FlurescenceMicroscopyinPharmacology荧光显微镜在药理学

FlurescenceMicroscopyinPharmacology荧光显微镜在药理学当前1页，总共39页。BasicsandIntroduction Fluorescence/Transmissionmicroscopy Advantage/Drawbackoflightmicroscopy FluorescenceDyes GFPsInstrumentalDevices Confocallaserscanmicroscopy(CLSM) Imaginginlivingcells DeconvolutionmicroscopyComparisonoftechniquesavailable当前2页，总共39页。FluorescenceMicroscopyIntroductionFluorescencemicroscopyAdvantages/disadvantages,limitationsFluorescencedyesVitaldyes,GFPandderivativesImmunofluorescenceTechnology2photonexcitationFRAPandFRETFluorescencelifetimeimagingConfocallaserscanningDeconvolutionandimagingExamples当前3页，总共39页。Limitationoflightmicroscopy5mmlimitofresolutionlbluePicture:S.Kohlwein,B&FB,Graz,Austria当前4页，总共39页。Fluorescence

microscopylightsourceobjectobjectivelens1stbarrierfilter(lex)2ndbarrierfilter(lem)beam-splittingmirroreyepiecePicture:S.Kohlwein,B&FB,Graz,Austria当前5页，总共39页。FluorescenceMicroscopyLifeCellandImmunoFluorescenceApplications-dyesOrganelle-specific,pH,membranepotential,ionConcentrationCagedcompounds

GFP,BFP,RFP,YFP;Aequorin;GFPandFRETSamplePreparation

当前6页，总共39页。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)

当前7页，总共39页。ImmunofluorescenceMicroscopy

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proteinlocalization 3dreconstruction resolution>lifecells(nodynamics)

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

当前8页，总共39页。Applications-dyes

Organelle-specific pH membranepotential ionselective ....

(MolecularProbes)

当前9页，总共39页。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当前10页，总共39页。Cholesteroldistributionin3T3cells(fillipin)Pictures:W.FGraier,MBC&MB,Graz,Austria当前11页，总共39页。Pictures:W.FGraier,MBC&MB,Graz,AustriaDiOC6deconvoluteddeconvoluted当前12页，总共39页。Pictures:W.FGraier,MBC&MB,Graz,Austria当前13页，总共39页。Blue/Green/Yellow/Redfluorescentproteins

/当前14页，总共39页。GreenFluorescentProteinCloningStrategiesN,C-terminalfusions<–>targetingsignals!endogenous<–>heterologouspromoter!steadystate-distribution<–>"pulse-chase"!function!当前15页，总共39页。

GFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRYFGGFPkanMXYFGGFPkanMXPCRtransformationG418selectionGFPC-terminalchromosomalfusionpUGplasmidtemplate当前16页，总共39页。FluorescenceDyesConjugatesSubstratesAgonistsChelators当前17页，总共39页。ConjugatesPrinciples:primaryantibodysecondaryantibody (dyecoupled)Samples: Alexa,Cy-XImmunfluorescencePictures:MolecularProbes当前18页，总共39页。4,5-Diaminofluorescein(DAF)Substrates当前19页，总共39页。AgonistsBODIPY-RyanodinePictures:W.FGraier,MBC&MB,Graz,Austria当前20页，总共39页。ChelatorsTargetingofchelatorsbyspecificgroups(e.g.fattyacids)Ca2+Na+H+K+Cl-......Fura-2当前21页，总共39页。FluorescenceMicroscopyTechnologyDeconvolutionMicroscopyConfocalLaserScanningMicroscopy2PhotonMicroscopy;time-resolvedFMFRAPfluorescencerecoveryafterphotobleachingFRETfluorescenceresonanceenergytransfer当前22页，总共39页。FluorescenceMicroscopy2PhotonExcitationMicroscopy1Photon2Photon当前23页，总共39页。FluorescenceMicroscopyTime-resolvedfluorescencemicroscopyfluorescencetime(nsec)dye1(e.g.background)dye2timewindow当前24页，总共39页。FluorescenceMicroscopyFRAPFRET(Cameleon)BFPGFPCa++ocalmdulinlexBFPlemBFPlemGFPBFPGFPCalmodulin/M13Ca++lemBFPlexBFP当前25页，总共39页。ER-tagged-CameleonsMi-tagged-CameleonsPictures:W.FGraier,MBC&MB,Graz,AustriaOrganell-specificexpressionofanCa2+-sensitiveproteineCameleons(developedbyR.Y.Tsien)当前26页，总共39页。 (localconcentration!) sensitivity

resolution rec.speed 100x100x300nm msec–sec

ElectronicLightMicroscopycellviability,structuredynamics当前27页，总共39页。TheConfocalPrincipleopticalresolution:>100nm(x/y)>300nm(z)PointsourceObjectivelensFocalplaneSpecimenDichroicmirrorIlluminatingapertureConfocaldetectoraperturePhotomultiplierin-focusraysoutoffocusrays当前28页，总共39页。TheConfocalPrincipleSingleopticalsectionmultipleopticalsections3dreconstructionzzxypictureelement(pixel;e.g.60x60nm)当前29页，总共39页。TheConfocalPrinciplecoverslidedepth‘‘focalspot‘‘Picture:S.Kohlwein,B&FB,Graz,Austria当前30页，总共39页。YeastLightMicroscopy100x当前31页，总共39页。MicrofluorometryPictures:W.FGraier,MBC&MB,Graz,Austria当前32页，总共39页。MicrofluorometryPictures:W.FG