Development and characterization of optical imaging probes

目前，主要有两个项目： 我们正在与国家癌症研究所放射肿瘤学分部的Martin Brechbiel和国家卫生研究院成像探针开发中心的Rolf Swenson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(560 Nm)激发并在远红光谱(680 Nm)发射，与较短的波长相比，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。 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。使用FND作为明亮和稳定的基准标记，实现了一种新的高分辨率多蛋白质定位和成像方法，该方法基于顺序添加和移除标记有相同荧光团的特定抗体，该抗体非常适合高分辨率测量(STORM)。由于这些实验是基于时分复用的，它们需要针对漂移的超高稳定性，而FND提供了这一点。利用这项技术(称为MADStorm)，25种不同蛋白质在固定细胞中的位置和局部结构已经确定。

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.