Correlative light and volume electron microscopy: using focused ion beam scanning electron microscopy to image transient events in model organisms

• Published in: Methods in cell biology Vol. 111; pp. 357 - 382
• Main Authors: Bushby, Andrew J, Mariggi, Giovanni, Armer, Hannah E J, Collinson, Lucy M
• Format: Journal Article
• Language: English
• Published: United States 2012
• Subjects: Animals; Blood Vessels - ultrastructure; Electron Microscope Tomography; Embryo, Nonmammalian - blood supply; Embryo, Nonmammalian - ultrastructure; Epoxy Resins - chemistry; Imaging, Three-Dimensional; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microscopy, Video; Microtomy; Neovascularization, Physiologic; Plastic Embedding; Software; Tissue Fixation; Zebrafish
• ISSN: 0091-679X
• DOI: 10.1016/B978-0-12-416026-2.00018-2

The study of a biological event within a live model organism has become routine through the use of fluorescent labeling of specific proteins in conjunction with laser confocal imaging. These methods allow 3D visualization of temporal events that can elucidate biological function but cannot resolve the tissue organization, extracellular and subcellular details of the tissues. Here, we present a method for correlating electron microscopy image data with the light microscopy data from the same sample volume to reveal the 3D structural information: "correlative light and volume electron microscopy." The methods for live video confocal microscopy, fixation and embedding of the tissue for electron microscopy, the focused ion beam scanning electron microscopy method for sequentially slicing and imaging the volume of interest, and the treatment of the resulting 3D dataset are presented. The method is illustrated with data collected during the angiogenesis of blood vessels in a transgenic zebrafish embryo.