WO2023078732A1

WO2023078732A1 - Sted microscope

Info

Publication number: WO2023078732A1
Application number: PCT/EP2022/079759
Authority: WO
Inventors: Andreas Popp; Torsten Mans
Original assignee: Trumpf Lasertechnik Gmbh; Amphos GmbH
Priority date: 2021-11-03
Filing date: 2022-10-25
Publication date: 2023-05-11
Also published as: DE102021128556A1

Abstract

The invention relates to a STED microscope (1) comprising: an ultrashort pulse laser (2) for generating a pulsed laser beam (3); a gas-filled optical hollow-core fiber (4) into which the pulsed laser beam (3) is coupled at one end and which is designed in such a way that the laser pulses of the pulsed laser beam (3) are spectrally extended during propagation through said gas-filled optical hollow-core fiber (4) as a result of non-linear optical effects; a wavelength-selective switch (5) for generating a pulsed excitation laser beam (6) and a pulsed de-excitation laser beam (7) from the pulsed laser beam (8) coupled out of the gas-filled optical hollow-core fiber (4) at the other end; and at least one optical transport fiber (9) for transporting the pulsed excitation laser beam (6) and the pulsed de-excitation laser beam (7).

Description

STED microscope

The invention relates to a STED microscope. STED microscopes are special light microscopes whose resolution is not diffraction limited. To be more precise, STED microscopy is a special form of fluorescence microscopy. Characteristic of STED microscopy is the use of two laser radiate, a so-called excitation laser beam and a so-called deactivation laser beam. These two laser beams are radiated into a sample to be imaged in a focused manner, with their foci spatially overlapping. The focus of the depletion laser beam is characterized by a central zero point in the spatial intensity distribution. A focus with this property is referred to below as a de-excitation focus. For example, a de-excitation focus can be approximately ring-shaped.

The imaging is now performed by detecting fluorescent light that is emitted when the sample is scanned with the excitation and deactivation laser beam. The processes that take place can be outlined as follows: As a result of the irradiation of the excitation laser beam, fluorescence molecules in the sample are excited from the ground state in the focus of the excitation laser beam. The excited fluorescent molecules can return to their ground state via spontaneous emission, i.e. by emitting fluorescent light. Even before spontaneous emission occurs, however, the fluorescence molecules in the depletion focus are de-excited via stimulated emission. The emission of the fluorescent light is thus limited to a central area around the zero point of the deactivation focus, which (if the deactivation laser beam is of sufficient intensity) is significantly smaller than the focus of the excitation laser beam. This explains the increased resolution of the method, which is unlimited in the idealized theory, assuming arbitrarily high de-excitation intensities. Practical limits are due, among other things, to the fact that the intensity in the center of the de-excitation focus only falls approximately to zero.

In many cases, the excitation laser beam and the deactivation laser beam are pulsed laser beams, which has the advantage of a lower average intensity.

DE 11 2015 001 640 T5 describes a STED microscope device with a STED light source and an excitation light source. A spatial light modulator is used to shape the STED light into a ring shape by phase modulation. By means of an optical system, a region of an observation Object irradiated with the excitation light and the STED light after the phase modulation. A detector is used to detect the fluorescence emitted from the region.

In principle, the excitation laser beam and the deactivation laser beam of a STED microscope can also be guided in a common optical fiber, but still separately from one another. A corresponding optical system for STED microscopy with an excitation radiation source, a deactivation radiation source and a polarization-maintaining optical fiber is known, for example, from WO 2019/169368. The polarization-maintaining optical fiber has a central mode in which the excitation radiation propagates. Furthermore, it has one or more external modes in which the depletion radiation propagates. Before being coupled into the optical fiber, the depletion radiation impinges on a spatial light modulator which serves to shape the beam in a Herr ite-Gauss mode (TEMw). In a Mach-Zehnder interferometer, the beam profile is rotated by 90° in one of the arms and then the non-rotated portion and the excitation radiation are superimposed. The excitation and de-excitation radiation coupled out of the optical fiber is collimated by means of a first objective and radiated into the sample by means of a second objective.

A STED microscopy system with two light sources and an optical fiber with a core region in which the excitation light propagates and a ring region in which the de-excitation light propagates is also described in WO 2017/210679.

DE 10 2007 048 135 B4 also discloses a fluorescence microscope in which light of different wavelengths is available in order to convert different fluorescent dyes from one state to another. Specifically, the fluorescence microscope includes an excitation light source that provides excitation light for exciting a fluorescent dye. Furthermore, de-excitation light is used to de-excite the fluorescent dye again outside of a measuring point of interest by stimulated emission. To form the de-excitation light, the fluorescence microscope includes a laser that emits light, which is coupled into an optical fiber. In the optical fiber, stimulated Raman scattering leads to the formation of several red-shifted Stokes lines. One of the Stokes lines is selected with a wavelength-selective element and forms the depletion light. The optical fiber is preferably a conventional, non-tapered single-mode fiber. In one embodiment, however, the optical fiber is a microstructured fiber with a gas-filled hollow core.

In the case of STED microscopes with two separate pulsed light sources, ie a pulsed excitation light source and a separate pulsed deactivation light source, these must be synchronized. Such synchronization is expensive.

However, STED microscopes with a common pulsed light source are also known from the prior art. For example, WO 2014/029978 describes a STED microscope that has a titanium-sapphire laser that emits a pulsed laser beam. The pulsed laser beam is divided into an excitation beam path and a deactivation beam path. A photonic crystal fiber is arranged in the excitation beam path, at the output of which there is a white light spectrum. An acousto-optic tunable filter is used for wavelength selection based on this white light spectrum. A light modulator, which serves as a phase mask and enables aberration correction, is also arranged in the deactivation beam path.

A STED microscope with a titanium-sapphire laser, an excitation beam path, a deactivation beam path and a photonic crystal fiber in the excitation beam path is also described in WO 2013/167479.

In contrast, the invention was based on the object of providing a STED microscope which is characterized by a high degree of flexibility, in particular with regard to the use of different fluorescence molecules, and which at the same time has a particularly simple and cost-effective structure. This object is achieved by a STED microscope, comprising an ultrashort pulsed laser for generating a pulsed laser beam, a gas-filled optical hollow-core fiber into which the pulsed laser beam is coupled at one end and which is designed in such a way that the laser pulses of the pulsed laser beam during propagation through the gas-filled optical hollow-core fiber can be spectrally expanded via non-linear optical effects, a wavelength-selective switch for generating a pulsed excitation laser beam and a pulsed depletion laser beam from the pulsed laser beam decoupled from the other end of the gas-filled optical hollow-core fiber, at least one optical transport fiber for transporting the pulsed excitation laser beam and the pulsed depletion laser beam , wherein either the STED microscope comprises a phase modulation device for modulating the phase of the pulsed depletion laser beam coupled out of the at least one optical transport fiber, which is designed in such a way that the phase-modulated, pulsed depletion laser beam is focused in a depletion focus, or the at least an optical transport fiber is exactly one optical transport fiber that has a central mode for the propagation of the pulsed excitation laser beam and at least one higher mode for the propagation of the pulsed depletion laser beam, a lens for joint focusing of the decoupled, pulsed excitation laser beam and the phase-modulated or the optical transport fiber coupled out, pulsed depletion laser beam into a sample and for collecting fluorescent light emitted from the sample, and a detection means for detecting the collected fluorescent light.

Ultrashort pulse lasers are lasers that emit laser light in the form of laser pulses with pulse durations in the picosecond or femtosecond range or less. Examples of ultrashort pulse lasers are mode-locked fiber lasers or hybrid lasers, in which a mode-locked fiber laser is used in combination with a solid-state amplifier.

Hollow-core optical fibers are optical fibers that are characterized by a hollow fiber core. Examples of hollow-core optical fibers are corresponding photonic crystal fibers (hollow-core photonic crystal fibers), revolver Hollow core fibers (also known as tubular lattice fibers), nested tube fibers and kagome fibers. Revolver hollow-core bevels are characterized by a plurality of rings in cross section, which are preferably arranged discretely rotationally symmetrically around the center point of the optical hollow-core fiber, the arrangement of which clearly corresponds to the arrangement of cartridges in the barrel of a revolver. Nested tube fibers are characterized by nested rings in the cross section of the fiber. The mechanism of light guidance can be based, for example, on a photonic band gap or the discrimination of the coupling in higher-order modes. During the propagation of the laser pulses of the pulsed laser beam through the gas-filled optical hollow-core fiber, non-linear optical effects result in a spectral expansion. The nonlinear optical effects include the Kerr effect, which leads to self-phase modulation, and stimulated Raman scattering. Furthermore, the dispersion properties of the gas-filled hollow-core optical fiber play an important role. The spectral expansion is to be understood in such a way that new spectral components are formed which the laser pulses coupled into the optical hollow-core fiber do not have. In particular, a spectral expansion in the sense of a broadening of the spectrum can be achieved.

Advantages of using gas-filled optical hollow-core fibers include the fact that relatively low particle densities are sufficient to excite the non-linear optical effects due to the confinement to a small beam cross-section and relatively long propagation lengths. By choosing the gases used (e.g. atomic Ne, Ar, Kr, Xe or molecular H2, N2, N2O, C2H2F4, C2H4F2) or gas mixtures (in particular from one or more of the gases mentioned), the gas pressure and the parameters of the pulsed laser light source can be altered In addition, a large number of spectra can be generated in a controlled manner. Compared to solids, which have long been used as non-linear optical media, the undesired absorption in gas-filled hollow-core optical fibers is significantly reduced and the destruction thresholds are much higher.

An essential purpose of the spectral expansion or spectral broadening is to use a large number of possible central wavelengths for the pulsed excitation depletion laser beam and the pulsed depletion laser beam. Starting from the laser pulses of the ultra-short pulse laser, which have a specific central wavelength, laser pulses with a central wavelength that can be selected from a large wavelength range can be generated after spectral broadening. As a result, both the central wavelength of the excitation laser beam and the central wavelength of the depletion laser beam can be flexibly adapted to different fluorescence molecules.

A wavelength-selective switch is a device for wavelength-dependent switching of light. Light entering via an input is divided into its wavelength components, which can then be influenced in parallel and independently of one another and output in a targeted manner via one or more outputs. Wavelength-selective switches with one or two outputs are particularly suitable for the present STED microscope. Suitable wavelength components for the pulsed excitation laser beam and the pulsed depletion laser beam are selected from the spectrally expanded spectrum and output together via one output or separately from one another via the two outputs. Generating the pulsed excitation laser beam and the pulsed deactivation laser beam from a common source eliminates the complex synchronization that is necessary in the case of two different laser sources. As a result, the structure is simpler and cheaper.

The pulsed excitation laser beam and the pulsed depletion laser beam can be supplied to a desired location in a simple manner by means of the at least one optical transport fiber. This enables a spatial separation between the location of the image, where the sample and the lens (and possibly the actual microscope body) are located, and the other components of the STED microscope, in particular the ultrashort pulse laser. The result is a particularly high level of flexibility in use. For example, additional space for sample preparation can be available at the location of the image. Compared to the delivery of the pulsed excitation or depletion laser beam via one or more mirrors, e.g. dielectric mirrors, can also be dispensed with a complex structure and an expensive adjustment of the beam path. After all, this is what makes it possible to use it in applications in which accessibility is severely restricted, for example use on living animals or in large systems in an industrial environment.

The phase of the pulsed deactivation laser beam is spatially influenced by means of the phase modulation device in such a way that a deactivation focus is obtained when focusing, that is to say that the intensity in the center of the focus drops to zero or approximately to zero. The phase modulator can be static or dynamic. In the first case, the phase modulation device can be, for example, a phase mask, in particular a phase mask with a spiral profile, or a diffractive optical element (DOE). In the latter case, the phase modulation device can be a spatial light modulator, for example.

In the case of propagation of the pulsed excitation laser beam in a central mode and the pulsed depletion laser beam in at least one higher mode of the optical transport fiber, the wavelength-selective switch is preferably designed to couple the pulsed excitation laser beam and the pulsed depletion laser beam into the corresponding modes. In order to facilitate the coupling of the pulsed depletion laser beam into the at least one higher mode, the wavelength-selective switch can include a device for influencing the spatial intensity profile of the pulsed depletion laser beam. The optical transport fiber is preferably designed in such a way that the central mode and the at least one higher mode have a low intensity overlap.

If the pulsed excitation laser beam propagates in a central mode and the pulsed depletion laser beam in at least one higher mode of the optical transport fiber, the phase modulation device is also omitted, which in this case is not necessary to achieve the depletion focus. Before the joint focusing by means of the objective, the pulsed excitation and deactivation laser beam decoupled from the optical transport fiber are preferably a further optical element, for example a lens or an arrangement of several lenses, is collimated.

In one embodiment, at least one pulse property, in particular a pulse duration, of the laser pulses of the pulsed excitation laser beam and/or the laser pulses of the pulsed depletion laser beam is controlled by means of the wavelength-selective switch via a modulation of the spectral phase and/or the spectral amplitude of the pulsed output from the gas-filled hollow-core optical fiber Adjustable laser beam. In addition to the central wavelength, the wavelength-selective switch can also be used to set other pulse properties with individual pulse precision, in particular if this allows modulation of the spectral phase in addition to modulation of the spectral amplitude. In particular, the pulse properties also include the pulse shape. The pulse properties of the laser pulses of the pulsed excitation laser beam and the pulsed deactivation laser beam can preferably be adapted to the specific requirements of the excitation or deactivation of the fluorescent molecules used. Typically, this results in different pulse properties of the laser pulses of the excitation pulsed laser beam and the depletion pulsed laser beam. For example, they can have different pulse durations. The pulse duration of the laser pulses of the excitation and/or deactivation laser beam can preferably be adjusted within the fluorescence lifetime. Controlling the pulse duration increases flexibility and expands the microscopic possibilities. A suitable setting of the pulse properties can also be used, for example, to compensate for pulse broadening due to dispersion due to propagation of the pulsed output laser beam through one or more optical elements that follow in the beam path.

In particular, the wavelength-selective switch also allows the temporal structure, in particular the pulse repetition frequency and/or the temporal sequence, of the laser pulses of the excitation laser beam and the depletion laser beam to be adjusted in the picosecond or femtosecond range by suitable modulation of the spectral phase. In this way, for example, the laser pulses of the excitation laser beam and the depletion laser beam can also be chronologically offset. be tuned to optimally utilize the fluorescence conditions in the sample. The wavelength-selective switch can thus also be referred to as a transient wavelength-selective switch.

The temporal structure of the laser pulses of the excitation laser beam and the deactivation laser beam can alternatively or additionally already be set in the ultrashort pulse laser. The laser pulses of the excitation laser beam and the deactivation laser beam can be controlled in time with the oscillator frequency of the laser oscillator. In particular, a temporal structure in the nanosecond range can already be impressed in a driver laser using a pulse picker. A corresponding procedure is common, for example, for generating pulse bursts in the field of material processing of glasses.

In a further embodiment, the STED microscope comprises the phase modulation device and a dichroic mirror and the at least one optical transport fiber is exactly one optical transport fiber, the pulsed depletion laser beam decoupled from the optical transport fiber being deflected in front of the phase modulation device by means of the dichroic mirror can be separated from the decoupled, pulsed excitation laser beam. If both the pulsed excitation laser beam and the pulsed depletion laser beam propagate in a superimposed manner, i.e. not in different modes, through exactly one optical transport fiber, they must be separated from one another after being coupled out of the optical transport fiber. This separation preferably takes place via the central wavelengths of the laser pulses, which differ from one another, by means of the dichroic mirror. A dichroic mirror is an optical element that reflects or transmits light depending on the wavelength. For example, the dichroic mirror may reflect the depletion pulsed laser beam and transmit the excitation pulsed laser beam. Only the pulsed deactivation laser beam separated from the pulsed excitation laser beam subsequently passes through the phase modulation device. In a further embodiment, the STED microscope comprises the phase modulation device and the at least one optical transport fiber is two optical transport fibers, the wavelength-selective switch being designed to direct the pulsed excitation laser beam into the first optical transport fiber and the pulsed depletion laser beam into the second optical transport fiber to couple. The phase modulation device is arranged in such a way that only the pulsed depletion laser beam coupled out of the second optical transport fiber passes through the phase modulation device.

In a further embodiment, the at least one optical transport fiber is an optical hollow core fiber, preferably a Kagome fiber, revolver hollow core fiber or nested tube fiber.

The main advantages of using an optical hollow-core fiber as a transport fiber are the low absorption over a large spectral range, including in the ultraviolet, infrared and far infrared ranges, high destruction thresholds, the weak development of non-linear effects and the weak or adjustable compared to conventional optical fibers dispersion.

Kagome fibers are special hollow-core optical fibers that have a kagome structure in their cross-section. The use of Kagome fibers is particularly advantageous because they concentrate the laser light particularly well in the hollow core and thus allow particularly high laser power.

In a further development, no or only weak nonlinear optical effects occur in the at least one optical transport fiber, with a vacuum or a pressure of less than 200 mbar, particularly preferably less than 100 mbar, in particular less than 10 mbar, preferably in the at least one optical transport fiber prevails and/or the at least one optical transport fiber is filled with a gas with a particularly low non-linearity, in particular with He, or a gas mixture comprising He. The pressure in the at least one A transport optical fiber is then typically lower than in the gas-filled hollow-core optical fiber. Weak non-linear optical effects are understood to mean in particular those which lead to an increase in the pulse duration of less than 50 fs or a B integral of less than 0.1 rad.

In another embodiment, the gas-filled hollow-core optical fiber is a Kagome gas-filled fiber, a revolver hollow-core gas-filled fiber, or a nested-tube gas-filled fiber.

In a further embodiment, the gas-filled hollow-core optical fiber is designed to spectrally expand the laser pulses of the pulsed laser beam to form a supercontinuum.

In a further embodiment, the gas-filled hollow-core optical fiber is designed to spectrally expand the laser pulses of the pulsed laser beam to form a Raman comb. The Raman ridge arises as a result of stimulated Raman scattering in the gas-filled hollow-core optical fiber. The Raman comb includes spectral lines on both sides of the wavelength of the pulsed laser beam, so-called Stokes lines or anti-Stokes lines. Typically, there are a large number of spectral lines that are close together. The excitation and de-excitation wavelengths can be adjusted individually to the respective fluorescence marker. Due to the spectral width of the comb, many different fluorescent markers can be used without having to modify the laser or the STED microscope. Whether a Raman comb or a continuous, broadened spectrum is created depends on the gas pressure and the properties of the pulsed laser beam. A flexible, continuous switching between a supercontinuum and a Raman comb is also possible, for example, by varying the pulse duration of the laser pulses of the pulsed laser beam.

An advantage of a Raman comb compared to a continuous, broadened spectrum is that in the former case, a significant part of the power goes to the spectral lines. With the same power of the ultrashort pulse laser, the spectral power density of the spectral lines of the Raman comb is much higher than the spectral power density of a continuously broadened spectrum. Relative to the relevant narrow wavelength ranges, higher intensities are therefore available for excitation and de-excitation.

In another embodiment, the gas-filled hollow-core optical fiber is filled with a gas from a first group comprising: Ne, Ar, Kr, Xe, or a gas from a second group comprising: H2, N2, N2O, C2H2F4, C2H4F2, or with a Gas mixture comprising two or more of the gases from the first and / or the second group filled.

In a further embodiment, the wavelength-selective switch has at least one dispersive optical element and a device for location-dependent phase and/or amplitude modulation. In this case, a modulation of the spectral phase and/or the spectral amplitude is achieved in that the different wavelength components of the pulsed laser beam coupled out of the gas-filled hollow-core optical fiber are first spatially separated by means of the dispersive optical element or one of the dispersive optical elements and then by means of the Device for location-dependent phase and / or amplitude modulation in phase and / or amplitude are influenced. The wavelength components influenced in this way can then be recombined using the same dispersive optical element or another of the dispersive optical elements, whereupon they are coupled into the at least one optical transport fiber. The wavelength components of the pulsed excitation laser beam and the pulsed depletion laser beam can be brought together at the same location or spatially separated from one another. In the latter case, the pulsed excitation laser beam and the pulsed depletion laser beam can each be coupled into their own optical transport fiber.

The at least one dispersive optical element is at least one prism, for example. In this case, the division or combination of the different wavelength components results from the wavelength dependency of the refraction angles, which is a consequence of the wavelength dependency of the refractive index of the prism. Alternatively, the at least one dispersive optical element can also be at least one diffraction grating. In this case, the division or combination of the different wavelength components results from the wavelength dependency of the interference effects leading to diffraction. It is also possible to use diffraction gratings in which the light is diffracted almost completely into a specific diffraction order, for example blazed gratings.

The device for location-dependent phase and/or amplitude modulation is based, for example, on a spatial light modulator, commonly referred to as an SLM. Spatial light modulators often have a thin liquid crystal layer. In this case, the location-dependent phase and/or amplitude modulation is achieved via the adjustable alignment of the liquid crystals in a pixel array.

The device for location-dependent phase and/or amplitude modulation preferably has a liquid-crystal-on-silicon element. Liquid-crystal-on-silicon elements are special spatial light modulators built for use in reflection. This design has the advantage over other spatial light modulators that the placement of conductor tracks in the beam path can be avoided. With liquid-crystal-on-silicon elements, a thin liquid crystal layer is applied to a silicon substrate. The liquid crystal layer is used to modulate the reflected light, while control electronics are implemented on the silicon substrate using CMOS technology. An electric field can now be set in the liquid crystal layer in a pixel array. With this, the alignment of the liquid crystals in the liquid crystal layer and thus the phase difference of the reflected light can be controlled independently for each pixel.

Preferably, the wavelength selective switch allows simultaneous and independent modulation of both spectral phase and spectral amplitude. Such is, for example, through the use of a liquid Crystal-on-silicon elements with a two-dimensional pixel array in combination with a coupling of the light reflected from the liquid-crystal-on-silicon element into at least one optical fiber, for example the at least one optical transport fiber, is achieved. An axis of the two-dimensional pixel array is aligned along the spatial splitting direction of the different wavelength components. The spectral phase can be modulated by setting a phase difference along this axis. In addition, the coupling efficiency into the at least one optical transport fiber can now be adjusted as a function of wavelength via the liquid-crystal-on-silicon element, for example by this acting as a diffraction grating, with which the spectral amplitude can be modulated.

However, the wavelength-selective switch can also be implemented without a dispersive optical element. A corresponding wavelength-selective switch can be based, for example, on a programmable acousto-optical filter (AOPDF), by means of which the manipulation of both the spectral phase and the spectral amplitude is possible. Here, the diffraction of an acousto-optical wave takes place with different efficiencies and at different locations along the propagation direction (of laser pulse and acoustic wave).

Further advantages of the invention result from the description and the drawing. Likewise, the features mentioned above and those detailed below can be used according to the invention individually or collectively in any combination. The embodiments shown and described are not to be understood as an exhaustive list, but rather have an exemplary character for the description of the invention.

The invention is illustrated in the drawings and is explained in more detail using exemplary embodiments. Show it:

1 shows a schematic representation of an exemplary embodiment of a STED microscope according to the invention with exactly one optical transport fiber, 2 shows a schematic representation of an exemplary embodiment of a STED microscope according to the invention with two optical transport fibers,

3 shows a schematic representation of an exemplary embodiment of a STED microscope according to the invention with exactly one optical transport fiber, with a pulsed excitation laser beam propagating in a central mode and a pulsed depletion laser beam in at least one higher mode of the optical transport fiber, and

4 shows a schematic detailed view of a wavelength-selective switch of the STED microscope shown in FIG.

In the following description of the drawings, identical reference symbols are used for identical or functionally identical components.

The Figs. 1 to 3 each show an example of a STED microscope 1 with an ultrashort pulse laser 2 for generating a pulsed laser beam 3, a gas-filled hollow-core optical fiber 4 into which the pulsed laser beam 3 is coupled at one end and which is designed in such a way that the laser pulses of the pulsed laser beam 3 are spectrally expanded during propagation through the gas-filled hollow-core optical fiber 4 via non-linear optical effects, a wavelength-selective switch 5 for generating a pulsed excitation laser beam 6 and a pulsed depletion laser beam 7 from the other end of the gas-filled hollow-core optical fiber 4 coupled pulsed laser beam 8 and at least one optical transport fiber 9,9',10 for transporting the pulsed excitation laser beam 6 and the pulsed depletion laser beam 7.

In the STED microscope 1 shown in FIG. 1, the at least one optical transport fiber 9 is exactly one optical transport fiber. Furthermore, the STED microscope 1 shown in FIG. 1 additionally comprises a phase modulation device 11 in the form of a phase mask and a dichroic mirror 12. Deviating from this, the phase modulation device 11 can also be trained differently. For example, the phase modulation device 11 can be a spatial light modulator. The optical transport fiber 9 is a Kagome fiber here, for example, but it can also be another hollow-core optical fiber or another optical fiber. A pressure of less than 10 mbar prevails in the optical transport fiber 9, but a higher pressure, for example a pressure of less than 200 mbar or less than 100 mbar, or a vacuum can also prevail in the optical transport fiber 9. The optical transport fiber 9 can also be filled with a gas with a particularly low non-linearity, in particular with He or a gas mixture comprising He.

The pulsed deactivation laser beam 7' decoupled from the optical transport fiber 9 is separated by means of the dichroic mirror 12 from the pulsed excitation laser beam 6' decoupled from the optical transport fiber 9. In the example shown, but not necessarily, the decoupled, pulsed deactivation laser beam 7' is reflected, while the decoupled, pulsed excitation laser beam 6' is transmitted. The decoupled, pulsed depletion laser beam 7′ then strikes the phase modulation device 11 via a mirror 13 and passes through it. This leads to a modulation of its phase in such a way that a focusing of the phase-modulated, pulsed deactivation laser beam 7″ results in a deactivation focus. The phase-modulated, pulsed deactivation laser beam 7″ is then superimposed by a further dichroic mirror 15 with the decoupled, pulsed excitation laser beam 6′, which in the example shown has previously been deflected by another mirror 14 .

In the STED microscope 1 shown in FIG. 2, the at least one optical transport fiber 9, 10 is two optical transport fibers. The wavelength-selective switch 5 is designed here to couple the pulsed excitation laser beam 6 into the first optical transport fiber 9 and the pulsed deactivation laser beam 7 into the second optical transport fiber 10 . The dichroic mirror 12 shown in FIG. 1 for separating the decoupled excitation laser beam 6' and the decoupled deactivation laser beam 7' is omitted here. The depletion laser beam 7′ coupled out of the second optical transport fiber 10 strikes the phase modulation device 11 and passes through it, the phase being modulated such that the phase-modulated, pulsed depletion laser beam 7″ is focused in a depletion focus. The phase-modulated, pulsed deactivation laser beam 7″ is deflected by a mirror 16 and is then superimposed by means of a dichroic mirror 15 with the pulsed excitation laser beam 6′ coupled out of the first optical transport fiber 9 .

In the STED microscopes 1 shown in FIGS. 1 and 2, the decoupled excitation laser beam 6' and the phase-modulated, pulsed depletion laser beam 7" superimposed thereon are focused by means of an objective 17 into a sample 18 to be imaged.

In the STED microscope 1 shown in FIG. 3, the at least one optical transport fiber 9' is exactly one optical transport fiber which has a central mode for the propagation of the pulsed excitation laser beam 6 and at least one higher mode for the propagation of the pulsed Depletion laser beam 7 has. The wavelength-selective switch 5 is designed here to couple the pulsed excitation laser beam 6 into the central mode and the pulsed depletion laser beam 7 into the at least one higher mode. The spatial intensity distribution of the at least one higher mode preferably has a ring shape with a central zero point. The excitation laser beam 6' coupled out of the optical transport fiber 9' and the deactivation laser beam 7' coupled out of the optical transport fiber 9' are focused into a sample 18 to be imaged by means of an objective 17.

In the STED microscopes 1 shown in FIGS. 1 to 3, the gas-filled hollow-core optical fiber 4 is, for example, a gas-filled Kagome fiber. Deviating from this, however, it can also be another gas-filled hollow-core optical fiber. Furthermore, the gas-filled hollow-core optical fiber 4 is designed here by way of example, the laser pulses of the pulsed laser beam 3 spectrally to a Ra to expand man crest. However, the propagation of the laser pulses of the pulsed laser beam 3 in the gas-filled optical hollow-core fiber 4 can also lead to a different type of spectral expansion. For example, the gas-filled hollow-core optical fiber 4 can also be designed to expand the laser pulses of the pulsed laser beam 3 into a supercontinuum.

The gas-filled hollow-core optical fiber 4 is also filled with helium here, for example. Deviating from this, the gas-filled hollow-core optical fiber 4 can also be filled with another gas from a first group, including Ne, Ar, Kr, Xe, or a gas from a second group, including H2, N2, N2O, C2H4, C2H2F4, C2H4F2, or with a gas mixture comprising two or more gases from the first and/or the second group.

The objective 17 is also used to collect fluorescent light 19 emitted by the sample 18 . To separate the collected fluorescent light 19 from the rest of the beam path, the STED microscopes 1 include a further dichroic mirror 20 here, for example. The collected fluorescent light 19 is then detected by means of a detection device 21 . The detection device 21 includes, for example, an avalanche photodiode, not shown here. Deviating from this, the detection device 21 can also be designed differently, for example it can comprise a spectrometer or a monochromator.

To image the sample 18, it is typically scanned. For this purpose, the STED microscope 1 can comprise a corresponding device, not shown here, for example a scanner head based on galvanometer mirrors or acousto-optical deflectors. Alternatively or additionally, the sample 18 can also be moved by means of piezo actuators, for example.

FIG. 4 shows a schematic detailed representation of the wavelength-selective switch 5 of the STED microscope 1 shown in FIG Diffraction grating, a cylindrical mirror 23 and a device 24 for location-dependent phase and amplitude modulation with a liquid-crystal-on-silicon element 25 on.

The pulsed laser beam 8 decoupled from the gas-filled optical hollow-core fiber 4 strikes the diffraction grating 22 via the cylindrical mirror 23 and is split into its wavelength components 8', 8". The cylinder axis of the cylindrical mirror 23 is perpendicular to the splitting direction of the different wavelength components 8', 8". The different wavelength components 8′, 8″ strike the liquid-crystal-on-silicon element 25 via the cylindrical mirror 23 and are reflected there. The liquid-crystal-on-silicon element 25 has a two-dimensional pixel array, with the phase difference that is impressed on the different wavelength components 8', 8" being individually adjustable for each pixel.

The wavelength components 26′, 26″ modulated in this way now strike the dispersive optical element 22 again via the cylindrical mirror 23 and are combined as a pulsed excitation laser beam 6 and a pulsed deactivation laser beam 7 . The pulsed excitation laser beam 6 and the pulsed deactivation laser beam 7 are then coupled into the optical transport fiber 9 via the cylindrical mirror 23 .

In the example shown, an axis of the two-dimensional pixel array of the liquid-crystal-on-silicon element 25 is aligned along the spatial splitting direction of the different wavelength components 8′, 8″. The spectral phase of the pulsed laser beam 8 coupled out of the gas-filled optical hollow-core fiber 4 can be modulated by adjusting the phase difference along this axis. In addition, the coupling efficiency into the optical transport fiber 9 can be adjusted as a function of wavelength via the phase difference impressed with the liquid-crystal-on-silicon element 25 and individually adjustable for each pixel. In this way, the spectral amplitude of the pulsed laser beam 8 coupled out of the gas-filled optical hollow-core fiber 4 can also be modulated. The result here is, for example, by means of the wavelength-selective switch 5, at least one Pulse property, in particular a pulse duration of the laser pulses of the pulsed excitation laser beam 6 and/or the laser pulses of the pulsed deactivation laser beam 7 can be adjusted.

Deviating from the case presented here, the dispersive optical element 22 does not have to be a diffraction grating. For example, it can also be a prism. The wavelength-selective switch 5 can also have no, two or even more than two dispersive optical elements and/or no cylindrical mirror. Furthermore, the device 24 for location-dependent phase and/or amplitude modulation does not have to have a liquid-crystal-on-silicon element. For example, this can also include an array of adjustable mirrors in the form of a microelectromechanical system (MEMS).

In order, as shown in FIG. 2, to couple the pulsed excitation laser beam 6 into the first optical transport fiber 9 and the pulsed depletion laser beam 7 into the second optical transport fiber 10, the wavelength-selective switch 5 shown in FIG. 4 can be modified accordingly. For this purpose, the end facets of the first optical transport fiber 9 and the second optical transport fiber 10 can be arranged spatially close together. Using the two-dimensional pixel array of the liquid-crystal-on-silicon element 25, the in-coupling efficiencies in the first optical transport fiber 9 and the second optical transport fiber 10 can then be adjusted as a function of wavelength via a wavelength-dependent variation of location and angle, analogously to the adjustment of the in-coupling efficiency described above into the one optical transport fiber 9.

In order to couple the pulsed excitation laser beam 6 into the central mode and the pulsed depletion laser beam 7 into the at least one higher mode of a suitable optical transport fiber 9' in accordance with the case shown in FIG. 3, an analogous procedure can be followed. Even small variations in location and angle are sufficient here to achieve coupling into a higher mode instead of into the central mode or vice versa.

Claims

Claims STED microscope (1), comprising

- an ultra-short pulse laser (2) for generating a pulsed laser beam (3),

- a gas-filled hollow-core optical fiber (4) into which the pulsed laser beam (3) is coupled at one end and which is designed in such a way that the laser pulses of the pulsed laser beam (3) produce non-linear optical effects during propagation through the gas-filled hollow-core optical fiber (4). be spectrally expanded,

- a wavelength-selective switch (5) for generating a pulsed excitation laser beam (6) and a pulsed depletion laser beam (7) from the other end of the gas-filled hollow-core optical fiber (4) decoupled, pulsed laser beam (8),

- at least one optical transport fiber (9,9', 10) for transporting the pulsed excitation laser beam (6) and the pulsed depletion laser beam (7), either the STED microscope (1) having a phase modulation device (11) for modulating the phase of the comprises at least one optical transport fiber (9, 10) decoupled, pulsed depletion laser beam (7'), which is designed in such a way that focusing of the phase-modulated, pulsed depletion laser beam (7") results in a depletion focus, or the at least one optical transport fiber (9') is exactly one optical transport fiber which has a central mode for the propagation of the pulsed excitation laser beam (6) and at least one higher mode for the propagation of the pulsed deactivation laser beam (7),

- A lens (17) for joint focusing of the decoupled, pulsed excitation laser beam (6 ') and the phase-modulated relation ing the pulsed deactivation laser beam (7',7") decoupled from the optical transport fiber (9') into a sample (18) and for collecting fluorescent light (19) emitted by the sample (18), and - a detection device (21) to detect the collected fluorescent light (19). STED microscope according to claim 1, characterized in that by means of the wavelength-selective switch (5) at least one pulse property, in particular a pulse duration, of the laser pulses of the pulsed excitation laser beam (6) and / or the laser pulses of the pulsed depletion laser beam (7) via a modulation of the spectral The phase and/or the spectral amplitude of the pulsed laser beam (8) coupled out of the gas-filled optical hollow-core fiber (4) can be adjusted. STED microscope according to Claim 1 or 2, characterized in that it comprises the phase modulation device (11) and a dichroic mirror (12) and the at least one optical transport fiber (9) is exactly one optical transport fiber, the optical transport fiber (9) decoupled, pulsed excitation laser beam (7 ') in front of the phase modulation device (11) by means of the dichroic mirror (12) from the decoupled, pulsed excitation laser beam (6') is separable. STED microscope according to Claim 1 or 2, characterized in that it comprises the phase modulation device (11) and the at least one optical transport fiber (9,10) is two optical transport fibers, the wavelength-selective switch (5) is designed to couple the pulsed excitation laser beam (6) into the first optical transport fiber (9) and the pulsed depletion laser beam (7) into the second optical transport fiber (10). STED microscope according to one of the preceding claims, characterized in that the at least one optical transport fiber (9, 9', 10) is a hollow core optical fiber, preferably a Kagome fiber, revolver hollow core fiber or nested tube fiber. STED microscope according to Claim 5, characterized in that no or only weak non-linear optical effects occur in the at least one optical transport fiber (9, 9', 10), with preferably in the at least one optical transport fiber (9, 9', 10) a vacuum or a pressure of less than 200 mbar, particularly preferably less than 100 mbar, in particular less than 10 mbar, prevails and/or the at least one optical transport fiber (9, 9', 10) with a gas with a particularly low non-linearity, is filled in particular with He or a gas mixture comprising He. STED microscope according to one of the preceding claims, characterized in that the gas-filled hollow-core optical fiber (4) is a gas-filled Kagome fiber, a gas-filled hollow-core revolver fiber or a gas-filled nested-tube fiber. STED microscope according to one of the preceding claims, characterized in that the gas-filled hollow-core optical fiber (4) is designed to spectrally expand the laser pulses of the pulsed laser beam (3) to form a supercontinuum. STED microscope according to one of the preceding claims, characterized in that the gas-filled hollow-core optical fiber (4) is designed to spectrally expand the laser pulses of the pulsed laser beam (3) to form a Raman comb. STED microscope according to one of the preceding claims, characterized in that the gas-filled hollow-core optical fiber (4) is filled with a gas from a first group comprising: Ne, Ar, Kr, Xe, or a gas from a second group comprising: H2, N2, N2O, C2H2F4, C2H4F2, or filled with a gas mixture comprising two or more of the gases from the first and/or the second group. STED microscope according to one of the preceding claims, characterized in that the wavelength-selective switch (5) has at least one dispersive optical element (22) and a device (24) for location-dependent phase and / or amplitude modulation. STED microscope according to Claim 11, characterized in that the device (24) for location-dependent phase and/or amplitude modulation has a liquid-crystal-on-silicon element (25).