Introduction

Electron probe microanalysis is a sensitive tool to localise elements in biological cells and tissues. In comparison to most specimens studied by this method in materials sciences, cells are not static objects, but vary in their elemental composition, depending on their functional state. Therefore, intracellular components, in particular diffusible ions, have to be immobilised in the defined functional state to be investigated. Rapid freezing, also called cryofixation, is the most promising approach to meet this requirement. This chapter points to the crucial role of appropriate freezing techniques for biological electron probe microanalysis. This is in particular important for X-ray microanalytical studies of the following biological features: (1) intracellular element compartmentation, (2) cell viability and membrane damage, (3) ion transport systems and (4) ion shifts related to dynamic processes in cells.

Preparation paths for electron probe microanalysis

Cells and tissues to be studied in an electron microscope have to be converted into a solid state specimen which is compatible with vacuum. Chemical preparation methods established for morphological investigations are based on fixation with aldehydes, staining by heavy metal salts, dehydration by alcohol and embedding in resin. Thereby, diffusible substances such as electrolyte ions are re-distributed and washed out (Zierold & Schäfer, 1988). As an alternative, low temperature preparation protocols as sketched in Fig. 7.1 were developed. They all start with cryofixation or with specimen sampling which means the appropriate handling of the specimen before rapid freezing to ensure the arrest of the functional state of interest.