Stable Isotope-Labeled Single-Cell Raman Spectroscopy Revealing Function and Activity of Environmental Microbes

Microorganisms play a key role in driving the global element (C, N, H, P, and S) cycling. However, the function and activity of environmental microbes remain largely elusive because the vast majority of them are yet uncultured. Recent achievements in single cell stable isotope-labeled Raman spectroscopy enable direct investigation of function and activity of individual microbes in complex environmental communities. Here, this protocol describes a workflow to investigate environmental microbes in soil and water by combining 15N, 2D, and 13C stable isotope labeling with different single-cell Raman techniques, including normal Raman, resonance Raman (RR), and surface-enhanced Raman spectroscopy (SERS). Their applications in investigating functional bacteria driving the N and C cycles, and metabolically active cells are described.