FlurescenceMicroscopyinPharmacology熒光顯微鏡在藥理學

FlurescenceMicroscopyinPharmacology熒光顯微鏡在藥(Yao)理學第一頁，共三十九頁。BasicsandIntroduction Fluorescence/Transmissionmicroscopy Advantage/Drawbackoflightmicroscopy FluorescenceDyes GFPsInstrumentalDevices Confocallaserscanmicroscopy(CLSM) Imaginginlivingcells DeconvolutionmicroscopyComparisonoftechniquesavailable第二頁，共三十九頁。FluorescenceMicroscopyIntroductionFluorescencemicroscopyAdvantages/disadvantages,limitationsFluorescencedyesVitaldyes,GFPandderivativesImmunofluorescenceTechnology2photonexcitationFRAPandFRETFluorescencelifetimeimagingConfocallaserscanningDeconvolutionandimagingExamples第三頁，共三十九頁。Limitationoflightmicroscopy5mmlimitofresolutionlbluePicture:S.Kohlwein,B&FB,Graz,Austria第四頁，共三十九頁。Fluorescence

microscopylightsourceobjectobjectivelens1stbarrierfilter(lex)2ndbarrierfilter(lem)beam-splittingmirroreyepiecePicture:S.Kohlwein,B&FB,Graz,Austria第五頁，共三十九頁。FluorescenceMicroscopyLifeCellandImmunoFluorescenceApplications-dyesOrganelle-specific,pH,membranepotential,ionConcentrationCagedcompounds

GFP,BFP,RFP,YFP;Aequorin;GFPandFRETSamplePreparation

第六頁，共三十九頁。LifeCellMicroscopy

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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)

第七頁，共三十九頁。ImmunofluorescenceMicroscopy

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proteinlocalization 3dreconstruction resolution>lifecells(nodynamics)

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

第八頁，共三十九頁。Applications-dyes

Organelle-specific pH membranepotential ionselective ....

(MolecularProbes)

第九頁，共三十九頁。Microscopicanalysisofyeastorganellesinvivomitochondria(DASPMI,Mito-TrakerMi)lipidparticles(NileRed)nucleus(DAPI,SYTO)endoplasmicreticulum(DiOC6,Mito-TrakerER)vacuoles(FM4-64,CDCFDA)endocyt.vesicles(FM4-64)membranesCholesterol:filipinpotential-sensitivedyes:bis-oxonol第十頁，共三十九頁。Cholesteroldistributionin3T3cells(fillipin)Pictures:W.FGraier,MBC&MB,Graz,Austria第十一頁，共三十九頁。Pictures:W.FGraier,MBC&MB,Graz,AustriaDiOC6deconvoluteddeconvoluted第十二頁，共三十九頁。Pictures:W.FGraier,MBC&MB,Graz,Austria第十三頁，共三十九頁。Blue/Green/Yellow/Redfluorescentproteins

/第十四頁，共三十九頁。GreenFluorescentProteinCloningStrategiesN,C-terminalfusions<–>targetingsignals!endogenous<–>heterologouspromoter!steadystate-distribution<–>"pulse-chase"!function!第十五頁，共三十九頁。

GFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRYFGGFPkanMXYFGGFPkanMXPCRtransformationG418selectionGFPC-terminalchromosomalfusionpUGplasmidtemplate第十六頁，共三十九頁。FluorescenceDyesConjugatesSubstratesAgonistsChelators第十七頁，共三十九頁。ConjugatesPrinciples:primaryantibodysecondaryantibody (dyecoupled)Samples: Alexa,Cy-XImmunfluorescencePictures:MolecularProbes第十八頁，共三十九頁。4,5-Diaminofluorescein(DAF)Substrates第十九頁，共三十九頁。AgonistsBODIPY-RyanodinePictures:W.FGraier,MBC&MB,Graz,Austria第二十頁，共三十九頁。ChelatorsTargetingofchelatorsbyspecificgroups(e.g.fattyacids)Ca2+Na+H+K+Cl-......Fura-2第二十一頁，共三十九頁。FluorescenceMicroscopyTechnologyDeconvolutionMicroscopyConfocalLaserScanningMicroscopy2PhotonMicroscopy;time-resolvedFMFRAPfluorescencerecoveryafterphotobleachingFRETfluorescenceresonanceenergytransfer第二十二頁，共三十九頁。FluorescenceMicroscopy2PhotonExcitationMicroscopy1Photon2Photon第二十三頁，共三十九頁。FluorescenceMicroscopyTime-resolvedfluorescencemicroscopyfluorescencetime(nsec)dye1(e.g.background)dye2timewindow第二十四頁，共三十九頁。FluorescenceMicroscopyFRAPFRET(Cameleon)BFPGFPCa++ocalmdulinlexBFPlemBFPlemGFPBFPGFPCalmodulin/M13Ca++lemBFPlexBFP第二十五頁，共三十九頁。ER-tagged-CameleonsMi-tagged-CameleonsPictures:W.FGraier,MBC&MB,Graz,AustriaOrganell-specificexpressionofanCa2+-sensitiveproteineCameleons(developedbyR.Y.Tsien)第二十六頁，共三十九頁。 (localconcentration!) sensitivity

resolution rec.speed 100x100x300nm msec–sec

ElectronicLightMicroscopycellviability,structuredynamics第二十七頁，共三十九頁。TheConfocalPrincipleopticalresolution:>100nm(x/y)>300nm(z)PointsourceObjectivelensFocalplaneSpecimenDichroicmirrorIlluminatingapertureConfocaldetectoraperturePhotomultiplierin-focusraysoutoffocusrays第二十八頁，共三十九頁。TheConfocalPrincipleSingleopticalsectionmultipleopticalsections3dreconstructionzzxypictureelement(pixel;e.g.60x60nm)第二十九頁，共三十九頁。TheConfocalPrinciplecoverslidedepth‘‘focalspot‘‘Picture:S.Kohlwein,B&FB,Graz,Austria第三十頁，共三十九頁。YeastLightMicroscopy100x第三十一頁，共三十九頁。MicrofluorometryPictures:W.FGraier,MBC&MB,Graz,Austria第三十二頁，共三十九頁。MicrofluorometryPictures:W.FGra