Exploring the Potential of Stable Isotope (Resonance) Raman Microspectroscopy and Surface-Enhanced Raman Scattering for the Analysis of Microorganisms at Single Cell Level

Raman microspectroscopy is a prime tool to characterize the molecular and isotopic composition of microbial cells. However, low sensitivity and long acquisition times limit a broad applicability of the method in environmental analysis. In this study, we explore the potential, the applicability, and the limitations of stable isotope Raman microspectroscopy (SIRM), resonance SIRM, and SIRM in combination with surface-enhanced Raman scattering (SERS) for the characterization of single bacterial cells. The latter two techniques have the potential to significantly increase sensitivity and decrease measurement times in SIRM, but to date, there are no (SERS-SIRM) or only a limited number (resonance SIRM) of studies in environmental microbiology. The analyzed microorganisms were grown with substrates fully labeled with the stable isotopes (13)C or (2)H and compounds with natural abundance of atomic isotopes ((12)C 98.89% or (1)H 99.9844%, designated as (12)C or (1)H, respectively). Raman bands of bacterial cell compounds in stable isotope-labeled microorganisms exhibited a characteristic red-shift in the spectra. In particular, the sharp phenylalanine band was found to be an applicable marker band for SIRM analysis of the Deltaproteobacterium strain N47 growing anaerobically on (13)C-naphthalene. The study of G. metallireducens grown with (13)C- and (2)H-acetate showed that the information on the chromophore cytochrome c obtained by resonance SIRM at 532 nm excitation wavelength can be successfully complemented by whole-organism fingerprints of bacteria cells achieved by regular SIRM after photobleaching. Furthermore, we present here for the first time the reproducible SERS analysis of microbial cells labeled with stable isotopes. Escherichia coli strain DSM 1116 cultivated with (12)C- or (13)C-glucose was used as a model organism. Silver nanoparticles synthesized in situ were applied as SERS media. We observed a reproducible red-shift of an adenine-related marker band from 733 to 720 cm(-1) in SERS spectra for (13)C-labeled cells. Additionally, Raman measurements of (12)C/(13)C-glucose and -phenylalanine mixtures were performed to elucidate the feasibility of SIRM for nondestructive quantitative and spatially resolved analysis. The performed analysis of isotopically labeled microbial cells with SERS-SIRM and resonance SIRM paves the way toward novel approaches to apply Raman microspectroscopy in environmental process studies.