Changes in mitochondrial morphology after H₂O₂ treatment.

<p>(A) Confocal microscopy images of hepatic cells stained with Mitotracker (upper panel). Microscopy images show hepatic cells treated with H₂O₂ for 30 min and then post-incubated for 2 h. Mitotracker images were filtered and thresholded to obtain segmented images (middle panel) using MicroP software. The imagesegmentation method extracts each individual mitochondrial object from Mitotracker images to identify mitochondrial subtypes (lower panel; higher magnification of the boxed area in Segmented Image). MicroP software classifies morphological features as different colours according to their shape and size: blue, small globules; yellow, swollen globules; green, linear tubules; orange, twisted tubules; purple, branched tubules; and red, looped tubules. In this subtyping, fragmented mitochondria are represented by small globules (shown in blue). The composition of mitochondrial subtypes in the H₂O₂ treated cells was clearly modified with respect to the control cells. Scale bar in Mitotracker images: control, 10 μm; 3.5 mM H₂O₂, 18 μm. Scale bar in mitochondrial subtypes: control, 5 μm; 3.5 mM H₂O₂, 8 μm. (B) Bar graphs showing the percentage of type I (small globular; left graph) and type II (tubular including linear, twisted, branched and looped forms; right graph) mitochondrial morphology quantified with MicroP software in non-treated and H₂O₂ treated cells. (C) Bar graph showing the value of the mitochondrial elongation index (relation between major and minor axis lengths) in non-treated and H₂O₂ treated cells. Mitochondrial axis lengths were quantified with MicroP software. (D) Bar graph showing mitochondrial area obtained with MicroP analysis in non-treated and H₂O₂ treated cells. At least 7,000 mitochondria were analysed with MicroP software in each condition and in each experiment in (B), (C) and (D). Bars show the mean ± SEM of three independent experiments. Significant differences with respect to non-treated cells: *<i>P</i> < 0.05 and ***<i>P</i> < 0.001.