Ogawa Takashi, Gang Geun Won, Thieu Minh Thu, Kwon Hyuksang, Ahn Sang Jung, Ha Tai Hwan, Cho Boklae
Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea.
Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea; Chungnam National University, Daejeon 34134, Republic of Korea.
Micron. 2017 May;96:65-71. doi: 10.1016/j.micron.2016.12.009. Epub 2017 Feb 27.
Utilization of graphene-supporting films and low-voltage scanning transmission electron microscopy (LV-STEM) in scanning electron microscopy (SEM) is shown to be an effective means of observing unstained nanobio materials. Insulin amyloid fibrils, which are implicated as a cause of type II diabetes, are formed in vitro and observed without staining at room temperature. An in-lens cold field-emission SEM, equipped with an additional homemade STEM detector, provides dark field (DF)-STEM images in the low energy range of 5-30keV, together with secondary electron (SE) images. Analysis based on Lenz's theory is used to interpret the experimental results. Graphene films, where the fibrils are deposited, reduce the background level of the STEM images compared with instances when conventional amorphous carbon films are used. Using 30keV, which is lower than that for conventional TEM (100-300keV), together with low detection angles (15-55mrad) enhances the signals from the fibrils. These factors improve image quality, which enables observation of thin fibrils with widths of 7-8nm. STEM imaging clearly reveals a twisted-ribbon structure of a fibril, and SE imaging shows an emphasized striped pattern of the fibril. The LV-STEM in SEM enables acquisition of two types of images of an identical fibril in a single instrument, which is useful for understanding the structure. This study expands the application of SEM to other systems of interest, which is beneficial to a large number of users. The method in this study can be applied to the observation of various nanobio materials and analysis of their native structures, thus contributing to research in materials and life sciences.
研究表明，在扫描电子显微镜（SEM）中利用石墨烯支撑膜和低电压扫描透射电子显微镜（LV-STEM）是观察未染色纳米生物材料的有效手段。胰岛素淀粉样纤维被认为是II型糖尿病的病因之一，可在体外形成并在室温下无需染色即可观察到。配备额外自制STEM探测器的透镜内冷场发射SEM，在5-30keV的低能量范围内提供暗场（DF）-STEM图像以及二次电子（SE）图像。基于伦兹理论的分析用于解释实验结果。与使用传统非晶碳膜的情况相比，沉积有纤维的石墨烯膜降低了STEM图像的背景水平。使用低于传统透射电子显微镜（100-300keV）的30keV以及低检测角度（15-55mrad）可增强来自纤维的信号。这些因素提高了图像质量，从而能够观察到宽度为7-8nm的细纤维。STEM成像清楚地揭示了纤维的扭曲带状结构，而SE成像显示了纤维强调的条纹图案。SEM中的LV-STEM能够在一台仪器中获取同一纤维的两种类型图像，这有助于理解其结构。本研究将SEM的应用扩展到其他感兴趣的系统，对大量用户有益。本研究中的方法可应用于各种纳米生物材料的观察及其天然结构的分析，从而为材料科学和生命科学研究做出贡献。
