3-DultrastructuralimagingofroutinepathologysamplesbyarraytomographyusingtheASHserialsectioningdeviceandfieldemissionscanningelectronmicroscopy(FESEM)

MurrayC.KillingsworthPhD1-4andTzipiCohenHyamsPhD1-3

1InghamInstituteforAppliedMedicalResearch2UniversityofNewSouthWales,Sydney3WesternSydneyUniversity4NewSouthWalesHealthPathology

Abstract:

Ultrastructuralexaminationofhumanrenalbiopsytissueisimportantfortheprovisionofmorphologicaldataeithertoestablishadiagnosisortocomplementothercontributorytestsincludingimmunofluorescencemicroscopyandhistology.3-Dmicroscopytodatehasnotplayedasignificantroleinrenalbiopsyassessmentprobablyduetotechnicaldifficultiesinobtainingserialsectionsforarraytomographyortherequirementforadedicatedandexpendablespecimenforserialblockfaceimaging.Hereweshowthegenerationof3-Dultrastructuralimagesfrommouserenaltissueprocessedroutinelytoepoxyresinasforexaminationbytransmissionelectronmicroscopy.Serialsections200nmthickwerecutusinganultramicrotomefittedwiththeASHserialsectioningdeviceanda“jumbo”diamondknife.Ribbonsofserialsectionsspanningadepthofapproximately18microns,orthethicknessoftwocells,werethenattachedtoanITOcoverslip.ThesectionswereimagedusingafieldemissionscanningelectronmicroscopefittedwithaBSDdetectorandanautomatedscangeneratorat25nmperpixelresolution.Eachsectionimagewasthenhand-segmentedwithfinal3-DrenderingdoneusingaPCworkstationrunningcommercialsoftware.3-Dvisualisationofglomerularultrastructureisusefulforglobalassessmentofchangesincellularity.Importantly,withthistechniquenoultrastructuraldataislostfromthesample.Oncesectionsarecollectedandmountedonthesubstrateitispossibletogobackandre-imageanysectionusedinthevolumeseries,oralternativelyimageother3-Dvolumesasrequired.

Methods:

Thetissueusedhereforarraytomography(Ref:1)wasprocessedroutinelyasfortransmissionelectronmicroscopy(TEM)examination.Brieflythisincludedfixationin2.5%glutaraldehydein0.1MsodiumcacodylatebufferpH7.4,osmication,uranylacetatestaining,dehydrationandembeddinginSpurrresin.Serialsections200nmthickwerecutfromablockfacewithareaofapproximately1.0mm2usinganPowertomePCultramicrotomefittedwiththeASHserialsectioningdevice(RMCBoeckeler,USA)anda3.0mmdiamondknifefittedwitha“jumbo”waterbath(Diatome,Switzerland).AdhesivemadefromKwik-GripTMglue(Selleys,USA)diluted1:1withxylenewasplacedalongtheloweredgeoftheblocktoallowsectionstosticktogethertoformribbons.

TheASHsystemallowedprecisemanipulationoftheITOcoverslipintothewaterbathanditspositioningclosetothecuttingedgeoftheknife.Thestableholdingofthecoverslipinthispositionallowstheoperatorthentousebothhandsforsectioning.Ribbonsof10-12serialsectionswereobtainedandthenattachedtotheITOcoatedglasscoverslip(RMCBoeckeler,USA)atthewatermeniscus.OncetherequirednumberofsectionswereobtainedtheASHsystemwasusedtopreciselyliftthecoverslipoutofthewatertostartthedryingprocess.Sectionswereair-driedonly.

ImagingacquisitionfromtheserialsectionswasthencarriedoutusingaGeminiSEM300fieldemissionscanningelectronmicroscope(FESEM)fittedwithanAtlas5.1scangeneratorsystem(CarlZeiss,Germany).Aninitiallowmagnificationsetofimageswasacquiredtoprovideanoverviewofthekidneycortexcomprisingglomeruliandtubuleswithimageresolutionof25nmperpixel.Eachsectionimagewasmadeupof63individualimagesautomaticallytiledtogetherbytheAtlassystem.Thisoperationfor90sectionstook5daysor120hoursandresultedinafinalfilesizeof51GB.Then,high-resolutionscanningofaglomeruluswasperformedataresolutionof6nmperpixelwithanimagemadeof30tilesandtotalfilesizeof24GBandscanninglengthof3days.Alignment,imagesegmentationand3-DrenderingwasdonetohighlighttheglomerularbasementmembraneinasingleglomerulussurroundedbyBowman’scapsule.SegmentationwasdonebyhandonsequentialsectionimageswithvisualisationbyAmira6.5.0,(ThermoFisher,USA).

Results:

Proof-of-principle3-Dultrastructuralimaginghasbeenobtainedfromroutinelyprocessedmouserenaltissue.UseoftheASHsystemfacilitatedalignmentandsteadymountingofanITOcoverslipwithin20mmofthediamondknifecuttingedge.Thiswasfoundtobeimportanttominimisetheworkingdistancethroughwhichcutsectionshadtobemanipulatedbefore“pinning”tothedrycoverslipsurfacebeyondthewatermeniscus.TheASHserialsectioningdeviceallowedefficientproductionofuptoseveralhundredserialsectionsfroma1mm2blockfacethatwerewellsuitedtoarraytomography.Pinningribbonsofupto12-14sectionstoITOcoverslipsallowedapproximately100sectionspercoverslip.

ImageacquisitionusinganautomatedscangenerationsystemcoupledwithaFESEMwaslargelyhandsfreebutdidrequireoccasionalinterventiontoresetfocusandimagecontrast.Resolutionof25nmperpixelwasfoundtobesuitableformid-resolutionultrastructuralcontextand6nmperpixelwasusedfordetailedexaminationsuchasrequiredforcellorganelles.

3-Dultrastructuralimagingisusefulforvisualisingthespatialrelationshipbetweenpodocytes,theglomerularbasementmembraneandoverallglomerulardimensionsasindicatedbyBowman’scapsule.Thiswillbeausefulmeansofassessingglobalglomerularshrinkageasoccursinfocalandsegmentalglomerulosclerosisandotherchangesthatresultincelldeletionorreplication.

Figure1:TheASHserialsectioningdeviceattachedtotheultramicrotomeallowsprecisemanipulationoftheITOcoverslipintheknifewaterbath.A)ASHmountedonarailinfrontoftheultramicrotomeB)ITOcoverslipinwaterbathC)cutsectionsatdiamondknifeedgeD)serialsections“pinned”toITOcoverslip.

Figure2:Automatedimageacquisitionfrom90serialsectionsusingtheAtlas5.1scangeneratorcoupledtotheGeminiSEM300FESEM.

Figure3:3-Dtomogramsproducedbyarraytomographyusingserialsectionsfromasampleofmouserenalcortex.Bowman’scapsuleoutliningaglomerulus(green),adjacenttubules(yellow),theglomerularbasementmembrane(magenta,blue)andglomerularcells(blue)withabackgroundBSD“TEM-like”imagefromthefinallayer.

Conclusion:

Cuttingserialsectionsforarraytomographyrequiresstableconditionsparticularlyinthediamondknifewaterbathtoenablesectionribbonstobemanipulatedontosupportingsubstrates.Alignment,positioningandsteadyholdingofanITOcoverslipbytheASHserialsectioningdeviceenabledcollectionof90serialsectionsfromanepoxyresinblockfor3-Dimagereconstruction.Ultrastructuralimagingofaselectedregionofinterest(ROI)ineachserialsectionwascentredonaglomerulusfromaspecimenofmousekidneycortex.ImageacquisitionofTEM-likeimagesbyBSDwasdoneusingautomaticscangenerationandtilinginaFESEMwiththearrayof90sectionimages,eachmadeupofmultiple“tiles”,taking5days.Currentlimitationsofthetechniqueare(i)manualsegmentation/annotationofultrastructuralfeatureswithintheimagedROIisgenerallyrequiredtodiscriminatestructureswithsimilargreylevels(ii)reconstructionsfrommorethan100sectionimagesrequiresignificantcomputerprocessingpowerandmemory.

References:

1. MichevaKD,SmithSJ.Arraytomography:anewtoolforimagingthemoleculararchitectureandultrastructureofneuralcircuits.Neuron(2007),55(1):25-36

2. CollanYetal.Valueofelectronmicroscopyinkidneybiopsydiagnosis.UltrastructuralPathology(2005),29(6):461-8

Abouttheauthors:

AssociateProfessorMurrayC.Killingsworth,PhD,FRMS,FFSc(RCPA)

MurrayKillingsworthisHeadoftheElectronMicroscopyLaboratory,NewSouthWalesHealthPathology,Liverpool,Sydney.HeisaConjointAssociateProfessoroftheSouthWesternSydneyClinicalSchool,UniversityofNewSouthWalesandtheSchoolofMedicine,WesternSydneyUniversity.Hisresearchinterestsarefocusedonthecellbiologyofchronicinflammatoryprocessesinrenaldisease,retinaldiseaseandcancer.In2010MurraywasawardedaFoundingFellowshipoftheFacultyofScienceintheRoyalCollegeofPathologistsofAustralasia.Hehaspioneeredtheuseofnanoparticlesincorrelativelightandelectronmicroscopy(CLEM)studiesofcellfunctioninhumanpathologytissue.HeiscurrentlyClinicalSciencesStreamLeaderandHeadofthenewCorrelativeMicroscopyFacilityattheInghamInstituteforAppliedMedicalResearch,SydneyAustralia.

DrTzipiCohenHyamsPhD

TzipiistheMicroscopyfacilitymanagerattheInghamInstitute.ShecompletedherPhDinMaterialsSci.andEng.attheTechnion,Israel.ShethencontinuedwithpostdoctoralresearchatUCBerkeley(FulbrightFellowship)specialisinginin-situRamanspectroscopy.Afterfinishingthepostdocfellowship,shewasappointedasastaffscientistattheNanotechnologyResearchandDevelopmentInstituteinIsrael.DuringherstudiesandworkexperienceTzipihasgainedmanyyearsofexperienceinadvancedmicrostructuralcharacterisation,includingFIBexpertisewithnanopatterningandfailureanalysisprocesses.ShealsogainedextensiveTEMsamplepreparationexperienceinareasincludingchemicalanalysis.Inaddition,Tzipihasguidedgraduateandundergraduatestudentsprovidingtrainingwithavarietyofcharacterisationtools.Shehasdevelopednewmicroscopy

applicationsandcollaboratedwithdifferentlocalandinternationalresearchgroupsresultinginvariouspublicationsandtalks.

Otherimages:

Image:MCKandTCH