Fluorochromesativ123-13536-00Rev.01第一页，共三十五页。Fluorochromes•Smallorganicmoleculesexamples:FITC,BDHorizonTMV450,Cy7•Fluorescentproteinsexamples:PE,APC,PerCP•Tandemdyes—typically,thecouplingofafluorescentproteindonorwithasmallorganicmoleculeacceptorexamples:PE-Cy7,PerCP-CyTM5.5•Nanocrystals(Qdots)—inorganicsemiconductorsexamples:Qdot565,Qdot605223-13536-00Rev.01第二页，共三十五页。FluorochromesFluorochromeTypeofmoleculeFluoresceinisothyocyanate(FITC)SmallorganicAlexaFluor®488SmallorganicPhycoerythrin(PE)ProteinPE-CyTM5ProteintandemPeridininchlorophyllprotein(PerCP)ProteinPerCP-CyTM5.5ProteintandemPE-CyTM7ProteintandemAllophycocyanin(APC)ProteinAlexaFluor®647SmallorganicAPC-CyTM7ProteintandemBDTMAPC-H7ProteintandemBDHorizonTMV450SmallorganicPacificBlueTMSmallorganicBDHorizonTMV500SmallorganicAmCyanProtein323-13536-00Rev.01第三页，共三十五页。FluorochromesAdvantages–Lowmolecularweight–Easytoconjugate—directattachmenttofreeaminogroupsonmAb•Excellentstability•ExtremelyconsistentemissionspectraDisadvantages•SmallStokesShift(50−100nm)•Tendtobelessbright423-13536-00Rev.01第四页，共三十五页。FluorochromesAdvantages–Goodstability–Consistentemissionspectra–MediumStokesShift(75−200nm)•TendtobemorebrightDisadvantages•Highmolecularweight•Moredifficulttoconjugate—intermediariesneededtoattachtomAb523-13536-00Rev.01第五页，共三十五页。FluorochromesAdvantages–VerylargeStokesShift(150−300nm)•Tendtobeverybright—oftenbrighterthanthefluorescentproteindonorDisadvantages•Highmolecularweight(similartofluorescentprotein)•Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)•Hardertoconjugate(sameasfluorescentprotein)•Sometandemshavepoorstability623-13536-00Rev.01第六页，共三十五页。FluorochromesAdvantages–LargeStokesShift(100−500nm)•Tendtobeverybright•Emissionpeaksareconsistentandnarrow,anddonotchangewithvariationsintheexcitationsource•Highlyresistanttophotobleaching•NanocrystalssharebiophysicalandconjugationpropertiesDisadvanta...
