The goal of our workshop is to introduce Photoactivated Localization Microscopy (PALM) and single particle tracking (sptPALM) imaging techniques, which are based on the sequential detection and localization of genetically encoded photo-transformable fluorescent proteins, allowing non-invasive labeling of a target protein. Collected data are used for a reconstruction of better-resolved images or protein trajectories, and with a further in-depth analysis can provide insights into protein distribution, clustering, dynamics, and binding-unbinding events. However, PALM/ sptPALM imaging and data analysis are not always straightforward. Experiments are complicated by motion blur, the relatively low photon budget of fluorescent proteins and their complex photophysics/blinking. These challenges as well as specific technical requirements for successful imaging (e.g. use of a sCMOS camera, advanced control of laser pulse sequence) will be addressed and discussed during the workshop. We will perform PALM and sptPALM acquisitions to unveil the localization and dynamics of the nucleoid associated protein HU from Deinococcus radiodurans. HU is one of the most conserved proteins in bacteria. In our group, we are interested in its role in nucleoid reorganization during recovery from DNA damage. To localize HU we labeled it with mEos4b, one of the best performing photoconvertible fluorescent protein. Furthermore, we will discuss strong and weak points of PALM versus other Single Molecule Localization Microscopy technics and how to adapt and optimize the experimental protocol for particle tracking. In particular, we previously showed that addition of low intensity 488 nm light reduces the blinking duration of mEos4b and thereby can increase the track length in sptPALM experiments. Who should attend: Our workshop is aimed at students and researchers who are new to the field of PALM and sptPALM imaging and researchers who want to optimize their imaging conditions.