3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of...
Transcript of 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of...
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