Raman Microscopic Analysis of Single Microbial Cells

Abstract

We demonstrate the utility of the Raman confocal microscope to generate a spectral profile from a single microbial cell and the use of this approach to differentiate bacterial species. In general, profiles from different bacterial taxa shared similar peaks, but the relative abundances of these components varied between different species. The use of multivariate methods subsequently allowed taxa discrimination. Further investigations revealed that the single-cell spectra could be used to differentiate between growth phases of a single species, but these differences did not obscure the overall interspecies discrimination. Finally, we tested the efficacy of the method as a means to identify cells responsible for the uptake of a specific substrate. A single strain was grown in media containing incrementally varying ratios of (13)C(6) to (12)C(6) glucose, and it was found that (13)C incorporation shifted characteristic peaks to lower wavenumbers. These findings suggest that Raman microscopy has significant potential for studies requiring the taxonomic identity and functioning of single microbial cells to be determined.

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community

“…Raman micro-spectroscopy was employed to quantify 13 C-incorporation of MRCs at the single cell level (Huang et al, 2004(Huang et al, , 2007(Huang et al, , 2009). MRCs were harvested from the above treatments.…”

“…SCRS of MRCs were acquired to examine their 13 C incorporation based on the fact that some Raman bands of 13 C-labelled cells shift to lower wavenumbers upon the incorporation of 13 C from a growth substrate (Huang et al, 2004(Huang et al, , 2007(Huang et al, , 2009Li et al, 2013). SCRS of MRCs from 12 C-phenol treatments were used as controls.…”

Localizing and Quantifying Carotenoids in Intact Cells and Tissues

“…Recent literature shows that Raman spectroscopic imaging is emerging as a key technology for single-cell analysis, including in vivo lipidomics [19], chemical composition of bacterial cells [20], lignin in plant cell walls [21], and metabolism in combination with stable isotope incorporation [22][23][24]. Researchers have also begun capitalizing on the speciic beneits of resonance Raman spectroscopic imaging to localize carotenoids in individual cells and tissues.…”

Who eats what, where and when? Isotope-labelling experiments are coming of age

“…The scattered laser light is captured by a charge-coupled device camera, and a spectrum is obtained that has discernable peaks for different biological molecules in the cell. When tested on single microbial cells, the peaks in the Raman spectra corresponding to phenylalanine, proteins and nucleic acids were 'red-shifted' by the incorporation of 13 C-labelled substrate (Huang et al, 2004). The magnitude of this red shift was independent of bacterial species tested and corresponded to the proportion of label.…”