Correlative imaging of single carbon nanotubes and fluorescently labelled neuronal structures in the extracellular space of live brains

The brain extracellular space (ECS) is a complex network that constitutes a key microenvironment for cellular communication, homeostasis, and clearance of toxic metabolites(1). Signaling molecules, neuromodulators, and nutrients transit via the ECS, therefore mediating the communication between cells. Despite the relevance of this important part of the brain, its dynamics and structural organization at the nanoscale is still mostly unknown(2). We have recently demonstrated that single-walled carbon nanotubes (SWCNTs) can be used to image and probe live brain tissue, providing super-resolved maps of the brain ECS and quantitative information on the local diffusion environment(3,4). Here, we propose an important refinement of this approach by implementing a structured illumination technique (named HiLo microscopy(5)) to image fluorescently labelled neuronal structures in parallel to SWCNT NIR imaging. This technique is based on speckle illumination and relies on the acquisition of one structured and one uniform illumination image to obtain images deep into tissues with good optical sectioning. Having access to spatially resolved SWCNT diffusivity around specific neuronal structures will provide more precise insights about the heterogeneity of the brain environment.