MICROSCOPE WITH AN ACOUSTOOPTIC DEVICE

Description

[0001] The invention relates to a scanning microscope for observing fluorescence light emitted from a sample, comprising an acousto-optic device which uses a mechanical wave, characterized by a frequency that is preferably adjustable, to remove from a polychromatic and collinear detection beam proportions of illumination light scattered and / or reflected from a sample of a wavelength associated with the frequency.

[0002] Such a scanning microscope can be designed in particular as a confocal scanning microscope.

[0003] In scanning microscopy, a sample is illuminated with a light beam to observe the reflected or fluorescent light emitted by the sample. The focus of the illumination beam is moved in the object plane using a controllable beam deflection device, generally by tilting two mirrors. The deflection axes are usually perpendicular to each other, so that one mirror deflects in the x-direction and the other in the y-direction. The tilting of the mirrors is achieved, for example, using galvanometer actuators. The power of the light coming from the object is measured as a function of the scanning beam's position. The actuators are typically equipped with sensors to determine the current mirror position.

[0004] Specifically in confocal scanning microscopy, an object is scanned in three dimensions using the focus of a light beam. A confocal scanning microscope generally comprises a light source, focusing optics that focus the light from the source onto a pinhole aperture—the so-called excitation aperture—a beam splitter, a beam deflection device for beam control, microscope optics, a detection aperture, and detectors for detecting the detection or fluorescence light. The illumination light is coupled in, for example, via a beam splitter.

[0005] The fluorescent light emanating from the object passes through the beam deflection device back to the beam splitter, through it, and is then focused onto the detection aperture, behind which the detectors are located. Detection light that does not originate directly from the focus region takes a different path and does not pass through the detection aperture, thus providing point information which, through sequential scanning of the object, leads to a three-dimensional image.

[0006] In fluorescence microscopy, the components of illumination light reflected from and scattered by the sample must be removed from the detection light in order to detect only the fluorescence light. For this purpose, a dichroic filter, known as a main beam splitter, is used in a conventional microscope.

[0007] Instead of such a beam splitter, an optical arrangement designed as an acousto-optic component can also be provided for coupling the excitation light from at least one light source into the microscope and for blocking the excitation light scattered and reflected at the object, or the excitation wavelength, from the light coming from the object via the detection beam path. This is known, for example, from German Patent Application DE 199 06 757 A1. The optical arrangement known from this publication is characterized, for variable configuration with the simplest possible construction, by the fact that excitation light of different wavelengths can be blocked by the spectrally selective element. Alternatively, such an optical arrangement is characterized by the fact that the spectrally selective element can be adjusted to the excitation wavelength to be blocked.

[0008] The functionality of such an acousto-optic component is essentially based on the interaction of the illumination light with a mechanical wave. In some acousto-optic components, such as an AOTF, the mechanical wave must have a very specific frequency so that the Bragg condition is fulfilled precisely for light of the desired illumination wavelength. Light for which the Bragg condition is not fulfilled is not deflected by the mechanical wave in these acousto-optic components. If light of several wavelengths is to be used to illuminate the sample, then several different mechanical waves must be generated simultaneously.

[0009] Acousto-optic components typically consist of an acousto-optic crystal to which an electrical transducer (often referred to as a transducer in the literature) is attached. The transducer usually comprises a piezoelectric material and an electrode positioned above and below it. By electrically applying radio frequencies, typically in the range of 30 MHz to 800 MHz, to the electrodes, the piezoelectric material is set into vibration, generating an acoustic wave, i.e., a sound wave, which, after its generation, travels through the crystal. Most often, after passing through an optical interaction region, the acoustic wave is absorbed or reflected away at the opposite side of the crystal.

[0010] Acousto-optic crystals are characterized by the fact that the resulting sound wave alters the crystal's optical properties. The sound induces a type of optical grating or a comparable optically active structure, such as a hologram. Light passing through the crystal undergoes diffraction at the optical grating. Consequently, the light is deflected in different diffraction orders and directions. There are acousto-optic components that influence all incident light more or less independently of its wavelength. Examples include components such as AOM (acousto-optic modulator), AOD (acousto-optic deflector), and frequency shifters. Furthermore, there are already components that selectively affect individual wavelengths depending on the incident radio frequencies (AOTF; acousto-optic tunable filter).Acousto-optic elements often consist of birefringent crystals, such as tellurium dioxide, whereby the optical effect of the respective element is determined in particular by the position of the crystal axis relative to the direction of incidence of the light and its polarization. These relationships are known, for example, from DE 10 2006 053 187 A1.

[0011] The operation of a scanning microscope that incorporates an acousto-optic component as its main beam splitter is described in detail in DE 101 37 155 A1. This document also describes how the mechanical wave of a first AOTF, which deflects the tangentially polarized illumination light, can only completely remove those components of the illumination light wavelength with tangential linear polarization from the detection light. However, the detection light contains components with both sagittal and tangential polarization.

[0012] For this reason, a compensation element, also an acousto-optic component, is connected downstream of the acousto-optic component. This additional acousto-optic component is also an AOTF and is driven by another high-frequency source with a different electromagnetic high-frequency wave. The RF frequency of this additional electromagnetic high-frequency wave is chosen such that the components of the detection light that have the wavelength of the illumination light and a sagittal polarization direction are filtered out. This approach is necessary because, for the sagittal-polarized light to be filtered out of the detection light, the Bragg condition for diffraction by a mechanical wave is only fulfilled if the mechanical wave has a different frequency than that of the light with a tangential polarization direction.Therefore, the microscope must provide two different RF frequencies for each illumination wavelength to simultaneously generate two mechanical waves of different frequencies. Furthermore, an additional acousto-optic component with another sound generator is essential. For example, if illumination with light of four wavelengths is required, fluorescence detection is only possible if eight electromagnetic RF waves are provided simultaneously and, distributed across two acousto-optic components, eight mechanical waves of different frequencies are generated from them.

[0013] DE 10 2010 033722 A1 describes an arrangement for eliminating unwanted radiation components from detected light coming from an illuminated sample, wherein the light coming from the sample contains components of light excited in the sample (excitation light) and illumination light, and wherein at least one acousto-optic tunable filter (AOTF) is provided in the detection beam path and the sample light is split into polar components (polar components), preferably perpendicular to each other, via at least one polarization element, wherein at least one polar component passes through the same AOTF at least twice or passes through a first and at least one second AOTF.

[0014] The article by Thomas J. Fellers "Olympus Fluoview Resource Center: Acousto-Optic Tunable Filters" describes the operating principle of an AOTF in the context of confocal microscopy.

[0015] WO 00 / 37985 A2 discloses an arrangement of a light-diffracting element for separating excitation and emission light in a microscopic beam path, particularly in a laser scanning microscope, wherein the light-diffracting element is traversed by both the excitation light and the emission light and influences at least one wavelength of the excitation by diffraction, while other wavelengths emitted by the sample pass through the element unaffected and are thereby spatially separated from the excitation light. The light-diffracting element can be an AOTF.

[0016] It is therefore the object of the present invention to provide a microscope with an acousto-optic component that allows flexible fluorescence light detection with less effort.

[0017] The problem is solved by a scanning microscope with the features of claim 1. Advantageous embodiments are specified in the dependent claims. According to the invention, it was first recognized that coupling two mechanical waves per wavelength of illumination introduces a particularly high heat input into the crystal(s), which ultimately reduces the diffraction efficiency. Furthermore, the unavoidable temperature fluctuations also cause variations in the deflection directions and the light intensities.

[0018] Furthermore, practical experience has shown that disturbing streaks often appear in the image of a scanning microscope. In this regard, it was recognized according to the invention that beat frequency can occur when the wavelengths of the required sound waves overlap, which ultimately leads to periodic fluctuations in the illumination light intensity when illumination light is also directed onto an illumination beam path via the acousto-optic component.

[0019] In accordance with the invention, it was also discovered that the crystal, the mechanical wave, and the detection light beam incident on the crystal can be aligned with each other in such a way that one and the same mechanical wave can simultaneously deflect detection light of the same wavelength but with different linear polarization directions. Although the deflection occurs in different directions depending on the linear polarization direction, this is irrelevant with regard to the microscope according to the invention, because only the remaining detection light is to be detected anyway, and the portion removed from the detection light is no longer needed.

[0020] The invention has the advantage that for each wavelength of illumination used, only a single RF frequency is required to generate a single mechanical wave for removing the unwanted illumination light reflected or scattered at the sample, whereas previously two RF frequencies were required, namely one for one polarization direction and one for the other polarization direction.

[0021] The invention has the further advantage that, compared to microscopes known from the prior art with the same RF generator, twice the number of illumination wavelengths can be removed from the detection light and, as is explained in detail below, it also enables the possibility of directing illumination light of twice the number of wavelengths onto an illumination beam path.

[0022] Most commercially available acousto-optic components have no more than eight channels in their RF generators, so that the microscope according to the invention enables the removal of illumination light of eight wavelengths (instead of only four wavelengths previously) from the detection light coming from the sample. In a particular embodiment, the same mechanical waves can simultaneously couple illumination light of eight wavelengths (instead of four previously) onto an illumination beam path.

[0023] A further advantage of the microscope according to the invention is that the heat input into the crystal described above is halved and the problem of the sometimes occurring beat frequencies is completely avoided. In this respect, the microscope according to the invention has the advantage that the risk of temperature-related fluctuations in light intensity is at least reduced and the problem of fringes in the image described above is completely eliminated.

[0024] In a particular embodiment of the microscope, the first linear polarization direction is the linear polarization direction of ordinary light with respect to a birefringent property of the crystal, and / or the second linear polarization direction is the linear polarization direction of extraordinary light with respect to a birefringent property of the crystal. Alternatively or additionally, the first or second linear polarization direction may be arranged in the plane defined by the propagation direction of the mechanical wave and the propagation direction of the detection light beam.

[0025] In a particularly advantageous embodiment, the remaining portion of the detection light beam exits the crystal collinearly. This can be achieved, for example, by a suitable angular orientation of the entrance surface through which the detection light beam enters the crystal and a suitable angular orientation of the exit surface through which the detection light beam exits the crystal. Alternatively or additionally, it is also possible to achieve collinearity of the remaining portion of the detection light beam using an additional dispersive optical component, which is described in more detail below.

[0026] In a particularly advantageous embodiment of a microscope according to the invention, the acousto-optic device with the mechanical wave splits illumination light of a wavelength corresponding to the frequency of the mechanical wave from at least one polychromatic and preferably collinear primary light beam and directs this light onto an illumination beam path for the illumination of a sample.

[0027] The primary light can originate from a single light source. However, it is also possible for the primary light to originate from multiple light sources. In particular, it is possible for the primary light beams from different light sources to be combined into a collinear primary light beam.

[0028] In particular, the light source may be designed as a broadband white light source. Such a light source may, in particular, include a special optical element, such as a microstructured optical element and / or a tapered fiber and / or a photonic crystal fiber and / or a photonic crystal and / or a photonic band-gap material and / or a comparable optical nonlinearity-inducing element, which spectrally broadens the incident primary light, especially the light from a pulsed laser. Such a light source can provide primary light with a spectral width of several hundred nm, so that the user can individually select from this broad spectrum, using the acousto-optic component, precisely the illumination light with the wavelength(s) required for their experiment.

[0029] In particular, it can be advantageously provided that the unpolarized light from such a white light source is split into two components whose linear polarization directions are perpendicular to each other. Advantageously, one component can be coupled in opposite directions to the first diffraction order of the ordinary light (relative to the diffracted component of the detection light), while the other component can be coupled in opposite directions to the second diffraction order of the extraordinary light (relative to the diffracted component of the detection light). The illumination light diffracted from the two components is thus collinear.

[0030] In this way, all the primary light from the white light source is available to direct illumination light of a specific wavelength or wavelengths onto the microscope's illumination beam path and thus to the sample, using the acousto-optic device. This has the particular advantage over prior art acousto-optic main beam splitters that all the primary light from such a light source can be utilized. Naturally, the same applies to other light sources that emit unpolarized light.

[0031] In addition to such a white light source, several other light sources may be present, whose primary light beams are combined with the primary light beam of the white light source.

[0032] According to an independent inventive concept, a particularly advantageous embodiment of the acousto-optic device with the mechanical wave provides that illumination light of the wavelength corresponding to the frequency of the mechanical wave is split off from both a first polychromatic and preferably collinear primary light beam with a first linear polarization direction, and from a second polychromatic and preferably collinear primary light beam with a second linear polarization direction perpendicular to the first linear polarization direction, and directed onto an illumination beam path to illuminate a sample. In this way, with a single mechanical wave, precisely the proportion of the desired illumination wavelength can be selected from the two primary light beams with different linear polarization directions and used to illuminate a sample.

[0033] This embodiment has the distinct advantage that the entire primary light of a light source, such as the primary light of a white light source with a microstructured element, which—unlike, for example, a laser—emits unpolarized light, can be used. According to the invention, this is achieved by splitting the unpolarized primary light into the first and second primary light beams, for example, using a polarization beam splitter, and then coupling the two primary light beams (generally at different coupling points and / or at different angles of incidence) into the acousto-optic device, in particular into the crystal.Preferably, the portions of the two primary light beams selected for illuminating the sample are split off from the same mechanical shaft with the desired illumination wavelength and directed onto the preferably common illumination beam path for illuminating a sample.

[0034] Compared to microscopes known from the prior art, the microscope according to the invention has the advantage that unpolarized primary light can also be used without losing half the light power.

[0035] Of course, the microscope according to the invention can also be designed in such a way that, as already indicated in some places, several mechanical waves are used simultaneously to remove illumination light of several different wavelengths from the detection light and / or to direct illumination light of several different wavelengths onto the illumination beam path, whereby, however, advantageously only a single mechanical wave is generated in the crystal for each wavelength of illumination light.This is described in detail below: In a particular embodiment, the acousto-optic device is provided for by several mechanical waves, each characterized by different frequencies, that remove from the detection light beam portions of illumination light of several wavelengths scattered and / or reflected from a sample, wherein each frequency is assigned one of the illumination wavelengths, and wherein each of the mechanical waves deflects both the portion with a first linear polarization direction corresponding to its frequency and the portion with a second linear polarization direction perpendicular to the first linear polarization direction, thereby removing it from the detection light beam.

[0036] In particular, it can be provided that the mechanical waves have the same direction of propagation and / or are generated by the same sound source. The sound source is preferably attached to an outer surface of the crystal, wherein the direction of propagation of the mechanical wave relative to the crystal and its lattice structure is determined by the orientation of this outer surface relative to the crystal body, which can be determined by the crystal's shape.

[0037] It can also be advantageously provided that the acousto-optic device with the multiple mechanical waves splits illumination light of several wavelengths from polychromatic primary light of at least one light source and directs it onto an illumination beam path for illuminating a sample. In particular, it can be provided that the acousto-optic device with the multiple mechanical waves splits illumination light of several wavelengths from a polychromatic and preferably collinear primary light beam for illuminating the sample and directs it onto an illumination beam path for illuminating the sample.

[0038] As already described above with regard to a mechanical wave, it can be advantageously provided that the acousto-optic device with the several mechanical waves splits illumination light of several wavelengths from both a first polychromatic and preferably collinear primary light beam with a first linear polarization direction, and from a second polychromatic and preferably collinear primary light beam with a second linear polarization direction perpendicular to the first linear polarization direction, and directs it onto an illumination beam path to illuminate a sample, wherein one of the illumination light wavelengths is assigned to the frequency of the respective mechanical wave (in particular to fulfill the Bragg condition) for both polarization directions of the portion to be split off.

[0039] As already mentioned, it may be advantageous to provide that the first primary light beam and the second primary light beam have different polarization directions and / or that the first primary light beam and the second primary light beam are created by spatial splitting of a primary-primary light beam, for example with the help of a polarization beam splitter.

[0040] In particular, it can be advantageously provided that the first primary light beam and the second primary light beam are generated by spatial splitting of an unpolarized primary-primary light beam, for example a white light source, with a polarization beam splitter.

[0041] As already mentioned, it can be advantageously provided that the first primary light beam has the same linear polarization direction as the ordinary light diffracted by the mechanical wave in the first diffraction order, while the second primary light beam has the same linear polarization direction as the extraordinary light diffracted by the mechanical wave in the second diffraction order. In this way, it is achieved that both the first and the second primary light beams can be coupled into the acousto-optic device simultaneously, and that illumination light of the desired wavelength, corresponding to the frequency of the mechanical wave, can be directed from both primary light beams onto an illumination beam path using a mechanical wave for both polarization directions.

[0042] As previously explained, multiple mechanical waves can also be used to direct the light from each of the two primary light beams, specifically the light with the desired wavelengths, onto the illumination beam path. The advantage in every case is that only a single mechanical wave is required for each wavelength of illumination light.

[0043] In a particularly advantageous embodiment, the emitted illumination light exits the acousto-optic device and / or the crystal as a collinear beam of illumination light. This embodiment has the advantage that the illumination light can be focused onto or into the sample with a single objective lens, preferably illuminating the entire objective pupil.

[0044] In a particular embodiment, the crystal has an entrance surface for the detection light, wherein it can be provided, in particular, that the detection light enters at an angle of incidence of zero degrees. It can also be provided, in particular, that the crystal has an exit surface for the detection light, through which the detection light preferably leaves the crystal at an angle of reflection of zero degrees.

[0045] In a special design, the crystal has an entrance surface and an exit surface aligned parallel to it for the detection light.

[0046] In a particularly advantageous embodiment, the crystal has at least one entrance surface for the primary light from at least one light source. As already explained, the light source can advantageously be a light source that emits primary light with a broad spectrum, for example, using a microstructured fiber and / or a PBG fiber, so that the acousto-optic device can direct the light components of the desired wavelengths onto the illumination beam path of the microscope. Preferably, the desired illumination light is deflected by diffraction at the mechanical wave (or waves, if light of several wavelengths is desired as illumination light) into the first diffraction order, while the remaining light passes into a beam trap without interacting with the mechanical wave (or waves).

[0047] In a particularly advantageous embodiment, the crystal is provided to have at least one entrance surface for the primary light of at least one light source, which is simultaneously the exit surface for the detection light.

[0048] In particular, it may also be provided that the crystal has an exit surface for the illumination light which is simultaneously the entry surface for the detection light and / or that the crystal has an entry surface for primary light of at least one light source and an exit surface for the split illumination light which are aligned to each other in such a way that the illumination light deflected by the mechanical wave hits the exit surface at an angle of incidence of zero degrees.

[0049] A particularly advantageous embodiment is one in which the crystal has an entrance surface for primary light from at least one light source and an exit surface for the split illumination light, which are aligned to each other in such a way that the primary light can be coupled into the crystal as a collinear primary light beam and the illumination light deflected by the mechanical wave leaves the crystal as a collinear illumination light beam.

[0050] In order to achieve collinearity of the relevant light beams, an advantageous embodiment provides that the acousto-optic device has at least one dispersive optical component that compensates for a (at least partially) spatial color splitting of the primary light and / or the illumination light and / or the detection light caused by the crystal.

[0051] Alternatively or additionally, the acousto-optic device may also include at least one dispersive optical component that compensates for (at least partially) spatial color splitting of the primary light and / or the illumination light and / or the detection light caused by the crystal and that has several entrance surfaces; in particular, a first entrance surface for light with a first linear polarization direction and a second entrance surface for light with a second linear polarization direction perpendicular to the first linear polarization direction.

[0052] These embodiments have the particular advantage that the acousto-optic device can be equipped with a crystal that can have a relatively simple basic shape. For example, the entrance surface for the detection light can be designed to be parallel to the exit surface for the detection light. A spatial color splitting of the primary light from a light source, coupled in through the exit surface for the detection light, can be compensated for by the crystal, for example, by first splitting the primary light spatially in the opposite direction before it strikes the crystal, and then reversing this spatial splitting.

[0053] The microscope can be designed in a particularly advantageous way, for example, as a scanning microscope, especially as a confocal scanning microscope.

[0054] As already mentioned, the acousto-optic device according to the invention can in particular be designed as an acousto-optic main beam splitter for a microscope according to the invention.

[0055] In particular, it may be provided that the acousto-optic device is designed as an AOTF (Acousto Optical Tunable Filter) and / or has at least one AOTF.

[0056] The design of the acousto-optic device, in particular the orientation of the crystal, the propagation direction of the mechanical wave(s) and the propagation direction of the detection light beam relative to each other, and also the orientation of the inlet and outlet surfaces relative to each other and to the optical axis of the crystal, can – especially for an embodiment in which the emitted illumination light leaves the acousto-optic device and / or the crystal as a collinear illumination light beam – be developed, for example, according to the iterative method described below, whereby the method is preferably not carried out using real components, although this would also be possible, but rather in a computer simulation until the individual parameters of the crystal shape, the orientation of the surfaces and the crystal lattice,The orientation of the propagation direction of the mechanical wave(s) and the propagation directions of the detection light and / or the illumination light must meet the desired requirements. If all relevant parameters have been determined in this way in a computer simulation, the crystal can then be manufactured in a further step.

[0057] First, an acousto-optic component with a known, commercially available crystal cut and orientation is inserted into the beam path of a microscope such that the detection light beam strikes the crystal's entrance surface at an angle of incidence of 90 degrees. The crystal is then preferably rotated in the plane defined by the incident detection light beam and the propagation direction of the mechanical wave, thereby changing the angles between the propagation direction of the mechanical wave and the crystal axes, until both linear polarization components of the illumination wavelength(s) are deflected from the detection light beam and thus removed from the detection light beam.

[0058] However, rotation typically results in the loss of collinearity of the emerging detection light. Therefore, in the next iteration step—without rotating the crystal—its shape is modified so that the entrance surface is again perpendicular to the incident detection light beam. In a further step, the exit surface for the detection light is aligned—without rotating the crystal, but by changing its shape—so that the detection light exits the crystal as a collinear beam.

[0059] However, changes to the crystal shape generally result in the mechanical wave no longer deflecting both linear polarization components of the illumination wavelength(s) and thus removing them from the detection light beam. For this reason, the crystal is rotated again until this condition is met. Then the further iteration steps already described are repeated.

[0060] The process continues with as many iteration cycles as necessary until the conditions for simultaneous deflection of both linear polarization components and collinear light emission are met. The method typically converges very quickly, so the goal is achieved after only a few iteration cycles.

[0061] In a particular embodiment, when rotating the crystal, care is taken to ensure that, with respect to one of the linear polarization directions of the detection light, all first-order diffracted light exhibiting the wavelengths of the illumination light exits the crystal collinearly. Such an embodiment not only has the advantage that both components of differing linear polarization can be removed from the detection light beam with a single mechanical wave, but also that multicolored illumination light can be diffracted collinearly onto the illumination light beam via the light path of the first diffraction order, where the collinearity described above exists.

[0062] The invention is schematically depicted in the drawing and is described below with reference to the figures, where elements with the same effect are designated with the same reference numerals. The figures show: Fig. 1 shows an embodiment of a microscope according to the invention and Fig. 2 shows an embodiment of an acousto-optic device of a microscope according to the invention.

[0063] Fig. 1 shows an embodiment of a microscope according to the invention designed as a confocal scanning microscope.

[0064] The microscope includes several light sources 1, whose light is combined to form a collinear primary light beam 2. Specifically, the microscope includes a diode laser 3, a preferably diode-pumped solid-state laser 4 (DPSS laser), a helium-neon laser 5, and an argon-ion laser 6, whose emission beams are combined using dichroic beam splitters 7. The combined emission beams then pass to an AOTF 8 (acoustic optical tunable filter), which allows light of specific wavelengths to be selected and directed from the combined emission beam. For this purpose, the AOTF 8 is stimulated with electromagnetic high-frequency waves from a high-frequency source 9, whereby the user can specify the wavelength or wavelength by inputting a command via a PC 10.The wavelengths of the transmitted light are specified, and the frequency of the high-frequency source 9 is automatically adjusted accordingly by the microscope. Within the AOTF 8, mechanical waves are generated using electromagnetic high-frequency waves to diffract the desired light.

[0065] The microscope also features an acousto-optic beam combiner 11, which receives on the one hand the light passed on by the AOTF 8 and on the other hand the light from a white light source 12.

[0066] Such a white light source 12 can, in particular, comprise a special optical element, such as a microstructured optical element and / or a tapered fiber and / or a photonic crystal fiber and / or a photonic crystal and / or a photonic band-gap material and / or a comparable optical nonlinearity-inducing element, which spectrally broadens the incident primary light, in particular the light from a pulsed laser. Such a light source can provide primary light with a spectral width of several tens of nm, in particular several hundred nm.

[0067] The acousto-optic beam combiner 11 is also stimulated by electromagnetic high-frequency waves supplied by the high-frequency source 9. The acousto-optic beam combiner 11 includes an acousto-optic element in which mechanical waves for deflecting or diffracting light beams can be generated using the electromagnetic high-frequency waves such that the light transmitted by the AOTF 8 and the light from the white light source 12 leave the acousto-optic beam combiner 11 as a combined primary light beam collinear with each other.

[0068] The microscope also includes an acousto-optic device 13, which functions as the main beam splitter. In this respect, the acousto-optic device 13 has the task, on the one hand, of directing illumination light 14 of a desired wavelength or illumination light 14 of several desired wavelengths onto an illumination light beam path 15, and on the other hand, of removing from the polychromatic and collinear detection light beam 18 (shown as a dashed line) emanating from the illuminated sample 16, which is arranged on a stage 17, the portions of illumination light 14 that are scattered and / or reflected by the sample 16.

[0069] The illumination light 14, directed by the acousto-optic device 13 onto the illumination beam path 15, reaches a beam deflection device 24 which includes a first galvanometer mirror 25 and a second galvanometer mirror 26. The remaining part of the primary light 2 is not affected by the mechanical wave(s) and enters beam traps (not shown).

[0070] After leaving the beam deflection device 24, the illumination light 14 reaches the scan lens 27, then the tube lens 28 and finally an objective 29, which focuses the illumination light 14 onto or into the sample 16.

[0071] The beam deflection device 24 guides the focus of the illumination light 14 preferably in a meandering pattern over or through the sample 16. Here, the galvanometer mirror 25 is responsible for the deflection in the X direction, while the second galvanometer mirror 26 is responsible for the deflection in the Y direction.

[0072] The acousto-optic device 13 is also subjected to at least one electromagnetic high-frequency wave in order to generate at least one mechanical wave of a specific frequency. By changing the frequency of the electromagnetic high-frequency wave, the frequency of the mechanical wave can be changed. The generation of the mechanical wave can be achieved, for example, using a piezoelectric sound generator.

[0073] A crystal (not shown in this figure) of the acousto-optic device 13, in which the mechanical wave propagates, and the propagation direction of the mechanical wave are aligned with the detection light beam 18 coming from the sample 16 such that the acousto-optic device 13 deflects with the mechanical wave both the portion of the detection light beam 18 having the illumination wavelength and a first linear polarization direction, and the portion of the detection light beam 18 having the illumination wavelength and a second linear polarization direction perpendicular to the first linear polarization direction, and thereby removes it from the detection light beam 18.The remaining portion 19 of the detection light beam 18 exits the crystal collinearly and, after passing through an AONF 20 (acoustic optical notch filter), an imaging optic 21, and a detection aperture 22, reaches a detector 23, which is preferably designed as a multiband detector. The electrical signals from the detector 23 are transferred to the PC 10 for further processing and evaluation.

[0074] The microscope is designed such that not only light of one wavelength can be directed as illumination light 14 onto the illumination beam path 15, and not only light of one wavelength can be removed from the detection light beam 18 coming from the sample 16, but that several mechanical waves are used simultaneously to remove illumination light of several different wavelengths - and both linear polarization directions - from the detection light and / or to direct illumination light of several different wavelengths - and both linear polarization directions - onto the illumination beam path, wherein, however, advantageously only a single mechanical wave is generated per illumination light wavelength in the crystal of the acousto-optic device 13.

[0075] The acousto-optic device 13 is supplied with a separate electromagnetic high-frequency wave, generated by the high-frequency source 9, for each illumination wavelength desired by the user. For the sake of completeness, it should be mentioned that the different high-frequency waves provided by the high-frequency source 9 for the AONF 20, the acousto-optic beam combiner 11, the AOTF 8, and the acousto-optic device 13 generally have different frequencies. However, it is also possible to design the acousto-optic components in such a way that two high-frequency waves of the same frequency can be used for at least two acousto-optic components.

[0076] Fig. 2 Figure 1 schematically shows an embodiment of an acousto-optic device 13 of a microscope according to the invention, which can function as a main beam splitter.

[0077] The acousto-optic device 13 includes a polarization beam splitter 33 which receives unpolarized primary light of several wavelengths, in particular unpolarized, broadband primary light 2, for example from a white light source. The polarization beam splitter 33 spatially splits the primary light 2 into a first primary light beam 34 and a second primary light beam 35, wherein the light of the primary light beams 34, 35 has linear polarization directions perpendicular to each other.

[0078] The acousto-optic device 13 comprises a crystal 30 with an entrance surface 31 for the detection light beam 18 (shown as a dashed line) coming from a sample (not shown here) and with an exit surface 32 for the remaining portion 19 of the detection light beam 18, which is ultimately directed to a detector (not shown here). The exit surface 32 for the remaining portion 19 is simultaneously the entrance surface for coupling the first primary light beam 34 and the second primary light beam 35 into the crystal 30.

[0079] A piezoelectric sound generator 36 is arranged on the crystal 30. This generator is subjected to an electromagnetic high-frequency wave of one frequency or to several electromagnetic high-frequency waves of several frequencies to generate a mechanical wave or several different mechanical waves. Using the mechanical wave or the multiple mechanical waves, the light components of a desired illumination wavelength or the light components of several desired illumination wavelengths can be deflected by diffraction from both the first primary light beam 34 and the second primary light beam 35, and thus directed collinearly onto an illumination beam path 15. Each frequency of the respective mechanical wave (in particular, to fulfill the Bragg condition) is assigned to one of the illumination wavelengths – simultaneously for both linear polarization directions.

[0080] The illumination light 14 leaves the crystal 30 through the entrance surface 31 for the detection light beam 18, which is thus simultaneously the exit surface for the illumination light 14.

[0081] Simultaneously, the component of light exhibiting the wavelength(s) of the illumination light 14 is removed from the detection light beam 18 (shown with a dashed line) by means of the mechanical wave(s). The crystal 30 and the propagation direction of the mechanical wave(s) are aligned with the detection light beam 18 coming from the sample 16 such that each mechanical wave deflects both the component of the detection light beam 18 exhibiting the illumination wavelength and a first linear polarization direction, and the component of the detection light beam 18 exhibiting the illumination wavelength and a second linear polarization direction perpendicular to the first linear polarization direction, thereby removing it from the detection light beam 18. The remaining component 19 of the detection light beam 18 exits the crystal 30 collinearly through the exit surface 32.The orientation of the crystal structure is schematically indicated in the figure by drawing the crystal axes

[001] and

[110] .

[0082] The first primary light beam 34 is coupled into the crystal 30 opposite to the direction of the first diffraction order of the ordinary light (with respect to the diffracted component of the detection light), while the second primary light beam 35 is coupled into the crystal 30 opposite to the direction of the first diffraction order of the extraordinary light (with respect to the diffracted component of the detection light). In this way, the entire primary light 2 is available to direct illumination light 14 of a specific wavelength or wavelengths from this primary light 2 onto the illumination beam path of the microscope and thus to the sample using the acousto-optic device, whereby only a single mechanical wave of a single frequency is required for each wavelength.

[0083] In order to achieve collinearity of the illumination light beam 14 emerging from the crystal 30, a dispersive optical component 37 is placed upstream of the crystal 30, which spatially splits the first primary light beam 34 and the second primary light beam 35 spectrally, wherein the extent of the spatial splitting (in particular by selecting the angles and / or the optical path lengths) is determined in such a way that it is reversed by the crystal 30.

[0084] The dispersive optical component 37 has an input and output coupling window 38 for the first primary light beam 34. Initially, the first primary light beam 34 enters the dispersive optical component 37 through the input and output coupling window 38 and, after passing through the dispersive optical component 37, is reflected by a first mirror 39, which is attached to a surface opposite the input and output coupling window 38, before the first primary light beam 34, spatially and spectrally split, exits the dispersive optical component 37 through the input and output coupling window 38.

[0085] Similarly, the dispersive optical component 37 has a further input and output window 40 for the second primary light beam 35, which is deflected by a deflecting mirror 42. First, the second primary light beam 35 enters the dispersive optical component 37 through the further input and output window 40 and, after passing through the dispersive optical component 37, is reflected by a second mirror 41, which is attached to a surface opposite the further input and output window 40, before the second primary light beam 35, spatially and spectrally split, exits the dispersive optical component 37 through the further input and output window 40.

[0086] The dispersive optical component 37 also has an input coupling surface 43 and a parallel output coupling surface 44 for the remaining portion 19 of the detection light beam 18. The remaining portion 19 of the detection light beam 18 passes perpendicularly through both the input coupling surface 43 and the parallel output coupling surface 44 and therefore does not undergo any spectral splitting.

[0087] The invention has been described with regard to a particular embodiment. However, it is understood that modifications and adaptations can be made without departing from the scope of protection of the following claims.

Claims

1. A scanning microscope for observing fluorescent light, emitted by a sample, with an acousto-optical device (13), which removes, with a mechanical wave, which is characterized by a, preferably adjustable, frequency, from a polychromatic and collinear detection light beam (18), portions of illumination light (14), scattered and / or reflected from a sample (16), of an illumination light wavelength, associated with the frequency, wherein the acousto-optical device (13) contains a crystal (30) with an entrance surface (31) and an opposite exit surface (32) for the detection light beam (18), characterized in that the crystal (30) of the acousto-optical device (13), in which the mechanical wave propagates, and the propagation direction of the mechanical wave are aligned relative to each other and relative to the detection light beam (18), incident into the crystal (30), such that the acousto-optical device (13) with the mechanical wave deflects both the portion of the detection light beam (18) having the illumination wavelength and a first linear polarization direction, as well as the portion of the detection light beam (18) having the illumination wavelength and a second linear polarization direction perpendicular to the first linear polarization direction, and thereby removes them from the detection light beam (18), and that the remaining portion of the detection light beam (18) leaves the crystal (30) collinearly through the exit surface (32).

2. The scanning microscope according to claim 1, characterized in that a. the first linear polarization direction is the linear polarization direction of the ordinary light, related to a birefringence property of the crystal (30) and / or that b. the second linear polarization direction is the linear polarization direction of the extraordinary light, related to a birefringence property of the crystal (30) and / or that c. the first or the second linear polarization direction is arranged in the plane, spanned by the propagation direction of the mechanical wave and the propagation direction of the detection light beam (18).

3. The scanning microscope according to claim 1 or 2, characterized in that the remaining part of the detection light beam (18) leaves the crystal (30) collinearly.

4. The scanning microscope according to any one of claims 1 to 3, characterized in that the acousto-optical device (13) with the mechanical wave splits off illumination light (14) of an illumination light wavelength, associated with the frequency of the mechanical wave, from at least one polychromatic and preferably collinear primary light beam (34, 35) of at least one light source and directs it into an illumination beam path (15) for illuminating a sample (16).

5. The scanning microscope according to any one of claims 1 to 4, characterized in that the acousto-optical device (13) with the mechanical wave splits of both from a first polychromatic and preferably collinear primary light beam (34) of at least one light source (3, 4, 5, 6) with a first linear polarization direction, and from a second polychromatic and preferably collinear primary light beam (35) of at least one light source (3, 4, 5, 6) with a second linear polarization direction different from the first linear polarization direction, each illumination light (14) of the illumination light wavelength, assigned to the frequency of the mechanical wave, and directs it into an illumination beam path (15) for illuminating a sample (16).

6. The scanning microscope according to any one of claims 1 to 5, characterized in that the acousto-optical device (13) with several mechanical waves, each characterized by different frequencies, removes portions of illumination light (14) of several illumination light wavelengths, scattered and / or reflected from a sample (16) from the detection light beam (18), wherein each frequency is assigned one of the illumination light wavelengths and wherein each of the mechanical waves deflects both the portion having the illumination wavelength, assigned to its frequency, with a first linear polarization direction and also the portion with a second linear polarization direction perpendicular to the first linear polarization direction and thereby removes it from the detection light beam (18).

7. The scanning microscope according to claim 6, characterized in that the mechanical waves have the same propagation direction and / or are generated by the same sound generator (36).

8. The scanning microscope according to claim 6 or 7, characterized in that the acousto-optical device (13) with the plurality of mechanical waves splits off illumination light (14) of several illumination light wavelengths from polychromatic primary light of at least one light source, and directs it into an illumination beam path (15) for illuminating a sample (16).

9. The scanning microscope according to any one of claims 6 to 8, characterized in that the acousto-optical device (13) with the plurality of mechanical waves splits off illumination light (14) of several illumination light wavelengths from a polychromatic and preferably collinear primary light beam (34, 35) for illuminating the sample (16) and directs it into an illumination beam path (15) for illuminating a sample (16).

10. The scanning microscope according to any one of claims 6 to 9, characterized in that the acousto-optical device (13) with the plurality of mechanical waves splits of, from a first polychromatic and preferably collinear primary light beam (34) of at least one light source (3, 4, 5, 6) with a first linear polarization direction, as well as from a second polychromatic and preferably collinear primary light beam (35) of at least one light source (3, 4, 5, 6) with a second linear polarization direction perpendicular to the first linear polarization direction, respective illumination light (14) of several wavelengths and directs it into an illumination beam path (15) for illuminating a sample (16), wherein each of the illuminating light wavelengths is assigned to the frequency of the respective mechanical wave.

11. The scanning microscope according to any one of claims 4 to 10, characterized in that a. the first primary light beam (34) and the second primary light beam (35) have different polarization directions and / or that b. the first primary light beam (34) and the second primary light beam (35) are created by spatial splitting off a primary light beam and / or that c. the first primary light beam (34) and the second primary light beam (35) are created by spatially splitting off a primary-primary light beam with a polarization beam splitter and / or that d. the first primary light beam and the second primary light beam are created by spatially splitting off an unpolarized primary light beam (2) with a polarization beam splitter (33).

12. The scanning microscope according to any one of claims 4 to 11, characterized in that the splitt-off illumination light (14) leaves the acousto-optical device (13) and / or the crystal (30) as a collinear illumination light beam (14).

13. The scanning microscope according to any one of claims 1 to 12, characterized in that a. the crystal (30) has an entrance surface (31) for the detection light, through which the detection light enters at an angle of incidence of zero degrees and that b. the crystal (30) has an exit surface (32) for the detection light, through which the detection light leaves the crystal (30) with an angle of emergence of zero degrees.

14. The scanning microscope according to any one of claims 1 to 13, characterized in that a. the crystal (30) has at least one entrance surface (32) for the primary light of at least one light source (3, 4, 5, 6) and / or that b. the crystal (30) has at least one entrance surface (32) for the primary light of at least one light source (3, 4, 5, 6), which is simultaneously the exit surface (32) for the detection light. c. the crystal (30) has an exit surface (31) for the illumination light (14), which is simultaneously the entrance surface (31) for the detection light and / or that d. the crystal (30) has an entrance surface (32) for primary light from at least one light source (3, 4, 5, 6) and an exit surface (31) for the split-off illumination light (14), which are aligned with one another in such a way that the illumination light (14), deflected by the mechanical wave, strikes the exit surface (31) at an angle of incidence of zero degrees and / or that e. the crystal (30) has an entrance surface (32) for primary light from at least one light source (3, 4, 5, 6) and an exit surface (32) for the split-off illumination light (14), which are aligned with each other in such a way that the primary light can be coupled into the crystal (30) as a collinear primary light beam and the illumination light (14), deflected by the mechanical wave, leaves the crystal (30) as a collinear illumination light beam.

15. The scanning microscope according to any one of claims 1 to 14, characterized in that a. the acousto-optical device (13) has at least one dispersive optical component (37) which compensates for a spatial colour splitting of the primary light (2) and / or the illumination light (14) and / or the detection light (18) caused (at least partially) by the crystal (30) and / or that b. the acousto-optical device (13) has at least one dispersive optical component (37) which compensates for a spatial colour splitting of the primary light (2) and / or the illumination light (14) and / or the detection light (18) caused (at least partially) by the crystal (30) and which has a plurality of entry surfaces (38, 40) and / or that c. the acousto-optical device (13) has at least one dispersive optical component (37) which produces a spatial colour splitting of the primary light (2) caused (at least partially) by the crystal (30) and / or of the illumination light (14) and / or the detection light (18) and has a first entrance surface (38) for light with a first linear polarization direction and a second entrance surface (40) for light with a second linear polarization direction, perpendicular to the first linear polarization direction.

16. The scanning microscope according to any one of claims 1 to 15, characterized in that the scanning microscope is a confocal scanning microscope.