FBS Colloquia No.355JEOL YOKOGUSHI Research Alliance Laboratories

Approach to in situ structural analysis using JEOL Cryo-FIB-SEM and CRYO ARM

JEOL YOKOGUSHI Research Alliance Laboratories has been participating in the AMED BINDS structural analysis support program and providing structural analysis support using JEOL transmission electron cryomicroscope (cryoTEM) CRYO ARM^TM 300 and CRYO ARM^TM 300 II. The main methods include single particle analysis for isolated and purified biological samples, cryo-tomography for cells, tissues, and microorganisms, and micro-ED (Electron Diffraction) for micro-crystals of small molecules and macromolecules. Among these methods, tomography can allow observation of the structures of tissues, cells, and proteins under physiological conditions. However, because the penetration depth of electron is limited, most of cells and tissues cannot be imaged by TEMs as they are, and it is necessary to cut the samples into 100 to 300 nm-thick sections. Cryo-FIB-SEM (cryo-focused ion beam-scanning electron microscope) is an equipment developed to prepare such thin frozen cell sections (lamella) by irradiating vitrified biological specimens with focused ion beams in cryo-SEM. To advance in situ structural analysis of proteins functioning in cells, we have installed a JEOL Cryo-FIB-SEM (JIB-4700Fx) and developed an efficient workflow for the preparation of frozen cell lamellae. The lamellae can be introduced directly into the JEOL CRYO ARM 300 II, enabling high-throughput tomography data collection. In this talk, we will introduce the workflow from sample preparation to data collection, using the data we have collected as examples.