Small sections of leaves (1mm2) were cut with a razor blade on a modelling wax plate in a drop of 3% glutaraldehyde in 0.06M Sørensen phosphate buffer at pH 7.2. Sections were then evacuated with a water jet vacuum pump for a maximum of 10 seconds in a vial filled with the above described fixative solution.
Subsequently, the specimens were transferred into small baskets with a mesh width of about 200μm. These baskets were then stacked on top of each other and transferred into the mono-mode chamber of the Leica EM AMW which already contained a vial filled with the above mentioned fixative solution. Microwave fixation was then started about 2 minutes after the cutting of the samples by starting the previously programmed protocol.
Sample preparation for transmission electron microscopy (TEM) was performed in order to develop a standard protocol that would reduce sample preparation time for TEM-investigations. Therefore the overall and fine structure of leaf cells prepared with the Leica EM AMW were compared with leaf cells that were prepared with a conventional fixation protocol at room temperature. Additionally, the diameter of membranes from different cell compartments (chloroplasts, nuclei and plasmamembrane) was determined by using quantitative computer supported transmission electron microscopy.
