title: Estimating the size and number of autophagic bodies by electron microscopy.
abstract: Much recent and ongoing research is focused on understanding the mechanisms and regulation of autophagy, a cellular self-degradation pathway with many links to human health. Although many assays exist to measure the total magnitude of autophagy, electron microscopy remains the tool of choice for the determination of the size and the number of autophagosomes formed in a given mutant or under given induction conditions. Here we present a detailed protocol for measuring autophagic bodies in the yeast Saccharomyces cerevisiae by electron microscopy. Furthermore, we present an improved mathematical method for estimating body size and a new method for estimating body number. Finally, we include a discussion of the merits and limitations of these methods and an example of their application to autophagic bodies formed in the ume6∆ strain.
authors.affiliation: Life Sciences Institute; University of Michigan; Ann Arbor, MI USA.
authors.forename: Steven K
authors.initials: SK
authors.lastname: Backues
authors.affiliation: School of Mathematical Sciences and LPMC; Nankai University; Tianjin, China.
authors.forename: Dachuan
authors.initials: D
authors.lastname: Chen
authors.affiliation: School of Mathematical Sciences and LPMC; Nankai University; Tianjin, China; State Key Laboratory of Medicinal Chemical Biology; Nankai University; Tianjin, China.
authors.forename: Jishou
authors.initials: J
authors.lastname: Ruan
authors.affiliation: School of Medicine; Nankai University; Tianjin, China.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Xie
authors.affiliation: Life Sciences Institute; University of Michigan; Ann Arbor, MI USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Klionsky
mesh_terms: D001343:Autophagy; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods*; D009154:Mutation; D010588:Phagosomes / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000648:ultrastructure*
keywords: autophagosome; autophagy; electron microscopy; stereology; vacuole; yeast