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1、MOBILITYMEASUREMENTBYANALYSISOFFLUORESCENCEPHOTOBLEACHINGRECOVERYKINETICSD.AXELROD,D.E.KOPPEL,J.SCHLESSINGER,E.ELSON,andw.W.WEBBFromtheSchoolofAppliedandEngineeringPhysics,andDepartmentofChemistry,CornellUniversity,Ithaca,NewYork14853ABSTRACTFluorescencephotob
2、leachingrecovery(FPR)denotesamethodformea-suringtwo-dimensionallateralmobilityoffluorescentparticles,forexample,themotionoffluorescentlylabeledmoleculesin10jm2regionsofasinglecellsurface.Asmallspotonthefluorescentsurfaceisphotobleachedbyabriefexposuretoanin-te
3、nsefocusedlaserbeam,andthesubsequentrecoveryofthefluorescenceismoni-toredbythesame,butattenuated,laserbeam.Recoveryoccursbyreplenishmentofintactfluorophoreinthebleachedspotbylateraltransportfromthesurroundingsur-face.Wepresentthetheoreticalbasisandsomepractica
4、lguidelinesforsimple,rig-orousanalysisofFPRexperiments.InformationobtainablefromFPRexperimentsincludes:(a)identificationoftransportprocesstype,i.e.theadmixtureofrandomdiffusionanduniformdirectedflow;(b)determinationoftheabsolutemobilitycoeffi-cient,i.e.thediff
5、usionconstantand/orflowvelocity;and(c)thefractionoftotalfluorophorewhichismobile.Toillustratetheexperimentalmethodandtoverifythetheoryfordiffusion,wedescribesomemodelexperimentsonaqueoussolutionsofrhodamine6G.INTRODUCTIONThispaperdescribesexperimentalandtheore
6、ticalaspectsofarecentlydevelopedmethod,Fluorescencephotobleachingrecovery(FPR),formeasuringtransportoffluorescentmoleculesinsmallsystemssuchasindividuallivingcells.Thisworkwaspromptedbyourdesiretomeasureratesoflateraltransportofproteinsandlipidsincellmembranes
7、.Thereiscurrentlywidespreadinterestinthisproblembecauseoftheapparentroleoflateralmembranetransportasanindicatororeffectorofchangesinthephysiologicalstatesofcells(1-4).Inadditiontomeasurementsoftransportonmembranes,FPRprovidesabroadcapabilityforavarietyofothera
8、pplications.Thenewmethodissimplebothinconceptandinpractice.Asmallregionofasur-facecontainingmobilefluorescentmoleculesisexposedtoabriefintensepulseoflight(perhapsfocusedonthesurfac
