简介：Objective:Thepresentstudyaimedtoevaluatethepossibilityofusingcoherentanti-StokesRamanspectroscopy（CARS）microscopytodeterminethespecificmolecularmorphologyofcholesteatomabydetectingthenaturalvibrationalcontrastofthechemicalbondswithoutanystaining.Materialsandmethods:SpecimensfromthemastoidandtympanicmembranewithandwithoutcholesteatomawereanalyzedusingCARSmicroscopy,two-photonexcitedfluorescence（TPEF）microscopy,andthesecondharmonicgeneration（SHG）microscopy.Results:Incholesteatomatissuesfromthemastoid,astrongresonantsignalat2845cm-1wasobservedbyCARS,whichindicatedthedetectionoftheCH2hydro-carbonlipidbondsthatdonotgeneratevisiblesignalsat2940cm-1suggestiveofCH3bondsinaminoacids.Astrongresonantsignalat2940cm-1appearedinanareaofthesamespecimen,whichalsogeneratedabundantsignalsbyTPEFandSHGmicroscopyat817nm,whichwassuggestiveofcollagen.Inthetympanicmembranespecimenwithcholesteatoma,astrongresonantsignalwithcorrugatedmorphologywasdetected,whichindicatedthepresenceoflipids.AstrongsignalwasdetectedinthetympanicmembranewithchronicotitismediausingTPEF/SHGat817nm,whichindicatedcollagenenrichment.TheCARSandTPEF/SHGimageswereinaccordancewiththehistologyresults.Conclusion:TheseresultssuggesttheneedtodevelopanovelCARSmicroendoscopethatcanbeusedincombinationwithTPEF/SHGtodistinguishcholesteatomafrominflammatorytissues.