A cross-cutting goal for biological engineering of microorganisms and microbe association with plants is dynamic, non-invasive imaging of cellular and subcellular metabolic processes and responses to environmental fluctuations and heterogeneity. The most versatile approach involves the acquisition of intrinsic spectra of molecular ensembles from regions of interest with the sensitivity and discrimination necessary to identify target analytes from within complex cellular environments. To date, all such approaches fail to achieve the ultimate bioimaging goal: dynamic in situ detection and imaging of key metabolic processes in and among living plant and microbial systems, non-destructively and in real-time. Nuclear magnetic resonance (NMR) is one of the most powerful non-invasive methods for identification and characterization of molecules. Conventional NMR instruments permit rapid, quantitative, and reproducible discrimination of biomolecules in complex biologic fluids but require large and expensive superconducting magnets to increase signal. Dynamic nuclear polarization (DNP) can âhyperpolarizeâ nuclear spins at levels that are orders of magnitude greater than thermal polarization allowing operation at lower magnetic fields. Extending the recently discovered optically enabled DNP of 13C in nanodiamonds containing nitrogen-vacancy color centers, we aim to develop novel three-dimensional, in-cell, NMR sensors using targeted nanodiamonds (NDs). 13C nuclear spins, abundantly present in the targeting ND particles, will be employed as NMR sensors of the analyte nuclei in cellular and sub-cellular systems. The ND sensors can be brought in close proximity to sample analytes, allowing amplification of the NMR response by optical hyperpolarization of 13C, while simultaneously spatially localizing the NMR signal sources. Sensor hyperpolarization would allow direct 3D radiofrequency readout from NDs located at up to cm-depth. Hyperpolarized 13C diamond nuclei, multiply dipolar-coupled to each other, open a ready pathway to exploit nuclei entanglement, allowing access to large entangled 13C cluster states in ND to be exploited for NMR sensing. This approach will be integrated into a fluorescent microscope and scanning microcoil employed for RF readout of NMR signals to allow correlation to structural features along with direct optical hyperpolarization. Diamond nanoparticles will be functionalized to target cell surfaces for demonstration of detection of metabolic processes in real time in yeast cel
