Development and characterization of optical imaging probes

研究进展： 该项目与以下机构合作： 马丁·W Brechbiel放射肿瘤学分支，国立癌症研究所，国立卫生研究院。 James Sellers和Yasuharu Takagi，美国国立卫生研究院国家心肺血液研究所分子生理学实验室。 和 Gopalakrishnan Balasubramanian 马克斯·普朗克生物物理化学研究所 目前，有两个主要项目： 第一个项目涉及使用单分子技术来测量多模式体内成像探针的光学性质和特征。一类颗粒由二氧化硅壳中的铁芯组成，在合成过程中有机荧光团被封装在二氧化硅壳中。由于颗粒的复杂性，事实证明很难可靠地确定掺入的荧光团的平均数量以及使用传统系综测量技术标记的颗粒比例。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，我们在实验室中使用定制的基于棱镜的全内反射荧光（TIRF）显微镜和单分子成像能力来测量这些合成颗粒的荧光特性。通过测量单个颗粒的荧光作为时间的函数，我们能够直接观察颗粒中单个染料的光漂白。将荧光降低至背景水平的光漂白步骤的数量指示每个颗粒中的染料数量。强度的每个离散降低的幅度指示各个染料的亮度，而光漂白步骤之间的时间直接提供光漂白速率或染料的光稳定性。宽视场单分子TIRF装置允许收集数千个单独的荧光痕迹，提供出色的统计样本。在原理验证实验中，我们能够确定嵌入Alexa 555或Cy 5.5染料的15 nm铁核二氧化硅颗粒的每个颗粒的平均染料数。荧光迹线的进一步分析揭示，染料的包封增加了其荧光强度并增加了其光稳定性，如与游离染料相比更亮的发射和更长的漂白时间所证明的。每个颗粒的染料数的分布很好地描述了泊松分布。这使我们能够推断被标记的粒子的分数，这是很难确定的合奏方法。我们预计，这种相对简单，强大和快速的技术，需要微量的材料将普遍感兴趣的纳米粒子和分子成像领域。在正在进行的实验中，我们正在测试的统计模型的标记，通过直接确定荧光颗粒的分数，通过单粒子成像和TIRF显微镜的组合。 这一原理性工作的证明最近发表，我们正在将其扩展到量化荧光标记的化疗抗体的化学计量和标记效率。 这项研究的长期目标是建立工具，技术和方法，以准确有效地表征生物医学应用中使用的纳米材料的特性，这是该领域尚未满足的需求。 在第二个项目中，我们正在与Martin Brechbiel的放射肿瘤学分支，国家癌症研究所，国立卫生研究院和Gopalakrishnan Balasubramanian，马克斯普朗克生物物理化学研究所哥廷根，德国的功能化和表征氮空位荧光纳米金刚石（FND）用作多模态成像探针。这些对于体内和体外跟踪和成像研究来说是有吸引力的无颗粒荧光，因为它们是明亮的、不闪烁的荧光团，在绿色（532 nm）中激发并在远红光谱（700 nm）中发射，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。该项目的初始目标是建立功能化5 - 10 nm FND并将钆螯合物作为MR造影剂的方案。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。 最后，我们与国家心肺血液研究所分子生理学实验室的Jim Sellers和Yasuharu Takagi合作，正在测试FND在光学捕获实验中的使用。

Research in Progress: This project is in collaboration with: Martin W. Brechbiel Radiation Oncology Branch, National Cancer Institute, National Institutes of Health. James Sellers and Yasuharu Takagi, Laboratory of molecular physiology, National Heart, Lung, and Blood Institute, National Institutes of Health. and Gopalakrishnan Balasubramanian Max Planck Institute for Biophysical Chemistry Currently, there are two main projects: The first project involves the use of single-molecule techniques to measure the optical properties and characteristics of multimodal in vivo imaging probes. One class of particles consists of an iron core in a silica shell in which organic fluorophores are encapsulated during the synthesis process. Because of the complex nature of the particles, it has proved difficult to reliably determine the average number of incorporated fluorophores and the fraction of particles that are labeled using traditional ensemble measurement techniques. In collaboration with Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, we used the custom built prism-based total internal reflection fluorescence (TIRF) microscope and single-molecule imaging capabilities in our lab to measure the fluorescence properties of these synthesized particles. By measuring the fluorescence from single particles as a function of time, we are able to directly observe the photo-bleaching of individual dyes in the particles. The number of photo-bleaching steps that reduce the fluorescence to background levels is indicative of the number of dyes in each particle. The magnitude of each discrete decrease in intensity is indicative of the brightness of the individual dyes, whereas the time between photo-bleaching steps directly provides the photo-bleaching rate, or the photo-stability of the dye. The wide-field single-molecule TIRF set-up allowed the collection of thousands of individual fluorescence traces, providing excellent statistical samples. In a proof-of-principle experiment, we were able to determine the average number of dyes per particle for 15 nm iron core silica particles embedded with either Alexa 555 or Cy 5.5 dyes. Further analysis of the fluorescence traces revealed that encapsulation of the dye increased its fluorescence intensity and increased its photo-stability as evidenced by brighter emission and longer bleaching times as compared with free dye. The distribution of the number of dyes per particle was well described by a Poisson distribution. This allowed us to infer the fraction of particles that were labeled, which is difficult to ascertain by ensemble methods. We anticipate that this relatively simple, robust and rapid technique that requires trivial amounts of material will be of general interest to the nanoparticle and molecular imaging fields. In ongoing experiments we are testing the statistical models of labeling by directly determining the fraction of fluorescent particles, through a combination of single particle imaging and TIRF microscopy. This proof of principle work was recently published and we are extending it to quantify the stoichiometry and labeling efficiency of fluorescently tagged chemotherapeutic antibodies. The long term goal of this research is the establishment of tools, techniques and methodologies to accurately and efficiently characterize the properties of nanomaterials employed in bio medical applications, which is an established unmet need in this field. In a second project we are collaborating with Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health and Gopalakrishnan Balasubramanian, Max Planck Institute for Biophysical Chemistry Gottingen, Germany on functionalizing and characterizing nitrogen vacancy fluorescent nanodiamonds (FNDs) for use as multi-modal imaging probes. These are attractive fluorescence particless 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 5-10 nm FNDs and attach gadolinium chelates as MR contrast agents. 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. Fianlly, in collaboration with Jim Sellers and Yasuharu Takagi in the Laboratory of Molecular Physiology in the National Heart, Lung, and Blood Institute, we are testing the use of FNDs in optical trapping experiments.