Cryo-FIB-SEM serial milling and block face imaging: Large volume structural analysis of biological tissues preserved close to their native state

The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20 μm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues.