SamplepreparationforScanningelectronmicroscopy(SEM)
SEMisprimarilyusefulforgivingathree‐dimensionalimageofthesurfaceofthe
specimenandisforviewinglargeobjects.
AswithTEM,specimensareimagedwithabeamofelectrons,butinsteadofthe
electronsbeingtransmittedthroughthespecimen,thebeamis"scanned"across,
creatinganimageofthesurfaceofthesample,withexceptionaldepthoffield.This
imageisachievedviathedetectionof"secondary"electronsthatarereleasedfromthe
specimenasaresultofitbeingscannedbyveryhighenergy"primary"electrons(ie.
thoseemittedfromtheelectron"gun"intheSEM).Asmostbiologicalspecimensare
madeupofnon‐densematerialtheamountofsecondaryelectronsproducedistoolow
tobeofmuchuseincreatinganimageandthereforetheyareusuallycoatedwithavery
finelayerofametalwhichreadilyproducessecondaryelectrons.Thelargedepthoffield
achievablecanproduceanimageofgreatvisualdepthwithathree‐dimensional
appearance.
Theoperatingenvironmentofastandardscanningelectronmicroscopedictatesthat
specialistpreparationtechniquesareused.Typically,abiologicalspecimenis
chemicallyfixed,dehydratedthroughanacetoneorethanolseriesandthendriedatthe
criticalpoint‐amethodusedtominimizespecimendistortionduetodryingtensions.
Fordrysamples,thisprocessisnotnecessary.SEMcanalsobeusedtoinvestigate
smoothsurfacesofindustrialsamples.
Thesamplesaremountedonastubofmetalwithadhesive,coatedwith40‐60nmof
metalsuchasGold/Palladiumandthenobservedinthemicroscope.
Everysampleisdifferent.PleaseconsultwiththeEMStaffbeforestartingaproject.
Epidermoidcarcinomacells.
Bar:2µm
Side1
Epidermoidcarcinoma
cells.Bar:2µm
StaphylococcusAureus.
Bar:2µm
Smallintestine,villi.
Bar:200µm.
Smallintestine,microvilli Plastinatedglomerulusfrom
onthesurfaceofavillus. rat.Tissueisremoved.
Bar:50µm.
Fly
Bar:200µm.
Spider
Bar:300µm.
Hair–onthesurface.
Bar:10µm.
SEMservicesinclude:
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Fixationanddehydration
Criticalpointdrying
Dryingwithhexamethyldisilazane(HMDS)andt‐Butanol
CoatingwithGold/PalladiumusingSputtercoater
Imageprocessing(softwareScandium)
Side2