Electron microscopy of the early Caenorhabditis elegans embryo

Abstract

SummaryThe early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser-induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time-resolved fixation to arrest development at specific stages. The second method uses high-pressure freezing of whole worms followed by freeze-substitution (HPF-FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high-resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF-FS and electron tomography. This procedure combines the advantages of time-resolved fixation and superior ultrastructural preservation by high-pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.

Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy

“…While several approaches and machines have been devised to cryofix satisfactorily the samples (Hawes et al 2007;Hess 2003;Hess et al 2000;Hohenberg et al 1994Hohenberg et al , 1996Jiménez et al 2006;Kiss et al 1990;Lancelle and Hepler 1989;Marsh et al 2001;McDonald 1999;Mims et al 2003;Müller and Moor 1984;Müller-Reichert et al 2008;Neuhaus et al 1998;Reipert et al 2004;Studer et al 1989;Wild et al 2005), the definition of FS protocols which would be optimal for immunolabeling has not been critically evaluated. It is necessary to mention here that cryoimmobilized and freeze-substituted cells are embedded into acrylic resins which are better suited for immunocytochemical studies (Acetarin et al 1986;Roth 1989;Roth and Taatjes 1998;Roth et al 1981).…”

Ultrastructural analysis of Caenorhabditis elegans cilia

“…It can also be used for larvae and adults. Other protocols for embryo fixation are described in (Müller-Reichert, Mäntler, Srayko, & O'Toole, 2008;Sims & Hardin, 2007).…”

A method for preserving ultrastructural properties of mitotic cells for subsequent immunogold labeling using low-temperature embedding in LR White resin

“…The cryomethods for electron microscopy specimen preparation definitely provide the safest way for the observation of cells in close-to-native state, and they have undoubtedly overcome the conventional method of chemical fixation (Maupin and Pollard 1983;Murk et al 2003;Sawaguchi et al 2002;Szczesny et al 1996). Currently, the best available method for cryofixation is high-pressure freezing (HPF; McDonald and Morphew 1993;Müller-Reichert et al 2008;Studer et al 1995;Verkade 2008) that immobilizes whole cells or tissue fragments with a thickness up to a few hundred micrometers within milliseconds (Müller and Moor 1984) without ice crystal formation during freezing, i.e. by vitrification (McEwen et al 1998).…”