Automated Transmission-Mode Scanning Electron Microscopy (tSEM) for Large Volume Analysis at Nanoscale Resolution.docVIP

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Automated Transmission-Mode Scanning Electron Microscopy (tSEM) for Large Volume Analysis at Nanoscale Resolution.doc

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Automated Transmission-Mode Scanning Electron Microscopy (tSEM) for Large Volume Analysis at Nanoscale Resolution

AutomatedTransmission-ModeScanningElectron Microscopy(tSEM)forLargeVolumeAnalysisat NanoscaleResolution MasaakiKuwajima1,JohnM.Mendenhall1,LaurenceF.Lindsey1,KristenM.Harris1,2* 1CenterforLearningandMemory,TheUniversityofTexasatAustin,Austin,Texas,UnitedStatesofAmerica,2SectionofNeurobiology,TheUniversityofTexasatAustin, Austin,Texas,UnitedStatesofAmerica Abstract Transmission-modescanningelectronmicroscopy(tSEM)onafieldemissionSEMplatformwasdevelopedforefficientand cost-effective imaging of circuit-scale volumes from brain at nanoscale resolution. Image area was maximized while optimizingtheresolutionanddynamicrangenecessaryfordiscriminatingkeysubcellularstructures,suchassmallaxonal, dendritic and glial processes, synapses, smooth endoplasmic reticulum, vesicles, microtubules, polyribosomes, and endosomeswhicharecriticalforneuronalfunction.IndividualimagefieldsfromthetSEMsystemwereupto4,295mm (65.54mmperside)at2nmpixelsize,contrastingwithimagefieldsfromamoderntransmissionelectronmicroscope(TEM) 2 system,whichwereonly66.59mm (8.160 mperside)atthesamepixelsize.ThetSEMproducedoutstandingimagesand hadreduceddistortionanddrift relativetoTEM.AutomatedstageandscancontrolintSEMeasilyprovidedunattended serialsectionimagingandmontaging.LensandscanpropertiesonbothTEMandSEMplatformsrevealednosignificant nonlineardistortionswithinacentralfieldof,100mm2andproducednear-perfectimageregistrationacrossserialsections using the computational elastic alignment tool in Fiji/TrakEM2 software, and reliable geometric measurements from RECONSTRUCTTMorFiji/TrakEM2software.Axialresolutionlimitstheanalysisofsmallstructurescontainedwithinasection (,45nm).SincethisnewtSEMisnon-destructive,objectswithinasectioncanbeexploredatfineraxialresolutioninTEM tomographywithcurrentmethods.FuturedevelopmentoftSEMtomographypromisesthinneraxialresolutionproducing nearlyisotropicvoxelsandshouldprovidewithin-sectionanalysesofstructureswithoutchangingplatforms.Brainwasthe testsystemgivenourinterestinsynap