Fluorescence microscopy is a powerful tool in many life science research areas; thanks to its live-cell compatibility and its ability to observe only specifically labeled molecules of interest. However, in the field of virology, this technique has traditionally been of limited use, specifically due to the fundamental resolution limit associated with the diffraction of the visible light. The diffraction limit makes fluorescence microscopes unable to resolve details below approximately 200 nm in the focal plane and approximately 600 nm along the optical axis, thus making studies of approximately 100-nm-sized virus particles infeasible. Therefore, for a long time, virus visualization was performed solely via electron microscopy-based methods, which -with its subnanometre resolution -enabled for numerous insights into details of virus structures. On the other hand, electron microscopy-based methods require laborious preparation of biological sample (fixation or freezing) making them unsuitable for the study of dynamic processes of viruses and virus-cell interactions.However, this situation began to change with the development of super-resolution fluorescence microscopy (SRFM) or nanoscopy in the 1990s [1,2]. These advanced microscopy techniques surpass the diffraction limit of light, giving molecular-scale spatial resolution, and thus, for the first time, opening up the possibility to observe details of subdiffraction-sized viruses with fluorescence microscopy. Currently, there are many different SRFM techniques available, each with its own set of advantages and disadvantages [3][4][5]. The requirement for the observation of sub-100 nm sized virus structures limited virological studies to the use of SRFM approaches that routinely offered 10-100 nm spatial resolution, such as stimulated emission depletion (STED) microscopy and single molecule switching microscopy-based approaches like photo-activation localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM).HIV-1 was one of the earliest biological systems studied by SRFM, and currently SRFM-based HIV-1 studies have already provided numerous ground breaking insights into retroviral replication cycle [6,7]. However, only very recently have these studies began to transition from the observations of fixed samples into live-cell imaging, which now allows for the study of dynamic aspects of the virus structure and virus-cell interactions. This is due to the fact that, to achieve highest spatial resolutions, SRFM techniques usually require long acquisition times thus making live-cell imaging infeasible especially for observation of molecular dynamics in single viruses.One of the solutions that allows for live-cell studies of single molecules on the surface of sub-100 nm objects lies in combining the existing STED SRFM technique, with single-molecule-based spectroscopic tools such as fluorescence correlation spectroscopy (STED-FCS) [8]. This combination allows for the determination of molecular mobility for observation spot sizes below 60 nm in ...