Use of energy-filtering transmission electron microscopy for routine ultrastructural analysis of high-pressure-frozen or chemically fixed plant cells

Abstract

In the present study energy-filtering transmission electron microscopy by use of an in-column spectrometer is employed as a powerful tool for ultrastructural analysis of plant cells. Images of unstained very thin (50 nm) and thick (140 nm) sections of the unicellular green alga Micrasterias denticulata, as a model system for a growing plant cell, taken by conventional transmission electron microscopy are compared to those obtained from filtering at zero energy loss (elastic bright field) and to those generated by energy filtering below the carbon-specific absorption edge at about 250 eV. The results show that the high-contrast images produced by the latter technique are distinctly superior in contrast and information content to micrographs taken at conventional transmission electron microscopy mode or at elastic bright field. Post- or en bloc staining with heavy metals, which is indispensable for conventional bright-field transmission electron microscopy, can be completely omitted. Delicate structural details such as membranous or filamentous connections between organelles, organelle interactions, or vesicle and vacuole contents are clearly outlined against the cytoplasmic background. Also, immunoelectron microscopic localization of macromolecules benefits from energy-filtering transmission electron microscopy by a better and more accurate assignment of antigens and structures and by facilitating the detection of immunomarkers without renunciation of contrast.

Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions

“…Cryo-substitution was performed in a Leica EM AFS (Leica Mikrosysteme GmbH, Vienna, Austria) as described by Meindl et al (1992) and Aichinger and Lütz-Meindl (2004). After freeze substitution cells were infiltrated and embedded in Agar low viscosity resin (Agar Scientific, Essex, U.K.) and polymerized for 16–24 h at 60 °C.…”

3-D analysis of dictyosomes and multivesicular bodies in the green alga Micrasterias denticulata by FIB/SEM tomography

“…Cells were embedded in epoxy resin (Agar low viscosity resin; Agar Scientific, Essex, UK) and sectioned on a Leica UC7 ultramicrotome for TEM. 60–80 nm sections were mounted on formvar coated copper grids and investigated in a LEO 912 AB Omega TEM (Zeiss, Oberkochen, Germany) at 80 kV by using zero-loss energy filtering (Lütz-Meindl and Aichinger, 2004). Sections of all samples were viewed in TEM prior to analysis by FIB/SEM tomography.…”

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

“…Treated cells and controls were cryofixed in a Leica EMPACT high-pressure freezer, underwent freeze substitution in a Leica EM AFS (Leica Microsysteme GmbH, Vienna, Austria) as described by Meindl et al (1992) and Lütz-Meindl and Aichinger (2004), and were then embedded in Agar low viscosity resin. Ultrathin sections were placed on formvar coated copper grids for ultrastructural investigation.…”