Measurements of wet metaphase chromosomes in the scanning transmission X-ray microscope



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 ± 8% for V. faba chromosomes.