Currently, there are two main projects: We are collaborating with Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health on functionalizing and characterizing nitrogen vacancy center fluorescent nanodiamonds (FNDs) for use as multi-modal imaging probes. These are attractive fluorescence particles for in vivo and in vitro tracking and imaging studies as they are bright, non-blinking fluorophores that are excited in the green (532 nm) and emit in the far red spectrum (700 nm), which has superior tissue penetration and signal-to-noise characteristics compared with shorter wavelengths in biological samples. Moreover, diamond is inert and the fluorescence arises from the nitrogen vacancy so the core particle contains no organic dyes or other potentially toxic material that would be problematic for in vivo applications. Remarkably, the FNDs can be as small as 5 nm, which is also advantageous for biocompatibility and clearing. The initial goal of the project is to establish protocols to functionalize FNDs. This will be followed by in vivo tracking and biodistribution and clearing studies of the functionalized and labeled FNDs to establish feasibility and biocompatibility in an in vivo model. In parallel we will optimize the functionalization to facilitate in vitro protein labeling for single-molecule fluorescence tracking applications. We have recently demonstrated a coating and functionalization process that stabilizes nm sized FNDs in solution and allows them to be specifically attached to bio-molecules, enabling high-resolution, high speed single-molecule tracking of motion. In a related project, we have demonstrated the applicability of FNDs as robust, broad band fiducial markers for use in high-resolution microscopy. We have exploited the unique magnetic field dependence of FND fluorescence to develop a wide-field background free imaging technique based on magnetic modulation of the diamond fluorescence. With this technique we have demonstrated a 100-fold increase in signal to background for fluorescence measurements of the sentinel lymph-nodes in intact mice.
