Irsen, S.

Schulz, K.

Tröger, C.

MPG.PuRe

References:[1] Christopher J. P., Lucy M. C. Micron (2014), 61, 9-19[2] Emmons, S. W. Philos Trans B (2015), 370, 20140309 – 20140309[3] Pereira, A. F., Hageman, D. J., Garbowski, T., Riedesel, C., Knothe, U., Zeidler, D., & Knothe Tate, M. L. (2016). PLoS Computational Biology, 12(11)Contact: Carola.Troeger@caesar.deVarious techniques are used to record 3D volumes of neuronal tissue using EM [1]. Especially for larger volumes, the experiment time is a limiting factor in most of these techniques [2].Recent developments of multi-beam scanning electron microscopes (mSEM) [3] reduce the experiment time dramatically.Here we present our workflow for serial sectioning of tissue for mSEMapplications. We modified an ultramicrotome for optimized collection ofserial sections on wafer pieces. With our workflow it is possible to collectup to hundreds of wrinkle- and chatter-free sections in a short time.A: Si-wafer as substrate for serial sections, 4 -5 ribbons with a 25 slices on 1cm x 1cm wafer piece are possible to handleThe advantages of conductive Si-wafers are:+ Imaging conditions without tape-related issues+ A higher packing density of the collected sections+ Rigid and flat wafer surface allows less complicated staining procedures+ Facilitates loading into various microscopesImproved serial sectioning workflow for multi-beam SEM applicationsHow to produce the ideal section for Volume EMCarola Tröger, Kathrin Schulz, Stephan IrsenElectron Microscopy and Analytics, research center caesar, Bonn, Germany0 5 10 15 20 25distance / µm2.66 2.68 2.7 2.72 2.74 2.76 2.78 2.8 2.82 height / µm 38 nmAFM Measurements (1 slice)0102030405060700 5 10 15 20 25Measured Height in nmSlice numberAFM MeasurementsM eas urem entsM eanSample Membrane contrastA: ROTO 134B: ROTO + 20 min UAC 155C: ROTO + 20 min UAC + 20 min Pb-citrate 198D: TEM contrast 180Contrast along membraneSample Membrane contrastA: ROTO 98B: ROTO + 20 min UAC 73C: ROTO + 20 min UAC + 20 min Pb-citrate 65D: TEM contrast 180Contrast along ribbon synapseEDAAB CBA DCAModification of an ultramicrotome workspace for serial sectioningC BAABEA: trimmed trapezoid with 5on both sidesAAA: heating plate for drying, at 60C less then 1min + CHCl3-flowA: spectrum profile of 1 slice, analyzed by Gwyddion-softwareB: spectrum profile of 24 slices, analyzed by Gwyddion-softwareIntroductionE:  line plot analyses, analyzed by Fiji SoftwarePost staining procedureSubstrate for serial sectioningTrimming procedureDrying procedureMethod for quality assuranceImaging with mSEMDE15µmD: zoom1 of mFOV14 E: higher magnification of DA: overview image with light microscope, specimen: chicken retinaB: overlay of mFOVs, 1 sliceC: 18 mFOVS recorded with mSEMA: optimized working place on a vibration-cushioned tableB: Si wafer positioned on special holderC: Si wafer ready for sectioning - under water surfaceD: Si wafer before sectioningE: :Si wafer after sectioning – 5 ribbons on top

Improved serial sectioning workflow for multi-beam SEM applications – How to produce the ideal section for Volume EM

Abstract

Similar works

Full text

Available Versions

MPG.PuRe