Backscattered electron imaging as a tool for histochemically localizing calcium oxalate with the scanning electron microscope

Abstract

The scanning electron microscope and a backscattered electron detector were used for the first time to identify calcium oxalate in leaf tissues after labelling crystals with silver. Freehand sections fixed in formalin – acetic acid – alcohol or glutaraldehyde were treated with 5% acetic acid to remove background calcium carbonate and calcium phosphate and were silver stained by calcium-substitution techniques. Two standard staining techniques, Pizzolato (AgNO3–H2O2) and Yasue (AgNO3 – rubeanic acid) tests, were compared. Druses and raphides were identified morphologically and histochemically in the leaves of Anthurium scherzeranum and Dieffenbachia maculata. The Pizzolato procedure was the best method for staining calcium oxalate for scanning electron microscope studies. Excessive deposition of granular material in the Yasue staining method tended to obscure crystal morphology in scanning electron microscope preparations.