Brain research has made tremendous progress over the last few decades in nearly all areas of investigation with the exception of one: the extracellular space (ECS). It is however a key compartment defined as the web-like space between brain cells, filled with a myriad of molecules that enable brain functions and homeostasis. How molecules navigate in the ECS is a very important, yet unsolved, challenge that precludes conceptual advance in brain science and innovation in therapeutics (e.g. immunotherapy). The lack of knowledge is mainly due to the absence of dedicated investigation strategies for such a complex and finely structured biological entity. Our ground-breaking project (ENSEMBLE) will shed light on the conceptual and methodological roadblocks that have prevented us from understanding the fine architecture of the ECS and how molecules navigate within it throughout the brain. We posit that molecular diffusion in the ECS is locally regulated by the properties of the ECS, which is essential for brain functions. Four world-class scientists, L. Groc (molecular neuroscience), E. Bezard (systems neuroscience), L. Cognet (optics & nanoscience), and U.V. Nägerl (neurophotonics), team up to develop and apply unconventional investigation approaches, based on original nano-imaging strategies (super-resolution microscopy and carbon nanotube/nanoparticle tracking), to the in vivo brain. Yet, to consider and achieve such an experimental and multidisciplinary tour de force a side-by-side and daily interactive effort is necessary. Thanks to our complementary expertise and geographical proximity, ENSEMBLE will provide a unique opportunity to unveil in vivo the structure and functions of this crucial brain compartment and will offer a new theoretical and experimental framework to manipulate molecule navigation. The ENSEMBLE project will also crossfertilize the fields of nanoscience, optical imaging, organ pathophysiology and immunotherapy.