Development of Super Resolution Bio-Phase Transmission Electron Microscopy

由光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。由于样品容易被电子束照射破坏，因此只允许一个小的电子剂量获得图像。因此，对生物样品进行高分辨率观察是极其困难的。此外，由于缺乏理想的相位板，焦内图像的对比度很小。然而，直接观察未染色的分子结构似乎是一种很有前途的方法，可以识别单个分子的片段并了解其功能。提出了一种三维傅里叶滤波方法（3D-FFM）作为相位重建方法。该方法对记录的多幅透焦图像进行三维傅里叶变换，并对得到的三维傅里叶谱应用带像差校正因子的滤波形状函数。最后通过对校正后的三维傅里叶谱进行反三维变换得到相位图像。由于滤波形状函数只提取出现在埃瓦尔德球上的线性图像分量，因此通过排除图像中的非线性图像分量和量子噪声，可以很好地提高图像的信噪比。这似乎是在分子尺度上观察未染色的生物样品不可或缺的优势。本项目利用200 kV高分辨率透射电子显微镜（TEM），采用三维傅里叶滤波方法，在分子水平上观察了脱氧核糖核酸（DNA）的无像差相位图像。为了避免TEM样品的支撑膜产生的噪声，我们制备了一种特殊的带纳米孔的金薄膜。观察到DNA分子纤维悬挂在孔上。获得图像所需的总剂量约为200-300电子/Å^2。虽然DNA的分子结构似乎受到了严重的破坏，但螺旋状结构得到了部分确认，间隔为3.4-4.5Å的精细结构在螺旋状结构中得到了部分分解，这与J.D.Watson和f.h.c.c crick确定的DNA分子模型相似。综上所述，本项目可成功建立超分辨生物相透射电镜。少

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