Figure 3.

Spatiotemporal fluctuation analysis can superresolve single-molecule dynamics: a simulated experiment. A three-dimensional moving spherical object (in this case, a vesicle) with a diameter corresponding to the nominal measurement resolution (PSF) is filled with fluorescent molecules (see drawing in the inset). Both the vesicle and the molecules are free to diffuse, but the latter are 10 times faster than the vesicle and cannot cross the imposed spherical boundary. By applying spatiotemporal analysis of fluorescence fluctuations, one can measure the motion of the molecules within the vesicle (red dashed line) and the motion of the vesicle (blue dashed line) concomitantly, even if both are significantly smaller than the nominal imaging resolution. Further details about the simulations and data analysis are reported in Supporting Materials and Methods.