Development and characterization of optical imaging probes

目前，有两个主要项目： 我们正在与美国国家癌症研究所放射肿瘤学分支的Martin Brechbiel和美国国立卫生研究院成像探针开发中心的Rolf Swenson合作，对氮空位中心荧光纳米金刚石（FND）进行功能化和表征，以用作多模态成像探针。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，因为它们是明亮的、不闪烁的荧光团，其在绿色（560 nm）中激发并在远红光谱（680 nm）中发射，与较短波长相比，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。 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。 使用FND作为明亮和稳定的基准标记物，实现了一种新的高分辨率多蛋白定位和成像方法，该方法基于顺序添加和去除标记有相同荧光团的特异性抗体，该荧光团非常适合高分辨率测量（STORM）。 由于这些实验是基于时间多路复用，它们需要FND提供的超高漂移稳定性。 利用这种技术（术语为MADstorm），已经在固定的细胞中确定了25种不同蛋白质的位置和局部结构。

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.