X-ray fluorescence microscopy (XFM) is a technique that allows visualization of chemical elements and their distribution within the structure of a living plant specimen. The technique is powerful, but also challenging, as it requires very strict protocols for specimen collection, preparation, and imaging. Failure to meet these requirements can lead to artefacts such as ultrastructural changes and the redistribution of elements. Another factor that needs to be considered is the radiation dose limit that a living specimen can be exposed to before this too can cause subtle and potentially difficult-to-recognize experimental artefacts. Until now, such a limit has not been established based on empirical evidence.
In a recent article published in Annals of Botany, Michael W.M. Jones and colleagues set out to put in place clear guidelines on the acceptable radiation dose for living plant material during XFM analysis. The researchers used fresh leaf and root samples from sunflowers, as well as the same tissues cryogenically frozen to compare damage levels. Sunflowers were chosen because their high hydration should make them more susceptible to radiation damage, giving a conservative estimate for other plant species. The authors evaluated both the immediate effects of varying radiation doses and their ongoing effects nine days after imaging.
The experiments suggest that, though cryogenically frozen specimens are quite robust against it, radiation damage in hydrated specimens occurs at a significantly lower dose than had been supposed, in doses routinely used in imaging. Subtle changes in the distribution of elements were detectable far below the level of radiation that left obvious effects, highlighting the need for dosage guidelines. The damage was element-specific, with diffusible elements such as potassium and solute calcium more prone to movement, while ions such as silicon or crystalline calcium were unaffected. “Consequently,” the authors write, “if the main objective of a study is to reveal K distribution or that of other diffusible ions such as Na, Rb, Cs, Cl or Br in vacuoles, radiation doses must be kept low. Conversely, if the objective is to reveal localization of Si or crystalline Ca in trichomes or in Ca oxalate crystals, higher radiation doses may be considered.”
The authors point out that many published XFM studies report only the incident beam energy and dwell time, but not the X-ray flux at the specimen, which is critical. “Without a record of the incident flux, it is impossible to assess the dose experienced by the specimen and therefore the likelihood of damage affecting the results.”