second lighting beam Optical axis S Sound propagation direction QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] US 2018 / 0259458 A1

[0007] US 2020 / 0393378 A1

[0007] US 2006 / 0140462 A1

[0014] US 2023 / 0288715 A1

[0019] Cited non-patent literature

[0000] Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes” by F. Balzarotti, Y. Eilers, KC Gwosch, AH Gynnå, V. Westphal, FD Stefani, J. Elf and SW Hell, Science 355, 606-612 (2017

[0006] MINSTED fluorescence localization and nanoscopy“ von M. Weber, M. Leutenegger, S. Stoldt, S. Jakobs, T.S. Mihaila, A.N. Butkevich und S.W. Hell, Nature Photonics 15, 361-366 (2021

[0008] Übersichtsartikeln „Acousto-Optic Devices - Optical Elements for Spectroscopy“ von C.D Tran, Analytical Chemistry 64, 971-981 (1992) und „Acousto-optic systems for advanced microscopy“ von M. Duocastella, S. Surdo, A. Zunino, A. Diaspro und P. Saggau, J. Phys. Photonics 3, 01 2004 (2021

[0015] A review of physical principles and applications of acousto-optic deflectors on the basis paratellurite“ von S.N. Antonov und V.A Kotelnikov, Phys Astron Int. J. 3(6), 235-249 (2019

[0021] Acousto-optic systems for advanced microscopy“ von M. Duocastella, S. Surdo, A. Zunino, A. Diaspro und P. Saggau, J. Phy. Photonics 3 (2021), 01 2004

[0022]

Claims

[1] Scanning device (1) for a light microscope (100) comprising at least one acousto-optic deflector (10) configured to deflect a first illumination light beam (L1) of a first wavelength and a second illumination light beam (L2) of a second wavelength, which differs from the first wavelength, wherein the scanning device (1) comprises a control unit (13) configured to generate sound waves of a first frequency range and a second frequency range separate from the first frequency range in the acousto-optic deflector (10), wherein the first frequency range and the second frequency range are selected such that the first illumination light beam (L1) is deflected by the sound waves of the first frequency range and the second illumination light beam (L2) is deflected by the sound waves of the second frequency range independently of the first illumination light beam (L2). [2] Scanning device (1) according to claim 1, characterized by , that the scanning device (1) is configured to detect the first illumination light beam (L1) at a first angle of incidence (θ i,1 ) to direct the acousto-optic deflector (10) and the second illumination light beam (L2) at a second angle of incidence (θ i,2 ) to direct onto the acousto-optic deflector (10), where the first angle of incidence (θ i,1 ) is tuned to a first zero position frequency within the first frequency range, which results in a zero position of the first illumination light ray (L1) within an image field, and wherein the second angle of incidence (θ i,2 ) is tuned to a second zero position frequency within the second frequency range, which results in a zero position of the second illumination light beam (L2) within the image field. [3] Scanning device (1) according to claim 2, characterized by, that the first illumination light ray (L1) and the second illumination light ray (L2) have a relative angle to each other when they strike the acousto-optic deflector (10), wherein the relative angle θr=λ1v⋅FN,1−λ2v⋅FN,2 is, where θ r the relative angle, λ1 the first wavelength, λ2 the second wavelength, F N,1 the first zero position frequency and F N,2 the second zero-position frequency. [4] Scanning device (1) according to any one of the preceding claims, characterized by that there is a separation range between the first frequency range and the second frequency range. [5] Scanning device (1) according to claim 4, characterized by , that the separation range is chosen such that for all frequency pairs (f1, f2) the conditions 2f1 - f2 < f u , 2f2 - f1 < f u , 2f1 - f2 > f o and 2f2 - f1 > f oare satisfied, where f1 are the frequencies from the first frequency range, f2 are the frequencies from the second frequency range, f u a lower cutoff frequency of a working range of the acousto-optic deflector and f o denotes an upper cutoff frequency of the operating range of the acousto-optic deflector. [6] Scanning device (1) according to claim 5, characterized by , that the separation range extends from a first threshold frequency to a second threshold frequency, wherein the first threshold frequency fs,1=2fu+fo3 is and the second threshold frequency fs,2=fu+2fo3 is, where f s,1 the first threshold frequency, f s,2 the second threshold frequency, f u the lower cutoff frequency and f o the upper cutoff frequency. [7] Scanning device (1) according to any one of the preceding claims, characterized by, that the first frequency range has lower frequencies than the second frequency range, wherein the first illumination light beam (L1) has a higher wavelength than the second illumination light beam (L2). [8] Scanning device (1) according to one of the preceding claims, characterized by , that the scanning device (1) comprises a first acousto-optic deflector (10a) and a second acousto-optic deflector (10b), wherein the first acousto-optic deflector (10a) is configured to deflect the first illumination light beam (L1) and the second illumination light beam (L2) independently of each other in a first direction, and wherein the second acousto-optic deflector (10b) is configured to deflect the first illumination light beam (L1) and the second illumination light beam (L2) independently of each other in a second direction, in particular wherein the first direction is perpendicular to the second direction. [9] Scanning device (1) according to any of the preceding claims, characterized by , that the control unit (13) is designed to adjust the frequencies of the sound waves for deflecting the first illumination light beam (L1) and the second illumination light beam (L2) in a range by a predetermined ratio, so that the first illumination light beam (L1) deflected by the acousto-optic deflector (10) and the second illumination light beam (L2) deflected by the acousto-optic deflector (10) maintain a relative distance in a focal plane in a predetermined range. [10] Light microscope (100) comprising a scanning device (1) according to any one of claims 1 to 9. [11] Method for deflecting a first illumination light beam (L1) with a first wavelength and a second illumination light beam (L2) with a second wavelength that differs from the first wavelength, in particular by means of a scanning device (1) according to any one of claims 1 to 9 or a light microscope (100) according to claim 10, wherein sound waves of a first frequency range and a second frequency range separate from the first frequency range are generated in an acousto-optic deflector (10), wherein the first frequency range and the second frequency range are selected such that the first illumination light beam (L1) is deflected by the sound waves of the first frequency range and the second illumination light beam (L2) is deflected independently of the first illumination light beam (L1) by the sound waves of the second frequency range.

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