Time lapse imaging reveals the contribution of photobleaching. (a) Stroboscopic illumination enables the excitation energy to be spread out over time. In this example, a fluorophore disappears after three exposures; when under continuous illumination (top) this takes 0.6 s; however, if there is a 0.4 s gap between frames (middle), then the same exposure takes 1.2 s; when the time between exposures is further extended to 1 s (bottom), then only after 3 s will the fluorophore disappear. The images used are representative and not actual images recorded during such a stroboscopic image series. (b) The kinetics of this process are represented by two unidirectional rate constants, therefore the rate of loss of the fluorescent spot is k_off+ k_bl. (c) Plot of the dependence of k_obs*τ_exp on the time lapse (τ_tl) reveals a linear relationship from which the intercept provides the photobleaching rate constant (0.13 ± 0.05 s⁻¹). Time lapses used were 200 ms (n = 1,689), 500 ms (n = 3,520), 1 s (n = 1,312), and 2 s (n = 1,455).