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Micro-X-ray fluorescence (μ-XRF) is a rapidly evolving analytical technique which allows visualising the trace level metal distributions within a specimen in an essentially nondestructive manner. At second generation synchrotron radiation sources, detection limits at the sub-parts per million level can be obtained with micrometer resolution, while at third generation sources the spatial resolution can be better than 100 nm. Consequently, the analysis of metals within biological systems using micro- and nano-X-ray fluorescence imaging is a quickly developing field of research. Since X-ray fluorescence is a scanning technique, the elemental distribution within the sample should not change during analysis. Biological samples pose challenges in this context due to their high water content. A dehydration procedure is commonly used for sample preparation enabling an analysis of the sample under ambient temperature conditions. Unfortunately, a potential change in elemental redistribution during the sample preparation is difficult to verify experimentally and therefore cannot be excluded completely. Creating a cryogenic sample environment allowing an analysis of the sample under cryogenic condition is an attractive alternative but not available on a routine basis. In this article, we make a comparison between the elemental distributions obtained by micro-SR-XRF within a chemically fixed and a cryogenically frozen Daphnia magna, a model organism to study the environmental impact of metals. In what follows, we explore the potential of a dual detector setup for investigating a full ecotoxicological experiment. Next to conventional 2D analysis, dual detector X-ray fluorescence cryotomography is illustrated and the potential of its coupling with laboratory absorption micro-CT for investigating the tissue-specific elemental distributions within this model organism is highlighted.

Dual detection X-ray fluorescence cryotomography and mapping on the model organism Daphnia magna