Topological assignment of genetically and biochemically identified macromolecular assemblies with in the living cell requires fast and efficient immobilization of the participating components. Adhesion zones between inner and outer membranes of E. Coli have been observed after aldehyde fixation of cells and electron microscopy. Slam-freezing (cryo-fixation) however revealed no such adhesions, rather a wide open periplasm. Other cryofixations, such as plunge freezing in liquid propane preserved some of the adhesions after UV crosslinking with a photographic flash. To prevent effects of conventional fixation such as molecular diffusion we used rapid (nanosecond) crosslinking with a 266 nm pulse from a NdYAG laser as pretreatment to aldehyde fixation and cryofixation of cell envelope membranes. We examined the structurally ill-defined periplasmic space of three strains of E. Coli. Over 70% of the cells showed a multiplicity of thin (6-8 nm) strands crossing the pe riplasm a nd connecting the inner and outer membrane of the slightly plasmolized cell. These structures have not been described before, even after the use of millisecond UV crosslinking and cryofixation. Presence of connecting strands would support several models proposing protein or protein-lipid bridges facilitating many processes of macromolecular uptake (for example (TonB functions) and members of multi drug resistance.
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