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FlurescenceMicroscopyinPharmacology荧光显微镜在药理学目前一页\总数三十九页\编于十三点BasicsandIntroduction Fluorescence/Transmissionmicroscopy Advantage/Drawbackoflightmicroscopy FluorescenceDyes GFPsInstrumentalDevices Confocallaserscanmicroscopy(CLSM) Imaginginlivingcells DeconvolutionmicroscopyComparisonoftechniquesavailable目前二页\总数三十九页\编于十三点FluorescenceMicroscopyIntroductionFluorescencemicroscopyAdvantages/disadvantages,limitationsFluorescencedyesVitaldyes,GFPandderivativesImmunofluorescenceTechnology2photonexcitationFRAPandFRETFluorescencelifetimeimagingConfocallaserscanningDeconvolutionandimagingExamples目前三页\总数三十九页\编于十三点Limitationoflightmicroscopy5mmlimitofresolutionlbluePicture:S.Kohlwein,B&FB,Graz,Austria目前四页\总数三十九页\编于十三点Fluorescence
microscopylightsourceobjectobjectivelens1stbarrierfilter(lex)2ndbarrierfilter(lem)beam-splittingmirroreyepiecePicture:S.Kohlwein,B&FB,Graz,Austria目前五页\总数三十九页\编于十三点FluorescenceMicroscopyLifeCellandImmunoFluorescenceApplications-dyesOrganelle-specific,pH,membranepotential,ionConcentrationCagedcompounds
GFP,BFP,RFP,YFP;Aequorin;GFPandFRETSamplePreparation
目前六页\总数三十九页\编于十三点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)
目前七页\总数三十九页\编于十三点ImmunofluorescenceMicroscopy
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proteinlocalization 3dreconstruction resolution>lifecells(nodynamics)
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limitsofresolution(wavelengthoflight) samplepreparation,preparationartifacts(fixation, Abspecificity) deadcells! bleaching
目前八页\总数三十九页\编于十三点Applications-dyes
Organelle-specific pH membranepotential ionselective ....
(MolecularProbes)
目前九页\总数三十九页\编于十三点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目前十页\总数三十九页\编于十三点Cholesteroldistributionin3T3cells(fillipin)Pictures:W.FGraier,MBC&MB,Graz,Austria目前十一页\总数三十九页\编于十三点Pictures:W.FGraier,MBC&MB,Graz,AustriaDiOC6deconvoluteddeconvoluted目前十二页\总数三十九页\编于十三点Pictures:W.FGraier,MBC&MB,Graz,Austria目前十三页\总数三十九页\编于十三点Blue/Green/Yellow/Redfluorescentproteins
/目前十四页\总数三十九页\编于十三点GreenFluorescentProteinCloningStrategiesN,C-terminalfusions<–>targetingsignals!endogenous<–>heterologouspromoter!steadystate-distribution<–>"pulse-chase"!function!目前十五页\总数三十九页\编于十三点
GFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRGFPkanMX6ERGChromosomalfusionviahomologousrecombinationGFPkanMX6ChromosomeGFPkanMX6PlasmidERGChromosomePCRYFGGFPkanMXYFGGFPkanMXPCRtransformationG418selectionGFPC-terminalchromosomalfusionpUGplasmidtemplate目前十六页\总数三十九页\编于十三点FluorescenceDyesConjugatesSubstratesAgonistsChelators目前十七页\总数三十九页\编于十三点ConjugatesPrinciples:primaryantibodysecondaryantibody (dyecoupled)Samples: Alexa,Cy-XImmunfluorescencePictures:MolecularProbes目前十八页\总数三十九页\编于十三点4,5-Diaminofluorescein(DAF)Substrates目前十九页\总数三十九页\编于十三点AgonistsBODIPY-RyanodinePictures:W.FGraier,MBC&MB,Graz,Austria目前二十页\总数三十九页\编于十三点ChelatorsTargetingofchelatorsbyspecificgroups(e.g.fattyacids)Ca2+Na+H+K+Cl-......Fura-2目前二十一页\总数三十九页\编于十三点FluorescenceMicroscopyTechnologyDeconvolutionMicroscopyConfocalLaserScanningMicroscopy2PhotonMicroscopy;time-resolvedFMFRAPfluorescencerecoveryafterphotobleachingFRETfluorescenceresonanceenergytransfer目前二十二页\总数三十九页\编于十三点FluorescenceMicroscopy2PhotonExcitationMicroscopy1Photon2Photon目前二十三页\总数三十九页\编于十三点FluorescenceMicroscopyTime-resolvedfluorescencemicroscopyfluorescencetime(nsec)dye1(e.g.background)dye2timewindow目前二十四页\总数三十九页\编于十三点FluorescenceMicroscopyFRAPFRET(Cameleon)BFPGFPCa++ocalmdulinlexBFPlemBFPlemGFPBFPGFPCalmodulin/M13Ca++lemBFPlexBFP目前二十五页\总数三十九页\编于十三点ER-tagged-CameleonsMi-tagged-CameleonsPictures:W.FGraier,MBC&MB,Graz,AustriaOrganell-specificexpressionofanCa2+-sensitiveproteineCameleons(developedbyR.Y.Tsien)目前二十六页\总数三十九页\编于十三点 (localconcentration!) sensitivity
resolution rec.speed 100x100x300nm msec–sec
ElectronicLightMicroscopycellviability,structuredynamics目前二十七页\总数三十九页\编于十三点TheConfocalPrincipleopticalresolution:>100nm(x/y)>300nm(z)PointsourceObjectivelensFocalplaneSpecimenDichroicmirrorIlluminatingapertureConfocaldetectoraperturePhotomultiplierin-focusraysoutoffocusrays目前二十八页\总数三十九页\编于十三点TheConfocalPrincipleSingleopticalsectionmultipleopticalsections3dreconstructionzzxypictureelement(pixel;e.g.60x60nm)目前二十九页\总数三十九页\编于十三点TheConfocalPrinciplecoverslidedepth‘‘focalspot‘‘Picture:S.Kohlwein,B&FB,Graz,Austria目前三十页\总数三十九页\编于十三点YeastLightMicroscopy100x目前三十一页\总数三十九页\编于十三点MicrofluorometryPictures:W.FGraier,MBC&MB,Graz,Austria目前三十二页\总数三十九页\编于十三点MicrofluorometryPictures:W.FGraier,MBC&MB,Graz,A
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