Grid preparation for cryo-electron microscopy.

Once 2D crystals suitable for electron crystallography have been obtained, grid preparation for cryo-EM is a critical step in obtaining high-resolution structural information. Specimens have to be prepared in a manner that prevents dehydration and disruption of the crystals in the vacuum of the electron microscope. Sugar embedding is an effective way to preserve specimens in the native and hydrated state. Preparation of almost perfectly flat specimens is another prerequisite. Imperfect specimen flatness is a crucial problem in the recording of images and diffraction patterns at higher tilt angles because it causes the blurring of spots perpendicular to the tilt axis. In this chapter, we describe the protocols of preparing 2D crystal specimen for electron crystallographical data collection. These protocols cover preparation of a flat carbon support film by sparkless carbon evaporation, sugar embedding using back injection, and the recently developed carbon sandwich technique.