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1、METHODSINMOLECULARBIOLOGYMETHODSINMOLECULARBIOLOGYTMTMVolume263FlowCytometryFlowCytometryProtocolsProtocolsSSECONDECONDEEDITIONDITIONEditedbyEditedbyTTeresaS.HawleyeresaS.HawleyRobertG.HawleyRobertG.Hawley1FlowCytometryAnIntroductionAliceL.GivanSummaryAflowcy
2、tometerisaninstrumentthatilluminatescells(orotherparticles)astheyflowindi-viduallyinfrontofalightsourceandthendetectsandcorrelatesthesignalsfromthosecellsthatresultfromtheillumination.Inthischapter,eachoftheaspectsofthatdefinitionwillbedescribed:thecharacteri
3、sticsofcellssuitableforflowcytometry,methodstoilluminatecells,theuseoffluidicstoguidethecellsindividuallypasttheilluminatingbeam,thetypesofsignalsemittedbythecellsandthedetectionofthosesignals,theconversionoflightsignalstodigitaldata,andtheuseofcomputerstocor
4、relateandanalyzethedataaftertheyarestoredinadatafile.Thefinalsectionofthechapterwilldiscusstheuseofaflowcytometertosortcells.Thischap-tercanbereadasabrief,self-containedsurvey.Itcanalsobereadasagatewaywithsignpostsintothefield.Otherchaptersinthisbookwillprovi
5、demoredetails,morereferences,andevensomecontroversyaboutspecifictopics.KeyWordsFlowcytometry,fluidics,fluorescence,laser.1.IntroductionAnintroductorychapteronflowcytometrymustfirstconfrontthedifficultyofdefiningaflowcytometer.TheinstrumentdescribedbyAndrewMol
6、davanin1934(1)isgenerallyacknowledgedtobeanearlyprototype.Althoughitmayneverhavebeenbuilt,indesignitlookedlikeamicroscopebutprovidedacap-illarytubeonthestagesothatcellscouldbeindividuallyilluminatedastheyflowedinsinglefileinfrontofthelightemittedthroughtheobj
7、ective.Thesignalscomingfromthecellscouldthenbeanalyzedbyaphotodetectorattachedinthepositionofthemicroscopeeyepiece.FollowingworkbyCoul-From:MethodsinMolecularBiology:FlowCytometryProtocols,2nded.Editedby:T.S.HawleyandR.G.Hawley©HumanaPressInc.,Totowa,NJ12Giva
8、nterandothersinthenextdecadestodevelopinstrumentstocountparticlesinsuspension(seerefs.2–5),adesignwasimplementedbyKamentskyandMelamedin1965and1967(6,7)toproduceamicroscope-basedflowcytome
