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

9
3-D ultrastructural imaging of routine pathology samples by array tomography using the ASH serial sectioning device and field emission scanning electron microscopy (FESEM) Murray C. Killingsworth PhD 1-4 and Tzipi Cohen Hyams PhD 1-3 1 Ingham Institute for Applied Medical Research 2 University of New South Wales, Sydney 3 Western Sydney University 4 New South Wales Health Pathology Abstract: Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either to establish a diagnosis or to complement other contributory tests including immunofluorescence microscopy and histology. 3-D microscopy to date has not played a significant role in renal biopsy assessment probably due to technical difficulties in obtaining serial sections for array tomography or the requirement for a dedicated and expendable specimen for serial block face imaging. Here we show the generation of 3-D ultrastructural images from mouse renal tissue processed routinely to epoxy resin as for examination by transmission electron microscopy. Serial sections 200 nm thick were cut using an ultramicrotome fitted with the ASH serial sectioning device and a “jumbo” diamond knife. Ribbons of serial sections spanning a depth of approximately 18 microns, or the thickness of two cells, were then attached to an ITO coverslip. The sections were imaged using a field emission scanning electron microscope fitted with a BSD detector and an automated scan generator at 25 nm per pixel resolution. Each section image was then hand- segmented with final 3-D rendering done using a PC workstation running commercial software. 3-D visualisation of glomerular ultrastructure is useful for global assessment of changes in cellularity. Importantly, with this technique no ultrastructural data is lost from the sample. Once sections are collected and mounted on the substrate it is possible to go back and re-image any section used in the volume series, or alternatively image other 3-D volumes as required.

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

Page 1: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

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.

Page 2: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

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).

Page 3: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

Results:

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

ImageacquisitionusinganautomatedscangenerationsystemcoupledwithaFESEMwaslargelyhandsfreebutdidrequireoccasionalinterventiontoresetfocusandimagecontrast.Resolutionof25nmperpixelwasfoundtobesuitableformid-resolutionultrastructuralcontextand6nmperpixelwasusedfordetailedexaminationsuchasrequiredforcellorganelles.

3-Dultrastructuralimagingisusefulforvisualisingthespatialrelationshipbetweenpodocytes,theglomerularbasementmembraneandoverallglomerulardimensionsasindicatedbyBowman’scapsule.Thiswillbeausefulmeansofassessingglobalglomerularshrinkageasoccursinfocalandsegmentalglomerulosclerosisandotherchangesthatresultincelldeletionorreplication.

Page 4: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

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

Page 5: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

Figure2:Automatedimageacquisitionfrom90serialsectionsusingtheAtlas5.1scangeneratorcoupledtotheGeminiSEM300FESEM.

Page 6: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

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.

Page 7: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

References:

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

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

Page 8: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

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

Page 9: 3-D ultrastructural imaging of routine pathology samples by ......Ultrastructural examination of human renal biopsy tissue is important for the provision of morphological data either

applicationsandcollaboratedwithdifferentlocalandinternationalresearchgroupsresultinginvariouspublicationsandtalks.

Otherimages:

Image:MCKandTCH