The brain is a highly dynamic structure with the extracellular space taking up almost a quarter of its volume. Signalling molecules, neurotransmitters and nutrients transit via the extracellular space, which constitutes a key microenvironment for cellular communication and clearance of toxic metabolites. Nevertheless, the extracellular space has not been characterized in detail in intact living samples because of the lack of appropriate tools allowing its study. Recent technological advances enhancing the luminescence properties and biocompatibility of carbon nanotubes opened the door to super-resolution imaging in the near-infrared in vivo. The luminescence efficiencies of single carbon nanotubes excited via various excitation strategies were compared and optimized for tissue imaging (e.g., targeting various excitonic transitions and through upconversion). The effects of tissue scattering, absorption, autofluorescence, and temperature increase induced by excitation light were systematically examined. Using carbon nanotube tracking, we revealed the hidden structure and viscoelastic properties of the extracellular space of brain slices. Local morphological and viscosity maps of the extracellular space of brain acute slices were reconstructed. A diversity of extracellular space dimensions down to ～40 nm and local viscosity maps were obtained. The rheological properties of the extracellular space are affected by chemical alterations of the extracellular matrix of the brains of live animals. Interestingly, these alterations are local and highly inhomogeneous in space. Probing the viscoelastic properties of the extracellular space is paramount to understand the spatiotemporal dynamics that regulate the cellular mechanisms ultimately influencing fundamental aspects of cell biology. These technological advances constitute the first milestone to generate super-resolution microscopy applications in the near-infrared to investigate live biological samples in situ.
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