PUBLICATION

Minimal resin embedding of multicellular specimens for targeted FIB-SEM imaging

Authors
Schieber, N.L., Machado, P., Markert, S.M., Stigloher, C., Schwab, Y., Steyer, A.M.
ID
ZDB-PUB-170523-11
Date
2017
Source
Methods in cell biology   140: 69-83 (Chapter)
Registered Authors
Stigloher, Christian
Keywords
Automated serial imaging, C. elegans, Platynereis, Sample preparation, Slice and view, Volume electron microscopy, Zebrafish
MeSH Terms
  • Animals
  • Caenorhabditis elegans/ultrastructure
  • Embryo, Nonmammalian/metabolism
  • Embryo, Nonmammalian/ultrastructure
  • Larva/ultrastructure
  • Microscopy, Electron, Scanning/methods*
  • Polychaeta/ultrastructure
  • Polymerization
  • Resins, Synthetic/chemistry*
  • Tissue Embedding/methods*
  • Zebrafish/embryology
PubMed
28528642 Full text @ Meth. Cell. Biol.
Abstract
Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy. We present here a simple workflow where multicellular specimens are embedded in minimal amounts of resin, exposing their surface topology that can be imaged by scanning electron microscopy (SEM). LM and SEM both benefit from a large field of view that can cover whole model organisms. As a result, targeting specific anatomic locations by focused ion beam-SEM (FIB-SEM) tomography becomes straightforward. We illustrate this application on three different model organisms, used in our laboratory: the zebrafish embryo Danio rerio, the marine worm Platynereis dumerilii, and the dauer larva of the nematode Caenorhabditis elegans. Here we focus on the experimental steps to reduce the amount of resin covering the samples and to image the specimens inside an FIB-SEM. We expect this approach to have widespread applications for volume electron microscopy on multiple model organisms.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping