[go: up one dir, main page]

WO2006114247A1 - Microscope à fluorescence - Google Patents

Microscope à fluorescence Download PDF

Info

Publication number
WO2006114247A1
WO2006114247A1 PCT/EP2006/003720 EP2006003720W WO2006114247A1 WO 2006114247 A1 WO2006114247 A1 WO 2006114247A1 EP 2006003720 W EP2006003720 W EP 2006003720W WO 2006114247 A1 WO2006114247 A1 WO 2006114247A1
Authority
WO
WIPO (PCT)
Prior art keywords
excitation light
light
sample
fluorescence microscope
microscope according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2006/003720
Other languages
German (de)
English (en)
Inventor
Stefan Hell
Johann Engelhardt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften
Publication of WO2006114247A1 publication Critical patent/WO2006114247A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes

Definitions

  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • tunable laser light sources are known, for. B. Titan-sapphire laser with downstream optically parameterized oscillator (OPO).
  • OPO optically parameterized oscillator
  • 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.
  • 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.
  • solid state lasers 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.
  • OPOs optically parameterized oscillators
  • 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.
  • the depletion light source is a mode-locked gas laser with a line width of the depletion of less than 2 nm.
  • 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.
  • mode-locked gas lasers as Abregungslicht provoke 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mode-locked gas lasers 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.
  • the de-excitation light can make use of the multitude of lines of excitation of a fluorescent dye of an ArKr gas laser.
  • 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.
  • 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.
  • 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.
  • 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
  • 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 Abregungsanders can consist of a single punctiform or linear portion of the sample or a pattern of a plurality of point or line-shaped portions.
  • 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.
  • Fig. 1 shows the energy levels of a fluorescent dye, in the context of his
  • Fig. 2 outlines the timing of the pulses of the excitation light
  • 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.
  • Fig. 1 shows the energy spectrum of a fluorescent dye.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the de-excitation light 7 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an objective 45 is provided between the dichroic mirror 44 and the sample 19.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • acousto-optical elements 51 and 52 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.
  • 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

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un microscope à fluorescence (12) doté d'une source (14) de lumière d'excitation (5), qui excite un colorant fluorescent dans un échantillon (19) pendant une durée limitée et dans une zone spatiale définie pour l'émission spontanée de lumière fluorescente (6) dont les longueurs d'ondes se situent dans une plage de longueurs d'ondes donnée. Ce microscope comporte une source (15) pulsée de lumière de désexcitation (7), qui désexcite le colorant fluorescent, mise à part une zone résiduelle réduite relativement à la zone spatiale définie. La lumière de l'échantillon (19) peut être mise en correspondance avec d'autres longueurs d'ondes que celles de la lumière d'excitation (5) et de la lumière de désexcitation (7) de l'émission spontanée de lumière fluorescente (6) de la zone résiduelle de la zone spatiale définie. L'invention est caractérisée en ce que la source (15) de lumière de désexcitation est un laser à gaz (13) à couplage de modes, la largeur de raie de la lumière de désexcitation (7) étant inférieure à 2 nm.
PCT/EP2006/003720 2005-04-27 2006-04-22 Microscope à fluorescence Ceased WO2006114247A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005020003.6 2005-04-27
DE200510020003 DE102005020003B4 (de) 2005-04-27 2005-04-27 Fluoreszenzmikroskop

Publications (1)

Publication Number Publication Date
WO2006114247A1 true WO2006114247A1 (fr) 2006-11-02

Family

ID=36648808

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2006/003720 Ceased WO2006114247A1 (fr) 2005-04-27 2006-04-22 Microscope à fluorescence

Country Status (2)

Country Link
DE (1) DE102005020003B4 (fr)
WO (1) WO2006114247A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7772569B2 (en) 2008-04-01 2010-08-10 The Jackson Laboratory 3D biplane microscopy
DE102009008646A1 (de) 2009-02-12 2010-08-19 Dodt, Hans-Ulrich, Dr. Vorrichtung zum optischen Abbilden einer Probe
US8039815B2 (en) 2007-10-05 2011-10-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Fluorescent light microscope for measuring a sample using red-shifted stokes lines
US8217992B2 (en) 2007-01-11 2012-07-10 The Jackson Laboratory Microscopic imaging techniques
US9759661B2 (en) 2008-02-12 2017-09-12 Hans-Ulrich Dodt Device for the optical imaging of a sample

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007025688A1 (de) 2007-06-01 2008-12-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Wellenlängen- oder polarisationssensitiver optischer Aufbau und dessen Verwendung
DE102010047237B4 (de) * 2010-08-13 2021-07-01 Leica Microsystems Cms Gmbh Verfahren zum Trennen von Detektionssignalen im Strahlengang einer optischen Einrichtung

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5814820A (en) * 1996-02-09 1998-09-29 The Board Of Trustees Of The University Of Illinois Pump probe cross correlation fluorescence frequency domain microscope and microscopy
DE19829944A1 (de) * 1998-07-04 2000-01-05 Zeiss Carl Jena Gmbh Verfahren und Anordnung zur Gerätekonfiguration von konfokalen Mikroskopen
DE10056384A1 (de) * 2000-11-14 2002-05-29 Leica Microsystems Verfahren und Vorrichtung zur Messung der Lebensdauer eines angeregten Zustandes in einer Probe
DE10063276A1 (de) * 2000-12-19 2002-07-04 Leica Microsystems Scanmikroskop
US20030107732A1 (en) * 2001-10-03 2003-06-12 Olympus Optical Co., Ltd. Laser scanning microscope
DE10231776A1 (de) * 2002-07-13 2004-01-22 Leica Microsystems Heidelberg Gmbh Verfahren zur Scanmikroskopie und Scanmikroskop
US20040186516A1 (en) * 2002-12-26 2004-09-23 Goodwin Paul C. System and method of illuminating living cells for imaging
DE10347712A1 (de) * 2003-10-14 2005-05-12 Leica Microsystems Rastermikroskop

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5731588A (en) * 1994-02-01 1998-03-24 Hell; Stefan Process and device for optically measuring a point on a sample with high local resolution
EP1055144B1 (fr) * 1998-02-19 2015-01-14 Leica Microsystems CMS GmbH Systeme optique a element spectroselectif
DE10105391B4 (de) * 2001-02-06 2004-11-25 Leica Microsystems Heidelberg Gmbh Scanmikroskop und Modul für ein Scanmikroskop

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5814820A (en) * 1996-02-09 1998-09-29 The Board Of Trustees Of The University Of Illinois Pump probe cross correlation fluorescence frequency domain microscope and microscopy
DE19829944A1 (de) * 1998-07-04 2000-01-05 Zeiss Carl Jena Gmbh Verfahren und Anordnung zur Gerätekonfiguration von konfokalen Mikroskopen
DE10056384A1 (de) * 2000-11-14 2002-05-29 Leica Microsystems Verfahren und Vorrichtung zur Messung der Lebensdauer eines angeregten Zustandes in einer Probe
DE10063276A1 (de) * 2000-12-19 2002-07-04 Leica Microsystems Scanmikroskop
US20030107732A1 (en) * 2001-10-03 2003-06-12 Olympus Optical Co., Ltd. Laser scanning microscope
DE10231776A1 (de) * 2002-07-13 2004-01-22 Leica Microsystems Heidelberg Gmbh Verfahren zur Scanmikroskopie und Scanmikroskop
US20040186516A1 (en) * 2002-12-26 2004-09-23 Goodwin Paul C. System and method of illuminating living cells for imaging
DE10347712A1 (de) * 2003-10-14 2005-05-12 Leica Microsystems Rastermikroskop

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8217992B2 (en) 2007-01-11 2012-07-10 The Jackson Laboratory Microscopic imaging techniques
US8039815B2 (en) 2007-10-05 2011-10-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Fluorescent light microscope for measuring a sample using red-shifted stokes lines
US9759661B2 (en) 2008-02-12 2017-09-12 Hans-Ulrich Dodt Device for the optical imaging of a sample
US7772569B2 (en) 2008-04-01 2010-08-10 The Jackson Laboratory 3D biplane microscopy
US7880149B2 (en) 2008-04-01 2011-02-01 The Jackson Laboratory 3D biplane microscopy
DE102009008646A1 (de) 2009-02-12 2010-08-19 Dodt, Hans-Ulrich, Dr. Vorrichtung zum optischen Abbilden einer Probe

Also Published As

Publication number Publication date
DE102005020003B4 (de) 2007-10-11
DE102005020003A1 (de) 2006-11-09

Similar Documents

Publication Publication Date Title
EP1055144B1 (fr) Systeme optique a element spectroselectif
DE102007039111B4 (de) STED-Fluoreszenzmikroskopie mit Zweiphotonen-Anregung
DE102007048135B4 (de) Fluoreszenzlichtmikroskopisches Messen einer Probe mit rotverschobenen Stokes-Linien
EP1058101B1 (fr) Sélection de zones spectrales par un diaphragme réfléchissant
EP1795938B1 (fr) Procédé et agencement destinés à l'analyse d'échantillons
DE10154699B4 (de) Verfahren und Vorrichtung zum räumlich eng begrenzten Anregen eines optischen Übergangs
EP3042169B1 (fr) Microscope à balayage et combinateur de faisceau acousto-optique pour microscope à balayage
EP3042232B1 (fr) Microscope à balayage et séparateur de faisceaux principaux pour microscope à balayage
EP2087391A1 (fr) Configuration optique, et procédé pour commander et influencer un faisceau lumineux
DE10228374A1 (de) Verfahren zur Mikroskopie und Mikroskop
DE10339311B4 (de) System und Verfahren zur Einstellung eines Fluoreszenzspektralmesssystems zur Mikroskopie
EP1085362A2 (fr) Arrangement optique pour microscope à balayage laser
EP2423727A1 (fr) Dispositif de déviation temporelle d'impulsions laser à lumière blanche
DE102005020003B4 (de) Fluoreszenzmikroskop
DE4015861C2 (de) Excimer-Laser
DE2831813A1 (de) Optisches filter
DE2020104B2 (de) Verstärkerkettenstufe für Laserlichtimpulse
EP2557445B1 (fr) Dispositif et procédé destinés à répartir la lumière d'éclairage et la lumière de détection dans un microscope
EP3252523B1 (fr) Procédé de réglage de l'intensité d'un rayonnement lumineux dans un agencement optique et agencement optique correspondant
WO2017207664A1 (fr) Disposition optique pour l'éclairage pulsé, procédé pour l'éclairage pulsé et microscope
DE19744302B4 (de) Vorrichtung zur Einkopplung der Strahlung von Kurzpulslasern in einem mikroskopischen Strahlengang
DE69020689T2 (de) Schmalband-Laservorrichtung.
DE102008028707A1 (de) Laser-Scanning-Mikroskop mit einer Laserdiode
EP1610173A1 (fr) Système laser à amplificateur optique paramétrique

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

NENP Non-entry into the national phase

Ref country code: RU

WWW Wipo information: withdrawn in national office

Country of ref document: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06753405

Country of ref document: EP

Kind code of ref document: A1