LOW-DOSE ENERGY-SLECTED ELECTRON MICROSCOPY WITH AN OMEGA FILTER

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We have investigated the potential of using an in-column omega filter in a conventional transmission electron microscope for elemental mapping of biological structures at low dose. Digital images were collected at a beam energy of 100 keV by means of a 1024 x 1024-pixel, phosphor-coupled, cooled slow-scan CCD camera on a Zeiss 912 electron microscope. Frozen-hydrated specimens of herpes simplex virus (vitrified by plunging into liquid ethane) were cryotransferred and freeze-dried before imaging in the region of the phosphorus L-edge at energy losses of around 130 eV. These digital micrographs were recorded from large numbers of viruses at a dose of less than 1000 electrons per square nanometer. Resulting noisy images were corrected for read-out noise and pixel-gain variations before cross-correlating, summing, and removing the noncharacteristic background image extrapolated from the pre-edge region of the EELS spectrum into the post-edge region. In this way, it was possible to build up an averaged projected distribution of phosphorus, i.e., nucleic acid, in the core of the herpes simplex capsid.