EP2185919A1 - Microscopie de fluorescence sted à excitation biphotonique - Google Patents

Microscopie de fluorescence sted à excitation biphotonique

Info

Publication number
EP2185919A1
EP2185919A1 EP08787227A EP08787227A EP2185919A1 EP 2185919 A1 EP2185919 A1 EP 2185919A1 EP 08787227 A EP08787227 A EP 08787227A EP 08787227 A EP08787227 A EP 08787227A EP 2185919 A1 EP2185919 A1 EP 2185919A1
Authority
EP
European Patent Office
Prior art keywords
excitation light
light
sample
fluorescent
pulses
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.)
Withdrawn
Application number
EP08787227A
Other languages
German (de)
English (en)
Inventor
Stefan W. Hell
Katrin Willig
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 eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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 eV filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Publication of EP2185919A1 publication Critical patent/EP2185919A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/636—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence

Definitions

  • the invention relates to a method having the features of the preamble of independent claim 1 and an apparatus having the features of the preamble of independent claim 11 for spatially high-resolution imaging of a structure labeled with a fluorescent dye in a sample.
  • the method defined in the preamble of independent claim 1 is also referred to as STED (Stimulated Emission Depletion) fluorescence light microscopy.
  • the method falls below the diffraction limit, which is normally the limit of resolution in far-field microscopic methods.
  • the diffraction-limited focus area in which the excitation light basically excites the fluorescent dye in the sample for spontaneous emission of fluorescent light, reduced to expansions below the diffraction limit by parts of the focus area are superimposed with de-excitation light, the excited fluorescent dye before the fluorescence emission again dissipates. This leaves only a measuring range which is smaller than the focal range and from which the fluorescent light spontaneously emitted by the fluorescent dye can originate.
  • the structure marked with the fluorescent dye in the sample can be imaged with a spatial resolution that exceeds the diffraction limit.
  • the spatial resolution is particularly good when the de-excitation light is directed in the form of an interference pattern on the sample, which is a zero at the location of the measuring range and otherwise reaches saturation in the de-excitation of excited with the excitation light state of the fluorescent dye.
  • the excitation light source can be a continuous wave laser (cw laser), while the depletion light source can be a pulse laser which is synchronized with the detector in order to detect the fluorescent light from the sample only after the Decay each one pulse of the de-excitation light register. In this way it is avoided that the detector detects reflected light reflected by the sample or also fluorescent light whose emission was stimulated by the de-excitation light and thus does not originate from the measuring range of interest.
  • the polarization of the excitation light and the polarization filtering of the light incident on the detector from the sample in the orthogonal direction to the polarization of the excitation light is described.
  • the average light intensity available for the de-excitation light is also to focus on pulses that are applied as soon as possible to the pulses of the excitation light on the sample in order to give the fluorescent dye outside the measuring range as little as possible opportunity to emit fluorescent light spontaneously.
  • This proximity to time can be achieved by an immediate sequence or even a partial or even complete temporal coverage.
  • a prerequisite for this sequence is a synchronization of the pulses of the de-excitation light with the pulses of the excitation light, if appropriate in addition to a synchronization of the detector with the pulses of the de-excitation light.
  • Fluorescent dyes below and above the focal plane are excited and thus be distracted by the de-excitation light or must be distracted.
  • Using a confocal pinhole in front of the detector can reduce the measuring range along the optical axis to the focal plane, it means that the
  • Fluorescent dye is uselessly excited and de-excitated outside the focal plane, resulting in significant bleaching of the fluorescent dye by the Abregungsstrahl and thus prevents the capture of 3D images in many dyes.
  • the excitation light can have portions of different wavelengths, and three or even more individual photons can be involved in the multiphoton process.
  • the excitation light can have portions of different wavelengths, and three or even more individual photons can be involved in the multiphoton process.
  • a two-photon excitation with excitation light only one wavelength at which the photons each contribute half of the photon energy required for the multiphoton process takes place.
  • the selectivity of the excitation at the focal plane is based here on the nonlinearity between the intensity of the excitation light and the excitation probability of the fluorescent dye in its fluorescent state over the - A -
  • Multiphoton process In a two-photon excitation, this excitation probability is dependent on the square of the intensity of the excitation light and thus focuses on the main diffraction peak of the focus region of the excitation light in the sample.
  • this temporal concentration of the excitation light and the concomitant increased photon concentrations in each individual pulse of the excitation light there is a markedly increased yield of fluorescent light compared to continuously, ie with excitation light incident on the sample with constant time intensity.
  • the fluorescent dye known to vary a time repetition distance of an incident on the sample optical signal in a range of at least 0.1 microseconds to 2 microseconds to maximize the fluorescent light output.
  • the optical signal from a continuous wave laser come when a scanning device is provided for spatially scanning the sample with the optical signal having a sampling rate that sets the desired repetition distance.
  • the pauses provided by the repeating interval allow the fluorescent dye to relax back to its singlet, fluorescent state from a dark state, particularly a triplet state, into which it is exposed to some extent on each exposure to the optical signal.
  • WO 2007/030835 A2 discloses a method for spatially high-resolution imaging of a structure labeled with a fluorescent dye in a sample and a device suitable therefor.
  • the fluorescent dye is a compound which has a dark state in which it is not excitable for fluorescence and a fluorescent state in which it is excitable with excitation light for fluorescence. From the Dark state, the connection with the switch-on light in the fluorescent state can be switched on, the transition to the fluorescent state is to take place via an intermediate state, from which the compound after a pulse of the switch-on with a pulse of down light back to the dark state should be transferred. With this downshift light, the switched-on state is reduced in relation to the focus range of the switch-on light to a measuring range.
  • excitation light which excites the compound in the fluorescent state to fluorescence.
  • This excitation light can have the same wavelength as the return light but has a different spatial distribution and in each case simultaneously acts as a switch-off light, which switches off the connection switched into the fluorescent state back into its dark state.
  • another pulse of the switch-on light must be incident on the sample with a subsequent pulse of the switch-back light.
  • the pulsed turn-on light may have a wavelength such that it turns on the compound via a multi-photon process in its fluorescent state.
  • the excitation light acting at the same time as the switch-off light can be provided with a continuous-wave laser, for example a diode laser, which can be switched on and off as required.
  • Declare light which again excites the fluorescence-excited compound before the spontaneous emission of fluorescent light, is not used according to WO 2007/030 835 A2.
  • the expense for the method known from this document and the apparatus used for this purpose are great, because light with at least three different wavelengths and / or spatial distributions, namely the switch-on light, the switch-back light and the excitation light which also acts as switch-off light, must be provided the switch-on light and the switch-back light are provided in exactly synchronized pulses, since the intermediate state of the switchable connection on which the switchback light can act is only short-lived, and the use of switchable fluorophores already represents a basic outlay that goes far beyond the expense goes beyond simple fluorescent dyes.
  • the invention has for its object to provide a method and apparatus for spatially high-resolution three-dimensional (3D) imaging of a marked with a fluorescent dye structure in a sample that can be realized with relatively little effort, which avoids premature bleaching of the sample by the Abregungsstrahl and yet allow a significant increase in the spatial resolution on the diffraction limit in all directions.
  • the object of the invention is achieved by a method having the features of independent claim 1 and by an apparatus having the features of independent claim 11.
  • Preferred embodiments of the new method are defined in the dependent claims 2 to 10, while the dependent claims 12 to 20 describe preferred embodiments of the new device.
  • the wavelength of the excitation light is selected so that the excitation light excites the fluorescent dye via a multi-photon process.
  • this is a two-photon process.
  • the excitation light for the multi-photon excitation is pulsed, which in a known manner has a favorable effect on the fluorescent light yield due to multiphoton excitation
  • the de-excitation light which has a shorter wavelength than the excitation light, becomes continuous or quasi-continuous over a plurality of pulses of the excitation light directed to the test.
  • the focus area in which an effective excitation of the fluorescent dye takes place, is clearly spatially limited compared to a possible excitation via a one-photon process.
  • this non-linear excitation is limited to a focal depth of typically -1 ⁇ m for high-aperture objectives that corresponds to the extent of its principal diffraction maximum along the optical axis. Because the excitation is spatially confined in 3D, less or no fluorescent dyes must be disturbed, which are located above and below the focal plane. In particular, fluorescent dyes that are outside the main focal maximum and that would otherwise be excited by a one-photon process need not be de-excited by the de-excitation light.
  • the wavelength of the depletion light is preferably selected to fall within the red end of the emission spectrum of the dye. Basically, therefore, applying a non-excited sample with de-excitation light does not lead to bleaching because its photon energy is too low to excite the fluorescent dye.
  • the de-excitation light can not only quench as desired fluorescence dyes that are already excited, but also bleach. Bleaching by the de-excitation light takes place only on already excited fluorescent dyes.
  • the excitation by the multiphoton excitation is narrowed from the outset in three dimensions to the main diffraction peak, the range in which fluorescent dyes must be reduced is also reduced. Because the number of unnecessary de-excitations per fluorescent dye molecule is reduced, the bleaching is also reduced, which considerably simplifies the nanoscale 3D imaging by STED fluorescence microscopy and even makes it possible with many dyes. If the multiphoton excitation is only in a 1 micron thick layer, the number of unnecessary de-excitation is reduced by a factor of 10 with a 10 micron thick sample. Accordingly, if the area of the sample in which the de-excitation light is needed can be significantly reduced from the measuring area, bleaching by the de-excitation light is correspondingly reduced. This is achieved by excitation of the fluorescent dye via a multiphoton process to fluorescence.
  • any synchronization of the detector for the fluorescent light spontaneously emitted by the fluorescent dye with any pulses of the illumination or de-excitation light can be completely eliminated.
  • the detector can continuously register the fluorescent light spontaneously emitted from the fluorescent dye over a plurality of pulses of the excitation light.
  • the detector, the fluorescent light z. B. by an edge filter or a narrow-band bandpass filter extract from the Abregungslicht or through a polarizing filter, to which then the de-excitation light is to polarize targeted.
  • the de-excitation light is provided with a diode laser.
  • diode lasers can be provided at approximately one-tenth of the cost of a conventional pulsed laser for providing de-excitation light in STED fluorescent light microscopy as a continuous wave laser.
  • the new method also has a positive effect that the effective multi-photon excitation of the sample by the excitation light at any time is also limited to a smaller spatial area and thereby also the total burden with the excitation light, which corresponds to the risk of bleaching the fluorescent dye, is reduced compared with the fluorescence yield.
  • the excitation light may in the new method with a comparatively high frequency of at least 80 MHz, preferably at least 100 MHz, more preferably at least
  • This range is 80 to 100 MHz. Due to the very high frequency of the pulses of the excitation light, the pauses between the pulses of the excitation light are very short. This prevents that very large portions of the de-excitation light fall onto the sample without there being very large proportions of the fluorescent dye in the ground state.
  • the individual pulses of the excitation light are, as in fluorescence light microscopy, under multi-photon excitation of the fluorescent dye compared to its pulse distance preferred. wise relatively short.
  • the individual pulses of the excitation light may have a duration of at most one-tenth, preferably at most one-hundredth, more preferably at most one-thousandth, and most preferably at most one ten-thousandth of their pulse spacing. As already stated in the introduction, this increases the relative yield of fluorescent light from the sample, while maintaining the average power of the excitation beam.
  • the new method it is possible, in particular, to scan the sample three-dimensionally with the measuring range or a plurality of similar measuring ranges, from which the fluorescent light spontaneously emitted by the fluorescent dye is spatially separated, the structure labeled with the fluorescent dye in the sample being three-dimensional is resolved.
  • a confocal pinhole in front of the detector and, in the case of multiple focal measurement areas, a confocal array of pinhole diaphragms.
  • Aperture diaphragms suppress stray light from both the depletion and excitation beams.
  • the fluorescent light is not returned via the optical scanning means, but registered in a simplified optical path, i. H. a "non-descanned detection", as is known in the art.
  • Abregungsetter interrupted after a plurality of pulses of the excitation light for a limited period of time. These interruptions have the purpose of relaxing the fluorescent dye from a dark state, in particular its Triplet state, to wait back to the fluorescent singlet state, after this dark state has been populated during the previous pulse of the excitation light by the excitation light already to a considerable extent.
  • This population of dark states results in a reduced yield of fluorescent light from the sample and may also be associated with an increased risk of bleaching the fluorescent dye.
  • the dark state decays again, so that corresponding interruptions of the excitation lead to an overall increased yield of fluorescence light from the sample.
  • the period of the plurality of pulses of the excitation light between the pauses of the excitation light over which the excitation light is continuously applied to the sample is typically from 100 ns to 50 ⁇ s and preferably 0.5 to 2 ⁇ s. The exact duration of this period depends on how fast the triplet state or the dark state practically populates.
  • the excitation light source irradiates the excitation light at such a wavelength that the excitation light excites the fluorescent dye through a multi-photon process, and the depletion light source continuously directs the depletion light having a shorter wavelength than the excitation light across a plurality of pulses of the excitation light to the test.
  • the detector of the device will also continuously register the fluorescent light spontaneously emitted by the fluorescent dye over a plurality of pulses of the excitation light.
  • the depletion light source has a continuously emitting diode laser.
  • the excitation light source has a pulse laser which excites the excitation light in pulses with the one explained above
  • a scanner which is provided in the new device to the sample with the measuring range or a
  • scanning is preferably carried out by displacing an optical element which counts both the optics focusing the excitation light into the specimen and the optic directing the excitation light into the specimen.
  • Various devices for the screening of a focused beam over the sample are known to the person skilled in the art sufficiently from the literature of laser scanning microscopy.
  • the new device may comprise a controller which is preferably adjustable to control the limited period and a period of the plurality of pulses between two such limited periods is set.
  • controllers preferably include acousto-optic or electro-optic beam modulators.
  • the detector of the new device need not be synchronized with the limited periods in which the excitation light and the excitation light are interrupted, but it can continuously register the fluorescent light spontaneously emitted from the fluorescent dye over these periods, but this does not occur.
  • Fig. 1 shows the schematic structure of an embodiment of the new device.
  • Fig. 2 shows at the top the schematic time course of the intensity of the excitation light, in the middle the schematic time course of the intensity of the depletion light and at the bottom the schematic time profile of the sensitivity of the detector for registering fluorescent light from a sample in one embodiment of the new method.
  • the device 1 sketched in FIG. 1 is used for spatially high-resolution imaging of a structure marked with a fluorescent dye in a sample 2.
  • an excitation light source 3 in the form of a pulsed laser 4 emits pulsed excitation light 5 through an optical system 6 into a focal region of an objective 7 in FIG Directed sample.
  • the fluorescent dye in sample 2 is excited by a Zweiprotonen Anlagen for fluorescence, ie the spontaneous emission of fluorescent light.
  • the focus area is defined here as the area in which an effective excitation of the fluorescent dye takes place via the two-photon process for the spontaneous emission of fluorescent light.
  • the fluorescent dye is immediately de-excited again by continuously applying depletion light 10 to the sample with a deenergizing light source 8 in the form of a diode laser 9.
  • the plane waves of the excitation light 10 are deformed with a phase modulator 11 in such a way that an intensity distribution of the excitation light 10 with a central zero point adjoins the intensity maxima covering the remainder of the focal region.
  • a detector 12 which registers fluorescent light spontaneously emitted by the fluorescent dye of the sample 2 only detects fluorescent light from the measuring range which has dimensions below the diffraction limit of the light with the wavelengths used.
  • the excitation light 5 is kept away from the detector 12 by a dichroic mirror 14 which detects the beam paths of the Excitation light 5 and the fluorescent light 13 separates.
  • the de-excitation light 10 is kept away from the detector 12 by a dichroic mirror 15 which separates the optical paths of the depletion light 10 and the fluorescent light 13.
  • a scanning device 16 can be provided to move an optical element 17 of the optics 6, through which the excitation light 5 as well as the excitation light 10 as well as the fluorescent light 13 from the sample passes, such that the structures labeled with the fluorescent dye in the sample have two is scanned three-dimensionally.
  • Fig. 2 shows the temporal progression of the intensity of the excitation light 5 above. Between limited periods 18, in which the intensity of the excitation light is zero, the excitation light consists of individual pulses with a pulse duration shorter than their distance. The limited periods 18 have a typical duration of 1 ⁇ s. The frequency of the pulses 19 of the excitation light 5 is above 120 MHz, their distance being at least 10 times greater than their duration. These numerical ratios are not fully reflected in FIG.
  • Fig. 2 shows the intensity profile of the depletion light 10 over time. The de-excitation light 10 is applied continuously, ie, with constant intensity, to the sample 2, except in the limited periods 18, in which its intensity is also zero.
  • Fig. 2 shows the sensitivity 20 of the detector 12 of Fig. 1 over time. This is continuous, ie also given over the limited periods 18 away.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Nonlinear Science (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne la représentation à haute résolution spatiale d'une structure marquée par un colorant fluorescent dans un échantillon. Des impulsions de lumière d'excitation (5) sont focalisées dans l'échantillon pour que le colorant fluorescent se trouvant dans la zone de focalisation émette spontanément de la lumière fluorescente. Une lumière de désexcitation (10), dont la longueur d'ondes est différente de celle de la lumière d'excitation (5), est dirigée vers l'échantillon pour désexciter le colorant fluorescent à l'extérieur d'une zone de mesure réduite par rapport à la zone de focalisation, avant l'émission spontanée de lumière fluorescente. La lumière fluorescente émise spontanément par le colorant fluorescent est enregistrée. Selon l'invention, la longueur d'ondes de la lumière d'excitation (5) est sélectionnée de manière à ce que la lumière d'excitation (5) excite le colorant fluorescent par un processus multiphotonique, et la lumière de désexcitation (10), dont la longueur d'ondes est inférieure à celle de la lumière d'excitation (5), est dirigée en continu vers l'échantillon, au-delà d'une pluralité d'impulsions (19) de la lumière d'excitation (5).
EP08787227A 2007-08-18 2008-08-14 Microscopie de fluorescence sted à excitation biphotonique Withdrawn EP2185919A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007039111.2A DE102007039111B4 (de) 2007-08-18 2007-08-18 STED-Fluoreszenzmikroskopie mit Zweiphotonen-Anregung
PCT/EP2008/060694 WO2009024529A1 (fr) 2007-08-18 2008-08-14 Microscopie de fluorescence sted à excitation biphotonique

Publications (1)

Publication Number Publication Date
EP2185919A1 true EP2185919A1 (fr) 2010-05-19

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Application Number Title Priority Date Filing Date
EP08787227A Withdrawn EP2185919A1 (fr) 2007-08-18 2008-08-14 Microscopie de fluorescence sted à excitation biphotonique

Country Status (6)

Country Link
US (1) US7863585B2 (fr)
EP (1) EP2185919A1 (fr)
JP (1) JP5269901B2 (fr)
CN (1) CN101821607B (fr)
DE (1) DE102007039111B4 (fr)
WO (1) WO2009024529A1 (fr)

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CN101821607B (zh) 2013-06-05
US7863585B2 (en) 2011-01-04
DE102007039111B4 (de) 2014-11-20
US20100176307A1 (en) 2010-07-15
WO2009024529A1 (fr) 2009-02-26
CN101821607A (zh) 2010-09-01
JP5269901B2 (ja) 2013-08-21
DE102007039111A1 (de) 2009-02-26

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