Analysis of mitochondrial morphology using the IMAGEJ 1.42 software

Mitochondria were labeled by incubating cells with MitoTracker Red (Invitrogen, 20 nM) for 20 min prior to acquisition. Digital images were captured on a confocal microscope, with a 63X oil immersion lens and subjected to a 2D deconvolution step, which is meant to compensate for various optical imperfections. Accordingly, the “Interative Deconvolution”, a plug-in written by Bob Dougherty for ImageJ was used. After image enhancements, mitochondrial shape metrics were reported by an ImageJ macro, “Morphometry”, described by Cribbs and Strack [36]. This macro allowed us to determine two parameters of mitochondrial morphology: form factor (FF) and aspect ratio (AR). The former takes into account the perimeter and area of a single mitochondrion and can therefore capture complex mitochondrial shapes. The latter, instead, despite being a useful shape metric for simple rod-like mitochondria, does not faithfully represent the shape of linked, branched, or highly interconnected mitochondria. Low values of FF and AR indicate circular mitochondria, whereas high values indicate elongated and highly interconnected mitochondria [36].