Main results of this work include, integration of Nitrogen Vacancy (NV) rich nanoscale diamond sensors into electronspun polymer nanofibers to replicate the physiological niche, particularly important for neuronal and stem cell culture. The electrospun fibre diamond sensing platform supported the growth of neural stem cells and their differentiated progeny and enabled collection of ODMR signals is a physiologically relevant niche. Experimental studies investigating cells cultured on planar diamond chips were also performed. The emphasis of this work was detection of paramagnetic contrast agents used in conventional MRI. NV sensing protocols were applied to image the extracellular pooling of contrast agents and internalisation of contrast agents in cells.
Sensing protocols were designed and exemplar studies performed to study cellular respiratory processes (live cells and mitochondrial extracts), discrimination of diseased cells from healthy cells based on detection of spin active species (Wild type Alzheimer’s cells vs Alzheimer’s cells with mutations), validation of detection of
hydroxyl radicals from cells (NV sensing protocols vs Electron Paramagnetic Resonance (EPR)) and super resolution imaging of nanodiamonds conjugated with antibodies for specific cell membrane proteins. Further studies demonstrated the successful integration of plannar diamond chips with functioning neuronal networks, as verified by conventional calcium reporters.
Instrumentation for optical detection of magnetic resonance and localisation of transmembrane receptors was developed. Here a new approach to localisation microscopy based on the intrinsic blinking of NV photoluminescence in a narrow wavelength emission band in physiologically relevant buffers used developed. Assessment of TMPs in fixed cells and tissue has been carried out using this imaging technique. Specifically, tissue studies were performed to attain single molecule localisation of ryanodine receptors in rat skeletal muscle sections. Additionally, cell studies enabled imaging of nuclear pore complexes, the multiprotein channels that connect the nucleus and cytoplasm of eukaryotic cells.
A new experimental system was developed enabling correlative optically detected magnetic resonance (ODMR) studies with electron microscopy. This approach involves drop casting nanodiamonds on a transmission electron microscope (TEM) finder grid such that samples of interest can be evaluated both at an atomic scale via TEM and their functional and paramagnetic profile assessed via NV based sensing. Several examplar studies were performed that demonstrated the ability to detect free radicals from nanoscale assemblies that will translate well for the study of sub-cellular components and transmembrane proteins.
