Simultaneous <i>In Situ</i> Detection of Ferroptosis-Related Multiple Molecules in Tissue Slices Using Thin-Layer Chromatography Tracing and Raman Microscopy

<i>In situ</i> detection of glutathione (GSH) and Fe²⁺ in tissue samples is extremely difficult using conventional mass spectrometry imaging owing to their instability as chemical species. To address this issue, we developed a method for the simultaneous <i>in situ</i> detection of ferroptosis-related molecules in frozen tissue sections using Raman spectroscopy, which is advantageous for multicomponent analysis. This methodology employs thin-layer chromatography (TLC) functionalized with a probe cocktail bearing Raman tags such as alkynes and nitriles. Frozen tissue sections were attached to a functionalized TLC plate, where GSH addition reactions and iron complex formation proceeded. We measured the Raman spectra of TLC plates after separation and found clear signals of the probes in the presence of targets. We also tracked changes in the levels of these components in mouse liver tissue. We successfully demonstrated a statistically significant decrease in GSH and an increasing trend in iron ions associated with acetaminophen (APAP)-induced ferroptosis. This analytical platform offers a powerful tool for studying the dynamics of ferroptosis-related molecules in tissue samples and has a wide range of potential applications from basic research to clinical diagnostics. Moreover, our findings open new possibilities for the multiplexed molecular detection of biological tissues.