Speaker: Or Shemesh, Ph.D.
Affiliation: Simons Postdoctoral Fellow, Synthetic Neurobiology Group, MIT

Talk Title: Membrane-targeted nanodiamonds for labeling neurons and sensing their activity
Abstract: Nanodiamonds (NDs) containing fluorescent Nitrogen-Vacancy (NV) defect centers are potentially useful tools for probing biological phenomena. This is attributed to their high photostability, low cytotoxicity, brightness, small size (as small as 5nm) and sensitivity to their local environment including electric and magnetic fields. Of particular interest to neuroscience is achieving voltage sensing using the fluorescence of NVs in NDs (NV/NDs). To this end one should produce: (a) voltage sensitive NV/NDs and (b) labeling of neuronal membranes with NDs.

In the first part of our study we found that the charge state and fluorescence dynamics of a single NV center can be controlled by an externally applied voltage in an electrochemical cell. We detected NV fluorescence modulation for voltage changes down to 100 mV with a single NV and down to 20 mV with multiple NV centers. These results offer new methods for optical recording of neuronal action potentials. In the second part of the project we targeted NV/NDs to the site of neural sensing: the membrane.  For this purpose we used micelles to deliver hydrophobic NV/NDs for stable delivery. We first functionalized the surface of 100 nm and 40 nm NV/NDs with octyl chains using triethoxy(octyl)silane (OTES). We then encapsulated these hydrophobic silanized NV/NDs (Si-NV/NDs) in PEG conjugated phosphoethanolamine (PEG-PE) micelles. Cell membrane delivery to primary hippocampal neurons was then achieved via a short 5-10 minute incubation with these micellated NV/NDs in neural culture. The NV/NDs followed the morphology of neurons, from soma through small neuritis, as verified by epi-fluorescence microscopy. Co-localization studies with cell membrane dyes confirmed the retention of above 90% the Si-NV/NDs on the membrane for at least 6-12 hours. Using TEM and Scanning Electron Microscopy (SEM) we found that hydrophobic NDs form a mono-disperse layer on the membrane while hydrophilic NDs form aggregates on top of cells. We then confirm good labeling detection of Si-NV/NDs on neurons using two-photon imaging in-vivo. In summary, we show micelle mediated delivery of hydrophobic, monodisperse, bright NV/NDs to cellular membranes for hours-long labelling of neurons. We are now in the process of testing activity sensing in neurons by NV/NDs.