Microscopic Analysis of Plant Leaf Structure

Light microscopy was performed as described before [72 (link)]. Cell size and number determination covered all parts of the leaf sparing cells surrounding the primary vein and at the edge of the leaves. For transmission electron microscopy, leaf samples were fixed in 2.5% glutaraldehyde, 0.1 M cacodylate buffer (pH 7.4), 5 mM calcium chloride for 4 h at 4°C, and post-fixed with 1% Os0₄ and 0.8% K₃Fe(CN)₆ for 2 h at 4°C. The samples were washed with water and post-stained with 2% aqueous uranyl acetate for 2 h. Subsequently, the tissue was dehydrated in a series of ethanol and propylene oxide and embedded in Spurr’s low viscosity epoxy resin. Ultrathin sections (60–70 nm) were cut with a Leica UC6 ultramicrotome using a diamond knife, stained with uranyl acetate and lead citrate and examined on an energy-filtering transmission electron microscope (EFTEM, Zeiss) at 120 kV.