Raman micro-spectroscopy reveals the spatial distribution of fumarate in cells and tissues

Aberrantly accumulated metabolites such as fumarate elicit intra– and inter-cellular pro-oncogenic cascades, yet current methods to measure them require sample perturbation or disruption and lack spatio-temporal resolution, limiting our ability to fully characterize their function and distribution in cells and within a tissue. Raman spectroscopy (RS) is a powerful bio-analytical tool that directly characterizes the chemical composition of a sample based solely on the optical fingerprint of vibrational modes. Here, we show for the first time that RS can directly detect fumarate in living cells in vivo and animal tissues ex vivo. Using the observed linear relationship between Raman scattered intensity and fumarate concentration, we demonstrate that RS can distinguish between Fumarate hydratase (Fh1)-deficient and Fh1-proficient cells based on their fumarate concentration. Moreover, RS reveals the spatial compartmentalization of fumarate within cellular organelles: consistent with disruptive methods, in Fh1-deficient cells we observe the highest fumarate concentration (37 ± 19 mM) in the mitochondria, where the TCA cycle operates, followed by the cytoplasm (24 ± 13 mM) and then the nucleus (9 ± 6 mM). Finally, we apply RS to tissues from an inducible mouse model of FH loss in the kidney, demonstrating that RS can accurately classify FH status in these tissues. These results suggest that RS could be adopted as a valuable tool for small molecule metabolic imaging, enabling in situ dynamic evaluation of fumarate compartmentalization.