Picea abies (L.) KARST - Sample Preparation for TEM

Application Note for Leica EM AMW - Life Science Research

January 23, 2017

Plants (5-years old) were grown in pots filled with soil and kept in greenhouse conditions. Five weeks before harvesting the plants were transferred into growth chambers and cultivated at a temperature of 20°C during daytime and 12°C overnight. The relative humidity was set at 60% and the photoactive radiation was 500 μmol m-2 s-1 during daytime. At harvesting (two hours after the onset of daylight) samples were taken from the middle of first year needles and prepared for electron microscopy with the Leica EM AMW.

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.

Fig.1: Overview
C = Chloroplast, ST = Containing starch, M = Mitochondria, N = Nucleus
Fig.2: Detail
C = Chloroplast, ST = Containing starch, M = Mitochondria, N = Nucleus