Application and Tissue Preparation of SBF-SEM for 3D Ultrastructural Analyses of Biological Specimens

Nobuhiko Ohno, Sei Saitoh, Yurika Saitoh and Shinichi Ohno

Abstract: Serial block face-scanning electron microscopy (SBF-SEM) repeats milling of tissue blocks by built-in ultramicrotome and subsequent observation of the tissue block surface with SEM. It facilitates rapid acquisition of serial electron microscopic images at a resolution of several nm from tissue areas larger than hundreds μm². This SEM approach requires adequate en bloc electron staining and also optimization of embedding, trimming and final observation conditions to reduce a specimen charging, increase contrast and stably acquire images of cells and tissues. Because of its rapid image acquisition, SBF-SEM is useful for high-throughput morphological analyses of various disease models and transgenic animals as well as “connectome” analyses in large tissue areas of the nervous system. Furthermore, its combination with molecular labeling can detect specific organelles and cells in 3-dimensional tissue architectures. Therefore, SBF-SEM will be widely used as a new approach for 3-dimensional analyses of ultrastructures in biological specimens which have been difficult to observe with conventional transmission electron microscopy.

Key words: SBF-SEM, Diamond knife, en bloc staining, 3D reconstruction