Figure 3
Pulse duration influence on de-excitation, bleaching and fluorescence induced by the STED beam at 750 nm. (a) De-excitation, bleaching and STED-light-induced fluorescence vs. pulse duration for ATTO647N. CW represents measurements for continuous-wave STED laser operation. The solid lines represent values calculated within the photobleaching model (Supplementary Information). (b) Bleaching vs. STED pulse duration for different dyes. The dashed lines represent the orders of photobleaching, with values already determined for pulses of 0.1–3 ps (Fig. 2). Deviations from this scaling indicate the dominant involvement of a different photobleaching pathway in these pulse-duration regimes (see text). (c) STED-light-induced fluorescence vs. STED pulse duration. For organic dyes typically used with the laser wavelengths applied in this study (ATTO647N, STAR635P), STED-light-induced fluorescence is mainly constant (flat dashed lines). For the orange-emitting dyes studied (ATTO590, STAR580), significant fluorescence emission induced by the STED beam occurs via two-photon absorption. Time-averaged STED power: 10 mW (Ep = 0.125 nJ). Measurements were performed for a low dye concentration (~0.5–10 μM).
