Development of bio electron microscopy under ultra low electron dose conditions

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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