庄小威超分辨率显微成像PPT

Super-resolutionfluorescenceimagingwithSTORMXiaoweiZhuang,HarvardUniversity,HHMIUSTCimagingcourse,2011Bio-imagingatvariouslengthsscales1m1mm1µm1nmPETMRICTEM,SPMX-raycrystallography,NMR,EM1Å10nm100nm10µm100µm10mm100mmLightMicroscopyLightmicroscopyAntonievanLeeuwenhoek(1632-1723)Lightmicroscopy1µm1nm10nm100nm10µm100µmAlbertsetal,MolecularBiologyoftheCellLMLightmicroscopy1µm1nm10nm100nm10µm100µmAlbertsetal,MolecularBiologyoftheCell~5nmLMDiffraction-limitedresolutionDiffraction-limitedresolutiond=200nmd=250nmd=350nmd=450nmd=550nmd=650nmd=750nmAbberesolution(diffraction)Limit:Afewhundrednanometers~Wavelength/2NAResolutionoflightmicroscopyAbberesolution(diffraction)Limit:AfewhundrednanometersToresolvemolecules:AfewnanometersDiscrepancy:TwoordersofmagnitudeResolutionoflightmicroscopySub-diffractionlimitimagingStimulatedEmissionDepletionMicroscopy–STEDReversiblesaturableopticalfluorescencetransitionmicroscopy–RESOLFT(Saturated)StructuredIlluminationMicroscopy–(S)SIMKlar&Hell,OptLett(1999)Hell,Science(2007)Gustafsson,PNAS(2005)Patterned  illumination  based  approaches  Sub-diffractionlimitimagingSingle-­‐molecule-­‐localization  based  approach    StochasticOpticalReconstructionMicroscopy--STORM(Fluorescence)PhotoactivationLocalizationmicroscopy--(F)PALMRustetal,NatMethods(2006)--STORMHessetal,Biophys.J(2006)--FPALMBetzigetal,Science(2006)--PALMSingle-moleculelocalizationGellesetal,Nature(1988)Thompsonetal,BiophysJ(2002)Yildiz,Selvinetal,Science(2003)σ≈σPSF/N1/2Yildizetal,Science(2003)Molecularmotor(MyosinV)Rustetal,NatMethods(2006)--STORMSTochasticOpticalReconstructionMicroscopyHessetal,Biophys.J(2006)--FPALMBetzigetal,Science(2006)--PALMσ≈σPSF/N1/245nmSTORMimageConventionalimageRust,Batesetal,NatMethods(2006)STochasticOpticalReconstructionMicroscopy45nmSTORMimageConventionalimageRust,Batesetal,NatMethods(2006)STochasticOpticalReconstructionMicroscopy45nmSTORMimageConventionalimageRust,Batesetal,NatMethods(2006)RecASTochasticOpticalReconstructionMicroscopyConventionalSTORMBatesetal,Science(2007)Photo-switchablecyaninedyesCy5RedlightUVlightRedUVBatesetal,PRL(2005)Dempseyetal,JACS(2009)Photo-switchablecyaninedyesCy5RedlightUVlightDempseyetal,JACS(2009)+DarkstateRedUVPhoto-switchablecyaninedyesCy5RedlightUVlightDempseyetal,JACS(2009)+DarkstateRed+DarkstateRed5µmSingleredlaserCommercialAlexa647(Cy5analog)labeledantibodyZhuang,NaturePhoton.(2009)ConventionalSTORM500nmPhoto-switchabledyesBatesetal,Science(2007)BluelightactivationRedlightexcitationGreenlightactivationvioletlightactivationCy5Cy5Cy5Cy2Cy3Alexa405█Clathrin█MicrotubuleBatesetal,Science(2007)Batesetal,Science(2007)█Clathrin█MicrotubuleBatesetal,Science(2007)Batesetal,Science(2007)Photo-switchabledyesRedlightexcitationNearIRlightexcitationGreenexcitationCy5Cy7Atto488Dempsey,Vaughan,Chenetal,NatMethods(2011)MitochondriaTubulinAcetylatedTubulinEndoplasmicreticulumDempsey,Vaughan,Chenetal,NatMethods(2011)3DSTORMEMCCD2γ(x,y,z)4002000-200-400z(nm)Huangetal,Science(2008)Infocus8006004002000z(nm)5µmHuangetal,Science(2008)3DSTORMHuangetal,Science(2008)2DSTORM3DSTORMHuangetal,Science(2008)100nmyxLocalizationprecision(SD):xy~8nm;z~20nmResolution(FWHM):xy20nm;z50nm2DSTORMzxyxClathrinFBP17200nmDynaminFBP17Wuetal,NatCellBiol(2010)EMSTORMSTORMHuang,Jonesetal,Nat.Meth.(2008)3µmEndoplasmicReticulum350nmZActinLocalizationprecision(SD):xy~4nm;z~8nmResolution(FWHM):xy10nm;z20nm-200nm2µmSTORMventrallayerSTORMdorsallayerConventional200nmx,yKeetal,NatMethods(inpress)350nmZActinLocalizationprecision(SD):xy~4nm;z~8nmResolution(FWHM):xy10nm;z20nm-200nm2µmSTORMventrallayerSTORMdorsallayerConventional200nmx,yVentrallayerDorsallayerKeetal,NatMethods(inpress)ClathrinTransferrinLive-cell3DSTORMzyxxzyxxPit3Pit4100nmzyxxzyxx100nmPit1Pit2Jones,Shimetal,NatMethods(2011)Nyquistresolution:~20nmTimeresolution:~1secFramerate:500Hz–1kHzAlexa647-clathrinAlexa647-clathrinAlexa568-transferrinNEBLabelinglivecellswithphoto-switchabledyesSNAP,CLIP,HALO,Peptidetags…Cy5/Alexa647~6000photons/cycleEosFP~1000photons/cycleclathrinclathrinLivecellimagingwithdifferentprobesAlexa647mEos2tdEosAtto655OregongreenTMRFluorescentproteinsCellpermeabledyesJones,Shimetal,NatMethods(2011)“HerewedemonstratetheconceptofSTORMusingthecyanineswitch,butinprincipleanysuitableopticallyswitchedfluorophorecouldbeused.”TheSTORMconceptisalsoapplicabletootherphotoswitchablefluorophoresandfluorescentproteins.”Rust,Bates,&Zhuang,NatMethods(2006)Post-activationex(nm)Post-activationem(nm)ReferencesPhotoactivatible–bluebsDronpa460504(Stieletal.,2007)Photoactivatible–green~495~515PA-GFP504517(PattersonandLippincott-Schwartz,2002)PS-CFP2490511(Chudakovetal.,2004)DronpaEYFP503515518527(Habuchietal.,2005)(Dicksonetal,1996;Biteenetal,2008)Photoactivatible–red~560~590PAmCherry564595(Subachetal.,2009)PAtagRFP562595(Subachetal.,2010)Photoconvertiblegreen-to-red550-570570-590tdEos/mEos2573584(Wiedenmannetal,2004;McKinneyetal.,2009)Dendra2553573(Gurskayaetal.,2006)Redcyaninedyes650-750670-800(Batesetal.,2007)Cy5orAlexa647Cy7647750665770(Bateset