2.6. TMRE staining and quantification

TMRE staining and quantification were performed as previously described [21] with the following modifications: C. elegans larvae were transferred onto plates containing 0.1 μM TMRE (Thermo Life Sciences T669) 24 h before imaging, and imaged at L4 stage using 60x (oil) objective of Nikon eclipse 80i microscope. Images were cropped to a fixed region between the posterior pharynx and vulva, and a segmentation algorithm for mitochondria and the worm body respectively were trained using Ilastik [28]. The worm body segmentation was subsequently used in a custom made Fiji [27] (supplementary file 3) to identify the worm in the original image and remove non-specific signal in the field of view. Within the identified worm, the signal intensity of TMRE was measured on the original image in the previously segmented mitochondria. Schematic representation of the analysis process is described in Fig. S2.