WO2006114247A1

WO2006114247A1 - Fluorescence microscope

Info

Publication number: WO2006114247A1
Application number: PCT/EP2006/003720
Authority: WO
Inventors: Stefan Hell; Johann Engelhardt
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2005-04-27
Filing date: 2006-04-22
Publication date: 2006-11-02
Also published as: DE102005020003B4; DE102005020003A1

Abstract

The invention relates to a fluorescence microscope (12) which comprises an excitation light source (14) for excitation light (5) in order to incite a fluorescent dye in a sample (19) during a limited period in a spatial area to spontaneously emit fluorescent light (6) having wavelengths in a wavelength range, and a pulsed de-excitation light source (15) for de-excitation light (7) in order to de-excite the fluorescent dye to a remaining area which is smaller than the spatial area. The light of the sample (19) can be associated with wavelengths other than those of the excitation light (5) and the de-excitation light (7) of the spontaneous emission of fluorescent light (6) from the remaining area of the spatial area. The invention is characterized in that the de-excitation light source (15) is a mode-coupled glass laser (13) having a line width of the de-excitation light (7) of less than 2 nm.

Description

FLUORESCENCE MICROSCOPE

The invention relates to a fluorescence microscope having an excitation light source for excitation light to excite a fluorescent dye in a sample for a limited period of time in a spatial region for spontaneously emitting fluorescence light having wavelengths in a wavelength region, and a pulsed depletion light source for deenergizing light to excite the fluorescent dye to de-excite a residual region which has been reduced in size compared with the spatial region, it being possible to assign light from the sample with wavelengths other than those of the excitation light and the depletion light to the spontaneous emission of fluorescent light from the remaining region of the spatial region.

The Wiederabregen the fluorescent dye can be operated so that the remaining area, d. H. the portion of the sample in which the fluorescent dye remains excited and from which spontaneously emitted fluorescent light exclusively originates from the sample becomes smaller than the diffraction limit. That is, with a fluorescence microscope of the type described above, the Abbe's diffraction limit can be undershot in the spatial resolution.

In particular, the invention relates to such a fluorescence microscope in which the de-excitation light source is provided to de-excite the fluorescent dye outside the remainder range by stimulated emission with the wavelength of the depletion light. Such a fluorescence microscope is also referred to as a STED fluorescence microscope, wherein the abbreviation STED stands for Stimulated Emission Depletion = depopulation (of the fluorescence-capable state) by stimulated emission. STATE OF THE ART

An STED fluorescence microscope of the type described above is known from WO 95/21393. A pulsed laser which can be used as the excitation light source is here stated to emit the excitation light preferably with a pulse width of 1 fs to 1 ns. On the other hand, a laser used as a depletion light source is intended to emit the depletion light at a preferred pulse width of 1 ps to 1 ns. With which lasers this is to be realized is not disclosed in WO 95/21393. Both specified pulse widths are significantly shorter than the lifetime of the fluorescent state of a typical fluorescent dye of a few nanoseconds.

In the practice of STED fluorescence microscopy, it has been found that the pulse width of the depletion light should preferably be longer than about 100 ps. Longer pulses lead to inefficiency in the stimulated emission, while shorter ones result in high energies per pulse required for complete de-excitation to sample damaging processes, presumably through multiple photon absorption. As de-excitation light sources for STED fluorescence microscopy various diode and solid state lasers are currently used. With diode lasers, the energies per pulse required for STED fluorescence microscopy are barely reached. For solid state lasers, z. As titanium-sapphire lasers, the pulse widths achievable in pulsed operation are significantly shorter than 100 ps and must be stretched by downstream systems, but affect the beam quality. In addition, the line width of pulsed solid-state lasers with a few nanoseconds is comparatively wide.

In the practice of STED fluorescence microscopy, a further requirement for the depletion light source and also for the excitation light source is to provide excitation light with different wavelengths for the use of different fluorescent dyes and corresponding de-excitation light with correspondingly different wavelengths. For this purpose, tunable laser light sources are known, for. B. Titan-sapphire laser with downstream optically parameterized oscillator (OPO). However, these tunable laser light sources are extremely complex as such and also have the fundamental disadvantages listed above for solid state lasers.

For a confocal fluorescence microscope, so a fluorescence microscope in which no increase in the spatial resolution by Wiederabregen the fluorescent dye in the It is known from WO 99/42884 to use acousto-optical elements as spectrally selective elements in order to separate the fluorescent light coming from the sample from the excitation light. Acousto-optical elements are characterized by a particularly high spectral selectivity. So it is possible with an acousto-optic element to hide light with a line width of 1 nm. Such narrow band light is provided in current confocal fluorescence microscopes of solid state lasers used as excitation light sources and operated as continuous wave lasers.

This filter width of acousto-optic elements can not be increased in order to adapt to light to be blended with greater line width.

In addition to pulsed diode and solid-state lasers, pulsed gas lasers in the form of so-called mode-locked gas lasers are also known. However, these are currently not commercially available. Currently only commercially available as continuous wave laser gas lasers are commercially available.

A well-known gas laser is the ArKr laser, the continuous wave laser z. B. is used in confocal fluorescence microscopy. An advantage of the ArKr laser is that it has a plurality of wavelengths in the spectral range useful to excite fluorescent dye. However, there is a fundamental tendency to replace all gas lasers for cost and stability reasons by solid state lasers. For the reasons of cost and stability mentioned above, it often makes sense to use several solid-state lasers, possibly combined with optically parameterized oscillators (OPOs), instead of an ArKr laser in order to provide excitation light with a different wavelength in a confocal fluorescence microscope.

OBJECT OF THE INVENTION

The invention has for its object to show a fluorescence microscope of the type described above, in particular a STED fluorescence microscope, are given in the principle particularly favorable conditions for separating the fluorescent light from a region of interest of the sample of other light from the sample. SOLUTION

The object of the invention is achieved by a fluorescence microscope with the features of independent claim 1. Preferred embodiments of the novel fluorescence microscope are described in the dependent claims 2 to 11.

DESCRIPTION OF THE INVENTION

In the new fluorescence microscope, the depletion light source is a mode-locked gas laser with a line width of the depletion of less than 2 nm. Surprisingly, it turns out that the cost of a commercially unavailable mode-locked gas laser and also the stability problems associated with its operation are outweighed by, in particular the de-excitation light having a line width of less than 2 nm can be provided. Mode-locked gas lasers are readily capable of providing pulsed de-excitation light with a linewidth of at most 1.nm and even significantly less. This makes it possible to separate light from the sample with the wavelength of the depletion light very narrow band from the spontaneously emitted fluorescent light from the sample. As a result, bandwidth losses are minimized in the detection of the spontaneously emitted from the respective residual region of the fluorescent light. However, with a mode-locked gas laser as Abregungslichtquelle further fundamental advantages are connected. The coherence length of mode-locked gas lasers is comparatively long, so that the targeted formation of interference patterns with the de-excitation light, as used for example in the context of so-called 4 Pi fluorescence microscopy for the spatial distribution of the depletion light around a region of interest of a sample, without critical adjustments in Beam path is possible. Mode-locked gas lasers also have an excellent transverse mode TEM00 with very good beam quality. In addition, mode-locked gas lasers for selectively influencing the emitted laser light can also be operated selectively in a higher mode, such as TEM01, TEM10, TEM11, etc. The available pulse energies are sufficiently high in a gas laser, and this usually with a plurality of lines available for the excitation light, in which the gas laser can be operated selectively or simultaneously. For example, the advantages of a mode-locked gas laser as a depletion light source are fully exploitable when the depletion light source is provided to de-excite the fluorescent dye outside the remainder of the sample by stimulated emission. The emission of the sample stimulated by the de-excitation light has the wavelength of the depletion light. That is, if the line width of the depletion light is particularly narrow, as in the mode-locked gas laser, this also applies to the stimulated emission of the sample. This can therefore be separated with a narrow-band filter together with reflected portions of the Abregungslicht from the spontaneously emitted fluorescent light. Therefore, even if the wavelength of the de-excitation light is within the wavelength range in which the fluorescent dye spontaneously emits, it remains at only small bandwidth losses in the detection of the fluorescence spontaneously emitted in a region of interest of the sample. However, since it has been found that the relative proportion of the stimulated emission of the sample to the light from the sample with the wavelength of the excitation light versus the portions of the depletion light reflected from the sample is generally negligible, substantial advantages of the novel fluorescence microscope are thereby obtained in that the de-excitation light is spectrally narrow-band and, accordingly, its portions of the fluorescent light reflected from the sample can be separated from the speculum in a narrow band, ie with low detection bandwidth losses, from the sample.

Another important advantage of mode-locked gas lasers is that they are capable of providing light with pulse widths in a range of 50 to 1000 ps and more particularly in a range of 100 to 500 ps. This fully covers the pulse width range of the depletion light of more than 100 to about 300 ps, which is ideal for STED fluorescence microscopy.

When the mode-locked gas laser of the new fluorescence microscope is an ArKr gas laser, the de-excitation light can make use of the multitude of lines of excitation of a fluorescent dye of an ArKr gas laser. In this case, the light source for the excitation light can be an ArKr gas laser. It is even possible to provide a single ArKr gas laser both as an excitation light source and as a depletion light source, emitting laser light of at least two different wavelengths at a time. In this case, the excitation light and the excitation light are preferably partially different beam paths to the sample to provide, wherein the path length of at least one of the two beam paths relative to that of the other beam path is variable to adjust the timing of the arrival of the excitation light and the de-excitation light in the sample.

However, the mode-locked gas laser as depletion light source of the new fluorescence microscope can also be synchronized in a conventional manner with another laser which forms the excitation light source. The other laser can, as related to the

ArKr gas laser has already been suggested to be another mode-locked gas laser. However, it may also be, for example, a diode or solid-state laser. A

Synchronization of the mode-locked gas laser of the new fluorescence microscope with one or more other lasers of the same or different design can also for

Purpose of multi-color applications.

In particularly preferred specific embodiments of the new fluorescence microscope, at least one acousto-optical element is provided, which separates light with the wavelength of the excitation light from the fluorescent light from the sample. Due to the narrow linewidth of the depletion light, in the new fluorescence microscope an acousto-optic element can be used to selectively mask the light from the sample with the wavelength of the depletion light. The acousto-optic element may be a so-called AOD (Acousto-Optical-Deflector) or an AOTF (Acousto-Optical-Tuneable-Filter).

Each acousto-optical element can also be provided at the same time for separating excitation light reflected from the sample from the fluorescent light from the sample. In this way, only the spontaneously emitted fluorescent light from the interesting portion of the sample remains.

At least one of the acousto-optic elements can also be used to form an acousto-optic beam splitter with which the excitation light and / or the excitation light is coupled into the beam path between a photodetector and the sample at the same time.

The residual area of the spatial area, whose spontaneous emission of fluorescent light, the light from the sample with wavelengths other than those of the excitation light and can be assigned to the Abregungslichts can consist of a single punctiform or linear portion of the sample or a pattern of a plurality of point or line-shaped portions. In other words, the reduced residual area due to the de-excitation light in relation to the spatial area in which the excitation of the fluorescent dye with the excitation light occurred can include both one or more punctiform partial areas, but also one or more linear partial areas, an improvement of the spatial resolution to below the diffraction limit is already achieved when the dimensions of the respective sub-regions fall below the diffraction limit in a single direction.

Advantageous developments of the invention will become apparent from the dependent claims and the entire description. Further features are the drawings - in particular the illustrated geometries and the relative dimensions of several components to each other and their relative arrangement and operative connection - refer. The combination of features of different embodiments of the invention or features of different claims differing from the chosen relationships is also possible and is hereby stimulated. This also applies to those features which are shown in separate drawing figures or are mentioned in their description. These features can also be combined with features of different claims.

BRIEF DESCRIPTION OF THE FIGURES

In the following the invention will be further explained and described with reference to preferred embodiments shown in the figures.

Fig. 1 shows the energy levels of a fluorescent dye, in the context of his

Use in STED fluorescence microscopy play a role.

Fig. 2 outlines the timing of the pulses of the excitation light and the

Diminishing light in STED fluorescence microscopy.

FIG. 3 outlines the wavelengths and wavelength spectra associated with FIG. Fig. 4 shows a block diagram of the new fluorescence microscope.

FIG. 5 outlines a realization of the fluorescence microscope according to FIG. 4; FIG. and

FIG. 6 shows an arrangement of two acousto-optical elements in the beam path between a sample and a photodetector according to FIG. 4 or 5.

DESCRIPTION OF THE FIGURES

Fig. 1 shows the energy spectrum of a fluorescent dye. By excitation light 5, the fluorescent dye passes from its ground state 1 within its electrical So state in an excited state 4 within its electrical Sr state. This excited state 4 decays into the fluorescent state 3 within the electrical state S _r . From the fluorescent state 3, the fluorescent dye passes under spontaneous emission of fluorescent light 6 back into a state within its electrical S ₀ state. However, the fluorescent dye can also be forced to stimulated emission by de-excitation light 7, in particular when the intensity of the depletion light 7 is above a saturation concentration for the stimulation of the fluorescent dye to the stimulated emission. Stimulation of the fluorescent dye to stimulated emission is used in STED fluorescence microscopy to excite a sample labeled with the fluorescent dye which, due to the Abbe's diffraction limit, excites spontaneous fluorescence with excitation light 5 only within a larger spatial area enclosing a region of interest can be de-energized outside the sub-area with the de-excitation light 7, whereby the spatial resolution during the measurement of the sample can be increased by detecting the fluorescence emitted from the sub-area to significantly exceeding the diffraction limit. Due to the lifetime of the fluorescent state 3 of typically a few nanoseconds, it is a technical challenge to separate the fluorescent light 6 of interest from the respective partial area of the sample both from reflected portions of the excitation light 5 and from reflected portions of the depletion light 7. In addition, although there is also additional light in the form of stimulated with the excitation light 7 emission of the fluorescent dye on; However, this stimulated emission has the same wavelength as the de-excitation light 7, so it is not from this and their intensity is many orders of magnitude smaller than that of the reflected portions of the de-excitation light 7. That is, this additional light is usually practically negligible, although it will be specifically discussed below.

2 shows an example of the time sequence of the intensities of the excitation light 5, the spontaneously emitted fluorescence light 6 and the depletion light 7 in STED fluorescence microscopy. A pulse 8 of the excitation light 5 is followed by a pulse 9 of the depletion light 7. The pulse 9 advantageously has a pulse duration of more than 100 ps up to about 300 ps in order to be significantly shorter than the time period over which the fluorescent light 6 is emitted Fluorescent dye is spontaneously emitted, and on the other hand, despite the desired saturation in the stimulated emission, the sample and the fluorescent dye are not burdened with excessive energy densities. The pulse 8 of the excitation light 5 may, as shown here, be shorter than the pulse of the de-excitation light 7. However, this is not mandatory. The time interval of the two pulses 8 and 9 is only to be understood as an example here. Thus, a substantial temporal overlap of the pulses 8 and 9 is possible. The temporal relative position of the pulses 8 and 9 is to be optimized in such a way that the signal of the fluorescent light 6 from the region of interest of the sample is particularly clear. However, due to the dense sequence of the pulses 8 and 9 and the fluorescence light 6 immediately adjacent thereto, a temporal separation of the fluorescent light 6 from the region of interest of the sample is at most possible with extreme effort and has not hitherto been realized.

In practice, the excitation light 5 and the depletion light 7 are separated from the fluorescent light 6 of interest by a spectral selection. Fig. 3 outlines the wavelengths lambda, which occur in the explained with reference to FIGS. 1 and 2 process. If one disregards negligibly small proportions of the spontaneously emitted fluorescent light 6, the excitation light 5 has the shortest wavelength, ie the highest quantum energy. The fluorescent light 6 also has 5 wavelengths Lambda from a comparatively extended wavelength range 10 of a few nanometers width in the case of spectrally narrow-band excitation light shown here. The wavelength of the depletion light 7 is longer than that of the excitation light 5 and here longer than the most occurring wavelengths of the spontaneously emitted fluorescent light 6. Thus remains a detection window 1 1 between the wavelengths of the excitation light 5 and the depletion 7, in the spontaneously emitted fluorescent light. 6 can be detected without simultaneously detecting excitation light 5 or depletion light 7 reflected by the respective sample or the stimulated emission having the same wavelength as the depletion light 7 from regions of the sample other than the subregion of interest. The spectral width of the detection window 11 depends strongly on how large the line widths of the excitation light 5 and the depletion light 7 actually are, which are reproduced here in a very narrow band. In particular, the line width of the depletion light 7 is of particular importance since its wavelength is generally much lower within the wavelength range 10, while the wavelength of the depletion light 5 is always at the outer edge of the wavelength range 10. In particular, when the wavelength of the depletion light 7 is even lower than shown in Fig. 3 in the wavelength range 10, it may be necessary to achieve a large detection bandwidth to detect also portions of the fluorescent light 6 with a longer wavelength than the wavelength of the depletion 7. For this purpose, the de-excitation light 7 must then be selectively faded out of a detection window 11 which extends further to larger wavelengths. This is only possible with very narrow band filters. As such narrow-band filters concrete acousto-optical elements are known with which light with a line width of about 1 nm can be selectively hidden. However, these spectrally narrow-band filters can only be usefully used if the de-excitation light 7 and the emission of the same wavelength stimulated by it have a correspondingly narrow linewidth. This is achieved in the fluorescence microscope described below.

The block diagram according to FIG. 4 gives an overview of the structure of a fluorescence STED microscope 12 as a concrete embodiment of a fluorescence microscope according to the invention. A mode-locked gas laser 13 is provided here both as an excitation light source 14 and as a depletion light source 15. The excitation light 5 and the de-excitation light 7 are emitted from the mode-locked gas laser 13 in simultaneous pulses. In a delay generator 16, a delay between the pulses of the de-excitation light 7 is brought about with respect to the pulses of the excitation light 5 (see Fig. 2). In a phase control device 17, the spatial distribution of the phases of the depletion light 7 is modulated in such a way that the desired intensity distribution of the depletion light 7 relative to the excitation light 5 results in the spatial area enclosing the region of interest of the sample. In a coupling device 18, the excitation light 5 and the Abregungslicht 7 coupled into the beam path of the STED fluorescence microscope 12 between the sample 19 and a photodetector 20. From the coupling device 18, the excitation light 5 and the de-excitation light 7 reaches the sample 19. In addition to reflected components of the excitation light 5 and the depletion light 7, the fluorescent light 6 of interest also comes from the spontaneous emission from the respective partial region of the sample the stimulated emission of the sample 19 with the same wavelength as the excitation light 7. A separation of the fluorescent light 6 takes place in a filter device 21, which is arranged in front of the photodetector 20. The coupling device 18 may also be involved in the filtering, so that only the fluorescent light 6 of interest reaches the detector 20.

Fig. 5 shows a realization of the STED microscope 12 according to the block diagram in Fig. 4. The mode-locked gas laser 13 is an ArKr gas laser 22, in whose laser cavity a mode coupler 23 is provided to the excitation light 5 and the de-excitation light 7 with the gas laser 13 to produce in the form of individual pulses. The laser cavity of the gas laser 13 is folded with a deflection mirror 24 in order to reduce the overall construction length of the gas laser 13. In order to bring about a dispersion compensation between the excitation light 5 and the depletion light 7 in the mode coupling, the laser cavity of the gas laser 13 is divided into two different arms for the excitation light 5 and the deenergizing light 7 beyond the mode coupler 13. In this case, the dispersive effect of the mode coupler 23 for dividing the animal laser cavity into the different arms for the excitation light 5 and the de-excitation light 7 may be sufficient or supported for example by an additional dispersive effect of the deflection mirror 24. The line specificity of the laser cavity for the excitation light 5 and the de-excitation light 7 is realized in each case by an apertured diaphragm 25 or 26 in front of the resonator mirrors 27 and 28 arranged at different distances from the deflecting mirror 24. The mode-locked gas laser 22 is operated in the transversal TEM00 mode in which it has a high beam quality with a high coherence length. In particular, in the area of the laser cavity of the gas laser 13 beyond the mode coupler 23, however, the mode can also be deliberately influenced so that the gas laser 13, z. B. for beam forming, specifically in a higher mode, such as TEM01, TEM10 or TEM11 operated. The delay generator 16 is realized in FIG. 5 by an arrangement with a dichroic mirror 29, two deflection prisms 31 and 32 and a deflection mirror 33. By the displacement of one of the deflection prisms 31 and 32, which here at the deflection prism 32nd is indicated by a double arrow 34, the path length for the de-excitation light 7 with respect to the excitation light 5 and thus the temporal position of the pulse 9 shown in FIG. 2 with respect to the pulse 8 shown in FIG. 2 changes over other deflection mirrors 34 and 35 and by a lens system From there, the excitation light 5 passes through a further lens system 39 from at least one lens to an acousto-optic beam splitter 40 and is there in the beam path between the Probe 19 and the photodetector 20 coupled. The de-excitation light 7 passes from the dichroic mirror 37 through a lens system 41 of at least one lens to a so-called Spatial Light Modulator 42 as realization of the phase control device 17. From there the modulated depletion 7 passes through another optical system 43 from at least one lens to one Dichroic mirror 44, which serves here for coupling the Abregungslicht 7 in the beam path between the sample 19 and the photodetector 20. In this beam path, an objective 45 is provided between the dichroic mirror 44 and the sample 19. By means of this objective 45, the fluorescent light 6 passes from the sample 19 together with reflected portions of the excitation light 5 and the depletion light 7 and the emission of the same wavelength stimulated by the depletion light 7 back to the dichroic mirror 44. From there, the light coming from the sample passes through an optical system 46 of at least one lens to the acousto-optical beam splitter 40, which decouples both light with the wavelength of the excitation light 5 and light with the wavelength of the Abregungslicht 7. The same applies to a further acousto-optical beam splitter 47, which here serves exclusively for filtering out further portions of light with the wavelengths of the excitation light 5 and the depletion light 7. From there, the fluorescent light 6 passes through an optical system of at least one lens 48 through a confocal to the region of interest of the sample 19 arranged aperture 49 and through a blocking filter 50 to the photodetector 20. The blocking filter 50 supplements the blocking effect of the acousto-optical beam splitter 40th and 47 for the de-excitation light 7 reflected from the sample 19 and the emission of the same wavelength stimulated by the de-excitation light 7 in the sample. The use of the acousto-optic beam splitters 40 and 47 for hiding both the excitation light 5 and the depletion light 7 and the emission of the same wavelength stimulated by this outside of the relevant portion of the sample is possible in the STED fluorescence microscope 12 because the ArKr Gas laser 22 both the excitation light 5 and the de-excitation light 7, each with a narrow line width of less than 1 nm. With such a narrow linewidth, acousto-optic elements have a good barrier effect for the wavelengths selected by their acoustic drive. Furthermore, the pulses of the mode-locked ArKr gas laser can be used directly in the fluorescence microscope 12 without having to be changed in their length. They have the optimum pulse width of a few 100 ps for STED fluorescence microscopy. By changing the positions of the pinhole apertures 25 and 26 used for the line selection, it is also possible, with simultaneous adjustment of the dispersion correction with the resonator mirrors 27 and 28, to select from the plurality of lines generally available for an ArKr gas laser the two which are suitable for the Excitation and de-excitation of a particular fluorescent dye in the sample 19 are particularly well suited.

FIG. 6 shows, as an enlarged detail of FIG. 5, the structure of the acousto-optical beam splitter 40. The acousto-optical beam splitter 40 has two acousto-optical elements 51 and 52. The acousto-optic element 51 is actively driven in order to couple the excitation light 5 via a deflecting mirror 53 into the beam path between the detector 20 and the sample 19 and, in return, both the excitation light 5 reflected by the specimen and the excitation light reflected by the specimen 7 as well as the stimulated by this emission of the same wavelength of the spontaneously emitted fluorescent light 6. Here, the second acousto-optical element 52 essentially serves to compensate for dispersion and birefringence effects, since the acousto-optical element 51 also spectrally splits the fluorescence light 6, which has no fixed wavelength (see FIG. D. h., The second acousto-optical element 52 aligns the individual spectral components of the fluorescent light 6 again parallel to each other. In addition to the input and in particular decoupling or filtering out, further portions of the excitation light 5 and the de-excitation light 7 can be used. The acousto-optic elements 51 and 52 used herein are known as Acousto-Optical Tuneable Filters. It is also possible to use so-called Acousto-Optical Deflectors as the acousto-optical elements 51 and 52. Furthermore, it is also possible to use known acousto-optical elements which comprise a special dispersion-free zeroth-order crystal, so that no dispersion correction is required, but instead a suppression of one or more lines of light from the sample can be operated with each acousto-optic element , The typical extinction of each AOTF realized acousto-optic element is for the selected lines with the line width of about 1 nm at about 95%. When using several active acousto-optic Elements 51 in conjunction with the notch filter 50 shown in FIG. 5, the excitation light 5 and the de-excitation light 7 and the stimulated by this emission of the same wavelength can be very effectively kept away from the photodetector 20.

LIST OF REFERENCE NUMBERS

Condition 11 detection window

Condition 12 STED fluorescence microscope

Condition 13 mode-locked gas laser

State 14 excitation light source

Excitation light 15 depletion light source

Fluorescent light 16 delay generator

Disengaging light 17 Phase control device

Pulse 18 coupling device

Pulse 19 sample

Wavelength range 20 detector

Filter device 31 deflecting prism

ArKr gas laser 32 deflection prism

Mode coupler 33 deflection mirror

Deflection mirror 34 deflection mirror

Aperture 35 deflection mirror

Aperture plate 36 lens system

Resonator mirror 37 Dichroic mirror

Resonator Mirror 39 Lens System Dichroic Mirror 40 acousto-optic beam splitter

Lens system 51 acousto-optic element

Spatial Light Modulator 52 acousto-optic element

Lens system 53 deflecting mirror dichroic mirror

lens

Lens system acousto-optical beam splitter

lens system

pinhole

cut filter

Claims

PATENT CLAIMS

A fluorescence microscope with an excitation light source for excitation light to excite a fluorescent dye in a sample for a limited period of time in a spatial region for spontaneously emitting fluorescent light having wavelengths in a wavelength region, and a pulsed deenergizing light source for deenergizing light to excite the fluorescent dye except one depleted residual region, wherein light from the sample with wavelengths different from those of the excitation light and the Abregungslicht the spontaneous emission of fluorescent light from the remaining region of the spatial area is assignable, characterized in that the de-excitation light source (15) is a mode-locked gas laser ( 13) having a line width of the depletion light (7) of less than 2 nm.

2. Fluorescence microscope according to claim 1, characterized in that the de-excitation light source (15) is provided to de-excite the fluorescent dye outside of the remaining region by stimulated emission with the wavelength of the depletion light (7) again.

3. fluorescence microscope according to claim 1 or 2, characterized in that the pulse width of the excitation light (7) is 100 to 500 ps.

4. Fluorescence microscope according to one of claims 1 to 3, characterized in that the mode-locked gas laser (13) is an ArKr gas laser (22).

5. Fluorescence microscope according to one of claims 1 to 4, characterized in that the mode-locked gas laser (13) is designed for a laser operation in the transverse fundamental mode or a higher mode.

6. Fluorescence microscope according to one of claims 1 to 5, characterized in that the mode-locked gas laser (13) is designed for a laser operation at a single, but from a plurality of possible wavelengths selectable wavelength.

7. fluorescence microscope according to one of claims 1 to 5, characterized in that the mode-locked gas laser is also provided as the excitation light source (14), wherein it is designed for simultaneous laser operation at two wavelengths.

8. Fluorescence microscope according to claim 7, characterized in that for the excitation light (5) and the de-excitation light (7) partially different beam paths to the sample (19) are provided, wherein the path length of at least one of the two beam paths relative to that of the other beam path is variable to set the timing of arrival of the excitation light (5) and the depletion light (7) in the sample (19).

9. fluorescence microscope according to one of claims 1 to 8, characterized in that at least one acousto-optical element (51) is provided, the light with the wavelength of the Abregungslicht (7) of the fluorescent light (6) from the sample (19 ) separates.

10. fluorescence microscope according to claim 9, characterized in that each acousto-optical element (51) also separated from the sample excitation light (5) from the fluorescent light (6) from the sample (19) separates.

11. Fluorescence microscope according to claim 10, characterized in that at least one acousto-optical element (51, 52) forms an acousto-optical beam splitter (40), at the same time the excitation light (5) and / or the de-excitation light (7) in the beam path between a photodetector (20) and the sample (19) couples.

12. A fluorescence microscope according to any one of claims 1 to 11, characterized in that the remaining region of the spatial region consists of a single point-shaped or line-shaped partial region of the sample or of a pattern of a plurality of dot-like or linear partial regions.