Impact of Fixation Protocols on Elemental Content in Human Sperm

X‐ray Fluorescence microscopy is a sophisticated advanced technique which can provide information regarding the concentration and the distribution of chemical elements within biological samples. It is increasingly used in the study of cells and tissues as it does not require the use of staining or fluorescent probes. For XRF measurements, sample preparation is a key step in the analytical process that affects all aspects of the analysis. Cells should be maintained in conditions that closely resemble physiological environments, but this can be challenging, especially under vacuum conditions. While numerous fixation protocols exist, finding one that works best for all cell types remains difficult. This study focuses on human spermatozoa, as they differ from other cell types in size, structure, and function. Furthermore, in human sperm, the presence and the balance of various chemical elements are crucial to their efficiency and success in reproduction. Our work aims at investigating the most suitable protocol for spermatozoa fresh cell fixation for XRF analyses at different energies; in particular, paraformaldehyde (PFA) 4% and methanol fixation are compared through XRF analyses at low and hard X‐ray energy excitation, revealing light and trace elements. Although PFA is the most widely used chemical fixative for biological sample preparation, our results suggest that methanol is an excellent alternative for fixing human sperm for X‐ray microscopy as it better preserves their elemental content while still maintaining an acceptably good morphology.