Designation:E1881−12StandardGuideforCellCultureAnalysiswithSIMS1ThisstandardisissuedunderthefixeddesignationE1881;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginaladoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscriptepsilon(´)indicatesaneditorialchangesincethelastrevisionorreapproval.1.Scope1.1ThisguideprovidestheSecondaryIonMassSpectrom-etry(SIMS)analystwithacryogenicmethodforanalyzingindividualtissueculturecellsgrowinginvitro.Thisguideissuitableforfrozen-hydratedandfrozen-freeze-driedsampletypes.Includedareproceduresforcorrelatingoptical,laserscanningconfocalandsecondaryelectronmicroscopiestocomplementSIMSanalysis.1.2Thisguideisnotsuitableforcellculturesthatdonotattachtothesubstrate.1.3Thisguideisnotsuitableforanyplasticembeddedcellculturespecimens.1.4Thisstandarddoesnotpurporttoaddressallofthesafetyconcerns,ifany,associatedwithitsuse.Itistheresponsibilityoftheuserofthisstandardtoestablishappro-priatesafetyandhealthpracticesanddeterminetheapplica-bilityofregulatorylimitationspriortouse.2.ReferencedDocuments2.1ASTMStandards:2E673TerminologyRelatingtoSurfaceAnalysis(Withdrawn2012)33.Terminology3.1Definitions:3.1.1SeeTerminologyE673fordefinitionsoftermsusedinSIMS.4.SummaryofGuide4.1Thisguidedescribesacryogenicfreeze-fracturemethodofsamplepreparationforcellculturespecimensforSIMSanalysis.Inbrief,cellculturesaregrownonaconductingsubstrate,suchassilicon.Whencellsreachabout80%confluency,theyarefastfrozenandfracturedbyusingasandwichmethod(1).4Thisallowsfreeze-fixationofcellularcontentsandremovaloftheEF-leafletoftheapicalplasmamembrane.Sincethiskindoffractureoccursingroupsofcellsgrowingtogether,fracturedcellsareeasilyrecognizedforoptical,SEMandSIMSimaging.4.2BycorrelativelaserscanningconfocalmicroscopyandSIMS,thesamefrozenfreeze-driedcellcanbeanalyzedfororganellelocalizationinrelationtoelementalcontent(2).5.SignificanceandUse5.1Thepresenceofcellgrowthmediumcomplicatesadirectanalysisof...
