Mikroskopické techniky a její praktické využití Eva Bártová Biofyzikální ústav Akademie věd ČR Brno Dendra2 photo-conversion Dendra2 is an improved version of a green-to-red photoswitchable fluorescent protein Dendra, derived from octocoral Dendronephthya sp. [Gurskaya et al., 2006]. Princip fluorescenčního mikroskopu Excitační filtr, (nastaven na modrou tj. 450-490 nm ) Bariérový filtr, (nastaven na zelenou tj. 520-560 nm ) Dichroické zrcadlo, odráží světlo pod 510 nm a propouští nad 510 nm Zdroj světla Okulár ObjektivPreparát Vstupní štěrbina, dělič svazků, dichroické zrcadlo, AOTF (akusto-optický dělič svazků) AOBS a AOTF Tandem scanning microscopes based on Nipkow disk Source of light Sample CCD rotation pinholes in the disk Dichroic mirror Scanning in 2D and 3D by confocal microscope Laser beam moves firstly along x axis and then starts with new line in y axis. y x y z Begin End x 2D 3D Finishing scanning of one thin optical slice in xy plane, the scanning plane is moved in z axis to other slice Optical resolution: conventional versus confocal Conventional Confocal Res = 0.61*λ / ΝΑλ / ΝΑλ / ΝΑλ / ΝΑ Res(xy) = 0.4*λ / ΝΑλ / ΝΑλ / ΝΑλ / ΝΑ Res(xz) = 0.45*λ /λ /λ /λ / n(1-cosα)α)α)α) Formulas by Kino 4PI and STED resolution are much higher… Inkubační komůrky pro přímý mikroskop Termistors in short tubes on the side of the chamber Partition with opening on the bottom of the chamber (for experiments under upright microscope) Cover glass 24x50 mm Glass tubes bent under the upper glass (to enable CO2 to flow above medium in the part II of the chamber) Part I – cells can be grown on both upper and lower sides of the chamber; this part is filled fully with medium Part II – medium reaches under the glass tubes; cells are not grown here JMJD2b(1-424)-GFP Experiments of Gabriela Galiová Využití UV laseru 355 nm ke studiu DNA reparace Experiments of Gabriela Galiová Experiments of Gabriela Galiová and Lenka Stixová Další metody využívané fluorescenční mikroskopií http://www.youtube.com/watch?v=pteO6FRWo3g 3D-FISH a konfokální mikroskopie Maximální obraz Všech řezů Galerie optických řezů 3D reconstrukce CT Weierich et al., (2003) in press Automatická analýza obrazuAutomatická analýza obrazu Comparative genome hybridization CGH onCGH on metaphasemetaphase spreadsspreads Transmisní Elektronový Mikroskop (TEM) Skenovací (Rastrovací) Elektronový Mikroskop 4pi: improved axial resolution. The typical value of 500- 700 nm can be improved to 100-150 nm which corresponds to an almost spherical focal spot with 5-7 times less volume than that of standard confocal microscopy. beam spliter The operation mode of a 4Pi microscope is shown in the figure. The laser light is divided by a beam splitter (BS) and directed by mirrors towards the two opposing objective lenses. At the common focal point superposition of both focused light beams occurs. Excited molecules at this position emit fluorescence light which is collected by both objective lenses, combined by the same beam splitter and deflected by a dichroic mirror (DM) onto a detector. There superposition of both emitted light pathways can take place again. Stimulated Emission Depletion microscopy, or STED microscopy, is a fluorescence microscopy technique that uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit imposed by diffraction with standard confocal laser scanning microscopes and conventional far-field optical microscopes. STED SP-5 LSCM Super-resolution microscope systems from Carl Zeiss ELYRA product family combines PAL-M (Photo-activated localization microscopy) and SR-SIM technology Fluorescence microscopy technique comparison Objective Petri Dish Oil WFM CLSMSDCM SD6000: ~ 0.84 µm SP5: ~ 0.50 µm TIRF: 0.1 – 0.3 µmwidefield: ~ 1.13 µm TIRF moderately thick 5 µm – 30 µm thick > 30 µm ! does not matter ! Only evanescent waves thin samples < 5 µm moderately thick + deconvolution samples Z resolution measured 4PI: 0.1 – 0.12 µm! Confocal Laser Scanning Microscopy – advanced systems Leica TCS SP5 – universal system for everything! Leica TCS SP5 STED Leica TCS SP5-X WLL Leica TCS 4PI Leica DM6000 CFS – Confocal Fixed Stage FRAP FRET AB, SE Live Data Mode ROI spectrophotometer APD SMD – FCS, FLIM, FCCS Spectral FLIM High Content Screening Auto 2-photon, 3-photon Unique Leica TCS SP5: the only broadband confocal Leica TCS SP5 basic features • full range of lasers: 355, 405, VIS, IR up to 1300 nm • conventional scanner up to 8192x8192 pxls • resonant scanner up to 29 f/s for 512x512pxls • AOBS – Acousto-Optical Beam Splitter • Up to 5 confocal spectral detectors • SuperZ Galvo and Pifoc Wide range of UNIQUE upgrades: • White Light Laser • Spectral FLIM • online ROI spectrometer • STED – superresolution in xy plane Separate UNIQUE systems • Leica DM6000 B CFS - electrophysiology • Leica 4PI – high resolution in z axis Unique Structure High resolution optical sections 3D Structures Correlation analysis Single Point Illumination Dynamic Movement analysis Kinetic measurements Leica TCS SP5: The Only Broadband Confocal Resolution and speed in one system Leica TCS SP5 – Leading in Multispectral imaging Ideal for multi-labelled specimens and fast events: Up to 5 SP Prism Spectrophotometer channels for sensitivity and flexibility AOBS – the dynamic beam splitter for sensitivity, selectivity and flexibility Spectral FLIM - a new dimension of experiments ROI-spectrometer captures dynamic spectral events Unique Leica TCS SP5 – Leading in Multispectral imaging Leica SP – Leica Spectral detection (1997) 5 spectral confocal detectors simultanously Arbitrary settings of spectra Low photobleaching, high efficiency Intuitive operation White Light Laser – set of lasers or just one tuneable source? No tunability Sub-optimal excitation Cross-excitation fixed Excitation Spectra 488nm 543nm 633nm (Alexa 488, Alexa 546, Alexa 568, Toto-3) Set of gas, DPSS or DL lasers Sophisticated merge module Expensive solution Only several combinations of wavelengths setting of excitation wavelength and intensity in software or at Panel Box „Smart Wavelength“ and „Smart Intensity“ Lambda Square Scan: lambda excitation and emission over whole visible spectra White Light Laser – new lambda scan, new wavelengths Leica STED – STimulated Emission Depletion SUPERRESOLUTION (subdiffraction) in xy plane Willig KI et al. Nature 2006 Kellner RR et al. Neurosience 2007 Sieber JJ et al. Biophy J 2006 Lin W et al. PNAS 2007 Kittel RJ et al. Science 2006 Seebach J Cardiovas. Res. 2007 Fitzner D et al. EMBO J 2006 Sieber JJ Science 2007 neurobiology membrane biology membrane rafts intracellular transport Hell, S. W. and J. Wichmann (1994). Opt. Lett. "Breaking the diffraction resolution limit by stimulated emission" Unique Concentration? Molecule motility? Molecule interaction? Diffusion behavior? Molecule identification? Reaction kinetics? Signal transduction? Explain phenomena! Predict models! Optimize agents! Improve procedures! Validate data! Understand life! Translocation? Verify assumptions! Quantify Life! – The Challenge Possibilities: FCS - FCCS - FLIM – FRET - FLCS - gated FCS