New Single Molecular Detection System from Three-Dimensional Tracking of Single Nanocrystals using Scanning Electron Microscope

Abstract

Recent technological progress in dynamical observations of individual functional single protein molecules in living cell has been achieved with several single molecular techniques and systems. In order to improve monitoring precisions and stability of the signal intensity from single molecular units under physiological conditions, we have proposed that single molecular techniques using shorten wavelength, for example, X-rays, electrons, neutron, and other accelerated ion probes. In this work, we demonstrate three-dimensional tracking of single nanocrystals using Scanning Electron Microscope. We called Diffracted Electron Tracking (DET).Diffracted X-Ray tracking (DXT) [1] has been developed for obtaining the information about the dynamics of single molecules. This method can observe the rotating motion of an individual nanocrystal, which is linked to specific sites in single protein molecules, using a time-resolved Laue diffraction technique. This method needs a very strong X-ray source, such as the SPring-8, so we began to develop a compact instrument for monitoring the rotation of the single protein molecules, using the electron beam instead of the X-ray.Instead of the Laue diffraction using white X-ray, the Electron Back-Scattered Diffraction Pattern (EBSP) is adopted to monitor the crystal orientation of the nano-crystals linked to the single protein molecules. For this purpose, it is necessary to realize (1) wet cell with very thin sealing film, (2) EBSP system with high sensitivity, (3) damage-less electron irradiation technique and (4) perfect gold nano-crystals.[1] H. Shimizu, M. Iwamoto, F. Inoue, T. Konno, Y.C. Sasaki, S. Oiki: Cell 132, 67-78 (2008).[2] T.Sagawa, T.Azuma, Y. C. Sasaki, Biochem. Biophy. Res. Commun. 335, 770-775 (2007).