No abstract

with energies between the carbon and oxygen K-edges Radiation damage to Vicia faba chromosome structure, as measured by the mass loss, was determined in the scanning transmission X-ray microscope for unstained specimens in both the wet and dry states. Dried specimens remain undamaged after either single or multiple images at doses up to 2400 Mrad at wavelengths of 3.15 or 3.64nm. In contrast, wet specimens are damaged irrespective of the imaging protocol. The damage induced by multiple exposures is greater than that seen in a single exposure of the same total dose. Thus, the rate of data collection is greater than or equal to the rate of damage. The damage during multiple exposures of wet chromosomes is influenced by several factors. First, the fixative used influences the extent of radiation damage. Wet chromosomes fixed with glutaraldehyde are more resistant than those fixed with formaldehyde or osmium tetroxide. A second factor is ionic strength. Damage to wet chromosomes increases if the ionic strength decreases below that at which chromatin undergoes a conformational transition. The mass of wet and dry chromosomes is the same, and consequently quantitative measurements can be made on wet specimens. Such measurements give a DNA mass fraction of 39 f 8% for V . faba chromosomes.(

Using a cryo scanning transmission X-ray microscope (Maser, et al. (2000) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc. 197, 68-79), we have obtained tomographic data-sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three-dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.