Substrate and standard evaluation for correlative elemental mapping of biological samples by X-ray fluorescence...

Analytical techniques that offer accurate, sensitive, and high-resolution elemental mapping have significantly advanced our understanding of the role of inorganic chemistry in vital biological processes. Among these, synchrotron-based X-ray fluorescence microscopy (XFM) is a particularly powerful tool for providing reliable, non-destructive quantitation of endogenous elements in biological specimens. However, its broader application is constrained by limited beamtime availability. Recent advancements in laboratory-based imaging techniques-such as laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS)-have significantly increased the availability and throughput of elemental mapping. Yet, quantitation with LA-ICP-TOF-MS is susceptible to matrix effects, making correlative mapping with XFM critical for validation. This presents a challenge: the two techniques require different sample preparations. LA-ICP-TOF-MS uses glass slides, while XFM requires thin, low-scatter substrates to minimize X-ray background signals. To address this, we evaluated twelve commercially available substrates previously reported for XFM to determine their suitability for LA-ICP-TOF-MS. Our goal was to identify substrates that (1) exhibit low elemental background and minimal interference with quantifying endogenous inorganic species, and (2) are compatible with both imaging modalities. As an initial step, we compared adjacent brain sections prepared on Ultralene (for XFM) and glass (for LA-ICP-TOF-MS) to establish a baseline correlative approach. Building on this, Ultralene, followed by Kapton film, emerged as the most promising candidates for enabling dual XFM and LA-ICP-TOF-MS workflows, offering low background, reliable XFM performance, and demonstrating robust elemental mapping in LA-ICP-TOF-MS. These findings support more accurate and accessible correlative imaging workflows for elemental mapping of biological samples with both modalities.