Advancing yeast cell analysis: A cryomethod for serial block-face scanning electron microscopy imaging in mitochondrial morphology studies.

Abstract

Conventional Transmission Electron Microscopy analysis of biological samples often provides limited insights due to its inherent two-dimensional (2D) nature. This limitation hampers a comprehensive understanding of the three-dimensional (3D) complexity of cellular structures, occasionally leading to misinterpretations. Serial block-face scanning electron microscopy emerges as a powerful method for acquiring high-resolution 3D images of cellular volumes. By iteratively removing ultrathin sample sections and capturing images of each newly exposed surface, Serial block-face scanning electron microscopy allows for the meticulous reconstruction of a comprehensive 3D volume. In this study, we investigate the 3D architecture of altered mitochondrial morphologies in Saccharomyces cerevisiae using Serial block-face scanning electron microscopy imaging. We have developed a novel cryomethod based on plunge freezing and a dedicated freeze-substitution protocol. This protocol enhances ultrastructural preservation enabling a more accurate understanding of mitochondrial defects observed in 2D electron microscopy. Our findings underscore the utility of Serial block-face scanning electron microscopy coupled with optimized sample preparation techniques in elucidating complex cellular structures in 3D.