Flurescence-Microscopy-in-Pharmacology熒光顯微鏡在藥理學

FlurescenceMicroscopyinPharmacology熒光顯微鏡在藥理學FlurescenceMicroscopyinPharmacology熒光顯微鏡在藥理學FlurescenceMicroscopyinPharmacology熒光顯微鏡在藥理學BasicsandIntroduction Fluorescence/Transmissionmicroscopy Advantage/Drawbackoflightmicroscopy FluorescenceDyes GFPsInstrumentalDevices Confocallaserscanmicroscopy(CLSM) Imaginginlivingcells DeconvolutionmicroscopyComparisonoftechniquesavailableFlurescenceMicroscopyinPhar1BasicsandIntroduction Fluorescence/Transmissionmicroscopy Advantage/Drawbackoflightmicroscopy FluorescenceDyes GFPsInstrumentalDevices Confocallaserscanmicroscopy(CLSM) Imaginginlivingcells DeconvolutionmicroscopyComparisonoftechniquesavailableBasicsandIntroduction2FluorescenceMicroscopyIntroductionFluorescencemicroscopyAdvantages/disadvantages,limitationsFluorescencedyesVitaldyes,GFPandderivativesImmunofluorescenceTechnology2photonexcitationFRAPandFRETFluorescencelifetimeimagingConfocallaserscanningDeconvolutionandimagingExamplesFluorescenceMicroscopyIntrodu3Limitationoflightmicroscopy5mmlimitofresolutionlbluePicture:S.Kohlwein,B&FB,Graz,AustriaLimitationoflightmicroscopy4Fluorescence

microscopylightsourceobjectobjectivelens1stbarrierfilter(lex)2ndbarrierfilter(lem)beam-splittingmirroreyepiecePicture:S.Kohlwein,B&FB,Graz,AustriaFluorescence

microscopylights5FluorescenceMicroscopyLifeCellandImmunoFluorescenceApplications-dyesOrganelle-specific,pH,membranepotential,ionConcentrationCagedcompounds

GFP,BFP,RFP,YFP;Aequorin;GFPandFRETSamplePreparation

FluorescenceMicroscopy6LifeCellMicroscopy

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dynamics! samplepreparation! 3dreconstruction-?multi-dimensional“ (3d+time,multiplewavelengths,reactionkinetics..)

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limitsofresolution(wavelengthoflight) viability,temperature,oxygen,phototoxicity, bleaching dynamicsofstructures(lossofresolution)

LifeCellMicroscopy7ImmunofluorescenceMicroscopy

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proteinlocalization 3dreconstruction resolution>lifecells(nodynamics)

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limitsofresolution(wavelengthoflight) samplepreparation,preparationartifacts(fixation, Abspecificity) deadcells! bleaching

ImmunofluorescenceMicroscopy8Applications-dyes

Organelle-specific pH membranepotential ionselective ....

(MolecularProbes)

Applications-dyes9Microscopicanalysisofyeastorganellesinvivomitochondria(DASPMI,Mito-TrakerMi)lipidparticles(NileRed)nucleus(DAPI,SYTO)endoplasmicreticulum(DiOC6,Mito-TrakerER)vacuoles(FM4-64,CDCFDA)endocyt.vesicles(FM4-64)membranesCholesterol:filipinpotential-sensitivedyes:bis-oxonolMicroscopicanalysisofyeast10Cholesteroldistributionin3T3cells(fillipin)Pictures:W.FGraier,MBC&MB,Graz,AustriaCholesteroldistributionin3T11Pictures:W.FGraier,MBC&MB,Graz,AustriaDiOC6deconvoluteddeconvolutedPictures:W.FGraier,MBC&MB12Pictures:W.FGraier,MBC&MB,Graz,AustriaPictures:W.FGraier,MBC&MB13Blue/Green/Yellow/Redfluorescentproteins

Blue/Green/Yellow/Redfluoresc14GreenFluorescentProteinCloningStrategiesN,C-terminalfusions<–>targetingsignals!endogenous<–>heterologouspromoter!steadystate-distribution<–>"pulse-chase"!function!GreenFluorescentProteinClon15

GFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRYFGGFPkanMXYFGGFPkanMXPCRtransformationG418selectionGFPC-terminalchromosomalfusionpUGplasmidtemplate

16FluorescenceDyesConjugatesSubstratesAgonistsChelatorsFluorescenceDyesConjugatesSub17ConjugatesPrinciples:primaryantibodysecondaryantibody (dyecoupled)Samples: Alexa,Cy-XImmunfluorescencePictures:MolecularProbesConjugatesPrinciples:Samples:I184,5-Diaminofluorescein(DAF)Substrates4,5-DiaminofluoresceinSubstrat19AgonistsBODIPY-RyanodinePictures:W.FGraier,MBC&MB,Graz,AustriaAgonistsBODIPY-RyanodinePictu20ChelatorsTargetingofchelatorsbyspecificgroups(e.g.fattyacids)Ca2+Na+H+K+Cl-......Fura-2ChelatorsTargetingofchelator21FluorescenceMicroscopyTechnologyDeconvolutionMicroscopyConfocalLaserScanningMicroscopy2PhotonMicroscopy;time-resolvedFMFRAPfluorescencerecoveryafterphotobleachingFRETfluorescenceresonanceenergytransferFluorescenceMicroscopy22FluorescenceMicroscopy2PhotonExcitationMicroscopy1Photon2PhotonFluorescenceMicroscopy1Photo23FluorescenceMicroscopyTime-resolvedfluorescencemicroscopyfluorescencetime(nsec)dye1(e.g.background)dye2timewindowFluorescenceMicroscopyfluores24FluorescenceMicroscopyFRAPFRET(Cameleon)BFPGFPCa++ocalmdulinlexBFPlemBFPlemGFPBFPGFPCalmodulin/M13Ca++lemBFPlexBFPFluorescenceMicroscopyFRAPFRE25ER-tagged-CameleonsMi-tagged-CameleonsPictures:W.FGraier,MBC&MB,Graz,AustriaOrganell-specificexpressionofanCa2+-sensitiveproteineCameleons(developedbyR.Y.Tsien)ER-tagged-CameleonsMi-tagged-C26 (localconcentration!) sensitivity

resolution rec.speed 100x100x300nm msec–sec

ElectronicLightMicroscopycellviability,structuredynamicsElectronicLightMicroscopycel27TheConfocalPrincipleopticalresolution:>100nm(x/y)>300nm(z)PointsourceObjectivelensFocalplaneSpecimenDichroicmirrorIlluminatingapertureConfocaldetectoraperturePhotomultiplierin-focusraysoutoffocusraysTheConfocalPrincipleopticalP28TheConfocalPrincipleSingleopticalsectionmultipleopticalsections3dreconstructionzzxypictureelement(pixel;e.g.60x60nm)TheConfocalPrincipleSingleo29TheConfocalPrinciplecoverslidedepth‘‘focalspot‘‘Picture:S.Kohlwein,B&FB,Graz,AustriaTheConfocalPrinciplecoversl30YeastLightMicroscopy100xYeastLightMicroscopy100x31MicrofluorometryPictures:W.FGraier,MBC&MB,Graz,AustriaMicrofluorometryPictures:W.F32MicrofluorometryPictures:W.FGraier,MBC&MB,Graz,AustriaMicrofluorometryPictures:W.F33Microfluorometry:

SimultaneouslyrecordingsofCa2+andioncurrentsPictures:W.FGraier,MBC&MB,Graz,AustriaMicrofluorometry:Pictures:W.F34FluorescenceImagingFluorescenceImaging35DeconvolutionmicroscopyPictures:W.FGraier,MBC&MB,Graz,AustriaDec