Robert Polster Classical STED Microscopy
STEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleSTED MicroscopyBreaking Abbes-LawTechnical DetailsState of the ArtExamplesRobert Polster20. Mai 2010
InhaltsverzeichnisSTEDRobert PolsterClassicalLimits of Resolution1Classical Microscopy and LimitsLimits of ResolutionModern Microscopes2STED-MicroscopyPrincipleTechnical DetailsState of the Art ExamplesModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamples
Abbes LimitSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamples
Abbes LimitSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesAn electromagnetic wave propagates as:Z Z 1 E 2E 0 · H dkx dky dx 0 dy 04π withH e i(kx x ky y kz z)kz can be expressed as:rq1222kz k kx ky 2π ρ2λ2kz describes an evanescent wave, if λ 1/ρ.
Abbes LimitSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesA electromagnetic Wave propagates as:Z Z 1 E 2E 0 · H dkx dky dx 0 dy 04π withH e i(kx x ky y kz z)kz can be expressed as:rq1222kz k kx ky 2π ρ2λ2kz describes an evanescent wave, if λ 1/ρ.
Abbes LimitSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesConclusions:The information of an evanescent wave vanishes in the far field.Because of the missing information we can not:reconstruct the scattering objectconstruct infinitely small focus pointBest resolution: x λ2
Optical ApertureSTEDRobert PolsterClassicalLimits of ResolutionThe resolution is λ/2, if you are able to collect every k-vector.As every microscope has a specific angle of acceptance theresolution is after the Rayleigh-Criterion:Modern MicroscopesSTEDMicroscopyPrinciple x 0.61Technical DetailsState of the ArtExamplesNA n sin αn refraction indexα angle of acceptanceλNA
Microscopy methodsSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesModern commercial approaches to reach the resolution limit:ordinary Far Field Fluorescence MicroscopyScanning Confocal Microscopyor even to break the resolution limit:Near Field Microscopes
Confocal MicroscopySTEDRobert PolsterStrahlteilerClassicalLimits of ResolutionLichtquelleModern MicroscopesPrincipleTechnical DetailsState of the l 200 nm (PSF )2longitudinal 500 nm1source: http://en.wikipedia.org/wiki/Confocal microscopyFokusebeneLochblendeSTEDMicroscopyWith a wavelength of: 500nm1
4PiSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamples2Resolution:transversal 200 nmBetter resolution because ofinterference pattern at probelongitudinal 80 nm2source: Bewersdorf, A. Egner, S.Hell ”4Pi Microscopy“. In: Handbook of Biological Confocal Microscopy, pp. 561-570, Ed.J. Pawley, Springer, New York. 2006
Near Field MicroscopeSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamples3tip: local scattererconverts near to far fieldtransversal resolution: 80 nm3source: http://nahfeldmikroskopie.de/allgemeines.htmlonly surface sensitivechallenging setup
STEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesSTED MicroscopyStimulated Emission Depletion Microscopy
Basic Principle Step 0STEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesThe goalMake an image of a cell with previously inserted dye molecules forfluorescence microscopy.
Basic Principle Step 1STEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesExcite the dyesAs in every fluorescence microscopy the dye molecules are excited bya Laser.
Basic Principle Step 2STEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesAt the edge of breaking the limitThe Laser excites too much dye molecules for a good resolution. Aswe could not distinguish the light of the different dye molecules.
Basic Principle Step 3STEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesDe-excitation of the dye moleculesAnother Laser with a donut shaped focus deexcite the molecules bystimulated emission.
Basic Principle Step 4STEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesFinishOnly selected molecules are still excited and can emit. HenceAbbes-Law is broken!
The DyeSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical Details4State of the ArtExamplesImportant attributes of the dye:2 level systemthe lower level has sub levels 1decay time of excited level(kFl) laser pulsesdecay rate of sub levels(kvib ) re excitation rate4source: S.W. Hell, Far-Field Optical Nanoscopy, www.sciencemag.org, 25/05/07
STED-Beam IntensitySTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesDepletion of the fluorescence level:dN1dt N1 σISTED / ω N0 σISTED / ω N1 kFl
STED-Beam IntensitySTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesDepletion of the fluorescence level:dN1dt N1 σISTED / ω N0 σISTED / ω N1 kFldN0dt N1 σISTED / ω N0 σISTED / ω N0 kvib
STED-Beam IntensitySTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsDepletion of the fluorescence level:dN1dt N1 σISTED / ω N0 σISTED / ω N1 kFldN0dt N1 σISTED / ω N0 σISTED / ω N0 kvibkvib State of the ArtExamplesσISTED / ω kFL N0 0
STED-Beam IntensitySTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsDepletion of the fluorescence level:dN1dt N1 σISTED / ω N0 σISTED / ω N1 kFldN0dt N1 σISTED / ω N0 σISTED / ω N0 kvibkvib State of the ArtExamplesσISTED / ω kFL N0 0 N1 e τ σISTED / ω
STED-Beam IntensitySTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsDepletion of the fluorescence level:dN1dt N1 σISTED / ω N0 σISTED / ω N1 kFldN0dt N1 σISTED / ω N0 σISTED / ω N0 kvibkvib State of the ArtExamplesσISTED / ω kFL N0 0 N1 e τ σISTED / ωWe can estimate N1 (ISTED ) as a step function which becomes 0 atISTED ωτσ
Area of Excited MoleculesSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesThe N1 occupation over ISTED :
Area of Excited MoleculesSTEDThe N1 occupation over ISTED :Robert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesThe N1 occupation folded with the spatial ISTED distribution:
New ResolutionSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesResolution of a STED microscope x rλI2·NA· 1 STEDISatAs the improvement is due to the limited area of light sourcesISTED must be the crucial parameter.
The Donut-ModeSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesThe minimum in the center of the STED Focus is made bygiving the half of the inner laying beams an phase shift of π
The STED SetupSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamples5The STED setup is similar to the setup of a confocal or 4Pimicroscope. With the difference that one more beam has to becoupled into the optical axes.5source: Imaging with the Leica TCS STED a Practical Guide
Proof of ConceptSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamples6Experiment details:dye: LDS 751ISTED 2.8GW /cm2λexc 540nmλSTED 700nmResults:a) predicted dependence N1 from ISTEDb) measured lateral FID7 without the STED beamc) reduced axial FID with the STED beamd) reduced lateral FID with the STED beam6source: Hell et al., Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission7FID stands for fluorescence light intensity distribution
STED Applications: BiophysicsSTEDRobert PolsterResolution:ClassicalLimits of Resolutiontransversal 16 nmModern MicroscopesSTEDMicroscopylongitudinal 32 nmPrincipleProbes:Technical DetailsState of the ArtExamplesa) microtubules in a neuronb) silica nanobeadsc) neurofilamented) cell membrane88source: S.W. Hell, Far-Field Optical Nanoscopy,www.sciencemag.org, 25/05/07
Record of ResolutionSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamples9Experiment details:dye: nitrogenvacancies in diamondISTED 3.7GW /cm2λexc 532nmλSTED 775nm9source: see next pageResults:resolution goes up to 8 nmwith ISTED 8.6GW /cm2 even 6 nm
Diamond Colour CentersSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamples10luminescent transitions arising from nitrogen vacancies inthe diamond structure10Hell et al, STED microscopy reveals crystal colour centers with nanometric resolution, nature photonics , 22/02/09
Diamond Colour CentersSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrinciple11Technical DetailsState of the ArtExamplesCenters with ms 0 emit more strongly, because thems 1 centers convert often to the metastable state 1 Eoptical measurement of spinsusable as magnetic field sensor or data storage11Hell et al, STED microscopy reveals crystal colour centers with nanometric resolution, nature photonics , 22/02/09
ConclusionSTEDRobert PolsterClassicalLimits of ResolutionModern MicroscopesSTEDMicroscopyPrincipleTechnical DetailsState of the ArtExamplesTheoretically no more resolution limits for far fieldmicroscopyPractically bounded by the destruction threshold or crosssection of the medium
n refraction index angle of acceptance. STED Robert Polster Classical Limits of Resolution Modern Microscopes STED- . Springer, New York. 2006. STED Robert Polster Classical Limits of Resolution Modern Microscopes STED-Microscopy . Excite the dyes As in every uorescence microscopy the dye molecules are excited by a Laser. STED Robert .
Practical fluorescence microscopy 37 4.1 Bright-field versus fluorescence microscopy 37 4.2 Epi-illumination fluorescence microscopy 37 4.3 Basic equipment and supplies for epi-illumination fluorescence . microscopy. This manual provides basic information on fluorescence microscopy
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Summary Technical Documentation (STED): . Template (CSDT) of the Asian Harmonization Working Party (AHWP) and the Association of Southeast Asian Nations (ASEAN) Medical Devices Product Working Group. Japan started trial use of the STED in 2002, based on discussion in GHTF SG1 and
Rapid STED manual - A Practical guide Contents (suite) 5.4 Contenu de l'atelier 35 5.4.1 Introduction de l'atelier 35 5.4.2 Session 1 - Introduction à l'atelier: objectif, impact visé, processus et cadre de diagnostic STED 36 5.4.3 Session 2 - Présentation et discussion sur les consultations et la recherche de fond 36
Rapid STED manual - A Practical guide Contents (suite) 5.3 Guidance on Conducting the Rapid STED Technical and Policy Foresight Workshop 33 5.3.1 Scope of Guidance 33 5.3.2 Workshop Materials 33 5.3.3 Workshop Location 33 5.3.4 Workshop Staffing 33 5.4 Workshop Content 34 5.4.1 Workshop Introduction 34
MULTIPHOTON LASER SCANNING MICROSCOPY Introduction to Multiphoton Laser Scanning Microscopy Carl Zeiss LSM 510 NLO 8-6 B 40-055 e 09/02 8.2 Introduction to Multiphoton Laser Scanning Microscopy Multiphoton laser scanning microscopy (MPLSM) has become an important technique in vital and deep tissue fluorescence imaging.
1. Static atomic force microscopy 958 2. Dynamic atomic force microscopy 959 III. Challenges Faced by Atomic Force Microscopy with Respect to Scanning Tunneling Microscopy 960 A. Stability 960 B. Nonmonotonic imaging signal 960 C. Contribution of long-range forces 960 D. Noise in the imaging signal 961 IV. Early AFM Experiments 961 V. The Rush .
graded readers end at around the 3,000 word-family level. Note that this figure differs from Nation (2001) who considered this level to con- tain only the first 2,000 word families. In Table 3, the mid-frequency vocabulary con-sists of around 6,000 word families, which when added to high-frequency vocabulary adds up to 9,000 word families. The reason for making the arbitrary cut-off point .
