Unraveling the spatio-temporal organization of dynamic cellular microcompartments requires live cell imaging techniques capable of resolving submicroscopic structures. While the resolution of traditional far-field fluorescence imaging techniques is limited by the diffraction barrier, several fluorescence-based microscopy techniques providing sub-100 nm resolution have become available during the past decade. Here, we briefly introduce the optical principles of these techniques and compare their capabilities and limitations with respect to spatial and temporal resolution as well as live cell capabilities. Moreover, we summarize how these techniques contributed to a better understanding of plasma membrane microdomains, the dynamic nanoscale organization of neuronal synapses and the sub-compartmentation of microorganisms. Based on these applications, we highlight complementarity of these techniques and their potential to address specific challenges in the context of dynamic cellular microcompartments, as well as the perspectives to overcome current limitations of these methods.