Individual filamentous phage imaged by electron holography
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An in-line electron hologram of an individual f1.K phage was recorded with a purpose-built low energy electron point source (LEEPS) microscope. Cryo-microscopic methods were employed to prepare the specimen so that a single phage could be presented to the coherent low energy electrons: An aqueous phage suspension was applied to a thin carbon membrane with micro-machined slits. The membrane was rapidly cooled to freeze the remaining water as an amorphous ice sheet, which was then sublimated at low temperatures and pressures to leave individual free-standing phages suspended across slits. An image of a phage particle, depicted as the amplitude of the object wave, was reconstructed numerically from a digitized record of the hologram, obtained using 88 eV coherent electrons. The reconstructed image shows a single phage suspended across a slit in a supporting carbon membrane, magnified by a factor of 100,000. The width and shape in the reconstructed image compared well with a TEM image of the same filament. It is thus possible to record and reconstruct electron holograms of an individual phage. The challenge now is to improve the resolution of reconstructed images obtained by this method and to extend these structural studies to other biological molecules.
KeywordsHolography Low energy electron microscopy Single molecule
The f1.K phage mutant was a generous gift of Jamie Scott (Simon Fraser University). We thank Daniel Steiner for helpful advice on growing and isolating the phages; Peter Wild and Elisabeth Schraner for the use of the TEM and help in obtaining the TEM micrographs; and Hiroshi Okamoto for helpful advice and discussions. This work was supported in part by the Swiss National Centre of Competence in Research (NCCR) ‘Nanoscale Science’ and in part by the European Union Framework 6 New and Emerging Science and Technology project ‘SIBMAR’.
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