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

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.(

X-ray microscopy

“…Turning now to the relation between dosage inflicted and damage done, recent work by Bennett, Forster, Buckley & Burge (1993), Williams et al (1993), Schneider (1994) and Jacobsen (private communication) has begun to reveal a situation roughly as follows. At approximately 106 rad, loss of cell function develops slowly (over minutes or hours following the exposure) in living biological cells; this can include ultimate death of the cell.…”

Synchrotron‐Based Near‐Edge X‐Ray Spectroscopy of Natural Organic Matter in Soils and Sediments

“…In general, beam damage is less of a problem in NEXAFS than, for example, in electron microscopy . The level of radiation that does not cause damage also depends on whether the specimen is dry or wet (Kirz et al, 1995) and increases in a wet stage due to the diffusion of radicals (Williams et al, 1993). Measurements under cryo conditions decreases morphological changes and mass loss, but has little effect on reducing beam damage of the functional group chemistry (Beetz and Jacobsen, 2003).…”

“…NEXAFS methods can be applied to nearly all elements, is not restricted to certain isotopes, does not typically exhibit interferences (e.g., paramagnetic iron with nuclear magnetic resonance (NMR) spectroscopy), and does not necessarily require sample drying (Williams et al, 1993;Kirz et al, 1995). An additional advantage is the ability of NEXAFS to directly interrogate the composition of minor or 1 Q PRINCIPLES 725 trace elements in NOM without extraction or chemical modifications (Brandes et al, 2007).…”

Cryogenic x-ray diffraction microscopy utilizing high-pressure cryopreservation

“…While the imaging of radiation-hardy samples, such as nanocrystals, has steadily expanded [13][14][15], early work in cellular imaging had been limited to stained or dried specimens [11,12,16]. The need to image hydrated specimens under conditions close to their living states is apparent; however, wet specimens are inevitably subject to radiation damage and require cryoprotection in high-resolution imaging [20,21]. Ongoing efforts have resulted * Corresponding author: enjulima@gmail.com in the demonstration of cryo XDM in imaging frozen-hydrated bacteria and yeast [22,23].…”