Cryométhodes en microscopie électronique à balayage en transmission (MEBT) appliquées a l'analyse cellulaire à l'échelle submicrométrique : mesure locale en eau par imagerie en champ sombre quantitative afin de déterminer des concentrations élémentaires dans l'état physiologique

Abstract

We describe a method which allows the determination of the water content at the subcellular level. This method uses the scanning transmission electron microscopy (STEM) dark field intensity of freeze-dried cryosections. The water mass content is simply deduced from the complement to 100% of the dry mass content. Freeze-dried cryosections of macromolecular solutions in known concentrations are used as standard to calibrate the relative dark field intensity versus the dry mass content. We present the different steps of the cryomethods: cryofixation, cryosection and cryoobservation after freeze-drying. We apply this method to isolated cells (erythrocytes) and cells in monolayer (osteoblasts). Moreover cryomethods and water content measurements combined with X-ray microanalysis allow to accede to elemental concentration of the biological tissues in the physiological state (expressed in mmol/L).