Fig. 5

Rapid time-lapse imaging of organelles with smart RESOLFT. a, b We imaged the continuous interplay of peroxisomes over 100 time points recorded at 2–5 Hz. Pex16-rsEGFP2 was expressed in U2OS cells. The decision was made with probe type 1. a The first 18 frames of the time lapse are shown. Each image has been smoothed with a 30 nm radius Gaussian filter. b Maximum projection of the entire 100 frames time-lapse. Few selected peroxisome trajectories are shown in different colors. c, d Mitochondria dynamics in U2OS cells. The mitochondrial outer membrane was labeled with rsEGFP2 with a targeting sequence of Omp25. c Time lapse recorded in the smart RESOLFT mode. The first frame is shown in the fire color bar. The related region of interest ROI I shows mitochondrial membranes fusion over time. d Maximum projection of the time-lapse mitochondria imaging. Each image is color coded and identify the fraction of mitochondria with minimal mobility (white) from the mobile one (colored) in the seconds time frames. e To detect real-time (30 Hz) dynamics of the individual mitochondria we performed a line recording xt-scan over thousands of time points. We observed sequential fission and fusion of the outer mitochondrial membrane with sub-80 nm spatial resolution. Scale bars are 1 µm (a−d), for (e) the vertical scale bar is 1 µm and the horizontal 1 s. RESOLFT REversible Saturable/Switchable Optical Linear Fluorescence Transitions