WO2017174100A1 - Parallélisation du procédé de microscopie sted - Google Patents

Parallélisation du procédé de microscopie sted Download PDF

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Publication number
WO2017174100A1
WO2017174100A1 PCT/EP2016/000581 EP2016000581W WO2017174100A1 WO 2017174100 A1 WO2017174100 A1 WO 2017174100A1 EP 2016000581 W EP2016000581 W EP 2016000581W WO 2017174100 A1 WO2017174100 A1 WO 2017174100A1
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WO
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Prior art keywords
diffractive optical
optical element
array
excitation
regions
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PCT/EP2016/000581
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German (de)
English (en)
Inventor
Karl-Heinz Brenner
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Universitaet Heidelberg
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Universitaet Heidelberg
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Priority to PCT/EP2016/000581 priority Critical patent/WO2017174100A1/fr
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Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems

Definitions

  • the present invention relates to a STED microscope and a ST E D method.
  • STED Microscopic stimulated emission and depletion
  • the object is illuminated not only with the focused excitation beam, but simultaneously with a second beam, the "switch-off beam", with an annular or donut-shaped light distribution
  • the excitation beam and the switch-off beam overlap so that the maximum intensity of the excitation beam in the center of the turn-off beam (ie, where the turn-off beam is dark) .
  • This causes the fluorescence molecules in the outer area of the excitation focus to be "switched off” in a targeted manner.
  • the fluorescence molecules in the central area remain unaffected. Details of the STED method are described in detail in the literature and are assumed to be known.
  • a first aspect of the invention relates to a STED microscope with diffractive elements.
  • the microscope comprises a light source or lighting device that is designed to generate an excitation beam and a switch-off beam.
  • the wavelength of the excitation beam may be suitably selected to excite the fluorescent dyes in the sample to be examined.
  • the wavelength of the turn-off beam (STED beam) can be suitably selected to achieve a stimulation of stimulated emission by deactivating or switching off excited fluorescent dyes.
  • the light source may e.g. pulsed or cw lasers, and possibly other optical components, such as beam splitters, collimators, lenses, etc.
  • an array of focused light spots or illumination spots is generated by means of the first diffractive optical element.
  • an array for example a one-dimensional or two-dimensional array, of annular or donut-shaped illumination spots (switch-off regions or depletion regions) is formed. Areas) for STED microscopy.
  • the centers of the point-shaped excitation regions preferably coincide with the centers of the annular or donut-shaped switch-off regions.
  • the first and / or the second diffractive optical element may be transmissive elements. This allows a particularly compact construction of the STED microscope.
  • the individual excitation regions are generated by the respective sub-elements of the first and second diffractive optical elements.
  • Each diffractive sub-element has a pattern which is designed to generate the corresponding point or donut-shaped intensity distribution in the respective excitation or switch-off region by means of diffraction.
  • the pattern can be generated, for example, by lithography or other suitable methods.
  • the STED microscope can comprise a detection unit for detecting the fluorescence emitted by a sample to be examined.
  • the detection unit may comprise a two-dimensional detector, e.g. a CCD camera.
  • An advantage of the proposed STED microscope is the "parallelization" of the STED method, as the object is simultaneously illuminated by multiple excitation and turn-off beams, creating multiple excitation and turn-off areas, in conventional STED methods based on a serial scan of the object , the illumination time (dwell time) per point is very short.
  • a much longer residence time eg, about 10,000 times
  • irradiation with significantly more light eg 10,000 times
  • the working distance (i.e., the focal length fo) of the STED microscope may be greater than that of conventional STED microscopes.
  • the working distance is equal to or greater than 1000 pm.
  • Another advantage is that by means of diffractive optical elements a Dahlfallt of Intensity curves can be generated, the individual parameters of the intensity distribution (such as ring radius and ring width) can be set independently. As a result, the STED microscope can be better and more flexibly adapted to the respective measurement situation.
  • r 0 ⁇ (x, y) a vector describing the displacement with respect to the origin of the excitation area (excitation focus) or the donut-shaped disabling area in the x-y plane, where the x-y is the plane perpendicular to the direction of light propagation z; J 0 is a Bessel function of zeroth order;
  • R 0 is the ring radius of the generated donut-shaped switch-off region.
  • the amplitude and the phase of the sub-elements of the first and second diffractive optical elements can each be obtained by means of Fourier transformation of the formula (2) or of the formula (3).
  • the Fourier transform of formula (2) or formula (3) thus provides an "ideal" diffractive sub-element for the first and second diffractive optical elements, respectively continuous phase up.
  • the complex amplitude of a binarized phase element can thus be described by the formula (4):
  • the diffractive optical elements or sub-elements defined by the formulas (2) and (3) are particularly suitable for producing optimal punctiform or donut-shaped light distributions.
  • a diffractive optical element or sub-element defined by formula (2) has a much sharper focus (in both the x and y directions) than a comparable refractive optical element (eg, a microlens made of glass or plastic). This allows a higher precision and a better resolution of the scan.
  • Each donut-shaped region (switch-off region) produced by means of a diffractive optical element according to the formula (3) has a ring width and a ring radius which are predetermined by the periodically arranged sub-elements of the first diffractive optical element ,
  • a particular advantage of the proposed diffractive optical element is that the ring radius can be chosen almost arbitrarily and independently of the ring width.
  • a step height of ⁇ is possible, so that the ring radius can not be selected or varied.
  • by modifying the Bessel function it is possible to set the intensity curve quantitatively and flexibly, which is not possible with a conventional spiral phase plate.
  • the array of sub-elements of the first diffractive optical element and / or the second diffractive optical element may have a period of less than 100 pm, for example a period between 10 and 100 pm.
  • the periods in two orthogonal directions e.g., in the x and y directions
  • the periods of the arrays of the first and second diffractive optical elements are the same.
  • the diffractive optical elements can be produced by lithography, in particular electron beam lithography.
  • the first and / or the second diffractive optical element may each comprise a glass plate, wherein at least one of the surfaces of the glass plate has elevations and / or depressions which form the diffractive patterns of the sub-elements.
  • An advantage of lithographic processes is the high accuracy with which the diffractive patterns can be generated.
  • the glass plate may e.g. be coated with a photoresist, which is exposed based on the produced or predetermined diffractive sub-elements or diffractive structures. Subsequently, the photoresist is developed so that, depending on the photoresist used, the exposed or unexposed areas of the photoresist dissolve.
  • One advantage of electronically controllable light modulators is that the generated excitation and / or switch-off regions can be changed quickly and flexibly and without the use of additional mechanical and / or optical parts and adapted to the respective measurement situation. Due to the production-related minimum size of the modulator pixels, a reduction optics can be used.
  • the STED microscope may further comprise: a first imaging system for imaging the array of excitation regions generated by the first diffractive optical element onto a sample to be examined; and a second imaging system for imaging the array of switch-off regions generated by the second diffractive optical element onto the sample to be examined.
  • the first and second imaging systems may include at least one lens. It is possible that the two imaging systems have common components (e.g., a common lens).
  • the first and second imaging systems may include other components, such as e.g. Beam splitters, spectral and / or spatial filters, aperture, etc., include. Preferably, all lenses are optimized for infinity rendering.
  • An exemplary illumination device comprises a first imaging system having a first objective, a second imaging system having a second objective and a third objective which is a component of both the first and the second imaging system.
  • the excitation beam and the switch-off beam can be superimposed and fed to the third objective.
  • the at least one beam splitter is non-polarizing and has a split ratio of 50:50.
  • the first and second diffractive optical elements and the objectives can be arranged and designed in such a way that a point in the object plane of the first imaging system is converted by the first objective into a (perfect) plane wave.
  • the third lens converts this plane wave back into a diffraction-limited point.
  • the combination of the first and the third lens thus causes an optical image.
  • the combination of the second and the third lens causes an optical image.
  • the first diffractive optical element thus generates an array of foci (excitation foci or excitation foci) in the object plane of the first imaging system, so that a corresponding image is formed on the sample.
  • the second diffractive optical element generates an array of donut-shaped off-switch areas in the object plane of the second imaging system. The image is also formed on the sample and is superimposed there with the array of foci or Foki (excitation focus or Excitation foci).
  • the first and / or second imaging system is / are adjusted such that the array of excitation regions substantially overlaps the array of switch-off regions on the sample, in particular such that each excitation region is overlapped with an associated switch-off region.
  • the alignment of the excitation regions and the donut-shaped switch-off regions with respect to each other can be achieved e.g. by moving the first and / or the second diffractive optical element. This can e.g. by a mechanical arrangement (e.g., including an actuator) or by a piezoelectric actuator.
  • the STED microscope may accordingly comprise adjusting means which are designed to displace the first and / or the second diffractive optical element in at least one direction in a plane which is perpendicular to the light propagation direction.
  • the adjusting means may further be designed to also displace the first and / or the second diffractive optical element along the light propagation direction. Due to the lower weight of diffractive optical elements, the realization of a mechanical translation or displacement with high accuracy is comparatively simple. If electronically controllable spatial light modulators are used, the alignment can be done electronically, e.g. by shifting the diffraction pattern generated by the light modulator.
  • the STED microscope may include a scanner adapted to move the arrays of excitation and turn-off regions to scan the sample with the arrays of excitation and turn-off regions.
  • the scan may be mechanical (eg, by mechanical movement of the first and second optical elements), by additional optical elements, or electronically. It is also possible to move the sample.
  • the STED microscope may further comprise a variable wheel, which comprises a plurality of different diffractive optical elements, and which makes it possible to replace the second diffractive optical element with another diffractive optical element. By replacing the second diffractive optical element can eg Ausschalte Symposium Edition with different ring widths and / or radii are generated. It is also possible to realize different depletion intensities.
  • a method for STED microscopy comprises the steps:
  • the method may further comprise aligning the first and second diffractive optical elements to achieve an overlap of the array of excitation regions and the array of switch-off regions.
  • the alignment or the adjustment takes place in such a way that each excitation area is overlapped or superposed with an associated switch-off area and that the maximum intensity of the respective excitation area substantially coincides with the center of the respective switch-off area.
  • the alignment of the first and second optical elements may be mechanical or electronic.
  • FIG. 2A is the amplitude and Fig. 2B is the phase of an exemplary "ideal" diffractive optical sub-element designed to produce a point-shaped light distribution;
  • FIG. 2C is the phase of an exemplary binary diffractive optical sub-element designed to produce a point-shaped light distribution;
  • 3A is a gray value representation of the phase of an exemplary diffractive optical subelement designed to generate a point-shaped light distribution
  • FIG. 3B illustrates the phase of an exemplary binary diffractive optical sub-element designed to produce a point-shaped light distribution
  • FIG. 3C shows the intensity of the diffraction pattern of an exemplary diffractive optical subelement designed to produce a donut-shaped light distribution
  • FIG. 3D shows the intensity of the diffraction pattern of an exemplary binary diffractive optical sub-element designed to produce a donut-shaped light distribution
  • FIG. 3C shows the intensity of the diffraction pattern of an exemplary diffractive optical subelement designed to produce a donut-shaped light distribution
  • FIG. 3D shows the intensity of the diffraction pattern of an exemplary binary diffractive optical sub-element designed to produce a donut-shaped light distribution
  • FIG. 4A illustrates the phase of an exemplary binary diffractive optical element configured to generate a periodic array of donut-shaped light spots
  • FIG. Fig. 4B shows an array of donut-shaped light spots (excitation regions) generated by the diffractive optical element shown in Fig. 4A.
  • Fig. 1 shows the basic structure of a STED microscope.
  • the STED microscope comprises a light source or illumination device (not shown) which generates an excitation beam and which generates a turn-off beam (depletion beam).
  • the light source or lighting device may e.g. include two pulsed or cw lasers.
  • the excitation beam is diffracted by the first diffractive optical element DOE1, so that a plurality of excitation beams is generated.
  • Each of the generated Ausschaltestrahlen is imaged by means of a first imaging system in a point-shaped spot (excitation area or excitation focus) for the STED microscopy.
  • the turn-off beam is diffracted by the second diffractive optical element DOE2, so that a plurality of turn-off beams are generated.
  • Each of the generated Ausschaltestrahlen is imaged by means of a second imaging system in an annular or donut-shaped Ausschplungs Symposium (depletion region) for the STED microscopy.
  • the first imaging system comprises a first objective M01 and a third objective M03
  • the second imaging system comprises a second objective M02 and the third objective M03.
  • the combination of the first objective M01 and the third objective M03 causes an optical imaging.
  • the first diffractive optical element DOE1 generates an array of foci (focus of focus or excitation focus) in the object plane of the first imaging system M01-M03, the image of this array being formed on the examined object 0.
  • the second diffractive optical element generates an array of donut-shaped spots (donut rings) in the object plane of the second imaging system M02-M03, the image of this array likewise being formed on the object and superimposed thereon with the array of foci ,
  • Alignment between focus and donut may be accomplished by translating DOE1 and DOE2 laterally relative to each other.
  • the lateral displacement can be realized in mechanical form (eg by an actuator) or in piezoelectric form.
  • the object is thus illuminated by an array 20 of punctiform light spots or excitation foci (excitation regions) and an array 30 of donut-shaped light spots (switch-off regions).
  • the beam splitter BS1 guides the signals generated by the diffraction at the first diffractive optical element DOE1 Excitation beams propagated by the first objective M01 and the excitation beams generated by the diffraction at the second diffractive optical element and propagated through the second objective M02.
  • the dichroic beam splitter BS2 separates the fluorescent light emitted by the object O from the excitation light.
  • the fluorescent light is registered by a camera 10, such as a CCD camera.
  • the objectives M01 to M03 are preferably optimized for imaging to infinity (or "infinity corrected").
  • the first diffractive optical element and the second diffractive optical element each comprise an array of diffractive optical sub-elements.
  • the arrays can be one-dimensional or two-dimensional arrays.
  • the individual sub-elements can be obtained as follows:
  • the formula (5) is exact in the case of a periodic pattern or a periodic wave field in the context of scalar optics (without vector character).
  • a diffractive sub-element obtained by a Fourier transform of the object spectrum according to formula (6) has an amplitude and a phase in continuous form and is capable of producing a point-shaped light distribution in the focus space or a focus. This point-shaped light distribution can be imaged by means of a suitable imaging system on the object to be examined.
  • Fig. 2A shows the amplitude and Fig. 2B the phase of such an exemplary "ideal" diffractive optical sub-element, which is obtained by means of a Fourier transform of formula (6) and is designed to generate a point-shaped light distribution.
  • a binary diffractive optical element Since the production of a diffractive element with continuous amplitude and / or phase is difficult, a binary diffractive optical element is proposed.
  • the amplitude of the Fourier transform can be normalized (e.g., set to 1).
  • the phase of the Fourier transform can be binarized.
  • Fig. 2C shows the phase of an exemplary binary diffractive optical element.
  • FIGS. 3A-3D illustrate the influence of the normalization and binarization operations on the focus or on the light distribution in the focal plane.
  • Fig. 3A shows the phase of an exemplary "ideal" diffractive optical element (ie without binarization) and
  • Fig. 3B the phase (+ ⁇ / 2, - ⁇ / 2) of a binary diffractive optical element
  • Fig. 3C shows the intensity of the diffraction image
  • Fig. 3D shows the same for the binarized diffractive optical element shown in Fig. 3 B.
  • the diffractive optical element or subelement (binary or continuous) obtained by means of a Fourier transformation of the formula (6) has a substantially sharper focus with the same parameters (diameter and focal length) (in both the x and y directions ) as a refractive optical element (eg, a microlens made of glass or plastic). This allows higher precision and better resolution of the scan.
  • the individual sub-elements of the second diffractive optical element can be obtained similarly to the sub-elements of the first diffractive optical element, using not the constant 1 (as in the first diffractive optical element) but the Fourier transform of the desired distribution as the pupil function. For an annular or donut-shaped distribution, this is the Bessel distribution Jo. The condition for a donut-shaped spot is thus:
  • Formula (8) gives the optimal object or element spectrum ü opt of a donut-shaped spot or a donut-shaped light distribution at ox .
  • the Fourier transform of the object spectrum given by formula (8) provides the single diffractive sub-element of the second diffractive optical element.
  • Such an "ideal" diffractive sub-element has an amplitude and a phase in continuous form and is suitable for generating a donut-shaped light distribution in the focus space
  • the periodic arrangement of several subelements forms the second diffractive optical element
  • Figure 4A shows the phase of an exemplary binary diffractive optical element designed to be a periodic array
  • the diffractive pattern of each subelement was obtained after binarizing the Fourier transform of formula (8).
  • FIG. 4B shows the generated array of donut-shaped light spots (excitation regions). Both the ring radius Ro and the ring position correspond to the design specification.
  • the xy distribution shown in Fig. 4B corresponds to the donut-shaped distribution produced with a spiral phase plate, with the difference that both the ring radius and the ring width are adjustable are. Other courses of depletion intensity could be selected as needed, which is not possible with a spiral phase plate.
  • detection unit e.g., CCD camera

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un microscope STED et un procédé de microscopie STED. Le microscope STED comprend : une source de lumière pour la production d'un faisceau d'excitation et d'un faisceau d'inactivation; un premier élément optique diffractif (DOE1) et un second élément optique diffractif (DOE2) comprenant chacun un agencement périodique de sous-éléments diffractifs, le premier élément optique diffractif (DOE1) étant conçu pour diffracter un faisceau d'excitation de manière à produire un réseau de zones d'excitation (20) punctiformes, et le second élément optique diffractif (DOE2) étant conçu pour diffracter un faisceau d'inactivation de manière à produire un réseau de zones d'inactivation (30) annulaire.
PCT/EP2016/000581 2016-04-08 2016-04-08 Parallélisation du procédé de microscopie sted Ceased WO2017174100A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020062609A1 (fr) * 2018-09-26 2020-04-02 中国科学院化学研究所 Système d'éclairage destiné à un microscope optique sted et microscope optique sted
CN111879737A (zh) * 2019-09-10 2020-11-03 之江实验室 一种产生高通量超衍射极限焦斑的装置和方法
WO2022028291A1 (fr) * 2020-08-07 2022-02-10 深圳大学 Dispositif et procédé d'imagerie microscopique à super-résolution à balayage de lumière structurée

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US20060176542A1 (en) * 2003-09-05 2006-08-10 Kawasaki Jukogyo Kabushiki Kaisha Optical control device and optical control method
US20070023686A1 (en) * 2005-07-22 2007-02-01 Ralf Wolleschensky Resolution-enhanced luminescence microscopy
WO2012045318A1 (fr) * 2010-10-05 2012-04-12 Universität Heidelberg Dispositif et procédé de concentration d'un rayonnement au moyen d'un réseau de phase
US20120257197A1 (en) * 2008-06-16 2012-10-11 The Regents Of The University Of Colorado, A Body Corporate Fourier domain sensing
WO2014005193A1 (fr) * 2012-07-05 2014-01-09 Martin Russell Harris Appareil et procédé d'endoscopie ou de microscopie
DE202013102039U1 (de) * 2013-05-10 2014-05-15 Picoquant Gmbh STED-Vorrichtung
US20140307299A1 (en) * 2011-10-26 2014-10-16 Hamamatsu Photonics K.K. Light modulation control method, control program, control device and laser beam irradiation device
US20160091799A1 (en) * 2009-01-24 2016-03-31 Ecole Polytechnique Federal De Lausanne (Epfl) High-resolution microscopy and photolithography devices using focusing micromirrors

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060176542A1 (en) * 2003-09-05 2006-08-10 Kawasaki Jukogyo Kabushiki Kaisha Optical control device and optical control method
US20070023686A1 (en) * 2005-07-22 2007-02-01 Ralf Wolleschensky Resolution-enhanced luminescence microscopy
US20120257197A1 (en) * 2008-06-16 2012-10-11 The Regents Of The University Of Colorado, A Body Corporate Fourier domain sensing
US20160091799A1 (en) * 2009-01-24 2016-03-31 Ecole Polytechnique Federal De Lausanne (Epfl) High-resolution microscopy and photolithography devices using focusing micromirrors
WO2012045318A1 (fr) * 2010-10-05 2012-04-12 Universität Heidelberg Dispositif et procédé de concentration d'un rayonnement au moyen d'un réseau de phase
US20140307299A1 (en) * 2011-10-26 2014-10-16 Hamamatsu Photonics K.K. Light modulation control method, control program, control device and laser beam irradiation device
WO2014005193A1 (fr) * 2012-07-05 2014-01-09 Martin Russell Harris Appareil et procédé d'endoscopie ou de microscopie
DE202013102039U1 (de) * 2013-05-10 2014-05-15 Picoquant Gmbh STED-Vorrichtung

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020062609A1 (fr) * 2018-09-26 2020-04-02 中国科学院化学研究所 Système d'éclairage destiné à un microscope optique sted et microscope optique sted
US11726309B2 (en) 2018-09-26 2023-08-15 Institute Of Chemistry Chinese Academy Of Sciences Illumination system for STED optical microscope and STED optical microscope
CN111879737A (zh) * 2019-09-10 2020-11-03 之江实验室 一种产生高通量超衍射极限焦斑的装置和方法
WO2022028291A1 (fr) * 2020-08-07 2022-02-10 深圳大学 Dispositif et procédé d'imagerie microscopique à super-résolution à balayage de lumière structurée
US12461354B2 (en) 2020-08-07 2025-11-04 Shenzhen University Point-scanning structured illumination-based super-resolution microscopic imaging system and method

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