Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue

The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles of brain ECS, its structure and dynamics remain largely inaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil. [Display omitted] •3D-STED and labeling of extracellular fluid for nanoscale imaging of live brain ECS•As negative imprint of all cells, it captures anatomical complexity of brain tissue•Activity-induced ECS remodeling and cell migration in visible neuropil is revealed•Super-resolution imaging paradigm immune to bleaching and with reduced toxicity Live tissue super-resolution 3D-STED microscopy combined with fluorescence labeling of the interstitial fluid reveals the complex spatial organization of the extracellular space of the brain.