A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture

Mitochondria exist in a dynamic cycle of fusion and fission whose balance directly influences the morphology of the ‘mitochondrial network’, a term that encompasses the branched, reticular structure of fused mitochondria as well as the separate, punctate individual organelles within a eukaryotic cell. Over the past decade, the significance of the mitochondrial network has been increasingly appreciated, motivating the development of various approaches to analyze it. Here, we describe the Mitochondrial Network Analysis (MiNA) toolset, a relatively simple pair of macros making use of existing ImageJ plug-ins, allowing for semi-automated analysis of mitochondrial networks in cultured mammalian cells. MiNA is freely available at https://github.com/StuartLab. The tool incorporates optional preprocessing steps to enhance the quality of images before converting the images to binary and producing a morphological skeleton for calculating nine parameters to quantitatively capture the morphology of the mitochondrial network. The efficacy of the macro toolset is demonstrated using a sample set of images from SH-SY5Y, C2C12, and mouse embryo fibroblast (MEF) cell cultures treated under different conditions and exhibiting hyperfused, fused, and fragmented mitochondrial network morphologies.