Development and characterization of optical imaging probes

光学成像探头的开发和表征

• 批准号: 10008794
• 负责人: Keir Neuman
• 金额: $ 33.69万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10008794
• 关键词: Address; Antibodies; Architecture; Binding; Biodistribution; Biological; Blinking; Cells; Characteristics; Chemical Structure; Development; Diamond; Dyes; Excision; Fluorescence; Goals; Image; In Vitro; Label; Location; Measurement; Methodology; Methods; Microscopy; Multimodal Imaging; National Cancer Institute; Nitrogen; Noise; Nucleosome Core Particle; Optics; Penetration; Property; Proteins; Protocols documentation; Radiation Oncology; Resolution; Scheme; Side; Signal Transduction; Techniques; Testing; Time; Tissues; United States National Institutes of Health; base; biomaterial compatibility; experimental study; fluorescence imaging; fluorophore; imaging approach; imaging probe; imaging study; improved; in vivo; in vivo Model; in vivo evaluation; in vivo imaging; interest; molecular imaging; nano; nanodiamond; nanoparticle; optical imaging; particle; single molecule

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.

目前，主要项目有两个： 我们正在与美国国家癌症研究所放射肿瘤科的 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，对用作多模态成像探针的氮空位中心荧光纳米金刚石 (FND) 进行功能化和表征。这些荧光颗粒对于体内和体外跟踪和成像研究来说是有吸引力的荧光颗粒，因为它们是明亮的、不闪烁的荧光团，在绿光 (560 nm) 中激发并在远红光谱 (680 nm) 中发射，与较短的波长相比，其具有优异的组织穿透性和信噪比特性。此外，金刚石是惰性的，荧光是由氮空位产生的，因此核心颗粒不含有机染料或其他可能对体内应用造成问题的潜在有毒材料。值得注意的是，FND 可小至 5 nm，这也有利于生物相容性和透明化。该项目的最初目标是建立 FND 功能化协议。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。与此同时，我们将优化功能化，以促进单分子荧光追踪应用的体外蛋白质标记。 在一个相关项目中，我们证明了 FND 作为稳健的宽带基准标记在高分辨率显微镜中的适用性。 使用 FND 作为明亮且稳定的基准标记，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用相同荧光团标记的特定抗体，非常适合高分辨率测量 (STORM)。 由于这些实验基于时间复用，因此需要超高的漂移稳定性，而 FND 可以提供这一点。 通过这项技术（称为 MADstorm），已经确定了固定细胞中 25 种不同蛋白质的位置和局部结构。