Blank Spectrum Correction as a Robust Solution to Artifacts in Quantitative X-ray Fluorescence Mapping.

X-ray fluorescence microscopy (XFM) continues to develop as a powerful quantitative technique for high-resolution, label-free, elemental mapping of biological, environmental, and material samples. Methods for rigorously fitting spectra, increasing throughput, accounting for background signals, and deconvoluting overlapping emission lines continue to evolve. We show here that quantitative fits of XFM data obtained after removing a baseline, calculated by peak stripping, can be unexpectedly dependent upon acquisition dwell-time and spectral aggregation, leading to differences in apparent elemental content. Using mouse preimplantation embryos and ovarian follicles as model samples, we demonstrate how these variables influence quantitative comparisons between samples. We find that subtracting an empirically measured blank spectrum instead of a baseline provides quantitative XFM elemental mapping results that are independent of dwell time and spectral aggregation dependencies.