Colloidal Nano-Particles for High Resolution Labeling

本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，同时识别和定位多个标记的分子物种在一个单一的标本。 这是对PA-00-084“高分辨率电子显微镜技术开发”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“...更好的试剂细胞内大分子的特异性标记和检测。“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通过使用具有不同发射波长的荧光染料部分地解决了这个问题。 不同的颜色用于明确地同时识别几个不同的标签。 标记系统，允许同时识别多个蛋白质种类的分子水平的分辨率，现在需要的电子显微镜研究。 目前可用于EM水平共定位的技术采用不同尺寸的电子“致密”胶体金纳米颗粒。 不幸的是，为了确保粒度没有重叠，通常一次只能使用所需分辨率范围内的一种或两种粒度。 空间问题的出现是因为较大颗粒的尺寸明显较大，并且在不同尺寸的标记物中检测的情况下也会出现差异。 因此，可以被识别和相对于彼此定位的不同分子种类的数量是极其有限的。这里提出的研究将采用两种新的方法。 第一种方法是合成具有相同尺寸但具有多种不同形状的胶体纳米颗粒，其在扫描电子显微镜和透射电子显微镜中容易区分。 第二种方法是合成和使用具有相似尺寸但不同元素组成的胶体纳米颗粒。 将采用能量过滤透射电子显微镜，根据与特定元素组成相关的电子能量损失谱区分不同组成的颗粒。 这两种方法，单独或组合，包括一个直接和快速的方法，允许同时识别大量的不同的分子种类或特定的表位在高水平的空间分辨率的单个分子。

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.