Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.H igh-resolution techniques relying on specific fluorescent probes (1-5) allow imaging at the nanometer scale. However, they impose severe constraints on tagging and cannot be easily combined with colocalization (6) (photoactivated localization microscopy or stochastic optical reconstruction microscopy). In general, these approaches improve resolution at the expense of a low image acquisition rate. Structured illumination microscopy (SIM), although not relying on particular properties of fluorescent probes such as photoconversion and being well suited for multicolor tagging (7,8), still requires the acquisition of a number of raw images (ranging from 9 to 15 images) to build a single full doubled resolved optical section. Consequently, despite recent advances (9), SIM remains poorly adapted to fast imaging of dynamical events. Finally, with the noticeable exception of sophisticated combinations (4), most of these methods use illumination configurations that expose the entire sample thickness to intense light radiation. Therefore, phototoxic effect of whole-cell illumination is often a limitation for live cell imaging.In total internal reflection fluorescence microscopy (TIRFM), fluorophores are excited with evanescent waves that intensity decays exponentially with the distance from the interface (10). The imaged section is therefore thinner (100-200 nm) in comparison with most optical sectioning techniques like confocal (11) or multiphoton microscopy (12), whose temporal resolutions are additionally limited. Therefore, TIRFM is particularly suitable for imaging the plasma membrane where fundamental cellular mechanisms related to cell/substrate contact regions, secretory and endocytic processes (13), binding of ligands to cell surface receptors, and dynamical remodeling of cytoskeleton elements take place. High temporal resolution is required as, for example, docking of vesicles to the plasma membrane may last for less than half a second (14) and fusion events can be complete in less than 300 ms (15). If classical TIRFM exhibits modulation patterns that prevent accurate quantification analysis, elimination of these artifacts can be achieved by varying the azimuthal angl...