Figure 2

Monitoring proton surface diffusion. (a) The membrane bound caged protons were released by a UV flash from the area in the red square (10 × 10 µm²) and their arrival was observed as a change in fluorescence intensity in the green square (10 × 10 µm²). The light emitted by the lipid-anchored pH sensor fluorescein was collected using a 40x water immersion objective and a 515 nm high-pass filter. (b) The proton concentration σ adjacent to the membrane is monitored as a function of the time that elapsed after the flash at the indicated distances x (in µm) from the observation site (19 °C). σ has been calculated from the fluorescence intensity of membrane anchored fluorescein according to a calibration curve (Supplementary Fig. S1). It reaches it maximum at time t _max, which according to equation (1) obeys: t _max = x ²/4D − k _off t _max ². When the last term is small, t _max depends linearly on x ², as shown in the inset. The colored traces are averaged data from at least 10 individual uncaging reactions each. The black lines represent a global fit to average traces at four distances of the non-equilibrium model. Therefore equation (1) was modified to take into account the finite sizes of release and detection zones (equation (S3), Supplementary Fig. S2). The global fit parameters, D _l = 5.1 × 10⁻⁵ cm² s⁻¹ and k _off = 2.3 s⁻¹, are common to all curves, whereas the amplitude A _neq was allowed to vary (±15%).