Raman spectroscopy in microfluidic chips reveals hyphal scale stress-associated metabolic responses in filamentous soil fungi

Understanding metabolic processes of soil fungi is essential for elucidating their ecological roles in biogeochemical cycles and responses to emergent environmental stressors. Here, we demonstrate the potential of using stable isotope probing Raman (SIP-Raman) microspectroscopy in microfluidics technology-based soil chips to trace glucose metabolism rates and stress responses in laboratory grown filamentous soil fungus Psilocybe cf. subviscida . The time evolution of Raman spectral band intensities resulting from deuterated glucose uptake in the fungal hyphae allowed us to assess glucose metabolism rates. Under excess copper (Cu) stress, we observed suppression of both glucose metabolic activity and growth. In addition, reduced spectral signatures of intracellular cytochrome c further implied impaired mitochondrial function and potential onset of cell death. However, laser-induced radiation damage hampered repeated Raman measurements, including multispectral mapping, on individual hyphae, especially when exposed to the Cu stress. To overcome this, we employed stimulated Raman scattering (SRS) microscopy, which offers much higher sensitivity and mapping speeds, and therefore much lower radiation doses. This enabled localization of the uptaken glucose at the inner edges of the P. cf. subviscida hyphae and Cu-induced formation of putative vacuolar structures. While integration of this approach with soil chips requires future modifications to the chip design for increased optical transparency and ensured sterility, overall, our results demonstrate the potential of Raman-based microspectroscopy for spatially resolved, in situ analysis of fungal primary metabolism and stress physiology. • Raman spectroscopy in soil chips enables tracing hyphal biochemistry in situ • Stable-isotope probing allows tracing hyphal metabolism rates and stress response • Cu exposure reduced hyphal glucose metabolism of fungus P. cf. subviscida • Reduced cytochrome c Raman signal indicates impaired mitochondria under Cu stress • SRS imaging of hyphae provides high sensitivity and reduced radiation damage