Quantifying mitochondrial content in living cells.

We describe a novel version of MitoGraph, our fully automated image processing method and software, dedicated to calculating the volume of 3D intracellular structures and organelles in live cells. MitoGraph is optimized and validated for quantifying the volume of tubular mitochondrial networks in budding yeast. We therefore include the experimental protocol, microscopy conditions, and software parameters focusing on mitochondria in budding yeast. However, MitoGraph can also be applied to mitochondria in other cell types and possibly other intracellular structures. We begin with our protocol and then include substantial discussion of the validation, requirements, and limits of MitoGraph to aid a wide range of potential users in applying MitoGraph to their data and troubleshooting any potential problems that arise. MitoGraph is freely available at the Web site http://rafelski.com/susanne/MitoGraph.