Fig. 1: Spatiotemporal manipulation and monitoring of ROS in single mitochondria.

a Schematic representation of all optical ROS generation and detection in mitochondria. b Imaging and analysis workflow to track spatiotemporal mitochondrial responses relative to single mitochondrial ROS photogeneration in HEK293T cells. c Representative images, where dotted line indicates a single cell expressing matrix-targeted HyPer7 and KR. Denoted circle indicates static spot stimulation used to pulse ROS photogeneration. d Inset from c highlighting transient elongation and contact of photostimulated mitochondria. e Single mitochondrial responses of HyPer7 and KR intensities normalized to area and baseline. f Comparison of HyPer7 and KR responses at first frame immediately following pulse 1, 10 min following first stimulation, immediately following pulse 2, and 10 min following second stimulation. Two-way ANOVA with Tukey post-hoc multiple comparisons. g Decay of HyPer7 following stimulation 1 and 2 fitted with a nonlinear variable plateau followed by single phase decay, mean ± SEM. Decay compared with one-sided extra sum-of-squares F test. h Mitochondrial regional proportion of proximal and distal subpopulations relative to total in the cell over time. i Area of individual mitochondria in subpopulations over time through ROS photostimulation. Two-way ANOVA with Holm-Šídák’s post-hoc multiple comparisons, mean ± SEM. j Form factor of individual mitochondria in subpopulations over time through ROS photostimulation. Two-way ANOVA with Tukey post-hoc multiple comparisons, mean ± SEM. Green bars indicate single frames of KR photostimulation. Mean ± SEM, N = 39 distal, 54 proximal, and 95 non-stimulated mitochondria per frame per group, on average. *p < 0.05, **p < 0.01, ***p < 0.001. Scalebars denote 5 µm.