Fig. 4: Photobleaching does not interfere with threshold-based segmentation of mitochondria in Imaris using mtGFP.

a Schematic of mitochondrial quantity measurement in animals expressing bcIs153. mtGFP (mean Integrated Density, Fiji) measurement within cell contours (white ROIs) at both metaphase and post-cytokinesis, either along multi-step (top) or two-step (centre) timeseries. Bottom: mitochondrial volume measurement (Imaris) at both metaphase and post-cytokinesis along multi-step timeseries. Quantifications were performed on z-stacks after image processing (fluorescence) or after 3D rendering (volume). b Mitochondrial quantities (post-cytokinesis) normalised to respective quantities at metaphase (n = 30, 24, 30). c Average mtGFP photobleaching from trends constructed on fluorescence values normalised to time 1 (n = 10, referring to panel a, top). d, e Linear regression modelling the relationship (see 'Methods') between log-transformed average grey value frequencies (n = 30) measured on 256 fixed or relative grey value classes both at metaphase and post-cytokinesis from processed images (see also Supplementary Fig. S9). f, g Average mtGFP grey value frequency distributions generated on 256 fixed or relative grey value classes from processed images (n = 30) (see also Supplementary Fig. S9) (error bars = SEM). Mean grey value classes to which Imaris Surface threshold values belong are shown. h, i Individual fixed or relative grey value classes to which Imaris Surface threshold values belong (n = 30). j Correlation between normalised Imaris Surface threshold values and normalised mtGFP fluorescence (photobleaching) at post-cytokinesis. P values are calculated using a Kruskal–Wallis non-parametric rank test with Benjamini, Krieger and Yekutieli correction (b), a Wilcoxon matched-pairs signed rank test (h) or a paired t test (i). Normality was tested with the Shapiro–Wilk test. *P value ≤ 0.05; **P value ≤ 0.01; ***P value ≤ 0.001; ****P value ≤ 0.0001. c We used aligned and subtracted z-stacks, while in (d–j) we used fully processed z-stacks. Red bars = mean (b, h, i).