Raman‐FISH: combining stable‐isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function
Top Cited Papers
Open Access
- 15 June 2007
- journal article
- Published by Wiley in Environmental Microbiology
- Vol. 9 (8), 1878-1889
- https://doi.org/10.1111/j.1462-2920.2007.01352.x
Abstract
We have coupled fluorescence in situ hybridization (FISH) with Raman microscopy for simultaneous cultivation-independent identification and determination of 13C incorporation into microbial cells. Highly resolved Raman confocal spectra were generated for individual cells which were grown in minimal medium where the ratio of 13C to 12C content of the sole carbon source was incrementally varied. Cells which were 13C-labelled through anabolic incorporation of the isotope exhibited key red-shifted spectral peaks, the calculated ‘red shift ratio’ (RSR) being highly correlated with the 13C-content of the cells. Subsequently, Raman instrumentation and FISH protocols were optimized to allow combined epifluorescence and Raman imaging of Fluos, Cy3 and Cy5-labelled microbial populations at the single cell level. Cellular 13C-content determinations exhibited good congruence between fresh cells and FISH hybridized cells indicating that spectral peaks, including phenylalanine resonance, which were used to determine 13C-labelling, were preserved during fixation and hybridization. In order to demonstrate the suitability of this technology for structure–function analyses in complex microbial communities, Raman-FISH was deployed to show the importance of Pseudomonas populations during naphthalene degradation in groundwater microcosms. Raman-FISH extends and complements current technologies such as FISH-microautoradiography and stable isotope probing in that it can be applied at the resolution of single cells in complex communities, is quantitative if suitable calibrations are performed, can be used with stable isotopes and has analysis times of typically 1 min per cell.Keywords
This publication has 64 references indexed in Scilit:
- Microbial analysis of soil and groundwater from a gasworks site and comparison with a sequenced biological reactive barrier remediation processJournal of Applied Microbiology, 2007
- The future of single‐cell environmental microbiologyEnvironmental Microbiology, 2007
- Studying Bacterial Metabolic States Using Raman SpectroscopyApplied Spectroscopy, 2006
- Microbial diversity in the deep sea and the underexplored “rare biosphere”Proceedings of the National Academy of Sciences of the United States of America, 2006
- Comparison of rRNA and Polar-Lipid-Derived Fatty Acid Biomarkers for Assessment of 13 C-Substrate Incorporation by Microorganisms in Marine SedimentsApplied and Environmental Microbiology, 2006
- Complete Sequence and Genetic Organization of pDTG1, the 83 Kilobase Naphthalene Degradation Plasmid from Pseudomonas putida strain NCIB 9816-4Journal of Molecular Biology, 2004
- The Uncultured Microbial MajorityAnnual Review of Microbiology, 2003
- Methane-Consuming Archaea Revealed by Directly Coupled Isotopic and Phylogenetic AnalysisScience, 2001
- Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4Gene, 1993
- Acidophilic and thermophilicBacillusstrains from geothermally heated antarctic soilFEMS Microbiology Letters, 1989