DE102010033722A1 - Method for eliminating unwanted radiation portions from light detected from illuminated sample by laser scanning microscope, involves passing acousto optical tunable filter through two pole components - Google Patents

Abstract

The method involves actuating an acousto optical tunable filter (AOTF). Illuminating light is faded out in a light case or a beam detour unit. A sample light is irradiated toward a detector. Unwanted light is faded out by the AOTF. The AOTF is passed through two pole components when irradiation takes place in two incident directions stretching along a plane, which is vertical to a propagation plane of a longitudinal wave of the AOTF and the irradiating directions symmetrical to the propagation direction. Independent claims are also included for the following: (1) a laser scanning microscope (2) a method for operating a laser scanning microscope.

Description

State of the art:

• Jang-Woo You et al., Opt. Express (2008), Vol. 16, no. 26 pp. 21505
• Donley et al., Rev. Sci. Instrum. (2005), Vol. 76, pp. 063112
• US 5377003
• US-6982818 B2
• US-6963398 B2
• US 6510001
• US 6654165
• US 2003/01 331 89

WO 99/42884 describes the use of an AOTF as the main color splitter in a laser scanning microscope. The coupling of illumination light and the hiding of excitation light take place via the first order of the AOTF. The sample light remains in the zeroth diffraction order. In DE 19936573 A1 something similar is described.

In DE 10241472 A1 a division of excitation and detection light is made using dispersive and polarization rotating elements.

The wavelengths are spectrally spatially separated.

shows the detection beam path of the ZEISS LSM 710 ( http://www.zeiss.de/C1256CFB00332E16/0/F7BE630BECC94A08C12573F3005483 F4 / $ file / 60-1-0001_e.pdf )

To represent a laser scanning microscope is still on DE 19702753 directed.

The light of two laser or laser groups LQ1 and LQ2 passes through main color splitter HFT1 and HFT2 for the separation of illumination and detection beam path, which can be configured as switchable dichroic filter wheels and can also be interchangeable to make the selection of wavelengths flexible, and reflect the illumination light first via a scanner, consisting preferably of two independent galvanometric scanning mirrors for the X and Y deflection, in the direction of a (not shown scanning optics SL and on this and the microscope objective in the usual way to the sample.) The sample light passes in the return direction through the divider HFT1 , HFT2 in the direction of detection D.

Here, the detection light passes first a pinhole PH via a Pinholeoptik upstream and downstream Pinholeoptik PHO and a filter arrangement F for narrow-band filtering unwanted radiation components, consisting for example Notchfiltern, and passes through a beam splitter BS, optionally with appropriate circuit via a transmissive component Extraction to external detection modules allows a mirror M and further deflections on a grid G for spectral splitting of the detection radiation.

The divergent spectral components split by the grating G are collimated by means of an imaging mirror IM and pass in the direction of a detector arrangement consisting of individual PMT1, PMT2 in the edge region and a centrally arranged multichannel detector MPMT.

Instead of the multichannel detector, a further single detector can also be used.

In front of a lens L1, there are two prisms P1, P2, which are displaceable perpendicular to the optical axis, in the edge region; which combine a part of the spectral components which are focused on the individual PMT1 and 2 via the lens L1. The remaining part of the detection radiation is collimated after passage through the plane of the PMT1 and 2 via a second lens L2 and spectrally separated directed to the individual detection channels of the MPMT.

By shifting the prisms P1, P2 it is possible to adjust in a flexible manner which part of the sample light is detected spectrally separated by the MPMT and which part is detected by the prisms P1, P2 summarized by PMT1 and 2.

problem

The contrast of the imaging in a laser scanning microscope is decisively determined by the efficiency of the suppression of the spectral components of the excitation radiation in the spectrum of the detection light. In essence, a suitable main color splitter enables the excitation light of one or more radiation sources to be threaded onto the detection beam path as well as to spatially separate the light reflected or scattered from the sample from the fluorescent light. Conventionally, main color separators consisting of one or more dichroic mirrors are used which are optimized for maximum suppression of a certain number of fixed regions in the spectrum of the transmitted radiation. However, the fixed filter properties of dichroic mirrors prove to be a disadvantage if the choice of excitation wavelengths is to be changed. On the other hand, although a main color divider which can be set to the desired excitation wavelengths ensures the desired flexibility with regard to the filter properties, However, such arrangements allow only a limited suppression of the excitation light in the detection beam path.

With the aid of an additionally arranged in the beam path of the detection light emission filter, it should be possible to hide the remaining spectral components of the excitation light from the detection beam path. In order to further ensure the flexibility of the overall system with regard to their filter properties, the emission filter should be selectively adjustable to the excitation wavelengths. Thus, the combination of a tunable main color divider and a tunable emission filter should enable a spectrally flexible and efficient suppression of the radiation components of the excitation light in the spectrum of the detection radiation.

The emission filter for use in a fluorescence microscope is an acousto-optical tunable filter (AOTF). This actively-switchable optical element makes it possible to remove the spectral components of the laser scattered or reflected by the sample from the detection beam path of the fluorescence. However, a one-time filtering only causes a suppression of the excitation radiation in the detection spectrum of less than 2OD (optical densities, corresponding to a transmission of 0.01). Furthermore, the radiation in conventional AOTFs due to the crystal cut by dispersion undergoes a spatial-spectral splitting. The single use of a single conventional AOTF requires additional post-filtering and optical correction of the spectral splitting.

Advantages, effects and description of the invention

An AOTF-based emission filter according to the invention, which allows the fluorescent light to pass through the AOTF twice, has the advantage that a high (> 3OD) and very narrow-band (<2 nm FWHM) suppression of excitation light in the spectrum of the fluorescent light is achieved In addition, the center wavelength to be filtered is freely adjustable to the laser wavelengths used. Furthermore, the arrangements according to the invention have a very high transmission of up to 90% for fluorescent light and allow the simultaneous filtering of several excitation wavelengths, wherein a drive frequency is switched for each polarization direction of a wavelength.

This is an advantage over known arrangements where the detection light is filtered only in a direction of polarization corresponding to the excitation radiation polarization. A comparable arrangement according to the prior art, based on dielectric notch filters, which are sequentially swiveled into the beam path depending on the laser wavelength used, would have a transmittance for fluorescent light of only 85% with simultaneous filtering of four excitation wavelengths and also significantly wider stop bands (ca. 10-25 nm FWHM depending on the wavelength) in the spectrum, which would be particularly at the expense of the detection of the fluorescence maximum.

The AOTF advantageously has mutually parallel entry and exit surfaces.

This has opposite the representation in the advantage that in the O. order a spectral splitting is avoided.

An AOTF according to 1a but with non-parallel entry and exit surfaces can be used for the invention when the dispersion in the zeroth order, for example by Kollimationsoptiken is compensated by appropriate expert-trained optics.

Furthermore, when using an AOTF after 1a the L1 and L2, for example in 2 on top of each other, which would not change your fade out.

Description of the pictures

List of used reference signs:

• AOTF, AOTF1, AOTF2:

  Acousto-optic tunable filter

  OA:

  Optical axis of the AOTF crystal

  D, D1, D2:

  Objektlicht

  ln:

  Laser 1 - n for sample illumination

  L1, L2:

  For example, two different wavelengths of Ln

  PBS:

  Polstrahlteiler

  s, p:

  polarization components

  M1-M12:

  mirror

  BB:

  Aperture, light blocker or light trap or light redirection

  BDP:

  Birefringent polarization prism

  QWP:

  Quarter-wave plate

  HWP:

  Half-wave plate

  F1, F2:

  Annular filter arrangement

schematically shows the principle of operation of an optical arrangement comprising an acousto-optically tunable filter (AOTF) and a spectrally flexible adjustable spatial separation of the selected spectral components of the incident light from the residual radiation allows. AW denotes therein the propagation direction of the acoustic wave in the AOTF.

An AOTF consists of an anisotropic birefringent optical crystal to which a piezocrystal is coupled. In general, the incoming unpolarized radiation undergoes a spatial separation into the perpendicularly linearly polarized ordinary and extraordinary rays as it traverses the crystal. By applying a radio frequency adapted to the selected wavelength and the polarization of the incident light to the piezoelectric crystal, an ultrasonic wave is generated in the AOTF crystal, which propagates through the crystal. In a non-collinear AOTF, the interaction of the propagating radiation with this sound wave causes a diffraction of a predetermined by the periodicity of the sound wave spectral component of the light, wherein the deflection is carried out depending on the polarization state in the first or minus first diffraction order. The remaining spectral components are transmitted and determine the zeroth diffraction order. Depending on the polarization state of the radiation, it is possible to select a plurality of narrow-band spectral regions from the light of a broadband source by means of an AOTF and to spatially separate them from the residual radiation. A suitable spatial filter makes it possible to hide these spectral components from the beam path. For the radiation in the zeroth diffraction order, an AOTF acts as a narrow-band, tunable notch filter, which suppresses selected components in the light spectrum.

In an inventive tunable emission filter is shown schematically.

Here, as in the other subsequent figures, the emission filter according to the invention in the detection beam path of an LSM as shown 11 by way of example, be arranged, advantageously behind the detection pinhole, in a collimated beam path (In 11 represented by the pinhole PH and the optics PHO).

Shown is sample light D with different polarization.

The portions of the illumination light In are exemplified by L1 and L2 having different wavelengths.

D and L together form the light D + L originating from the object.

Here, it is initially assumed that the illumination light generally has a linear polarization, which therefore only passes the pole splitter PBS in one direction.

This optical arrangement comprises a single AOTF which is traversed twice by the object light (D + L). The coupling of the object light (D + L) into the arrangement and the extraction of the radiation filtered by the unwanted spectral components in the direction of the detector takes place on a polarizing beam splitter cube (PBS). Here, the p-polarized object light (D + L) is transmitted and passes through the AOTF. By applying the appropriate frequencies to the AOTF, the radiation components of the excitation light (L1, L2) are diffracted into the first orders and masked out of the detection beam path by means of the suitable spatial filters (BB1). The filters BB can be light traps or beam deflecting units, which could possibly also enable a further utilization of the excitation light for the illumination of the sample. The filters BB are shown here in simplified form as room apertures with a passage for the sample light.

The p-polarized detection light (D1) passes through the PBS, remains in the zeroth order of diffraction of the AOTF and is reflected back into itself by means of a mirror (M1).

The light reflected at M1 traverses the AOTF a second time, allowing the detection light (D1) to be filtered again. In order to be able to separate the incoming and outgoing radiation at the polarizing beam splitter (PBS), an achromatic quarter wave plate (QWP) is arranged between the AOTF and the mirror (M) for rotation of the polarization plane of the p component. The detection light (D1) again traverses the AOTF, the polarization state of the reflected radiation portions being rotated by 90 ° as a result of a double pass through the quarter wave plate. A second set of frequencies, adapted to the resulting s-polarization of the selected spectral components, again causes a spatial separation of the radiation components of the excitation light (L1, L2) from the detection light (D1). By means of the spatial filter BB2, the radiation scattered or reflected by the sample is masked out of the detection beam path. Finally, the s-polarized detection light (D), which has been cleared of the spectral components of the excitation light, undergoes a deflection at the polarizing beam splitter (PBS) and is directed in the direction of the detector.

The s-polarized component of the radiation coming from the sample (D + L), which consists essentially of the sample light, in particular Fluorescence light is reflected on the PBS and with the aid of an arrangement consisting of a quarter wave plate (QWP) and a mirror (M2) rotated as p-polarized detection light (D2) coupled to the detection beam path again. In this case, it is assumed that the illumination radiation used for the excitation is p-polarized and the state of polarization is maintained after the interaction with the sample. Therefore, the s-polarized portion of the object light (D + L) is considered to be almost free of radiation components of the excitation light.

The now reflected p component reflected at M2 passes through the PBS in the direction of the detection DE.

In is a further inventive tunable emission filter in side view and plan view from above shown schematically.

In extension of Here both the s and the p portion of the sample light, preferably the fluorescent light, filtered by the AOTF.

This optical arrangement makes it possible, by means of a single AOTF, to efficiently clean both polarization components of the object light (D + L) from the radiation components of the excitation light (L1, L2). For this purpose, both polarization components of the object light (D + L) are spatially separated from one another at a polarizing beam splitter (PBS) and coupled from different angles into the AOTF.

The s component is reflected at the PBS and reaches the AOTF via a mirror M5 while the p component passes through the PBS onto the AOTF.

Particularly advantageous, this arrangement can be configured by the two planes, which are spanned on the one hand by the two incident rays (top view in the figure) and the other by the propagation direction of the sound wave and the optical axis (side view in the figure) perpendicular to each other stand. In this case, the phase matching of the input beams to the acoustic wave is not critical, so that the orientation of the input beams within the mentioned plane is arbitrary and subject only to the aperture fitting of the AOTF crystal.

The components can also be radiated symmetrically, ie at the same angle to OA as in also shown below. In this case, the phase matching for any orientations of the two mentioned levels is given to each other.

Thus, the coupling of both polarization components takes place under the same conditions for phase matching. By applying the acoustic frequencies adapted to the polarization components (p and s) of the excitation radiation (L1, L2), the radiation components of the excitation light (L1, L2) are diffracted into the first orders and masked out of the detection beam path by means of a spatial filter BB1. Both polarization components of the detection light (D1, D2) each remain in the zeroth order and are each reflected by means of a mirror (M3 and M4) in itself.

The reflected components pass through the AOTF a second time, whereby a new filtering of the portions of the detection light (D1, D2) by blanking L1, L2 to BB2 is possible. In order to be able to separate the incoming and outgoing radiation at the polarizing beam splitter (PBS), an achromatic quarter wave plate QWP is arranged between the AOTF and the mirrors (M3, 4) for both polarization components (D1, D2). Thus, both portions of the detection light (D1, D2) undergo a rotation of the polarization direction by 90 ° due to the double passage through the respective quarter wave plate (QWP). Both polarization components of the detection light (D1, D2) are again spatially superimposed on the polarizing beam splitter (PBS), reflected and coupled onto the detection beam path DE.

shows an extension of the in the presented arrangement. Here pass through both polarization components of the object light (D + L) separately each one in the described filter arrangement with advantageously identically constructed AOTF1 and 2.

It is therefore assumed that the excitation light Ln can also contain different polarization components s and p.

A division of D + Ln into polarization components s (reflected in direction AOTF1 via M8) and p (transmitted in direction AOTF2) takes place on PBS.

As in the object light is reflected at mirrors M2, M7, rotated by QWP, and passes through the AOTF1 and 2 twice each, filtering the excitation light Ln twice through BB1 and BB2, respectively.

After the second filtering in the return, the s and p polarization components of the detection light (corresponding to D1 and D2) are again superimposed on the polarizing beam splitter cube (PBS) and directed to the detector DE.

The s and p components are now a p and s component after the rotation and are reflected on PBS (s component) or transmitted (p component)

This makes it possible to efficiently filter both polarization components of the object light, regardless of the polarization state of the excitation light.

In an inventive tunable filter is shown which comprises two identical and advantageously identically controlled AOTF. Here, the object light is coupled into the optical arrangement by means of a polarizing beam splitter cube (PBS), wherein s and p polarization components pass through a ring arrangement formed by mirrors M9, AOTF1, mirrors M10, mirrors M11 and AOTF2 in the opposite direction. Each polarization component thus traverses sequentially (one behind the other) both AOTFs, which are set identically to both polarizations of the selected spectral components. During each pass through an AOTF, the spectral components L1, L2 of the excitation radiation from the detection light of both polarization states (D1 and D2) are masked out at BB1, BB2. After filtering, both polarization components s and p of the detection radiation (D1 and D2) are superimposed on the PBS and directed in the direction of the detector.

The quarter wave plates QWP can be dispensed with here, because the s component initially reflected at PBS is mirrored again in the direction DE after passage of AOTF1 and AOTF2 on the PBS, while the p portion initially transmitted to PBS returns to PBS after passing AOTF2 and AOTF1 in the opposite direction is transmitted in the direction of DE.

shows an inventive emission filter comprising a single AOTF.

Note that the orientation of the splitter mirror of PBS here in relation to the previous representations in 2 - 5 turned 90 degrees.

The p-polarization (polarization direction of the excitation light) of the object light is here reflected by means of a polarizing beam splitter cube (PBS) to PBS and coupled into an annular filter assembly (F1) consisting of a deflection mirror M12, a half-wave plate HWP, deflection mirrors M13, M14 and a AOTF exists.

The s-polarized object light is assumed to be almost free of the radiation components of the excitation light, reflected on PBS and guided directly in the direction of the detector DE. The p-polarization component of the object light passes through the AOTF as p1, the spectral components of the excitation light L1, L2 being blanked out of the beam path at BB as described above. By means of an achromatic half-wave plate (HWP), the polarization of the p-polarized detection light (D1) is rotated by 90 °, resulting in s1. As a result, s-polarized detection light (D1) s1 is reflected at the PBS and again passes through the AOTF and the half-wave plate (HWP) as s2, the p-polarized portions p2 passing through PBS after the second pass and thus out of the filter arrangement (F1) of the polarizing beam splitter (PBS) and are guided together with the unfiltered s-polarized object light S in the direction of the detector DE.

shows an extension of the in the arrangement shown to cases where it can not be assumed that a clear orientation of the polarization of the radiation components D and Ln. In D and Ln, therefore, there is a mixed polarization.

The orientation of the splitter mirror in PBS corresponds to that in ,

The p-polarized portions p of the object light (D + L) are transmitted at the polarizing beam splitter (PBS) and pass through the in the described filter arrangement (F1), which includes an AOTF1, because they are rotated by HWP in s shares and thus reflected on PBS and the AOTF1 go through again. As a result, the radiation components of the excitation light (L1, L2) are efficiently masked out of the detection light (D1). The cleaned radiation is then, in turn, a p-portion, coupled out by means of the polarizing beam splitter (PBS) from the ring assembly, wherein the p- and s-polarized components are spatially superimposed. In order to hide the possible radiation components of the excitation light from the s-polarized object light, the filter arrangement F1 is followed by a mirror M15, a second AOTF2 downstream, which is set to the spectral regions of the light used for excitation (L1, L2). The radiation components of the excitation light (L1, L2) are thereby masked out in the direction of the detection beam path both with respect to the s and the p polarized light. Overall, therefore, with this arrangement, the p-portion that arrives at the PBS can be filtered twice by the AOTF1 and once by AOTF2 and the s-portion once by AOTF2 with respect to the interfering radiation components. In order to increase the transmission of the arrangement with respect to fluorescent light, a circulation can be dispensed with in arrangement F1, since there is still a post-filtering by AOTF2. For this purpose, HWP is either removed or rotated by 45 °, which does not reorient the polarization.

shows a tunable emission filter according to the invention, an extension of the in represents described arrangement.

Here are two PBS provided with opposite orientation of the splitter layer.

The p-polarized portions of the object light (D + L) pass through the corresponding filter arrangement (F1) again twice, which includes an AOTF1. On the description of will be referred.

Finally, the detection radiation (D1) which is adjusted by the radiation components of the excitation light L1, L2 to BB is spatially superimposed again on the PBS1 with the s-polarized radiation components of D + Ln and arrives in the direction of PBS2. An achromatic half-wave plate (HWP) arranged downstream of the filter arrangement (F1) rotates the polarization state of the radiation consisting of s and p portions by 90 °.

As a result, the detection light (D1), which has been cleared of the radiation components of the excitation light, is reflected as s-polarized radiation on the second polarizing beam splitter (PBS2) and guided in the direction of the detector DE. The p-polarized portions of the object light are transmitted on PBS2 and into another, 6 corresponding annular filter assembly (F2) coupled. This includes a single AOTF2 and is identical in effect to F1.

The radiation thus passes through the arrangement F2 again twice, wherein the polarization state of the detection light (D2) is rotated by 90 ° with each pass by means of an achromatic half-wave plate (HWP).

The frequencies of the AOTF are adapted to the spectral regions of the excitation light (L1, L2) for both polarization states. Thus, the unwanted spectral components of the excitation light L1, L2 are blinded to BB at each pass from the detection beam path (D2) targeted. The p-polarized light (D2), which is likewise cleared of the radiation components of the excitation light, is transmitted at the polarizing beam splitter (PBS) and guided together with D1 in the direction of the detector DE.

shows an advantageous variant of, for example, mutatis mutandis in the described arrangement. The unpolarized object light (D + L) passes through a birefringent polarization prism, which is preferably designed as Kalkspatstab (BDP). As a result, the s- and p-polarized portions of the object light (D + L) are spatially separated and preferably parallel, wherein the polarization state of the s-component by means of an achromatic half-wave plate (HWP1), which is nachgeordet the birefringent element BDP, by 90 ° is turned. Both parallel, both p-polarized partial beams are using a polarizing beam splitter (PBS) in the filter assembly comprising a single AOTF and already in the was described, coupled. Here, the radiation components of the excitation light (L1, L2) are simultaneously masked out of the detection beam path (D) of both partial beams and the cleaned detection beams, now rotated by QWP, are reflected at the polarizing beam splitter (PBS). The polarization state of one of the partial beams is rotated by 90 ° by means of another achromatic half-wave plate HWP2. The two mutually orthogonally polarized partial beams are spatially superimposed in a second birefringent element, which is preferably designed as Kalkspatstab (BDP), to an unpolarized detection beam (D) and guided in the direction of the detector DE. Thus, this arrangement allows a simultaneous double filtering of the object light by means of a single AOTF.

The second birefringent element BDP2 may also be optionally provided and the radiation from PBS may also pass directly to the detector DE.

In contrast to the cited above US 6982818 Here, a direct coupling can be done by using the PBS.

shows an advantageous variant of, for example, mutatis mutandis in illustrated arrangement comprising a single AOTF. The linearly polarized detection light (D1) passes through the AOTF twice in the zeroth diffraction order, whereby the radiation components of the excitation light (L1, L2) are masked out of the detection beam path (D1) on each pass. As a result, an effective filtering of the object light (D + L) is possible.

If the diaphragm (BB) (one of the diaphragms) is replaced by a mirror (M), coupling of the polarized illumination radiation (L1, L2) in the direction of the sample over the first diffraction order is advantageously also possible.

In this case, this arrangement also surprisingly serves as a tunable main color splitter, wherein the detection light (D1) is filtered twice.

This is also advantageous in the other representations 2 - 9 possible and was shown in dashed lines there (as coupling example, s-polarized laser radiation). A Simultaneous coupling and decoupling is technically feasible and reasonable.

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 5,377,003 [0001]
• US 6982818 B2 [0001]
• US 6963398 B2 [0001]
• US 6510001 [0001]
• US 6654165 [0001]
• US 2003/0133189 [0001]
• WO 99/42884 [0002]
• DE 19936573 A1 [0002]
• DE 10241472 A1 [0003]
• DE 19702753 [0006]
• US 6982818 [0068]

Cited non-patent literature

• Jang-Woo You et al., Opt. Express (2008), Vol. 16, no. 26 pp. 21505 [0001]
• Donley et al., Rev. Sci. Instrum. (2005), Vol. 76, pp. 063112 [0001]
• http://www.zeiss.de/C1256CFB00332E16/0/F7BE630BECC94A08C12573F3005483 F4 / $ file / 60-1-0001_e.pdf [0005]

Claims (3)

Arrangement and / or method for eliminating unwanted radiation components from detected light which comes from an illuminated sample, with an illumination beam path and a detection beam path with at least one detector for detecting the sample light coming from the illuminated sample, the proportion of light excited in the sample (excitation light) and At least one acousto-optic tunable filter AOTF) is provided in the detection beam path and at least one polarization element splits the sample light into pole components (pole portions), preferably perpendicular to one another, wherein at least one pole component passes through the same AOTF at least twice or a first and at least one second AOTF goes through. Arrangement and / or method according to claim 1, characterized by the use or the use of at least one of the following features, method steps or structures: - Polarization element is a pole splitter - The second pass through an AOTF or the passage through a second AOTF, the polarization direction is rotated so that the pole parts both come in the direction of detection An AOTF is driven so that the polarized excitation light passes through the AOTF in zeroth order and the illumination light is faded out in the first order - Blanking of the illumination light in a light trap or a Strahlumlenkeinheit for further use - Combining both treated in an identical manner polarization directions of the sample light on a polarization beam splitter and forwarding the sample light in the direction of the detector - The blanking of unwanted light after the AOTF occurs both at the first and second entry into the AOTF or when entering a first AOTF and when entering a second AOTF, after the AOTF respectively - when passing through the AOTF in back and forth direction, a blanking is provided on both sides of the AOTF - The AOTF is traversed twice by two pole components, wherein the irradiation takes place before and after the AOTF in two directions of incidence, the plane is perpendicular to the propagation plane of the longitudinal wave of the AOTF and the irradiation directions are preferably symmetrical to the propagation direction - The AOTF is at least twice pass through at least one Polkomponente each An AOTF is traversed twice by at least one pole component An AOTF is run twice by at least one pole component, a second one once - For pole pitch and / or union of the pole components, a birefringent polarization prism, preferably formed as Kalkspatstab (BDP) is used, an AOTF is traversed twice by at least one component - About deflecting a ring assembly for the detection light is generated with one or more AOTF - A decoupling from the ring assembly via the pole divider - For use as a main color splitter is coupled via the first diffraction order in the AOTF illumination light Laser scanning microscope (LSM) and / or operating method for an LSM, according to at least one of the preceding claims.

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