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，有两个主要项目： 我们正在与国家癌症研究所，国家癌症研究所的马丁·布雷希比（Martin Brechbiel）合作，在国立卫生研究院成像探针开发中心的罗尔夫·斯文森（Rolf Swenson），用于功能化和表征氮空位中心荧光纳米木质纳米德（FND）（FNDS），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吸引人的荧光颗粒，因为它们是明亮的，非闪烁的荧光团，在绿色（560 nm）中激发并在远红色光谱（680 nm）中发射，与较短的波长相比，它们具有出色的组织穿透性和信噪比。此外，钻石是惰性的，荧光是由氮的空缺产生的，因此核心颗粒不含有机染料或其他潜在有毒物质，这对于体内应用而言是有问题的。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。随后将进行体内跟踪和生物分布以及对功能化和标记的FND的清除研究，以在体内模型中建立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，我们证明了FND作为可用于高分辨率显微镜的稳健，广泛的基准标记。 使用FND作为明亮和稳定的基准标记，可以基于基于连续添加和去除具有相同荧光团标记的特定抗体的新高分辨率多蛋白定位和成像方法，该抗体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的，因此它们需要针对FND提供的超高稳定性。 使用这种技术（称为Madstorm），在固定细胞中确定了25种不同蛋白质的位置和局部结构。