NOTE:This Powerpoint presentation also includes so far not published pictures and results. It has been released only for teaching the principles and possibilities of high resolution micrsocopy to graduate and post-graduate students. - Thank you very much for your fairness.If any other use is planed please contact:Prof. Wolfgang F. GraierDepartment of Medical Biochemistry and Medical Molecular BiologyKarl-Franzens University of GrazHarrachgasse 21/IIIA-8010 GrazTel. +43-316-380-7560Fax. +43-316-380-9615E- mail: wolfgang.graierkfunigraz.ac.at Basics and IntroductionFluorescence/TransmissionmicroscopyAdvantage/Drawback of light microscopyFluorescence DyesGFPsInstrumental DevicesConfocal laser scan microscopy (CLSM) Imaging in living cellsDeconvolution microscopyComparison of techniques availableFluorescence MicroscopyIntroductionFluorescence microscopyAdvantages/disadvantages, limitationsFluorescence dyesVital dyes, GFP and derivativesImmunofluorescenceTechnology2 photon excitationFRAP and FRETFluorescence life time imagingConfocal laser scanningDeconvolution and imagingExamplesLimitation of light microscopy5 m mmlimit ofresolutionl lbluePicture: S. Kohlwein, B & FB, Graz, AustriaFluorescencemicroscopylight sourceobjectobjective lens1st barrier filter(lex)2nd barrier filter(lem)beam-splittingmirroreyepiecePicture: S. Kohlwein, B & FB, Graz, AustriaFluorescence Microscopy Life Cell and Immuno Fluorescence Applications - dyesOrganelle-specific, pH, membrane potential, ionConcentration Caged compounds GFP, BFP, RFP, YFP; Aequorin; GFP and FRET Sample Preparation Life Cell Microscopy +dynamics !sample preparation !3d reconstruction - multi-dimensional“(3d + time, multiple wavelengths, reaction kinetics.) limits of resolution (wavelength of light)viability, temperature, oxygen, phototoxicity,bleachingdynamics of structures (loss of resolution) Immunofluorescence Microscopy +protein localization3d reconstructionresolution life cells (no dynamics) limits of resolution (wave length of light)sample preparation, preparation artifacts (fixation, Ab specificity)dead cells !bleaching Applications - dyesOrganelle-specificpHmembrane potentialion selective. http:/www.probes.com (Molecular Probes) Microscopic analysis of yeast organelles in vivomitochondria(DASPMI , Mito-Traker Mi)lipid particles(Nile Red)nucleus(DAPI, SYTO) endoplasmic reticulum (DiOC6, Mito-Traker ER)vacuoles(FM4-64, CDCFDA)endocyt. vesicles(FM4-64)membranesCholesterol: filipinpotential-sensitive dyes: bis-oxonolCholesterol distribution in 3T3 cells (fillipin)Pictures: W.F Graier, MBC & MB, Graz, AustriaPictures: W.F Graier, MBC & MB, Graz, AustriaDiOC6deconvoluteddeconvolutedPictures: W.F Graier, MBC & MB, Graz, AustriaBlue/Green/Yellow/Red fluorescent proteins http:/www.clontech.com/Green Fluorescent Protein Cloning StrategiesN, C-terminal fusions targeting signals !endogenous heterologous promoter !steady state-distribution pulse-chase !function ! GFPkanMX6ERGChromosomal fusionvia homologous recombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomal fusionvia homologous recombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomal fusionvia homologous recombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRYFGGFPkanMXYFGGFPkanMXPCRtransformationG418 selectionGFP C-terminal chromosomal fusionpUG plasmid templateFluorescence DyesConjugatesSubstratesAgonistsChelatorsConjugatesPrinciples:primary antibodysecondary antibody(dye coupled)Samples:Alexa, Cy-XImmunfluorescencePictures: Molecular Probes4,5-Diaminofluorescein(DAF)SubstratesAgonistsBODIPY- RyanodinePictures: W.F Graier, MBC & MB, Graz, AustriaChelatorsTargeting of chelators by specific groups (e.g. fatty acids)Ca2+Na+H+K+Cl-.Fura-2Fluorescence MicroscopyTechnologyDeconvolution MicroscopyConfocal Laser Scanning Microscopy2 Photon Microscopy; time-resolved FMFRAP fluorescence recovery after photo bleachingFRET fluorescence resonance energy transferFluorescence Microscopy2 Photon Excitation Microscopy1 Photon2 PhotonFluorescence MicroscopyTime-resolved fluorescence microscopyfluorescencetime (nsec)dye 1 (e.g. background)dye 2time windowFluorescence MicroscopyFRAPFRET (Cameleon)BFPGFPCa+ocalmdulinlexBFPlemBFPlemGFPBFPGFPCalmodulin/M13Ca+lemBFPlexBFPER-tagged-CameleonsMi-tagged-CameleonsPictures: W.F Graier, MBC & MB, Graz, AustriaOrganell-specific expression of an Ca2+-sensitive proteineCameleons (developed by R.Y. Tsien)(local concentration !)sensitivity resolutionrec. speed100 x 100 x 300 nmmsec secElectronic Light Microscopycell viability, structure dynamicsThe Confocal Principleoptical resolution:100 nm (x/y)300 nm (z)Point sourceObjective lensFocal planeSpecimenDichroicmirrorIlluminating apertureConfocal detectoraperturePhotomultiplierin-focus raysout of focus raysThe Confocal PrincipleSingle optical sectionmultiple optical sections3d reconstructionzzxypicture element (pixel; e.g. 60x60 nm)The Confocal Principlecover slidedepthfocal spotPicture: S. Kohlwein, B & FB, Graz, AustriaYeast Light Microscopy100 xMicrofluorometryPictures: W.F Graier, MBC & MB, Graz, AustriaMicrofluorometryPictures: W.F Graier, MBC & MB, Graz, AustriaMicrofluorometry:Simultaneously recordings of Ca2+ and ion currentsPictures: W.F Graier, MBC & MB, Graz, AustriaFluorescence ImagingDeconvolution microscopyPictures: W.F Graier, MBC & MB, Graz, AustriaPoint spread functionFocusOut-of-focus fluorescence3D reconstructionpixel (x-y plane) .voxel (x-y-z plane)2D reconstructionDeconvolution microscopy allows time resolvedtwo dimensional fluorescence recordings in high x-yresolution and app. 200 to 300 m thick slices (pixel) Confocal vs DeconvolutionOut-of-focus lightSignal to noise ratio Serial lines (time scan)Image acquisitionImaging qualityThick samplesExcitation lCostspinholelow (10 p/px) slow= f(object) 100 m# laser lines PSF & comput.high (104 p/px)n.a.fast= f(object) 100 mSpectral lamp