Morphologically, glutaraldehyde-fixed and -dried intermediate filaments (IFs) appear flexible, and with it width of 8 12 nm when observed by electron microscopy. Sometimes, the filaments are even unraveled on the carbon-coated grid and reveal a protofilamentous architecture. In this study, we have used atomic force microscopy to further investigate the morphology of IFs in a more physiological environment. First, we have imaged hydrated glutaraldehyde-fixed IFs adsorbed to a graphite support. in such conditions, human vimentin and desmin IFs appeared compact with a height or 5 8 nm and revealed either a beading repeat or a helical morphology. Second, we have analyzed the architecture of hydrated vimentin, desmin, and neurofilament IFs adsorbed to mica, graphite, and hydrophilic glass without the presence of fixative. On mica, vimentin IFs had it height of only 3 5 nm, whereas desmin IFs appeared as 8 10nm height filaments with a helical twist. Neurofilaments were 10 -12nm in height with a pronounced 30 50nm beading along their length. On graphite, the different IFs were either not adsorbing properly or their architecture was modified yielding, for example. broad. flattened filaments. Finally, hydrophilie glass Was the surface which seemed to best preserve the architecture of the three IFs, even if, in some cases, unraveled vimentin filaments were observed oil this support, These results are straightening the idea that mature IFs are dynamic polymers in vitro and that IFs can be distinguished front each others by their physicochemical properties. (c) 2005 Elsevier Inc. All rights reserved.