ELSEVIER Ultramicroscopy 65 (1996) 147-158 ultramicroscopy Method for controlled formation of vitrified films for cryo-electron microscopy Nikolai D. Denkov *, Hideyuki Yoshimura 1, Kuniaki Nagayama 2 Protein Array Project, ERATO, JRDC, Tsukuba Research Consortium, 5-9-1 Tokodai, Tsukuba 300-26, Japan Received 17 May 1996; revised 22 July 1996; accepted 20 August 1996 Abstract 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 ixm 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. I. Introduction Cryo-electron microscopy of thin vitrified films offers the unique possibility for a high resolution structural analysis of biosamples that are not suitable for direct X-ray or NMR investigation [1,2]. Vitrifi- * Corresponding author. Permanent address: Laboratory of Thermodynamics and Physico-chemical Hydrodynamics, Faculty of Chemistry, Sofia University, 1 "James Bouchier" Avenue, 1126 Sofia, Bulgaria. Fax: +359 2 9625643; E-mail: Denkov@Ltph.cit.bg. Present address: Department of Physics, School of Science and Technology, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki 214, Japan. Fax: +89 44 900 0421; E-mail: hyoshi @isc.meiji.ac.jp. 2 Present address: Department of Life Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153, Japan. Fax: + 81 3 5454 4332; E-mail: nagayama@orca.c.u-tokyo.ac.jp. cation ensures excellent structure preservation of hy- drated molecules and biomolecular complexes [3,4]. The vitrified samples do not need staining and fixa- tion reagents which could affect the molecular struc- ture and reduce the resolution. In addition, the radia- tion damage, caused by the electron beam, is de- creased several times due to the low working tem- perature [5-7]. Cryo-microscopy has yielded valu- able structural information for viruses [8-12], mem- brane proteins [13-17], and protein complexes [4,18-22]. The method is of particular use for visual- isation of soft, unstable when dried molecular aggre- gates, like lipid vesicles [23-27], microemulsions [28], and surfactant micelles [29-33]. Two procedures for preparation of vitrified films for cryo-electron microscopy are widely used. In the first, the film is spread on a hydrophilic carbon layer deposited on an electron microscope grid [1,13,34]. 0304-3991/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0304-3991 (96)00065-4