Colloidal Nano-Particles for High Resolution Labeling

这项研究的目的是开发一种新的标记技术，用于电子显微镜，使研究人员能够快速、可靠地同时识别和定位单个标本中的多个标记分子物种。这是对PA-00-084“高分辨率电子显微镜技术发展”的回应。该PA的优先事项包括“分析细胞中大分子的空间分布”和“……更好的试剂……对细胞中的大分子进行特殊标记和检测。”高分辨率研究的一个共同要求是多个分子物种的定位和共定位。基于光学显微镜的成像系统解决了这个问题，部分是通过使用发射波长不同的荧光染料。不同的颜色用于明确并同时识别几个不同的标签。现在的电子显微镜研究需要一种能够在分子分辨率水平上同时鉴定多个蛋白质物种的标记系统。目前可用于EM级共定位的技术，使用不同大小的电子“致密”胶体金纳米颗粒。遗憾的是，为了确保粒子大小没有重叠，通常一次只能使用所需分辨率范围内的一到两个粒子大小。立体问题的产生是因为较大颗粒的大小大大增加，并且在不同大小的标签中检测的情况也会出现差异。因此，能够相互识别和定位的不同分子物种的数量极其有限。这里提出的研究将采用两种新的方法。第一种方法是合成尺寸相同但形状不同的胶体纳米粒子，这些粒子在扫描电子显微镜和透射电子显微镜中很容易区分。第二种方法是合成和使用尺寸相似但元素组成不同的胶体纳米粒子。能量过滤电子显微镜将被用来根据与特定元素组成相关的电子能量损失谱来区分不同组成的粒子。这两种方法单独或结合起来，包括一种直接和快速的方法，允许在高空间分辨率水平上同时识别大量不同的分子物种或单个分子中的特定表位。

The purpose of this study is the development of a new labeling technology for use with electron microscopy that will permit investigators to rapidly, reliably, and simultaneously identify and localize multiple labeled molecular species in a single specimen. This is in response to PA-00-084 "Tachnology Development for High Resolution Electron Microscopy." Priorities for this PA include "analysis of spatial distribution of macromolecules in cells," and "..better reagents for.. The specific labeling and detection of macromolecules in cells." A common requirement of high resolution studies is the localization and co- localization of multiple molecular species. Light microscopic based imaging systems solve this problem, in part, through the use of flourescent dyes with differing emission wavelengths. The different colors serve to unambiguously and simultaneously identify several different labels. Labeling systems which permit the simultaneous identification of multiple protein species at molecular levels of resolution are required now for electron microscopic studies. Techniques currently available for EM level co-localization, employ electron "dense" colloidal gold nano-particles of differing sizes. Unfortunately, to ensure that there is no overlap in particle size, generally only one or two particle sizes within the resolution range needed can be employed at once. Steric problems arise because of the substantially greater size of the larger particle and differences in the case of detection in differently sized labels also occurs. Hence the number of different molecular species which can be identified and localized relative to one another is extremely limited. Studies proposed here will employ two new approaches. The first approach is to synthesize colloidal nano-particles of the same size but of multiple different shapes which are readily distinguishable in scanning electron microscopy and in transmission electron microscopy. The second approach is the synthesis of and use of colloidal nano-particles of similar size but of different elemental composition. The Energy Filtering Transmission Electron Microscope will be employed to differentiate the particles of different composition based on the electron energy loss spectra associated with the particular elemental composition. The two approaches, singly or in combination, encompass a direct and rapid methodology which permit simultaneous identification of a substantial number of different molecular species or specific epitopes within individual molecules at high levels of spatial resolution.