Quantifying Spatial Heterogeneity of Lead and Zinc In Rat Brain Tissues across Preparation Methods Using Micro-XRF
Abstract
Purpose
Laboratory-based micro-X-ray fluorescence(micro-XRF) offers micrometer-scale spatial profiling of elemental distribution in biological tissues. However, quantitative analysis of thin tissue sections can be influenced by instrumental acquisition parameters and tissue preparation methods. This study examines micro-XRF measurement of lead (Pb) and zinc (Zn) in hippocampal brain tissue from Pb-treated rat populations across two preparation protocols.
Methods
Paraformaldehyde (PFA)-perfused 40 µm sections (mounted, no coverslip, not dehydrated), and phosphate-buffered saline(PBS)-perfused 20 µm sections(formalin-fixed, paraffin-embedded) were evaluated with four Pb-treated conditions(control, low, medium, high). Initially, the whole sections were scanned at 50 kV and 1 mA current with pixel sizes of 10-16 µm and 80-110 ms per pixel. Elemental intensities were normalized by dwell time and rescaled to a common pixel area of 100 µm2. Mean intensity(cps), coefficient of variation (CV), and high-end tail contrast(99th percentile/mean) were computed. Pb/Zn ratio profiles were used to reduce sensitivity to absolute intensity scaling and preparation-dependent effects.
Results
Mean Pb and Zn intensity in hippocampal ROIs were higher in PBS-prepared sections (1265 ± 25 and 202 ± 37) than in PFA-prepared sections (930 ± 36 and 101 ± 3), while the mean Pb/Zn ratio showed an opposite trend(6.61 ± 0.13 vs 9.86 ± 0.20). PBS-prepared sections exhibited reduced heterogeneity, with lower CV values for Pb (0.27 ± 0.004 vs 0.31 ± 0.003), Zn (0.23 ± 0.004 vs 0.26 ± 0.002), and Pb/Zn (0.27 ± 0.004 vs 0.31 ± 0.004). High-end tail contrast for Pb was slightly higher in PBS-prepared sections, while Zn and Pb/Zn tail contrast were higher in PFA-prepared sections.
Conclusion
Micro-XRF enables joint Pb and Zn mapping and can be translated to future investigations of Pb effects on Zn-related processes in the hippocampus. Micro-XRF is also capable of extending this type of analysis to a wide range of other elements (Fluorine to Americium).