Designation:F2998−14GuideforUsingFluorescenceMicroscopytoQuantifytheSpreadAreaofFixedCells1ThisstandardisissuedunderthefixeddesignationF2998;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginaladoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscriptepsilon(´)indicatesaneditorialchangesincethelastrevisionorreapproval.1.Scope1.1Thisguidedescribesseveralmeasurementandtechnicalissuesinvolvedinquantifyingthespreadareaoffixedcells.Cellspreadingandthedistributionofcellspreadareasofapopulationofcellsaretheresultofabiologicalresponsethatisdependentonintracellularsignalingmechanismsandthecharacteristicsofcelladhesiontoasurface.Cellspreadareaisamorphologicalfeaturethatcanberesponsivetoalterationinthemetabolicstateorthestateofstressofthecells.Changesincellspreadareacanalsoindicateanalterationintheadhesionsubstratethatmaybeduetodifferencesinmanufacturingofthesubstratematerialorbeinresponsetoextracellularmatrixsecretions.Highqualitymeasurementofcellspreadareacanserveasausefulmetricforbenchmarkinganddetectingchangescellbehaviorunderexperimentalconditions.1.2Themeasurementdescribedinthisdocumentisbasedontheuseoffluorescencemicroscopyimagingoffixedcellsandtheuseofimageanalysisalgorithmstoextractrelevantdatafromtheimages.Toproducerobustcellspreadareameasurements,technicaldetailsinvolvedinsamplepreparation,cellstaining,microscopyimaging,imageanalysisandstatisticalanalysisshouldbeconsidered.Severaloftheseissuesarediscussedwithinthisdocument.1.3Thisstandardismeanttoserveasaguidefordevelopingmethodstoreliablymeasuretheareatowhichcellsspreadatasurface.Thissurfacecanbeconventionaltissueculturepoly-styreneorsophisticatedengineeredbiomaterialsurfaces.Anexampleofadetailedproceduretomeasurethespreadingareaofcellsonatissueculturepolystyrenesurfaceisprovidedintheappendixsection.1.4Cellmorphologyfeaturessuchascellspreadingareaandperimeteraregenerallyreportedinunitsoflength.Forexample,spreadingareapercell(thatis,ce...
