Minimizing Experimental Artefacts in Synchrotron-based X-ray Analyses of Fe Speciation in Tissues of Rice Plants

Iron (Fe) plays an important role within environmental systems. Synchrotron-based X-ray approaches, including X-ray absorption spectroscopy (XAS), provide powerful tools for in situ analyses of Fe speciation, but beam damage during analysis may alter Fe speciation during its measurement. XAS was used to examine whether experimental conditions affect the analysis of Fe speciation in plant tissues. Even when analyzed in a cryostat at 12 K, it was found that Fe can rapidly (within 0.5-1 min) photoreduce to Fe, although the magnitude of photoreduction varied depending upon the hydration of the sample, the coordination chemistry of the Fe, as well as other properties. For example, photoreduction of Fe was considerably higher for aqueous standard compounds than for hydrated plant-root tissues. The use of freeze-dried samples in the cryostat (12 K) markedly reduced the magnitude of this Fe photoreduction, and there was no evidence that the freeze-drying process itself resulted in experimental artefacts under the current experimental conditions, such as through the oxidation of Fe, although some comparatively small differences were observed when comparing spectra of hydrated and freeze-dried Fe compounds. The results of this study have demonstrated that Fe photoreduction can occur during X-ray analysis, and provides suitable conditions to preserve Fe speciation to minimize the extent of beam damage when analyzing environmental samples. All studies utilizing XAS are encouraged to include a preliminary experiment to determine if beam damage is occurring, and, where appropriate, to take the necessary steps (such as freeze drying) to overcome these issues.