Microglia-synapse interactions have been examined in a variety of contexts, in vitro and in vivo, which revealed an important role for microglia in synaptic pruning and loss. Nevertheless, it remains to be determined whether and how microglia are involved in the process of synapse formation under physiologic conditions. We hypothesize that microglia serve as a link between pre- and post-synapse by sensing neurotransmitter release at highly active pre-synaptic sites and actively modulate the formation of new dendritic spines.To examine this relationship, we target microglia-synapse communication under normal physiological conditions in the adult mouse hippocampus in vivo, while specifically knocking out neurotransmitter receptors on microglia. Thereby, we investigate if microglia lacking neurotransmitter receptors show changes in their synapse-contact rate, in their motility and how this impacts the formation and elimination of dendritic spines.Our methodological approach goes beyond the current state-of-the-art and for the first time yields microglia-synapse interactions with super-resolution time-lapse imaging in awake mice. We established two-photon (2P) stimulated emission depletion (STED) microscopy via a cranial window in vivo, which enables us to overcome the diffraction limit and therefore resolve synapses at the nanoscale. Our results show improved resolution of 2P-STED imaging of microglia-synapse interactions, as well as a relationship between dendritic spine formation and previous contact frequency by microglia. These findings will pave the way to visualize nanoscale anatomical structures during microglia-synapse communication in vivo and therefore allow longitudinal and correlative studies in combination with behavioral experiments.