Fig. S4.

Single-molecule bleaching measurements (Atto647N). The fluorescence signal (A) was acquired (integrating over 16 ms) while the STED laser was periodically blocked to acquire fluorescence signal (probe the fluorescent state). The data were then separated into a “confocal” part, where the STED laser was blocked, and the “STED” part. Note that no doughnut profile was used in these measurements, but (Gaussian) excitation and STED foci were overlaid to study the bleaching at different intensities (B). Data points with signal above a threshold were considered to belong to the fluorophores’ fluorescent state. The total time the molecule is in the fluorescent state (above threshold) before bleaching was averaged over ∼100 molecules (C). Alternatively, the signal of all measured molecules was summed and fit with an exponential decay yielding the bleaching constant (D).