Development of Fully Automated Lamellae Milling Scheme by Using the Cryo-FIB-SEM

Abstract

Cryo-electron tomography enables us to acquire the three-dimensional structure of quick-frozen specimens within a few nanometers spatial resolution. However, because of the limited penetration of the electron beam, cryo-EM observation of cellular specimens is restricted to only the periphery. To enable cryo-EM to access deep structures such as the nucleus, cryo-FIB-SEM has been developed for micromachining of the vitreous cellular specimens. The operation of cryo-FIB-SEM for the lamella preparation is a little bit complicated due to the multiple settings and handling. Therefore, the development of an automated lamella preparation scheme is required for stable lamellae preparation in daily experiments, especially for beginners. In this paper, we will introduce our fully automated lamella milling scheme using the cryo-FIB-SEM.