荧光显微镜在药理学.ppt

FluorescenceMicroscopyWolfgangGraier(wolfgang.graierkfunigraz.ac.at),F-actin,NFkB(activationbyH2O2),Pictures:W.FGraier,MBCAequorin;GFPandFRETSamplePreparation,LifeCellMicroscopy+dynamics!samplepreparation!3dreconstruction-multi-dimensional“(3d+time,multiplewavelengths,reactionkinetics.)limitsofresolution(wavelengthoflight)viability,temperature,oxygen,phototoxicity,bleachingdynamicsofstructures(lossofresolution),ImmunofluorescenceMicroscopy+proteinlocalization3dreconstructionresolutionlifecells(nodynamics)limitsofresolution(wavelengthoflight)samplepreparation,preparationartifacts(fixation,Abspecificity)deadcells!bleaching,Applications-dyesOrganelle-specificpHmembranepotentialionselective.(MolecularProbes),Microscopicanalysisofyeastorganellesinvivo,mitochondria(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,Cholesteroldistributionin3T3cells(fillipin),Pictures:W.FGraier,MBCtime-resolvedFMFRAPfluorescencerecoveryafterphotobleachingFRETfluorescenceresonanceenergytransfer,FluorescenceMicroscopy2PhotonExcitationMicroscopy,1Photon,2Photon,FluorescenceMicroscopyTime-resolvedfluorescencemicroscopy,f,l,u,o,r,e,s,c,e,n,c,e,time(nsec),dye1(e.g.background),dye2,timewindow,FluorescenceMicroscopy,FRAP,FRET(Cameleon),ER-tagged-Cameleons,Mi-tagged-Cameleons,Pictures:W.FGraier,MBCe.g.60 x60nm),TheConfocalPrinciple,focalspot,Picture:S.Kohlwein,B&FB,Graz,Austria,YeastLightMicroscopy,Microfluorometry,Pictures:W.FGraier,MBC&MB,Graz,Austria,Microfluorometry,Pictures:W.FGraier,MBC&MB,Graz,Austria,Microfluorometry:SimultaneouslyrecordingsofCa2+andioncurrents,Pictures:W.FGraier,MBC&MB,Graz,Austria,FluorescenceImaging,Deconvolutionmicroscopy,Pictures:W.FGraier,MBC&MB,Graz,Austria,Pointspreadfunction,Focus,Out-of-focusfluorescence,3Dreconstruction,pixel(x-yplane).voxel(x-y-zplane),2Dreconstruction,Deconvolutionmicroscopyallowstimeresolvedtwodimensionalfluorescencerecordingsinhighx-yresolutionandapp.200to300mthickslices(pixel),ConfocalvsDeconvolution,Out-of-focuslightSignaltonoiseratioSeriallines(timescan)ImageacquisitionImagingqualityThicksamplesExcitat