Confocal volumetric μXRF and fluorescence computed μ‑tomography reveals arsenic three-dimensional distribution nwithin intact Pteris vittata fronds

The fern Pteris vittata has been the subject of numerous studies because of its extreme arsenic hyper22 accumulation characteristics. However, information on the arsenic chemical speciation and distribution across cell types within intact frozen-hydrated Pteris vittata fronds is necessary to better understand the arsenic biotransformation pathways in this unusual fern. While 2D X-ray absorption spectroscopy imaging studies show that different chemical forms of arsenic, As(III) and As(V), occur across the plant organs, depth29 resolved information on arsenic distribution and chemical speciation in different cell types within tissues of Pteris vittata have not been reported. By using a combination of planar and confocal μ-X-ray fluorescence imaging and fluorescence computed μ-tomography, we reveal, in this study, the localization of Arsenic in the endodermis and pericycle surrounding the vascular bundles in the rachis and the pinnules of the fern. Arsenic is also accumulated in the vascular bundles connecting into each sporangium, and in some mature sori. The use of 2D X-ray absorption near edge structure imaging allows for deciphering arsenic speciation across the tissues, revealing arsenate in the vascular bundles and arsenite in the endodermis and pericycle. This study demonstrates how different advanced synchrotron X-ray microscopy techniques can be complementary in revealing, at tissue and cellular levels, elemental distribution and chemical speciation in hyperaccumulator plants