Figure 1.

Principle of LIESS–FCS: (A) sSTED–FCS data are usually generated from rapidly scanning with a diffraction-limited confocal (orange) or super-resolved STED (red) spot several times (time t axis) along a line (spatial x axis), yielding intensity traces for each pixel along the line that are then correlated to generate the final FCS data (correlation data G(τ) against correlation lag time τ) in confocal and STED separately (bottom plots). (B) In LIESS–FCS, confocal and super-resolved STED–FCS data are generated simultaneously by alternating confocal and STED modes in-between subsequent lines. Arrows: movement of the beam scanner. (C, D) Representative correlation carpets in (C) confocal and (D) STED for simulated data of free diffusion (measurement time of 40 s; d_STED = 100 nm and d_confocal = 240 nm; x axis: correlation lag time τ; y axis: line pixels, i.e., space; color code: normalized G(τ) decaying from red to blue). (E) Values of D_rat = D_STED/D_conf over space (pixel number) and (F) corresponding frequency histogram as obtained from the analysis of the correlation carpets of panels C and D, indicating fluctuation around D_rat = 1, i.e., free diffusion (red line in panel E).