The broad range of topics presented at this year’s Frontiers in Neurophotonics conference included novel techniques in acquiring, processing, and analyzing image datasets, new microscopic and fiber-optic tools to probe and manipulate neurons, and impressive advancements in the design of integral photosensitive proteins. Yet one of the hot topics of this symposium, which still remains a major issue for experts, was indisputably the quest to solve the trade-off between space and time resolution in living biological systems. Indeed, super-resolution imaging has become more and more popular in the last decade but is still often limited to fixed tissues. Thus, super-resolution microscopy has been mostly restricted to very simple biological systems and hardly combined with other techniques. Yet, recent advances have made easier the use of super-resolution techniques in more physiological and intact systems. The combination of super-resolution with other known techniques now allows the investigation of the nanoscopic molecular organization of single spine in its characterized environment. By doing STORM imaging following some electrophysiological recordings and confocal imaging, István Katona and his team (IEM, Budapest) elegantly showed how it is now possible to assess endocannabinoid receptors’ precise localization in axon terminals of electrophysiologically and anatomically characterized neurons. Tying the functional and structural connectivity of circuits has been a growing endeavor. Rainer Friedrich (FMI, Basel) presented serial block face-scanning electron microscopy and tedious three-dimensional reconstruction of the olfactory circuit of zebrafish following live calcium imaging in the same tissue exposed to odors, allowing detailed investigation of neuronal computation based on dynamic and structural measurements. Monitoring how synaptic plasticity-inducing stimuli impact the long-term fate of individual synapses has been quite challenging. Thomas Oertner (ZMNH, Hamburg) combined the control and sensing components of optogenetics in organotypic hippocampal slices to describe the fate, over seven days, of individual synapses following depression or potentiation protocols. These different advances in combining optical methods with high resolution will be revealed to be particularly important in the next years to decipher synaptic signaling and remodeling.