Correlative light and volume electron microscopy to study brain development.

Recent advances of volume electron microscopy (volume EM) have been driving our thorough understanding of the brain architecture. Volume EM becomes increasingly powerful when cells and their subcellular structures that are imaged in light microscopy are correlated to those in ultramicrographs obtained with EM. This correlative approach, called correlative light and volume electron microscopy (vCLEM), is used to link three-dimensional ultrastructural information with physiological data such as intracellular Ca2+ dynamics. Genetic tools to express fluorescent proteins and/or an engineered form of a soybean ascorbate peroxidase (APEX) allow us to perform vCLEM using natural landmarks including blood vessels without immunohistochemical staining. This immunostaining-free vCLEM has been successfully employed in two-photon Ca2+ imaging in vivo as well as in studying complex synaptic connections in thalamic neurons that receive variety of specialised inputs from the cerebral cortex. In this mini-review, we overview how volume EM and vCLEM have contributed to studying developmental processes of the brain. We also discuss potential applications of genetic manipulation of target cells using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and subsequent volume EM to analysis of protein localisation as well as to loss of function studies of genes regulating brain development. We give examples for the combinatorial usage of genetic tools with vCLEM that will further enhance our understanding of regulatory mechanisms underlying brain development.