Development of Super Resolution Bio-Phase Transmission Electron Microscopy

超分辨率生物相透射电子显微镜的研制

基本信息

批准号：
15360036
负责人：
TAKAI Yoshizo
金额：
$ 7.87万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15360036/
关键词：
Transmission Electron Microscope DNA Phase Electron Microscope Three Dimensional Fourier Filtering unstained biological sample Aberration-Free observation Spherical aberration Correction 位相像観察

项目摘要

Biological samples that are composed of light atoms show very little contrast in transmission electron microscopy(TEM). Only a small electron dose is allowed to obtain the image because the samples are easily damaged by the electron beam irradiation. Therefore, high resolution observation of biological samples is extremely difficult. In addition, an in-focus image shows little contrast due to the lack of an ideal phase plate. However, direct observation of unstained molecular structures seems to be a promising approach in identifying the segments of individual molecules and to understand their functions. We proposed a three-dimensional Fourier filtering method (3D-FFM) as a phase reconstruction method. In this method, a 3D Fourier transform is performed for a number of recorded through-focus images and a filtering shape function with aberration correction factors is applied to the obtained 3D Fourier spectrum. The phase image is finally obtained by performing an inverse 3D Fourier tran … More sform on the corrected 3D Fourier spectrum. Since the filtering shape function extracts only the linear image components that appear on the Ewald sphere, the image processing works well for improving the S/N ratio by excluding the non-linear image components and quantum noise in the images. This seems to be an indispensable advantage for observing unstained biological samples on a molecular scale.In the present project, aberration-free phase images of deoxyribonucleic acid(DNA) were observed on a molecular scale level by a three-dimensional Fourier filtering method using a 200 kV high-resolution transmission electron microscope(TEM). To avoid the noise produced by the supporting film of the TEM sample, we prepared a special gold thin film with nano-sized holes. The DNA molecular fibers hanging across the holes were observed. The total dose required to obtain the image was about 200-300 electrons/Å^2. Although the molecular structure of the DNA seems to be heavily damaged, a helical-like structure is partially confirmed and fine structures with intervals of 3.4-4.5Å are partially resolved in the helical-like structure, which is similar to the DNA molecular model determined by J.D.Watson and F.H.C.Crick. In conclusion, super resolution bio-phase transmission electron microscopy could be successfully established by the present project. Less

由轻原子组成的生物样品在透射电子显微镜（TEM）中显示的对比度很小，因为样品很容易被电子束照射损坏，因此需要高分辨率观察。此外，由于缺乏理想的相位板，聚焦图像几乎没有对比度。然而，直接观察未染色的分子结构似乎是识别单个分子片段和理解的一种有前途的方法。我们提出了一个三维的功能。作为相位重构方法的傅里叶滤波方法（3D-FFM）在该方法中，对多个记录的离焦图像执行3D傅里叶变换，并将具有像差校正因子的滤波形状函数应用于所获得的3D傅里叶频谱。由于滤波形状函数仅提取 Ewald 上出现的线性图像分量，因此最终通过对校正的 3D 傅立叶频谱执行逆 3D 傅立叶变换来获得相位图像。通过排除图像中的非线性图像成分和量子噪声，图像处理可以很好地提高信噪比，这似乎是在分子尺度上观察未染色的生物样本的一个不可或缺的优势。利用200 kV高分辨率透射电子显微镜（TEM），通过三维傅里叶滤波方法在分子尺度水平上观察脱氧核糖核酸（DNA）的无像差相位图像。在 TEM 样品的支撑膜上，我们制备了带有纳米尺寸孔的特殊金薄膜，观察了悬挂在孔上的 DNA 分子纤维，获得图像所需的总剂量约为 200-300 个电子/Å^2。尽管DNA的分子结构似乎被严重破坏，但部分证实了螺旋状结构，并且螺旋状结构中部分解析了间隔为3.4-4.5Å的精细结构，这与确定的DNA分子模型相似经过J.D.Watson 和 F.H.C.Crick 的结论是，本项目可以成功建立超分辨率生物相透射电子显微镜。