Method for controlled formation of vitrified films for cryo-electron microscopy

A novel method for controlled formation of large, unsupported vitrified films for cryo-electron microscopy is described. Liquid films of diameter between 40 and 150 μm are formed in the central hole of a thin, disc-shaped specimen holder. The bulk liquid, surrounding the film, is connected through a glass capillary to a microsyringe, which allows the fine control of the film radius. The processes of liquid film formation and subsequent thinning are observed in reflected, monochromatic light by means of a CCD-camera equipped with a long-focus magnifying lens. The film thickness is precisely determined from the intensity of light, reflected from the film. In this way the vitrification can be accomplished at desired film thickness and radius. The control of the liquid film radius and thickness is utilized to produce well ordered two-dimensional arrays of monodisperse particles (latex spheres and protein/lipid vesicles) just before the film freezing, without need of specific binding agents. At suitable conditions ordered bilayers and triple-layers of particles are obtained.