Multi-element stable isotope Raman microspectroscopy of bacterial carotenoids unravels rare signal shift patterns and single-cell phenotypic heterogeneity.

The combination of single-cell Raman microspectroscopy (SCRM) and stable isotope probing (SIP) enables in situ tracking of carbon or hydrogen fluxes into microorganisms at the single-cell level. Therefore, it has high potential for the analysis of metabolic processes and biogeochemical cycles. However, especially for high throughput applications such as imaging or cell sorting, it is hampered by low Raman scattering intensities (and therefore long acquisition times). In order to overcome these limitations, this study brings forward a systematic investigation of Resonance Raman (RR) enhanced SCRM for SIP of bacterial carotenoids. Dynamic carbon uptake from 13C-glucose was successfully monitored and quantified utilizing 13C stable isotope-induced red-shifts of RR signals. High single-cell phenotypic heterogeneity was revealed in terms of carbon uptake and, unlike in previous studies, clear evidence for de novo synthesis of carotenoids was found. For the first time, hydrogen uptake into carotenoids was systematically investigated by deuterium labeling (providing a direct probe for metabolic activity of single cells). In carotenoid single-cell Resonance Raman (SCRR) spectra, a unique pattern of signal red-shifts and apparent blue-shifts was observed and quantitatively evaluated. Finally, a novel combined approach for simultaneous monitoring of carbon and hydrogen uptake revealed complementary effects in carotenoid SCRR spectra that can be analyzed in parallel. Overall, it was shown that the high RR intensity, simplicity of spectral features and straightforward signal processing make microbial carotenoids an ideal target for quantitative multi-element SIP, with great potential for high throughput applications.

Effect of Plasma Gas and the Pressure on Spatially and Temporally Resolved Images of Copper Emission Lines in Laser Induced Plasma Optical Emission Spectrometry.

This paper investigated two-dimensional spatial and temporal images of a copper emission line in laser-induced breakdown spectroscopy (LIBS), in order to clarify the excitation/de-excitation processes occurring in a laser-induced plasma. The measurements were carried out under different plasma gases (argon, krypton, helium, and nitrogen), pressure levels (100 - 900 Pa) and delay times (100 - 1000 ns) with the aim of monitoring their effects on the behavior of the copper emission. Depending on the plasma gas type and the pressure level, large differences were found in the plasma shape and temporal intensity evolution of the copper emission profile. Namely, krypton produced the most compact plasma emitting larger intensities, compared to argon and helium, and an increase in the gas pressure made these plasmas to shrink, which could be related principally to the stopping power of the applied gases. Through temporally resolved analysis, the delay profiles could be obtained for each plasma gas, indicating that the helium plasma disappeared more rapidly than the argon and krypton plasmas. It was suggested that the variations in the emission intensity would be determined by interactions between gas particles and highly energetic particles in the plasma breakdown as well as interactions between excited gas particles and copper species during plasma expansion. These insights could prove to be useful in the understanding of the background of LIBS as well as the optimization of its practical applications.