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，Neil Billington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成像探针的光学性质和特征。 由于用于分子成像的纳米颗粒的复杂性质，已经证明难以可靠地确定掺入的荧光团的平均数量和使用传统的整体测量技术标记的颗粒的分数。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设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，我们确定了嵌入Alexa 555或Cy 5.5染料的15 nm铁芯二氧化硅颗粒的每个颗粒的平均染料数。荧光迹线的分析揭示，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。从每个颗粒的染料的数量的分布，我们可以推断出被标记的颗粒的分数，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。我们正在扩展这项技术，以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，对氮空位荧光纳米金刚石（FND）进行功能化和表征，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（600-700 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。 我们已经开发了一种涂层和功能化过程，其使溶液中的纳米尺寸的FND稳定，并提供了一种容易的功能化方案，使它们能够特异性地连接到生物分子上。 这些FND探针能够在不确定的时间段内实现高分辨率、高速的三维单分子跟踪，我们预计它们将被广泛采用作为单分子跟踪探针。 在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合作的第三个项目中，我们开发了一种新的基于FND的相位敏感无背景成像技术。 FNDS的荧光发射表现出弱的磁场强度依赖性。 我们利用这一现象来选择性地调制FND的发射，并使用相敏技术从背景荧光和噪声中提取金刚石荧光发射。 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，Neil Billington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, Neil Billington 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 imaging probes. Because of the complex nature of nano particles used for molecular imaging, 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 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 allows the collection of thousands of individual fluorescence traces, providing excellent statistical samples. In a proof-of-principle experiment, we determined the average number of dyes per particle for 15 nm iron core silica particles embedded with either Alexa 555 or Cy 5.5 dyes. 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. From the distribution of the number of dyes per particle we could 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. We are extending this technique 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 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 (600-700 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. We have developed a coating and functionalization process that stabilizes nm sized FNDs in solution and provides a facile functionalization scheme that allows them to be specifically attached to bio-molecules. These FND probes enable high-resolution, high speed three dimensional single-molecule tracking over indefinite periods and we anticipate that they will be widely adopted as single-molecule tracking probes. In a third project in collaboration with Martin Brechbiel of the Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, we have developed a new phase-sensitive background-free imaging technique based FNDs. The fluorescence emission of FNDS shows a weak magnetic-field strength dependence. We exploit this phenomenon to selectively modulate the emission of FNDs and use phase sensitive techniques to extract the diamond fluorescence emission from background fluorescence and noise. Fianlly, in collaboration with Jim Sellers, Neil Billington, 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.