Development and characterization of optical imaging probes

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Currently, there are two main projects: We are collaborating with Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, and Rolf Swenson at the Imaging Probe Development Center, 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 (560 nm) and emit in the far red spectrum (680 nm), which has superior tissue penetration and signal-to-noise characteristics compared with shorter wavelengths. 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. In a related project, we have demonstrated the applicability of FNDs as robust, broad-band fiducial markers for use in high-resolution microscopy. Using FNDs as bright and stable fiducial markers enabled a new high resolution multi-protein localization and imaging methodology based on the sequential addition and removal of specific antibodies labeled with an identical fluorophore that is well suited to high resolution measurements (STORM). Since these experiments are based on time multiplexing they require ultra-high stability against drift, which the FNDs provide. With this technique (Termed MADstorm) the locations and local architecture of 25 different proteins have been determined in a fixed cell.