Development of bio electron microscopy under ultra low electron dose conditions

由轻原子组成的生物样品在透射电子显微镜(TEM)中对比度很小，由于电子束辐照容易损坏结构，因此只能容忍较小的电子剂量拍摄图像。此外，由于传统的透射电子显微镜中缺少理想的相位板，聚焦图像显示的对比度很小。因此，生物样品的高分辨率观察是极其困难的，尽管直接观察未染色的分子结构似乎是识别单个分子的局域结构和了解其功能的有前途的方法之一。Ikuta和Kawasaki提出的三维傅立叶滤波法(3DFFM)似乎是实现这一目的的最有前途的方法。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，并将获得的3D傅立叶频谱乘以具有像差校正因子的滤波形状函数。最后对校正后的三维傅里叶光谱进行三维傅里叶逆变换得到相位图像。在预先设定的研究项目中，利用一种称为三维傅立叶滤波的相位重建方法，利用透射电子显微镜观察了脱氧核糖核酸(DNA)纤维的分子结构。尽管分子结构受到电子束辐照的破坏，但由于S/N比的改善和相重建的分辨率提高，特征螺旋结构和相邻碱基对的间距被部分分辨。在球差无像差位相图像中，有时可以观察到单个原子大小的光斑排列成正弦曲线，这似乎是主链上硫原子的对比。

Biological samples that are composed of light atoms show very little contrast in transmission electron microscopy (TEM) and only a small electron dose is tolerable for taking the image because the structures are easily damaged by electron beam irradiation. Furthermore, an in-focus image shows little contrast due to a lack of an ideal phase plate in conventional TEM. Therefore, high-resolution observation of biological samples is extremely difficult, although the direct observation of unstained molecular structures seems to be one of the promising approaches for identifying the localized structures of individual molecules and for understanding their functions. The three-dimensional Fourier filtering method (3DFFM) developed by Ikuta and Kawasaki seems to be the most promising approach for this purpose. In the method, a 3D Fourier transform is performed for a number of recorded through-focus images and the obtained 3D Fourier spectrum is multiplied by a filtering shape function with aberration correction factors. Finally, the phase image is obtained by performing an inverse 3D Fourier transform on the corrected 3D Fourier spectrum.In the preset research project, the molecular structure of deoxyribonucleic acid (DNA) fibers were observed by a phase reconstruction method called three-dimensional Fourier filtering using a transmission electron microscope. The characteristic helical structure and the spacing of adjacent base pairs of DNA were partially resolved due to an improved S/N ratio and resolution enhancement by the phase reconstruction although the molecular structure was damaged by the electron beam irradiation. In the spherical aberration-free phase images, the arrangements of single atom-sized spots forming sinusoidal curves were sometimes observed, which seem to be the contrast originating in the sulfur atoms along the main chains