SynECS started with the development of an innovative microscopy technique to correctly visualize synapses within a thick brain slice. This technique, called HiLo microscopy, was key to correlate morphological and diffusional properties of the brain ECS around fluorescently labelled synaptic regions and was used to achieve simultaneous visible and near infrared images. HiLo microscopy is based on speckle illumination and allow acquisition of optically sectioned images from a standard widefield microscope.
HiLo imaging was indeed used to correlate the position of single-walled carbon nanotubes (SWCNTs) with fluorescently-labelled synaptic regions. SWCNTs are thin and long carbon-based nanowires with excellent mechanical and photophysical properties. Indeed, these nanoprobes have attracted particular attention for deep-tissue microscopy, due to their unique brightness, photostability, and spectral imaging range in the near infrared region. SWCNTs were initially tracked in organotypic and acute brain slices up to 100 μm deep using a widefield microscopy approach at millisecond timescale. Super-localization analysis showed that the ECS is a maze of polymorphic channels with widths in the order of 50-500 nm. Importantly, tracking of individual SWCNTs could also provide simultaneous measurements of the local ECS diffusivity environment. Indeed, specific rheological properties of the space, ranging from low to high local diffusivity, were measured in brain slices. SWCNT trajectories were also correlated with the synaptic extracellular environment, showing a modulation of the diffusional NT rates depending on different conditions of neuron stimulation.
SWCNT super-localization analysis was also applied to a mouse model of α-synuclein-induced neurodegeneration. Our group firstly performed an integrative study on pathological animals, exploring the extracellular microenvironment as a whole in adult brain tissue and giving novel insights on Parkinson’s disease effects in the brain. The study revealed poor correlation between local ECS width and nanoscale diffusion, suggesting that the diffusive inhomogeneities were not only driven by geometrical factors, but also by the molecular composition of the space. Importantly, our study pointed out to hyaluronan as major actor for the local variations in ECS diffusivity properties, opening up new hypothesis for the connection between ECS and brain pathologies.
SynECS was supported by a strong international visibility throughout the 2 years of funding. The project was presented in 4 different international conferences, including Photonics West and SPIE photonics Europe, the largest biophotonics, biomedical optics, and imaging conferences in the World. Moreover, results from the project have been already published in 3 different high-impact journals (including Nature Communication). Two additional publications are on current preparation for imminent submission.
