Super resolution microscopy techniques are a useful tool to understand some biomolecular mechanisms, particularly in neurons. We have built such a STED (STimulated Emission Depletion) microscope for observing Nitrogen-Vacancy (NV) fluorescent defect in diamond, and have reached a resolution of 50 nm. In the longer term, this instrument will study the macromolecular organization of proteins involved in synaptic plasticity and marked with fluorescent nanodiamonds (ND). In this context, we studied the resolution limit of STED for ND of subwavelength size. Our experiments, conducted with the team of Stefan Hell (Max Planck Institute for Biophysical Chemistry) showed that the STED spot size of an NV in ND could reach 10 nm, which is similar to performances obtained in a macroscopic diamond. We can also resolve several NV centers, which are separated from only ~15 nm in the same ND. These results are in agreement with numerical simulations carried out by the team of Jean-Jacques Greffet (Laboratoire Charles Fabry). In parallel, we have demonstrated the spontaneous internalization of fluorescent ND in primary culture of cortical neurons from mouse embryos, and studied their colocalization with vesicles of the trans-Golgi network. Finally, we started the study of trafficking vesicles containing ND and showed that it depends on the microtubule network. The motion parameters are compatible with those of molecular motors, but we expect them to be different in the case of overexpression of proteins involved in traffic (work in progress).
Development of optical microscopes for super resolution imaging of fluorescent diamond nanocrystals as probes of functional anomalies of neurons