Development and characterization of optical imaging probes

目前，主要有两个项目： 我们正在与美国国立卫生研究院国家癌症研究所放射肿瘤学分部的Martin Brechbiel合作，将氮空位中心荧光纳米钻石(FND)功能化并进行表征，以用作多模式成像探针。这些荧光粒子在体内和体外跟踪和成像研究中具有吸引力，因为它们是明亮的、不闪烁的荧光团，在绿色(532 Nm)中激发并在远红光谱(700 Nm)中发射，与生物样品中的较短波长相比，具有更好的组织穿透性和信噪比特性。此外，钻石是惰性的，荧光来自氮空位，因此核心颗粒不包含有机染料或其他可能对体内应用造成问题的潜在有毒物质。值得注意的是，FND可以小到5 nm，这也有利于生物相容性和清晰度。该项目的最初目标是制定使FND发挥作用的议定书。随后将对功能化和标记的FND进行体内跟踪、生物分布和清除研究，以建立体内模型的可行性和生物相容性。同时，我们将优化功能化，以促进单分子荧光跟踪应用的体外蛋白质标记。我们最近展示了一种涂层和功能化工艺，可以在溶液中稳定纳米尺寸的FND，并允许它们特定地附着在生物分子上，从而实现高分辨率、高速的单分子运动跟踪。在一个相关的项目中，我们已经证明了FND作为用于高分辨率显微镜的坚固的宽带基准标记的适用性。 我们利用FND荧光独特的磁场依赖性，开发了一种基于钻石荧光磁调制的广域无背景成像技术。有了这项技术，我们已经证明了在正常小鼠的前哨淋巴结荧光测量中，信号与背景的信号比增加了100倍。

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.