Development and characterization of optical imaging probes

目前，有两个主要项目： 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支的Martin Brechbiel合作，对氮空位中心荧光纳米金刚石（FND）进行功能化和表征，以用作多模式成像探针。这些是用于体内和体外跟踪和成像研究的有吸引力的荧光颗粒，因为它们是在绿色（532 nm）中激发并在远红光谱（700 nm）中发射的明亮、不闪烁的荧光团，与生物样品中的较短波长相比，其具有上级组织穿透和信噪比特性。此外，金刚石是惰性的，并且荧光由氮空位产生，因此核颗粒不含有机染料或其他可能有毒的材料，这些材料对于体内应用是有问题的。值得注意的是，FND可以小至5 nm，这对于生物相容性和清除也是有利的。该项目的最初目标是建立功能化FND的协议。随后将进行功能化和标记的FND的体内追踪和生物分布以及清除研究，以确定体内模型的可行性和生物相容性。同时，我们将优化功能化，以促进体外蛋白质标记的单分子荧光跟踪应用。我们最近展示了一种涂层和功能化过程，该过程可以稳定溶液中的纳米级FND，并使它们能够特异性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稳健的宽带基准标记的适用性。 我们利用FND荧光的独特磁场依赖性，开发了一种基于金刚石荧光磁调制的宽场无背景成像技术。 用这种技术，我们已经证明了一个100倍的信号增加到背景的荧光测量的前哨淋巴结在完整的小鼠。

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.