A targeted 3D EM and correlative microscopy method using SEM array tomography

Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric Array Tomography for asymmetric samples and model organisms ( Caenorhabditis elegans, Drosophila melanogaster, Danio rerio ). We modified a diamond knife to simplify serial section arrays acquisition with minimal artifacts. After array acquisition, we transfer the arrays to a glass coverslip or silicon wafer support. Using light microscopy, we rapidly screen the arrays for initial recognition of global anatomical features (organs or body traits). Then, using SEM, we perform an in-depth study of the cells and/or organs of interest. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than do alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.