Monitoring autophagy in yeast using FM 4-64 fluorescence

The original observations and experiments dealing with autophagy were purely morphological in nature. Even though more and more molecular techniques have been introduced, experimenters are often asked to provide visual evidence of autophagic processes in order to back up data obtained via other means. In yeast as well, autophagosomes were initially defined morphologically and indirectly, by observing intravacuolar autophagic bodies that accumulate upon starvation. This can be achieved by electron microscopy, which affords very high resolution but is time consuming and costly, or by light microscopy, which is a relatively inaccurate method of scoring autophagy. A third alternative, which we present here, is to use the unique properties of the fluorescent dye FM 4-64 to follow the accumulation of autophagic bodies.