US20090294694A1 - Luminescence Microscopy with Enhanced Resolution - Google Patents

Luminescence Microscopy with Enhanced Resolution Download PDF

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Publication number
US20090294694A1
US20090294694A1 US12/442,093 US44209307A US2009294694A1 US 20090294694 A1 US20090294694 A1 US 20090294694A1 US 44209307 A US44209307 A US 44209307A US 2009294694 A1 US2009294694 A1 US 2009294694A1
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sample
laser radiation
radiation field
partial volume
luminescence
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US12/442,093
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Helmut Lippert
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Carl Zeiss Microscopy GmbH
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Carl Zeiss MicroImaging GmbH
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Assigned to CARL ZEISS MICROIMAGING GMBH reassignment CARL ZEISS MICROIMAGING GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LIPPERT, HELMUT
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    • 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
    • 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
    • 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
    • G02B21/008—Details of detection or image processing, including general computer control
    • G02B21/0084—Details of detection or image processing, including general computer control time-scale detection, e.g. strobed, ultra-fast, heterodyne detection

Definitions

  • the invention is directed to resolution-enhanced luminescence microscopy and particularly to a method in which a luminescing sample to be examined is illuminated by excitation radiation and an image of the sample that has been excited to luminescence is obtained.
  • the invention is further directed to a microscope for resolution-enhanced luminescence microscopy of a sample, means for exciting luminescence which irradiate the sample with excitation radiation, and means for acquiring an image of the excited sample.
  • Luminescence microscopy is a typical field of application of light microscopy for examining biological samples.
  • certain dyes phosphors or fluorophores, as they are called
  • the sample is illuminated by excitation radiation and the luminescent light that is excited in this way is acquired by suitable detectors.
  • the light microscope is usually provided with a dichroic beamsplitter combined with blocking filters which split off the fluorescence radiation from the excitation radiation and enable separate observation. This procedure makes it possible to display individual, differently colored cell parts in the light microscope.
  • more than one part of a specimen may also be dyed simultaneously with different dyes attaching themselves specifically to different structures of the specimen. This process is known as multiple luminescence. Samples which luminesce, per se, that is, without the addition of dye, can also be measured.
  • luminescence is used as an umbrella term for phosphorescence and fluorescence and embraces both processes.
  • LSM laser scanning microscopes
  • the optical resolution of a light microscope and of a LSM is diffraction-limited by physical laws.
  • Special illumination configurations such as the 4Pi arrangement or arrangements with standing wave fields are known for optimal resolution within these limits.
  • the resolution can be appreciably improved over a conventional LSM particularly in axial direction.
  • the resolution can be increased by up to a factor of 10 over a diffraction-limited confocal LSM by means of nonlinear depopulation processes.
  • GSD ground state depletion
  • S. W. Hell and M. Kroug stimulated emission depletion
  • a saturated excitation of the triplet state (hereinafter: GSD) or a saturated de-excitation of the fluorescing state (hereinafter: STED) makes it possible to deliberately quench the fluorescence of molecules which are not located in the immediate vicinity of the interference minimum. The radiation then proceeds only from the interference minimum.
  • GSD triplet state
  • STED saturated de-excitation of the fluorescing state
  • the up-conversion fluorescence depletion technique established by Iketaki et al. functions in a similar way [see T. Watanabe, Y. Iketaki, T. Omatsu, K. Yamamoto, M. Salkai and M. Fujii, “Two-point separation in super-resolution fluorescence microscope based on up-conversion fluorescence depletion technique”, Opt. Exp. 24, 3271-3276 (2003)].
  • DE 19908883 A1 proposes a direct saturation of the fluorescence transition as a nonlinear process.
  • the enhanced resolution is based on a periodically structured illumination of the sample so that there is a transfer of high object space frequencies in the range of the optical transfer function of the microscope.
  • the transfer can be achieved through costly postprocessing of data by computer.
  • a resolution-enhanced luminescence microscopy method in which a sample is excited to emission of luminescence radiation by irradiation with excitation radiation and an image of the luminescing sample is acquired, wherein a first partial volume of the sample is irradiated by a first laser radiation field of the excitation radiation and a second partial volume of the sample is irradiated by a second laser radiation field of the excitation radiation, wherein the first partial volume of the sample and the second partial volume of the sample overlap partially but not completely, only the first laser radiation field is modulated with a first frequency, and luminescence radiation is detected from the first partial volume of the sample with modulation filtering so that luminescence radiation from the second partial volume of the sample is suppressed.
  • the above-stated object is further met though a resolution-enhanced luminescence microscope with means for irradiating a sample with excitation radiation for exciting the emission of luminescence radiation and means for acquiring images of the luminescing sample, wherein the means for irradiating with excitation radiation have means for irradiating a first partial volume of the sample with a first laser radiation field and means for irradiating a second partial volume of the sample with a second laser radiation field, wherein the first partial volume of the sample and the second partial volume of the sample overlap one another partially but not completely, the means for irradiating the sample with the first laser radiation field have a modulator which modulates the first laser radiation field with a first frequency, and the means for acquiring images detect luminescence radiation from the first partial volume of the sample with modulation filtering so that the luminescence radiation from the second partial volume of the sample is suppressed by the filtering.
  • the method according to the invention and the corresponding arrangement are single-point techniques in which resolution is enhanced beyond the resolution of the laser radiation field irradiation by the nonlinear cooperation of at least two laser radiation fields.
  • Similar to DE 19908883 A1 direct saturation of the fluorescence transition can be applied as a nonlinear process. But simultaneous occupation of the triplet state no longer necessarily has negative results. It is essential that the fluorescence generated by the excitation laser and the fluorescence generated by the saturation laser are separated from one another by modulation marking (MMF: Modulation Marked Fluorescence) and suitable frequency-sensitive and/or phase-sensitive detection.
  • MMF Modulation Marked Fluorescence
  • two laser radiation fields are radiated in according to the invention for increasing resolution.
  • One of these two laser radiation fields is modulated.
  • This laser radiation field is referred to hereinafter as center beam, center radiation or center laser radiation.
  • a second laser radiation field whose radiation is not linearly modulated is radiated in so as to overlap this first laser radiation field but not completely cover it.
  • This second laser radiation field will be referred to hereinafter as the side laser radiation or side laser beam.
  • the two laser radiation fields are preferably structured in such a way that the maximum of the center laser beam coincides with the interference minimum of the side laser beam. In principle, the resolution is improved over the resolution at which the center laser beam and side laser beam are coupled in.
  • GSD is based on a saturation of the triplet state and therefore requires molecules with a high rate of intersystem crossing.
  • modulation-marked fluorescence because neither the side laser beam T 1,0 excitation nor the side laser beam S 1,0 excitation has an effect on the signal generated by the center laser beam. In the former case, there is no fluorescence, whereas in the latter case no modulated fluorescence occurs.
  • a drawback of the GSD method is the relatively long pixel dwell time required during scanning for image generation. In the first place, this is necessary to achieve the stationary equilibrium needed for triplet saturation (approximately 10 ⁇ s). In the second place, an initial relaxation of all molecules back into the ground state is required following the detection of a point for detecting the adjacent point (again approximately 10 ⁇ s). According to the invention, a saturation of the triplet state is not necessary, so that shorter dwell times can be used whose bottom limits are basically determined by the periods of the center laser beam modulations.
  • the intensities required within the framework of the invention are lower than those in STED.
  • a substantial advantage of the invention is the flexibility in the choice of dye. While the intersystem crossing required in GSD is limiting, the STED method requires molecules which allow the most efficient possible de-excitation of the S 1,0 state. By contrast, the invention makes it possible to use almost any dye whose level diagram corresponds approximately to that shown in FIG. 1 . Optimization of the reaction rates (e.g., with respect to moderately longer fluorescence lifetimes) is advantageous, but does not present a fundamental limitation of the method. It must be emphasized that the essential modification with respect to conventional techniques is to be found more in the type of excitation and detection than in the choice of the sample to be examined (in stark contrast to DE 10325460 A1, for example).
  • the modulation-marked fluorescence (MMF) according to the invention makes it possible to improve high-resolution optical imaging to an even greater degree. It presents an alternative to the two single-point methods GSD and STED which are already known. As in these known methods, the method according to the invention also works with at least two laser radiation fields (center laser beam and side laser beam). However, whereas the aim in GSD and STED is to completely suppress the fluorescence in the side laser beam area, in MMF the center sample area and the laterally excited sample area are distinguished, e.g., by modulated center laser beam excitation followed by phase-sensitive detection of the signal of interest, and can therefore be separated.
  • a modulation-frequency-sensitive detection e.g., by means of lock-in technology
  • Marked fluorescence in the side laser beam area i.e., excitation through photons of the center laser radiation field
  • a substantial advantage of MMF over the known methods of GSD and STED is the freedom of choosing the fluorophor and the possibility of operating the center laser and side lasers at the same wavelength.
  • FIG. 1 shows, by way of example, an energy diagram of a dye molecule or of a sample which is used within the framework of the invention
  • FIG. 2 shows a possible intensity distribution for a side beam and a center beam in the method according to the invention and in the device according to the invention
  • FIG. 3 shows, by way of example, a schematic for a device according to the invention
  • FIG. 4 shows the intensity ratio between the center beam and side beam for different embodiment forms of the invention
  • FIG. 5 shows the equilibrium population as a function of the radiated laser beam intensity for a dye that can be used, for example, within the framework of the invention
  • FIG. 6 shows the equilibrium population of the ground state along a normalized coordinate during irradiation by the side laser beam in the method according to the invention for three different possible peak intensities
  • FIG. 7 shows a diagram similar to that shown in FIG. 6 for other dye parameters.
  • FIG. 8 shows the population of the ground states and excited states as a function of the illumination period in a particular embodiment form of the invention.
  • FIG. 1 The typical arrangement, known per se, of the lowest energy level for a fluorescing dye molecule is shown schematically in FIG., 1 .
  • state S 0,0 approximately vibrational ground state in the lowest electronic state
  • S 1,v vibration-excited vibronic state
  • stimulated emission is, of course, also possible.
  • S 1,v a fast vibrational relaxation takes place in state S 1,0 and subsequently, as competing processes, either fluorescence or the transition to the triplet state T i,v with subsequent phosphorescence.
  • the excitation is carried out, according to the invention, by at least two different light fields which are arranged in the same way as the excitation laser radiation field and the saturation laser radiation field in the known GSD or STED method.
  • the use of lasers seems sensible but generally does not represent a limitation of the method.
  • the fields are designated in the following as center beam and side beam. They can have the same wavelength.
  • FIG. 3 shows an embodiment form of the device which in this case is constructed similar to a Mach-Zehnder.
  • a beamsplitter 2 divides the light into a center beam path 4 and a side beam path 3 after a light source 1 .
  • a unit for spatial beam shaping 5 is located in the side beam path 3 .
  • This unit can comprise, e.g., an annular aperture which is imaged on the sample 10 .
  • Other possibilities are described, for example, in T. A. Klar, E. Engel and S. W. Hell, “Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes”, Phys. Rev. E 64, 066613 (2001). Of course, two separate beam sources can also be used.
  • a modulation unit 6 is provided for the center beam path 4 , which is not subjected to spatial beam shaping, and modulates this beam with a frequency f c . After overlapping, the two beams are focused in the sample 10 in a diffraction-limited manner. An objective 9 is used for this purpose. In addition, the focus is displaced in two dimensions by a scan unit 8 .
  • the fluorescence excited in this way is recorded by a detector 12 , e.g., a confocal detector, via the objective 9 , scan unit 8 and a preferably dichroic beamsplitter 7 .
  • a control unit (not shown) controls the operation of the device.
  • the fluorescence signal measured in this way can be associated with the respective beam 3 , 4 by taking into account the modulation, i.e., the fluorescence is marked correspondingly.
  • the fluorescence signal generated by the center beam is then likewise modulated with the frequency f c .
  • This effect corresponds among others to that which is also applied in the phase method for measuring fluorescence lifetimes (see, e.g., M. J. Booth and T.
  • the side beam and center beam are each modulated with frequencies f s and f c , where f s ⁇ f c .
  • the modulation frequency f c can be optimized in accordance with the dye.
  • a lock-in amplifier ( 13 ) at frequency f c as is shown by way of example in FIG. 3 , the fluorescence signal generated by the center beam is extracted. Molecules which, in contrast, are excited (also) by the side laser beam show a non-modulated fluorescence and therefore do not contribute to the signal at the output 14 .
  • a polarization-sensitive detection can also take place making use of the fluorescence polarization.
  • a resolution in the molecular range can be achieved when it is ensured that the probability that molecules located in a sample area in which the side laser has an intensity other than zero will be excited by the side laser beam is as high as possible.
  • the lock-in technique is, of course, only one example of a phase-sensitive or frequency-sensitive detection method.
  • N p,s is the photon flux (of the side laser beam 5 ) and ⁇ is the absorption cross section of the optical transfer.
  • the intensity of the fluorescence radiation is proportional to N 1,v by which the nonlinear relationship between the intensity of the exciting light and that of the emitted light which was mentioned above as necessary for high resolution can be directly verified. For very high photon fluxes, equal occupation of the states and, therefore, saturation is achieved. Further, when the intensity of the modulated center laser beam 4 is very much smaller compared to the side laser beam 5 (i.e., N p,s >>N p,c ), the probability of fluorescence excitation by the center laser beam 4 differs substantially from zero only at the interference minimum. This state of affairs is shown clearly in FIG.
  • FIG. 5 shows the population of states S 0,0 and S 1,0 (N 0,0 and N 1,0 , respectively) as a function of the laser beam intensity.
  • FIG. 6 the specific shape of the curves in FIG. 6 depends among other things on the properties of the selected fluorophor or sample 10 .
  • the example above is based on a fluorescence lifetime of 2 ns. A more efficient saturation (and, therefore, lower intensities) can be realized by using dyes with longer lifetimes.
  • FIG. 7 corresponds to FIG. 6 and assumes a lifetime of 10 ns. It can clearly be seen that a flattening of the population curve occurs already at 20 MW/cm 2 .
  • FIG. 8 shows how the populations of states S 0,0 , S 1,0 and T 1,0 (see FIG. 1 ) change within 1 ⁇ s under these conditions assuming an irradiation intensity of 20 MW/cm 2 .
US12/442,093 2006-09-29 2007-09-10 Luminescence Microscopy with Enhanced Resolution Abandoned US20090294694A1 (en)

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DE102006046369A DE102006046369A1 (de) 2006-09-29 2006-09-29 Auflösungsgesteigerte Lumineszenzmikroskopie
DE102006046369.2 2006-09-29
PCT/EP2007/007882 WO2008040435A1 (de) 2006-09-29 2007-09-10 Auflösungsgesteigerte lumineszenzmikroskopie

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011131591A1 (de) * 2010-04-22 2011-10-27 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Bestimmen der verteilung einer substanz durch abtasten mit einer messfront
US20130015366A1 (en) * 2011-07-11 2013-01-17 Olympus Corporation Sample observation apparatus
US8704196B2 (en) 2008-11-03 2014-04-22 Carl Zeiss Microscopy Gmbh Combination microscopy
US20150042973A1 (en) * 2013-08-08 2015-02-12 Applied Materials, Inc. Photonic activation of reactants for sub-micron feature formation using depleted beams
US9383563B2 (en) * 2013-06-11 2016-07-05 Olympus Corporation Confocal image generation apparatus
US9384537B2 (en) * 2014-08-31 2016-07-05 National Taiwan University Virtual spatial overlap modulation microscopy for resolution improvement
US11181727B2 (en) * 2016-03-10 2021-11-23 University Of Notre Dame Du Lac Super-sensitivity multiphoton frequency-domain fluorescence lifetime imaging microscopy

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5699421B2 (ja) * 2008-09-16 2015-04-08 横河電機株式会社 顕微鏡装置
JP5589374B2 (ja) * 2009-06-22 2014-09-17 横河電機株式会社 顕微鏡装置
JP6003072B2 (ja) * 2011-02-08 2016-10-05 横河電機株式会社 顕微鏡装置
JP5609729B2 (ja) * 2011-03-18 2014-10-22 横河電機株式会社 顕微鏡装置、観察方法および試料搭載機構
WO2013007726A1 (en) 2011-07-11 2013-01-17 Facultes Universitaires Notre-Dame De La Paix Method for high resolution sum-frequency generation and infrared microscopy
DE102012017922B4 (de) * 2012-09-11 2024-03-14 Carl Zeiss Microscopy Gmbh Optikanordnung und Lichtmikroskop
ITTO20120929A1 (it) * 2012-10-23 2014-04-24 Fond Istituto Italiano Di Tecnologia Sistema di microscopia ottica basato su transizioni ottiche saturabili reversibili (resolft), in modulazione di frequenza.
US9897536B2 (en) 2013-01-04 2018-02-20 University Of Limerick Differential infra red nanoscopy system and method
DE102013100172A1 (de) * 2013-01-09 2014-07-10 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Verfahren zum räumlich hochaufgelösten Abbilden einer einen Luminophor aufweisenden Struktur einer Probe
DE102013100174A1 (de) * 2013-01-09 2014-07-10 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Verfahren zum räumlich hochaufgelösten Abbilden einer einen Luminophor aufweisenden Struktur einer Probe
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JP2016012114A (ja) * 2014-06-02 2016-01-21 オリンパス株式会社 照明装置、これを有する顕微鏡装置及び顕微鏡観察方法
DE102016117096C5 (de) * 2015-09-22 2023-11-30 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Verfahren zum hochaufgelösten lokalen Abbilden einer Struktur in einer Probe
EP3159676B1 (de) * 2015-10-23 2018-04-04 Abberior Instruments GmbH Verfahren und vorrichtung zum hochauflösenden abbilden einer mit fluoreszenzmarkern markierten struktur einer probe
JP6806161B2 (ja) * 2016-11-02 2021-01-06 株式会社ニコン 顕微鏡システム
IT201700118432A1 (it) 2017-10-19 2019-04-19 Fondazione St Italiano Tecnologia Metodo di microscopia a deplezione mediante emissione stimolata ad alta risoluzione spaziale
KR102105814B1 (ko) * 2018-12-21 2020-05-04 한국표준과학연구원 레이저 공간 변조 초고분해능 광학 현미경

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
US5866911A (en) * 1994-07-15 1999-02-02 Baer; Stephen C. Method and apparatus for improving resolution in scanned optical system
US20010045529A1 (en) * 2000-03-23 2001-11-29 Yoshinori Iketaki Double-resonance-absorption microscope
US20010045523A1 (en) * 1994-07-15 2001-11-29 Baer Stephen C. Superresolution in microlithography and fluorescence microscopy
US20020109101A1 (en) * 2000-12-19 2002-08-15 Juergen Hoffmann Scanning microscope
US20050162654A1 (en) * 1997-07-03 2005-07-28 Jon Opsal Systems and methods for evaluating semiconductor layers
US20070023686A1 (en) * 2005-07-22 2007-02-01 Ralf Wolleschensky Resolution-enhanced luminescence microscopy
US20070291353A1 (en) * 2004-08-09 2007-12-20 Olympus Corporation Super-Resolution Microscope

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4331570C2 (de) * 1993-08-17 1996-10-24 Hell Stefan Verfahren zum optischen Anregen einer Probe
DE19956620A1 (de) * 1999-11-25 2001-05-31 Zeiss Carl Jena Gmbh Verfahren zur Erfassung von Fluoreszenzerscheinungen in einem Mikroskop
DE10154699B4 (de) * 2001-11-09 2004-04-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Verfahren und Vorrichtung zum räumlich eng begrenzten Anregen eines optischen Übergangs
DE10231543B3 (de) * 2002-07-11 2004-02-26 Universität Siegen Konfokale 3D-Scanning Absorption
DE10340965A1 (de) * 2003-09-05 2005-03-24 Leica Microsystems Heidelberg Gmbh Rastermikroskop
WO2005029149A1 (de) * 2003-09-25 2005-03-31 Leica Microsystems Cms Gmbh Mikroskop mit evaneszenter beleuchtung
DE102004039035A1 (de) * 2004-04-07 2005-10-27 EuroPhoton GmbH Gesellschaft für optische Sensorik Verfahren und Vorrichtung zur Fluoreszenz-Lebensdauer-Imaging-Nanoskopie

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5866911A (en) * 1994-07-15 1999-02-02 Baer; Stephen C. Method and apparatus for improving resolution in scanned optical system
US20010045523A1 (en) * 1994-07-15 2001-11-29 Baer Stephen C. Superresolution in microlithography and fluorescence microscopy
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
US20050162654A1 (en) * 1997-07-03 2005-07-28 Jon Opsal Systems and methods for evaluating semiconductor layers
US20010045529A1 (en) * 2000-03-23 2001-11-29 Yoshinori Iketaki Double-resonance-absorption microscope
US20020109101A1 (en) * 2000-12-19 2002-08-15 Juergen Hoffmann Scanning microscope
US20070291353A1 (en) * 2004-08-09 2007-12-20 Olympus Corporation Super-Resolution Microscope
US20070023686A1 (en) * 2005-07-22 2007-02-01 Ralf Wolleschensky Resolution-enhanced luminescence microscopy

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8704196B2 (en) 2008-11-03 2014-04-22 Carl Zeiss Microscopy Gmbh Combination microscopy
WO2011131591A1 (de) * 2010-04-22 2011-10-27 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Bestimmen der verteilung einer substanz durch abtasten mit einer messfront
US9024279B2 (en) 2010-04-22 2015-05-05 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Determining the distribution of a substance by scanning with a measuring front
US20130015366A1 (en) * 2011-07-11 2013-01-17 Olympus Corporation Sample observation apparatus
US8933418B2 (en) * 2011-07-11 2015-01-13 Olympus Corporation Sample observation apparatus
US9383563B2 (en) * 2013-06-11 2016-07-05 Olympus Corporation Confocal image generation apparatus
US20150042973A1 (en) * 2013-08-08 2015-02-12 Applied Materials, Inc. Photonic activation of reactants for sub-micron feature formation using depleted beams
US9869938B2 (en) * 2013-08-08 2018-01-16 Applied Materials, Inc. Photonic activation of reactants for sub-micron feature formation using depleted beams
US10061208B2 (en) 2013-08-08 2018-08-28 Applied Materials, Inc. Photonic activation of reactants for sub-micron feature formation using depleted beams
US9384537B2 (en) * 2014-08-31 2016-07-05 National Taiwan University Virtual spatial overlap modulation microscopy for resolution improvement
US11181727B2 (en) * 2016-03-10 2021-11-23 University Of Notre Dame Du Lac Super-sensitivity multiphoton frequency-domain fluorescence lifetime imaging microscopy

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EP2067020A1 (de) 2009-06-10
EP2067020B1 (de) 2012-05-30
US20120097865A1 (en) 2012-04-26

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