Figure 1

Image analysis. The two-color STED images were collected with an interleaved pulse scheme. The first pair of excitation/STED pulses (at wavelengths of 650 and 755 nm) was followed by the second pair (at wavelengths of 570 and 710 nm) with a 40 ns delay. Using two detectors and a time-gated detection scheme, it was thus possible to detect the fluorescence emission following each pulse in two different wavelength bands, resulting in a set of four recordings. The four raw images were linearly unmixed to correct for channel crosstalk and were normalized. Segmentation was then performed with an iterative seeded region growing algorithm which extracted the regions of the image containing mitochondria. Only those regions were used in the subsequent calculation of the correlation coefficients. For better visibility the images are shown with a logarithmic color map.