NEW APPLICATIONS OF LIGHT AND ELECTRON MICROSCOPIC TECHNIQUES FOR THE STUDY OF MICROBIOLOGICAL INCLUSIONS IN AMBER

Abstract

Amber is a superb medium for the fossilization of delicate organisms. Besides light microscopy techniques for the study of insects in amber, scanning electron microscopy (SEM) in backscattered electron mode (SEM-BSE), low temperature SEM (LTSEM) and also confocal laser scanning microscopy (CLSM) were used to examine microcenosis and particulate plant remains (microdebris). We applied these techniques to such inclusions in amber Alava, northern Spain (Allaian: Early Cretaceous). Confocal microscopy provides a 3D image of partial microcenosis showing bifurcate fungal hyphae. The huge potential of SEM-BSE yields high resolution images, in which the relationship between protozoa and fungal hyphae may be observed and the characterization of further ultrastructural details in flagellates. According to the SEM-BSE images, food and pulsatile vacuoles appear better preserved than mitochondria and lipids in amber-embedded protozoa. A process of protozoan mineralization has led to the deposition of S and Fe in peripheral areas, and the Fe is also present in the core of surrounding fungal hyphae. Application of LTSEM for the study of protozoan inclusions produces images of their exteriors showing many vacuoles. Plant tissues under SEM-BSE show mummified cell walls, while the cytoplasm exhibits a bright appearance and is very rich in Fe and S. SEM in secondary electron mode (SEM-SE) also reveals a microbiota trapped in gas bubbles.