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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)中显示出很小的对比度。由于样品容易受到电子束辐照而损坏，因此只允许较小的电子剂量来获得图像。因此，生物样品的高分辨率观测是极其困难的。此外，由于缺少理想的相位板，聚焦图像显示的对比度很小。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了解其功能的一种有前途的方法。我们提出了一种三维傅立叶滤波方法(3D-FFM)作为一种相位重建方法。在该方法中，对多个记录的直通聚焦图像执行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傅立叶频谱。通过执行逆3D傅里叶变换…，最终获得相位图像校正后的3D傅立叶频谱有更多的变化。由于滤波形状函数只提取出现在埃瓦尔德球面上的线性图像分量，因此图像处理通过排除图像中的非线性图像分量和量子噪声来很好地提高S/N比。这似乎是在分子尺度上观察未染色生物样品不可缺少的优势。在本项目中，利用200千伏高分辨率电子显微镜，采用三维傅立叶滤波方法，在分子尺度上观察了脱氧核糖核酸(DNA)的无像差位相图像。为了避免透射电子显微镜样品支撑膜产生的噪声，我们制备了一种特殊的带有纳米孔的金薄膜。观察到横跨孔洞的DNA分子纤维。获得图像所需的总剂量约为200-300个电子/Å^2。虽然DNA的分子结构似乎受到了严重破坏，但部分确认了螺旋状结构，并部分分解了间隔为3.4-4.5Å的精细结构，这与J.D.Watson和F.H.C.Crick确定的DNA分子模型相似。综上所述，本项目成功地建立了超分辨生物相透射电子显微镜。较少