Investigating autophagy: quantitative morphometric analysis using electron microscopy

Autophagy is a compensatory pathway involving isolation and subsequent degradation of cytosolic material and organelles in eukaryotic cells.(1) The autophagic process can provide a "housekeeping" function by removing damaged proteins and organelles in a selective or nonselective fashion in order to exert a protective effect following stress.(2) Remarkably, after being discovered to be much more of a targeted process than a random one, the role of autophagy became implicated in many normal cellular and disease processes.(3) Several methodologies are routinely employed to monitor the entire autophagic process.(4) Microtubule-associated protein light chain 3, a mammalian homolog of yeast Atg8, has been widely used as a specific marker to monitor autophagy in numerous cell types.(5) While monitoring autophagic flux is extremely important, it is also beneficial to perform a detailed analysis by electron microscopy (EM) to evaluate changes in various autophagic structures, quantify the areas involved, and determine if any particular organelle(s) or area of the cell cytoplasm is being targeted for degradation.(6) The following article describes methods to localize and quantify subcellular areas of autophagy using transmission EM. Also discussed are methods for subcellular localization of specific proteins by employing immunogold EM; this method becomes particularly useful in detecting early changes in cellular homeostasis that may occur before later signs of cellular insult can be observed morphologically.