DE102014008098A1 - Spectrally flexible, fast-switching optical filter device - Google Patents

Abstract

The invention relates to an optical filter device (1) having a filter (5) which has regions with different spectral filter characteristics. However, the known variable filter devices have the disadvantage that the switchover between different spectral subregions to be removed takes a relatively long time, since the graduated filter has to be shifted and / or swiveled for this purpose. The invention is intended to enable rapid switching even with high spectral selectivity and strong suppression. For this purpose, two variably adjustable Strahlablenkvorrichtungen (6/7) are provided, wherein an incident on the first Strahlablenkvorrichtung light beam by means of the first Strahlablenkvorrichtung to different spectrally selective areas of the filter is deflected and the filter and the Strahlablenkvorrichtungen are arranged so that the deflected to the filter and Reflected or transmitted there light beam strikes the second beam deflecting device. This allows a more flexible, faster switching between different filter areas in less than 100 ms, since the beam deflecting devices, for example, in the embodiment as a rotatable mirror significantly smaller than the filter and thus much less sluggish so that they can be moved faster. fluorescence microscopy

Description

The invention relates to an optical filter device which comprises a filter, in particular with dichroic layers, which has regions ("spectrally selective regions") with different spectral filter properties arranged parallel to one of its (preferably planar) surfaces ("gradient filter"). The optical transmissivity of the filter is therefore different depending on the wavelength (and different in magnitude in the same range for different wavelengths) and for at least one wavelength in mutually different regions of the filter. The filter can be in one piece, or the different areas are arranged distributed on several pieces. Several pieces are expediently mechanically connected to each other, but need not be directly in contact with each other.

Spectral filter devices are used for the removal of a spectral subregion (which can also consist of several separate bands) from a light beam by spectrally selective reflection, transmission and / or absorption (suppression). For the purposes of the invention, the term light comprises any electromagnetic radiation which can be manipulated by optical means, that is to say in particular ultraviolet, visible and infrared radiation. A ray of light may be convergent, divergent or collimated.

Such filter devices can be used in particular in the microscopic imaging of luminescent (fluorescent or phosphorescent) samples. In fluorescence microscopy, the light coming from the sample ("sample light") contains on the one hand fluorescent light, on the other hand reflected and scattered illumination light ("excitation light"). Excitation light and fluorescent light have different wavelengths (colors), which allows their separation.

Dichroic mirrors are known in the art as optical filter devices. These can, for example, as in US 6,167,173 in a fluorescence microscope as a main color splitter for the optical coupling of the illumination beam path with the detection beam path (also referred to as "threading" of the illumination beam to the fluorescent beam) are used. Different geometries are possible, which affect the suppression factor for each unwanted light. This should be as high as possible.

In general, dichroic mirrors with vertical incidence (0 °) have maximum selectivity and suppression. In this case, however, no coupling of a plurality of beam paths is possible, so that a main beam divider deviates from 0 °, but is selected as small as possible. Originally as in US 6,167,173 used an angle of incidence of 45 °. Smaller but non-zero angles of incidence according to US 2008/0062511 A1 on the other hand, allow greater suppression of the spectral subrange to be removed in the transmission.

As a main color splitter (main beam splitter) such dichroic mirrors are usually used, which are designed as a notch filter. Long passes or broad band passes, on the other hand, are used as emission filters in the (pure) detection beam path. Emission filters may also be arranged at vertical incidence. For all applications, the spectral dependence of transmission and reflection at dichroic mirrors is static. This is particularly problematic in connection with spectrally adjustable or broadband emitting light sources, for example so-called multi-line or white-light lasers. In spectrally different excitation situations, only the instantaneous excitation light may be filtered prior to detection so as not to falsify the measurement result. This is especially true for the use of a suitable optical filter device as the main color splitter.

Necessary are optical filter devices that are variably adjustable with regard to the spectral subrange to be removed. They can be used for spectrally flexible illumination and / or for the flexible separation of illumination light and sample light by adapting the spectral subrange to be removed to the current illumination band (or the current illumination bands).

A color divider, which allows variability of the light source is, for example, in US 2010/188741 A1 described. It comprises a plurality of spectrally different filters, which are arranged on a wheel and thereby are selectively pivotable in the beam path. In this case, the color divider in the illumination direction may be preceded by a rotatable mirror for adjusting the angle of incidence on the color splitter to adjust the collinearity of at least one illumination laser to the optical axis. In particular, it may be a tunable laser.

Furthermore, in US 2002/0097485 A1 describes a filter device comprising a beam splitter and a mirror, wherein the beam splitter is formed as a dichroic mirror having a plurality of spectrally selective regions with different filter properties. Moving the beam splitter transversely to the light beam allows the Adjustment of the spectral characteristics of the main beam splitter to the illumination light. Due to the angle of incidence of 45 °, the suppression of the spectral portion to be removed is relatively small.

A stronger oppression allows the in US 2008/062511 A1 described filter device due to the there lower angle of incidence of the illumination light or the sample light of less than 45 °, in particular less than 20 °, on the surface of the main color splitter or disposed in the detection beam path further beam splitter. In this case, the further beam splitter can be designed as a variable graduated filter and arranged to be displaceable along the path. In addition to the shift is off DE 20 2008 016 287 U1 also a pivoting of the gradient filter relative to the light beam known to move the spectral position of the filter edges.

However, the aforementioned variable filter devices have the disadvantage that the switchover between different spectral subregions to be removed takes a relatively long time, since in each case at least one sluggish mechanical part has to be moved (filter has to be rotated in the filter wheel or the graduated filter has to be shifted and / or swiveled).

The invention has for its object to improve an optical filter device of the type mentioned in order to allow even with high spectral selectivity and strong suppression of the spectral portion to be removed fast switching between different to be removed spectral portions.

This object is achieved by an optical filter device having the features specified in claim 1.

Advantageous embodiments of the invention are specified in the subclaims.

According to the invention, two variably adjustable beam deflection devices are provided, wherein a light beam incident on the first beam deflection device can be deflected by the first beam deflection device to different spectrally selective regions of the filter and the filter and the beam deflection devices are arranged such that the light beam deflected to the filter and reflected or transmitted there (Irrespective of the location of these areas) encounters the second beam deflecting device. In the context of the invention, variably adjustable means that a beam deflecting device can be set with regard to the deflection direction (angular difference between the incoming and outgoing light beam).

Such a configured filter device allows a more flexible, faster switching between different filter areas in less than 100 ms, preferably even less than 10 ms, since the beam deflecting devices, for example in the case of the design as a rotatable mirror significantly smaller than the filter and thus significantly less sluggish, so that they can be moved faster than the filter itself. In this case, conventional, commercially available with little effort gradient filter and Strahlablenkvorrichtungen can be used. The second beam deflecting device serves to compensate for the directional error, which is impressed on the light beam by the deflection to different spectrally selective regions of the filter by the first beam deflecting device. The gradient filter can be designed so that the filter device allow continuous adjustment of the spectral beam splitting properties to the selected wavelength range to be separated without having to accept compromises with regard to the suppression in the filter direction. The transmission of a fluorescence signal is only insignificantly reduced by the additional optics in comparison to conventional, purely dichroic beam splitter solutions.

The invention can be used particularly advantageously in the image-wise multiple tracking of a plurality of different types of fluorescent dyes, since, in conjunction with rapid wavelength switching by the excitation light source, a different wavelength is set for each image acquisition passage and thus a different dye is excited can. On the one hand, this minimizes crosstalk in the excitation light among the dyes and, on the other hand, optimizes the image contrast since, for example, edge filters with a high suppression in the non-transmitted spectral range have a steep transition, typically narrower than 2 nm, between transmissions and blocking area. Thus, only small filter losses are added to the fluorescent light.

Preferably, the filter and the beam deflection devices are arranged such that the light beam transmitted to the filter or transmitted through the filter (for each of the different spectrally selective regions of the filter) is deflectable by the second beam deflection device so as to be parallel, in particular coaxial, to the incident light beam runs. Conveniently, the filter device may comprise a control unit, which is connected to the setting of the Strahlablenkvorrichtungen with these and is adapted to set the first beam deflecting device in response to a predetermined or predetermined parameter and the second beam deflecting device to adjust that it deflects the light beam reflected on the filter or transmitted through the filter so that it is parallel, in particular coaxial, to the incident light beam.

Preferably, the first and / or the second beam deflection device is a (respective) mirror, in particular based on a microelectromechanical system (MEMS), or a (respective) acousto-optical element, in particular an acousto-optically adjustable deflector (AOD). Thus, the adjustable beam deflection is possible with little effort and high accuracy. The use of an acousto-optic element, by virtue of its additional spectrally selective effect, enables greater suppression of the spectral subarea to be removed.

Embodiments in which at least one of the beam deflecting devices (in particular both beam deflecting devices) are rotatable about two different axes are particularly advantageous. Thereby, the light can be moved two-dimensionally over the filter, so that a larger number of switching positions is possible. The fact that then can be deflected synchronously over two axes, the new switch position on average also faster reach. In particular, the axes of rotation can lie in one plane, preferably with mutually orthogonal course. Due to the fixed pivot point thus provided, the deflection is possible with less local error. Conveniently, the fulcrum may be in the surface of the beam deflector to minimize the location error.

A larger number of filter configurations may be provided if the regions having different spectral filter characteristics are distributed two-dimensionally parallel to the surface of the filter. In particular, a combination with a drive for displacing the filter transversely to a direction of deflection achievable by means of the first beam deflecting device is possible, for example if the first beam deflecting device is only rotatable about exactly one axis.

A simplified construction is possible because the second beam deflection device is formed by the first beam deflection device. This multiple use of the first beam deflecting device allows for a more compact design and the use of less complex optical components. This is achieved, for example, by arranging a mirror, in particular formed by the filter itself, in such a way that it reflects light deflected by the first beam deflecting device onto the filter and then reflected by the filter in a direction parallel to the light deflected onto the filter to the first beam deflecting device, and / or by providing a retroreflector such that it reflects light deflected by the first beam deflector onto the filter and subsequently transmitted through the filter, in a direction parallel to the light deflected by the filter away from the filter or through a colorless region of the filter to the first beam deflector. The second beam deflector is then virtual by being provided from the perspective of the filter as an offset mirror image of the first beam deflector (or its backside).

Preferably, the filter device may comprise a third variably adjustable Strahlablenkvorrichtung, which is arranged so that those spectral components of the light beam from the first Strahlablenkvorrichtung ago striking the filter, which do not pass from the filter to the second Strahlablenkvorrichtung encounter the third Strahlablenkvorrichtung. Analogously to the second beam deflecting device, the third beam deflecting device serves to compensate for directional errors and resulting spatial errors which are impressed on the first deflecting device by the light beam deflected toward the filter. In the case of use as a main color splitter, the second beam deflecting device is responsible, for example, for the spectral portion corresponding to the excitation light to the sample light coming from the objective and the third beam deflecting device for the spectral portion of the sample light directed to the detector.

The output-side beam position can remain constant with higher accuracy if optically a (preferably around the same axis as the first Strahlablenkvorrichtung) variably adjustable Hilfsstrahlablenkvorrichtung or a Kollimationsoptik is disposed between the first Strahlablenkvorrichtung and the filter and / or optically between the second Strahlablenkvorrichtung and the filter further (preferably around the same axis as the second beam deflection) variably adjustable Hilfsstrahlablenkvorrichtung or for collimating optics inversely acting Dekollimationsoptik is arranged and / or optically between the third Strahlablenkvorrichtung and the filter another variably adjustable Hilfsstrahlablenkvorrichtung or for collimating optics inversely acting decollimation optics is arranged. The auxiliary beam deflection device is used to compensate for a local error imposed on the light beam by the first beam deflection device. Accordingly, by means of the other auxiliary beam deflection device, a positional error impressed on the light beam by the second beam deflection device can be compensated. By appropriate collimation and Dekollimationsoptiken such a location error can be avoided from the outset.

Advantageously, the filter device may comprise collection optics for reducing the beam cross section on the filter. As a result, the spectrally differently selective regions can be made smaller, so that the filter size can be reduced or a larger number of filter configurations can be provided with the same filter size. In addition, the surface area reduces over which the filter function is integrated during gradient filters. Accordingly, the selectivity of the arrangement increases when using graduated filters. In this case, optics for reversing the focusing effect of the collection optics can be arranged on the opposite side of the filter. Thus, an identical beam geometry can be achieved on the input and output side of the filter device.

The filter device described above can advantageously be used in a microscope, in particular for the detection of luminescence, preferably as a main color splitter for optically coupling an illumination beam path comprising a light source, and a detection beam path comprising an optoelectronic detector, which from the filter on the second beam deflecting incident by the second beam deflecting device into a lens of the microscope, in particular by a pupil of the lens, or as an excitation filter in the illumination beam path, in particular outside the detection beam path, or as a sub-color splitter in the detection beam path, in particular outside the illumination beam path, or as an emission filter in Detektionsstrahlengang, in particular outside the illumination beam path.

An embodiment of the microscope in which the filter is arranged in or at least approximately in an intermediate image in order to provide increased selectivity is advantageous. The beam deflecting devices are then preferably arranged in or at least approximately in (to the pupil plane of the objective) conjugate pupil planes. Alternatively, it may be advantageous if the filter is arranged in or at least approximately in a conjugate pupil plane and the (reflecting surfaces of) the beam deflecting devices in intermediate image planes. Although the selectivity is lower in this alternative, the Aufpunkte are constant and thus correctable defects.

Such a microscope may preferably comprise a tunable laser directed (directly or indirectly) to the first beam deflecting device of the filter device (which is used as a main color splitter). This allows different measurements within a wide spectral bandwidth with short switching times. The filter can be designed as a notch filter (with different spectral position of the notch in the different spectrally selective regions of the filter). This also applies to multiline lasers and spectrally broadband (non-tunable) emitting lasers.

For the purposes of the inventors, a tunable laser is present if a wavelength range can be selected exclusively by means of an internal means of the laser or an external means, for example a spectrally selective modulator.

In addition, the microscope may include a (static) color splitter and at least one other laser directed (directly or indirectly) at this color splitter, the further color splitter being arranged to reflect light from the further laser to the second beam deflector. In this way, excitation light of different wavelengths can be used simultaneously. This is particularly advantageous in living cell imaging, for example, if fast-running processes are to be investigated which, for example, already cause changes within an image acquisition time, so that the image information of two multi-tracking images no longer covers one another. Furthermore, two different highly efficiently filtered and rapidly switchable wavelengths can be provided for simultaneous excitation in special microscopic procedures such as Stimulated Emission Depletion (STED) or Coherent Anti-Stokes-Raman Scattering (CARS).

Alternatively or additionally, if the first and the second beam deflecting device are arranged in a first half space defined by the surface of the filter, the microscope can have another variably adjustable beam deflecting device which is arranged in a second half space (different from the first half space) a tunable laser directed (directly or indirectly) to the additional beam deflector, or a color splitter separating a spectral portion of the first tunable laser and directing (directly or indirectly) to the additional beam deflector, and a mirror separating one of the additional beam deflectors Beam deflection device deflected on the filter and reflected there light beam in its direction and reflected by the filter on the second beam deflecting comprise. This embodiment also makes it possible to simultaneously provide two different, quickly switchable excitation wavelengths with highly efficient filtering. When combined with a color divider as in the previous paragraph, three different excitation wavelengths can be provided simultaneously.

Particularly preferred embodiments are those in which the microscope as a light scanning microscope comprises at least one further beam deflecting device for scanning the light beam through a field of view of an objective of the scanning microscope, in particular with arrangement of the further beam deflecting device between the filter device and the objective, in particular in a common section of illumination beam path and detection beam path , and / or with a confocal aperture in the illumination beam path and / or with a confocal aperture in the detection beam path. In the sample-side focus, the light beam can have an arbitrary cross-sectional shape, for example punctiform or linear. In addition to spectral imaging with optical cutting, light-scanning microscopes enable a variety of correlative and manipulative examination methods, which are only limitedly possible, for example, in widefield microscopes. In addition, the manipulation of excitation and detection light in the light scanning microscope is much easier possible, because usually no image field is transmitted and these measures can be limited to blasting manipulations.

In addition, it is advantageous if the microscope comprises means for generating a plurality of disjoint (focused) illumination spots in the focus of the objective, which can be rastered simultaneously by the field of view by means of the further beam deflection device. By means of such a multi-spot scanning ("multi-spot scanning"), the recording can be accelerated or the load on the sample can be reduced. The illumination spots can have any (preferably identical) shapes, for example (quasi) punctiform or linear, and any (preferably regular) relative arrangements to one another, for example in the form of a matrix.

The gradient filter can be designed as a spectral longpass, shortpass, bandpass, bandstop, multi-bandpass, multi-bandstop filter or notch filter. These properties may be in transmission or reflection. In particular, the filter can have at least one filter region which is color-neutral and / or polarization-selective.

The invention also includes a method for operating a microscope, in particular as described above, with a filter device as described above and a tunable laser, wherein an adjustment of the tunable laser and an adjustment of the beam deflecting the filter device by means of a control unit synchronously, in particular simultaneously performed ,

The invention will be explained in more detail by means of exemplary embodiments.

In the drawings show:

1 a light scanning microscope with a first optical filter device as a flexible main color splitter and a single multispectral laser,

2 a light scanning microscope with a second optical filter device as a flexible main color splitter and a single multispectral laser,

3 a light scanning microscope with the second optical filter device as a flexible main color splitter and a further main static color splitter and a tunable laser and a plurality of static lasers,

4 a light scanning microscope with a third optical filter device as a flexible main color splitter and two tunable lasers,

5 a section of the ray trajectories of another light-scanning microscope including imaging elements,

6 a section of the ray trajectories of another light-scanning microscope including imaging elements,

7 a section of the ray trajectories of another light-scanning microscope including imaging elements,

8th a section of the ray trajectories of another light scanning microscope including imaging elements and

9 a section of the beam paths of another light scanning microscope including imaging elements.

In all drawings, like parts bear like reference numerals.

1 shows in highly schematic representation of an optical filter device 1 in a microscope 2 as a main color splitter HFT for the optical coupling of the illumination beam path B, the tunable laser as a light source 3 comprising, with a detection beam D, the one detector 4 includes. For reasons of clarity, no imaging elements such as lenses are shown. The filter device 1 here includes a dichroic gradient filter 5 with a plurality of spectrally selective areas with, for example, different short-pass filters, a first variably adjustable beam deflection device 6 in the form of a galvanometer mirror and a second variably adjustable Strahlablenkvorrichtung 7 in the form of a galvanometer mirror and not shown drives for the beam deflectors. In the detection beam D are also a confocal diaphragm 8th and for example an optional emission filter 9 arranged for spectral or polarization optical filtering.

In alternative embodiments (not shown), the deflection devices 6 and 7 each may be formed, for example, as a single MEMS mirror or as an AOD. In the case of a MEMS mirror, this can preferably be tilted over two orthogonal axes lying in the same plane. To the possibilities of 2-axis tiltable deflection devices 6 / 7 exploit the spectrally different selective areas of the gradient filter 5 be arranged in several columns and rows. With one scan direction, the column is selected, with the other the line. This makes it possible to increase the spectral resolution and / or to accommodate different spectral properties in several "floors" on a filter substrate. In the case of an AOD, a driver circuit should be provided instead of a drive. In this case, the high speed of acousto-optic scanners can be advantageously used with simultaneous improved filter suppression.

The gradient filter 5 can basically have all the spectral properties known from interference filters. Instead of a short pass (KP), it can be designed as longpass (LP), bandpass (BP), bandstop (BS, also called notch filter in very narrow BS), multi-bandpass (MBP) and multiband-lock (MBS).

The emission filter 9 may for example be designed as a motorized sliding gradient filter or as a selectable filter in a motorized filter wheel. In particular, it can also be designed as a multi-bandpass, the spectral on N positions of the Strahlablenkvorrichtungspaares 6 and 7 is tuned. Alternatively, filters 9 in combination with detector 4 also be designed as a spectrally resolving device.

The laser beam, which serves to luminescence excitation in the sample P, first meets the first scan mirror 6 which is capable of selectively changing the direction of gravity of the laser beam after reflection within a few milliseconds. It serves to direct the laser beam at any point of a gradient filter 5 to let strike. The gradient filter 5 is characterized by the fact that its spectrally selective property varies spatially, which is achieved, for example, by optical interference layers whose thickness changes continuously or in small steps. Ideally, when the location of the radiation to be filtered varies, only the spectral filter edges shift in proportion or at least monotonically to the location. The gradient filter used here by way of example 5 has the well-known feature of interference filters that the non-transmitted radiation is almost completely reflected, so that absorption can be neglected.

The location of the laser beam on the filter 5 is done with the help of scanner 6 adjusted so that the laser beam wavelength is just before the spectral edge of the short pass, so that the laser beam largely through the filter 5 is transmitted. Subsequently, the laser beam hits the second scanner 7 , which for example is identical to scanner 6 is. scanner 7 serves the purpose of scanner 6 compensate for directional change, causing the laser beam after reflection to scanner 7 always has the same propagation direction. The microscope 2 is for training as a light scanning microscope ("LSM") with an additional variably adjustable beam deflecting device 10 equipped, by means of which the excitation spot, for example, in two dimensions on the sample P is rasterbar. Thereafter, the laser beam is directed to the sample P.

The emitted by the sample luminescence, as a rule has a greater wavelength than the laser radiation. Therefore, the luminescence after the deflection at the LSM scanner 10 and on the scanner 7 from the gradient filter 5 reflected. The confocal aperture 8th serves to suppress extra-focal radiation. It consists for example of a pinhole, but can (not shown) also consist of a slot, a hole matrix, a grating aperture or an even more complex transmission structure.

Behind the confocal aperture 8th meets the luminescence radiation on the photosensitive detector 4 , which is designed, for example, as a photomultiplier (PMT). Alternate embodiments (not shown) may be a photodiode (PD), an avalanche photodiode (APD), or a camera.

The function of scanner 7 can alternatively (not shown) can be achieved by the laser beam once again via scanner 6 is passed, with the front or back of scanner 6 can be used. As a result, a scanner can be saved, but in turn can increase the complexity of the optical structure.

In 2 is again shown in highly schematic representation of an embodiment in which an LP gradient filter 5 (instead of KP as in 1 ) is used. Again, the first scanner steers 6 that of the laser 3 next laser beam on the gradient filter 5 , The point of impact of the laser beam is determined by means of a scanner 6 chosen so that the Laser beam wavelength just below the spectral edge of the LP gradient filter 5 lies. Therefore, the laser beam is reflected therefrom and via the second scanner 7 about the additional scanner 10 passed to sample P. In contrast to 1 Here is the luminescence radiation at the gradient filter 5 transmitted before going to the detector 4 arrives. For the rest, structure and functionality are in line with the plan 1 match. The illustrated embodiment has the advantage that the excitation light reflected or scattered by the sample P or by optical elements, for example objective lenses (not shown), from the gradient filter is of high efficiency 5 is trapped and thus not to the detector 4 arrives.

3 shows - also in highly schematic representation - an embodiment such as 2 , in which, however, excitation lasers of different wavelengths, here a tunable laser 3 and several static lasers 3A . 3B to 3N (N = C, D, E, ...), can be combined with each other simultaneously. In these cases, the spectrally selective areas of the filter point 5 instead of long-pass or short-pass filters spectrally different notch filters on, with the spectral position of the stopband with the position on the filter 5 varied. The tunable laser 3 This is done by means of the first scanner 6 to the position on the filter 5 steered, at which this a high reflectivity for those at the laser 3 has set wavelength of light. Thus, light of the tunable laser 3 deflected toward sample P for fluorescence excitation, using scanners 7 turn the deflection of scanner 6 counteracts. Because of the narrow band stopband of the selected notch filter, light of larger and lesser wavelengths passes through the filter 5 According to the prior art, a conventional dichroic can be used as another beam splitter 11 the lasers 3A . 3B . 3N Thread fixed wavelength on the illumination beam B. This is particularly advantageous for manipulation experiments such as FRAP or uncaging since fixed wavelength lasers in the visible part of the spectrum generally have a greater spectral power density. In this case, the other beam splitter 11 arranged in a Teilerrad (not shown) or as a sliding gradient filter or as a further filter device 1 be educated.

Another embodiment is in 4 presented in the most schematic way. subfigure 4B illustrated on the edge very schematically the part in figure 4A unrecognizable spatial dimension of the beam paths. Here are two independently tunable laser 3 . 12 aufgefädelt on the illumination beam B. For this purpose, the filter device 1 an additional beam deflector 13 and another expediently adjustable beam deflecting device M on. Using the scanner 6 and 13 the two resulting beams are at the spectrally selective areas corresponding to the wavelengths set on the filter 5 distracted. The scanner 7 compensates the deflection by the scanner 6 and 13 and directs the illumination light toward additional scanner 10 from. The fluorescence emitted by sample P passes through the filter twice in different spectrally selective regions in the opposite way 5 and thereafter the detection arrangement 4 fed without once again on the scanner 7 to be reflected, with the filter 5 for threading the of the selected emission wavelength of the tunable laser 3 different emission wavelength of the tunable laser 12 through scanners 13 is used in two different levels. That's why there are at least scanners 7 and scanners 13 designed for beam deflection in two dimensions.

In alternative embodiments (not shown), the laser may emit at least two relevant, independently adjustable wavelengths which are separated by a dichroic beam splitter.

By using imaging optics and folding the beam paths by means of plane mirrors and retroreflectors (not shown), the number of necessary scanners in 1 to 3 to one and in 4 reduce to two. In addition, by focusing the light beams on the gradient filter, the spectral resolution of the arrangement can be increased and the size of the filter substrate can be reduced.

5 shows a section of the ray trajectories in a microscope 2 with a filter device 1 in detail. It is a microscope as in 2 but with a third variable beam deflector 20 equipped to pass through the scanner 7 caused direction errors in the detection beam D to compensate. The scanners 6 . 7 and 20 are in optically conjugate planes. The scanner 7 is for example by means of a lens 14 both on scanner 6 as well as on scanners 20 displayed. Focusing the laser beam by means of an exemplary lens 15 in front of the scanner 6 reduces the accuracy requirement on the scanner 6 and has the advantage that the confocal pinhole 8th without additional optics in the "descanned" beam behind the scanner 20 can be arranged. A disadvantage here is that the gradient filter 5 is used for different excitation wavelengths at different angles. This causes a spectrally varying astigmatism in the convergent beam. To reduce this, there are small angles of incidence on the gradient filter 5 and a small numerical aperture of the focus on the pinhole 8th advantageous. The Lens 21 The excitation radiation propagated through the array collimates to cause the optical interface 10 for arranging the beam deflecting devices of the light scanning microscope 2 provide.

In 6 is a 5 corresponding embodiment shown, however, with collimating optics 16 . 17 and a decollimation optics 18 is equipped so that each filter 5 incident light beam is collimated. This will create an in 5 occurring astigmatism avoided.

7 shows by way of example how the number of necessary beam deflection devices can be reduced by folding the illumination beam path B. scanner 6 represents here by the reflection on the filter 5 also the function of the second beam deflection device (scanner 7 in 6 ) ready. Illumination and detection beam paths can be separated via the beam angle as illustrated or alternatively (not shown) over the beam location in a fourier inverse plane.

In 8th is shown as the third beam deflector (scanner 20 in 6 and 7 ) can be omitted. For this purpose, the scanner 6 directly on the confocal aperture 8th imaged and a two-dimensional spatially resolving detector 4 used. With a 2D sensor 4 behind the aperture 8th and without dispersive elements, different elements of the sensor can be read out without a deflection compensation.

In an extended embodiment, a spectrally resolved detection without "descanning" the detection beam path D can be realized when scanner 6 performs only a one-dimensional scan and a dispersive element (not shown) with a dispersion direction transverse to this scan direction in front of the detector 4 is arranged. The usable aperture of the dispersing element must be sufficiently large to accommodate the respective deflecting directions of the scanner 6 to be able to record. The resulting dispersed image then rests in the sensor plane of the detector 4 , For monofocal imaging, a linear sensor array is then used as the detector 4 sufficient. Multifocal imaging requires a 2D sensor as a detector 4 , The advantage of such an arrangement is the low number of optics and thereby an increase in the detection sensitivity to the fluorescent signal.

Finally shows 9 an embodiment with folding as in 7 in which a filter 5 can be used with a high density of spectrally different selective areas. For this purpose are optics 19 provided which cross section of the filter 5 Reduce the incident light rays by placing them on the filter 5 focus or focused light rays on the aperture 8th depict. The first scanner 6 is doing on the third scanner 20 displayed. This embodiment can also be realized without folding (not shown).

With the above-described filter devices as the main color splitter, methods such as rapid multi-tracking with a fast-switching, tunable laser are possible, as described, for example, in US Pat "High power ultra-wide tunable femtosecond pulses from a non-collinear optical parametric oscillator (NOPO)" by Lang et al. in Optics Express 20 (2012), p. 912 , wherein a clean separation of the fluorescence signals can be done and spectral crosstalk is significantly reduced. For this purpose, the laser and the main color splitter are controlled simultaneously in a corresponding image recording method. In this case, for example, a control of the laser by the main color splitter or its control unit and vice versa possible. Due to the high switching speeds that can be achieved, line-by-line multitracking is possible. This is particularly important for dynamic samples, as in the time between two full frames may have occurred changes in the sample, which are otherwise misinterpreted in the recorded images, for example in the context of a co-localization analysis.

In addition, the full spectral range of a tunable laser with a high spectral resolution and thus a nearly continuous adjustment of the spectral division behavior can be used. For example, this allows fine tuning to the best possible spectral excitation / emission position for a given dye. This maximum, which does not necessarily have to lie in the excitation maximum of the dye, can be determined adaptively, for example by means of a feedback algorithm, due to the high switching speed of the invention. For this purpose, the excitation wavelength in combination with the point used for filtering on the gradient filter is optimized to a maximum at one point of the sample containing the desired dyes.

LIST OF REFERENCE NUMBERS

1

filter means

2

microscope

3

light source

4

detector

5

filter

6

First variably adjustable beam deflection device

7

Second variably adjustable beam deflection device

8th

Confocal aperture

9

emission filter

10

Further beam deflecting device

11

Another beam splitter

12

Tunable laser

13

Additional beam deflector

14

lens

15

lens

16

collimating optics

17

(De-) collimating optics

18

Dekollimationsoptik

19

collection optics

20

Third variably adjustable beam deflection device

21

lens

C

control unit

B

Illumination beam path

D

Detection beam path

HFT

Main color splitter

M

Mirror / beam deflection

P

sample

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 6167173 [0004, 0005]
• US 2008/0062511 A1 [0005]
• US 2010/188741 A1 [0008]
• US 2002/0097485 A1 [0009]
• US 2008/062511 A1 [0010]
• DE 202008016287 U1 [0010]

Cited non-patent literature

• "High power ultra-wide tunable femtosecond pulses from a non-collinear optical parametric oscillator (NOPO)" by Lang et al. in Optics Express 20 (2012), p. 912 [0068]

Claims (17)

Optical filter device ( 1 ), which is a filter ( 5 ), in particular with dichroic layers, which has regions with different spectral filter properties arranged parallel to one of its surfaces, in particular for microscopes ( 2 ), characterized by two variably adjustable Strahlablenkvorrichtungen ( 6 . 7 ), wherein one on the first beam deflecting device ( 6 ) incident light beam by means of the first beam deflecting device ( 6 ) to different areas of the filter ( 5 ) is deflectable and the filter ( 5 ) and the beam deflecting devices ( 6 ) are arranged so that the filter ( 5 ) deflected and reflected there or transmitted light beam to the second beam deflecting device ( 7 ) meets. Filter device ( 1 ) according to claim 1, wherein the filter and the Strahlablenkvorrichtungen are arranged so that the filter is reflected or transmitted through the filter light beam from the second Strahlablenkvorrichtung deflected so that it runs parallel, in particular coaxial, to the incident light beam. Filter device ( 1 ) according to claim 2, comprising a control unit (C) which is connected to these for adjusting the beam deflecting devices and is adapted to set the first beam deflection device in response to a predeterminable or predetermined parameter and to set the second beam deflection device to control the first beam deflection device deflected at the filter or transmitted through the filter light beam deflects so that it runs parallel, in particular coaxial, to the incident light beam. Filter device ( 1 ) according to one of the preceding claims, wherein the first and / or the second beam deflection device is a mirror, in particular based on a microelectromechanical system, or an acousto-optical element, in particular an acousto-optically adjustable deflector. Filter device ( 1 ) according to one of the preceding claims, wherein at least one, in particular both, the beam deflecting devices are rotatable about two different axes, in particular with a position of these axes in a plane, in particular with mutually orthogonal course of the axes. Filter device ( 1 ) according to one of the preceding claims, wherein the regions with different spectral filter properties are distributed two-dimensionally parallel to the surface of the filter. Filter device ( 1 ) according to one of the preceding claims, wherein the second beam deflecting device is formed by the first beam deflecting device, in particular with a mirror, in particular formed by the filter itself, such that it realizes the light deflected by the first beam deflecting device onto the filter and subsequently reflected by the filter is reflected parallel to the light deflected onto the filter to the first beam deflection device, and / or with a retroreflector such that it deflects light deflected by the first beam deflecting device onto the filter and then transmitted through the filter, in real parallel to the light deflected onto the filter Filter or reflected by a color-neutral portion of the filter to the first beam deflecting device. Filter device ( 1 ) according to one of the preceding claims, comprising a third variably adjustable Strahlablenkvorrichtung, which is arranged so that those spectral components of the light beam from the first Strahlablenkvorrichtung ago striking the filter, which do not pass from the filter to the second Strahlablenkvorrichtung encounter the third Strahlablenkvorrichtung. Filter device ( 1 ) according to one of the preceding claims, wherein a variably adjustable Hilfsstrahlablenkvorrichtung or a Kollimationsoptik is optically arranged between the first beam deflecting device and / or optically between the second Strahlablenkvorrichtung and the filter another variably adjustable Hilfsstrahlablenkvorrichtung or a collimating optics inversely acting decollimation optics is arranged and / or optically arranged between the third beam deflecting device and the filter is a further variably adjustable auxiliary beam deflecting device or decollimating optics acting inversely to the collimation optics. Filter device ( 1 ) according to one of the preceding claims, comprising collecting optics for reducing the beam cross section on the filter, in particular comprising optics arranged on the opposite side of the filter, for reversing the focusing effect of the collecting optics. Microscope with an optical filter device ( 1 ) according to one of the preceding claims, in particular as a main color splitter for optically coupling an illumination beam path comprising a light source and a detection beam path comprising an optoelectronic detector, the light beam striking the second beam deflection device from the filter being transformed into an objective by means of the second beam deflection device of the microscope, in particular by a pupil of the lens, is deflectable, - Excitation filter in the illumination beam path, in particular outside the detection beam path, - Secondary color splitter in the detection beam path, in particular outside the illumination beam path, or - Emission filter in the detection beam path, in particular outside the illumination beam path. Microscope according to claim 11, comprising a tunable laser ( 3 ), which is directed directly or indirectly to the first beam deflection device of the filter device, in particular with formation of the filter as a notch filter. Microscope according to the preceding claim, comprising a color splitter ( 11 ) and at least one other laser ( 3A ... 3N ), which on the this color divider ( 11 ), wherein the color divider ( 11 ) is arranged so that it receives light from the further laser ( 3A ... 3N ) to the second beam deflecting device ( 7 ) reflected. Microscope according to one of claims 11 to 13, wherein the first and the second beam deflection device are arranged in a first half space defined by the surface of the filter, comprising - an additional variably adjustable beam deflection device ( 20 ), which is arranged in a second half space, - another tunable laser, which directly or indirectly to the additional beam deflecting device ( 20 ), or a color splitter which separates a spectral portion of the first tunable laser and directs it directly or indirectly to the additional beam deflecting device, and - a mirror which reverses in its direction a beam of light deflected and reflected by the additional beam deflecting device onto the filter and reflected by the filter onto the second beam deflecting device. Light scanning microscope according to one of claims 11 to 14, comprising at least one further beam deflecting device ( 10 ) for scanning the light beam through a field of view of an objective of the light scanning microscope, in particular with arrangement of the further beam deflecting device between the filter device and the objective, in particular in a common section of the illumination beam path and detection beam path, and / or with a confocal aperture in the illumination beam path and / or with a Confocal aperture in the detection beam path. Scanning microscope according to the preceding claim, comprising means for generating a plurality of disjoint illumination spots in the focus of the objective, which can be rastered by means of the further beam deflection device simultaneously through the field of view. Method for operating a microscope ( 2 ), in particular according to one of claims 11 to 16, with a filter device ( 1 ) according to one of claims 1 to 10 and a tunable laser ( 3 ), wherein an adjustment of the tunable laser ( 3 ) and a setting of the beam deflecting devices ( 6 . 7 ) of the filter device ( 1 ) by means of a control unit (C) synchronously, in particular simultaneously performed.

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