Monitoring autophagy by electron microscopy in mammalian cells

Electron microscopy remains one of the most accurate methods for the detection of autophagy and quantification of autophagic accumulation. Compared to fluorescence microscopy, the resolution of transmission electron microscopy is superior. In this chapter we describe the fine structure of early and late autophagic compartments in mammalian cells. Instructions are given for the preparation of samples for conventional electron microscopy using three different protocols suitable for cultured cells and animal tissues. We also introduce tomography as a tool. to study the three-dimensional morphology of autophagic organelles and show the morphology of a phagophore as an example. Finally, we describe a protocol for the quantification of autophagic compartments by electron microscopy and point counting.