Single-Slice XRF Mapping of Light Elements in Frozen-Hydrated Allium schoenoprasum via a Self-Absorption-Corrected Hyperspectral Tomographic Reconstruction Approach

3D and 2D-cross-sectional X-ray fluorescence analysisof biologicalmaterial is a powerful tool to image the distribution of elementsand to understand and quantify metal homeostasis and the distributionof anthropogenic metals and nanoparticles with minimal preparationartifacts. Using tomograms recorded on cryogenically prepared leavesof Allium schoenoprasum, the cross-sectionaldistribution of physiologically relevant elements like calcium, potassium,manganese, and zinc could be tomographically reconstructed by peakfitting followed by a conventional maximum-likelihood algorithm withself-absorption correction to reveal the quantitative cross-sectionalelement distribution. If light elements such as S and P are locateddeep in the sample compared to the escape depth of their characteristicX-ray fluorescence lines, the quantitative reconstruction becomesinaccurate. As a consequence, noise is amplified to a magnitude whereit might be misinterpreted as actual concentration. We show that atomographic MCA hyperspectral reconstruction in combination with aself-absorption correction allows for fitting of the XRF spectra directlyin real space, which significantly improves the qualitative and quantitativeanalysis of the light elements compared to the conventional methodas noise and artifacts in the tomographic reconstruction are reduced.This reconstruction approach can substantially improve the quantitativeanalysis of trace elements as it allows the fitting of summed voxelspectra in anatomical regions of interest. The presented method canbe applied to XRF 2D single-slice tomography data and 3D tomogramsand is particularly relevant for, but not limited to, biological materialin order to help retrieve self-absorption corrected quantitative reconstructionsof the spatial distribution of light elements and ultra-trace-elements.

Automated summary

3D and 2D-cross-sectional X-ray fluorescence analysis is a powerful tool for imaging element distribution in biological material and quantifying metal homeostasis. This study presents a method using peak fitting and self-absorption correction to reconstruct the cross-sectional distribution of elements in cryogenically prepared leaves. Additionally, the authors introduce an atomographic MCA hyperspectral reconstruction method that improves quantitative analysis of light elements compared to conventional methods.