PMID- 17635536
OWN - NLM
STAT- MEDLINE
DCOM- 20070913
LR  - 20121115
IS  - 1462-2912 (Print)
IS  - 1462-2912 (Linking)
VI  - 9
IP  - 8
DP  - 2007 Aug
TI  - Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ 
      hybridization for the single cell analysis of identity and function.
PG  - 1878-89
AB  - We have coupled fluorescence in situ hybridization (FISH) with Raman microscopy 
      for simultaneous cultivation-independent identification and determination of 
      (13)C incorporation into microbial cells. Highly resolved Raman confocal spectra 
      were generated for individual cells which were grown in minimal medium where the 
      ratio of (13)C to (12)C content of the sole carbon source was incrementally 
      varied. Cells which were (13)C-labelled through anabolic incorporation of the 
      isotope exhibited key red-shifted spectral peaks, the calculated 'red shift 
      ratio' (RSR) being highly correlated with the (13)C-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 (13)C-content 
      determinations exhibited good congruence between fresh cells and FISH hybridized 
      cells indicating that spectral peaks, including phenylalanine resonance, which 
      were used to determine (13)C-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.
FAU - Huang, Wei E
AU  - Huang WE
AD  - Biodiversity and Ecosystem Function Group, Molecular Microbial Ecology Section, 
      Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxford, OX1 3SR, UK.
FAU - Stoecker, Kilian
AU  - Stoecker K
FAU - Griffiths, Robert
AU  - Griffiths R
FAU - Newbold, Lyndsay
AU  - Newbold L
FAU - Daims, Holger
AU  - Daims H
FAU - Whiteley, Andrew S
AU  - Whiteley AS
FAU - Wagner, Michael
AU  - Wagner M
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Environ Microbiol
JT  - Environmental microbiology
JID - 100883692
RN  - 0 (Carbon Isotopes)
RN  - 0 (DNA, Bacterial)
RN  - 0 (DNA, Ribosomal)
RN  - 0 (Naphthalenes)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - 2166IN72UN (naphthalene)
SB  - IM
MH  - Bacteria/chemistry/genetics/*isolation & purification
MH  - Carbon Isotopes/*metabolism
MH  - DNA, Bacterial/genetics
MH  - DNA, Ribosomal/genetics
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Naphthalenes/metabolism
MH  - RNA, Ribosomal, 16S/genetics
MH  - Spectrum Analysis, Raman/instrumentation/*methods
MH  - *Water Microbiology
EDAT- 2007/07/20 09:00
MHDA- 2007/09/14 09:00
CRDT- 2007/07/20 09:00
PHST- 2007/07/20 09:00 [pubmed]
PHST- 2007/09/14 09:00 [medline]
PHST- 2007/07/20 09:00 [entrez]
AID - EMI1352 [pii]
AID - 10.1111/j.1462-2920.2007.01352.x [doi]
PST - ppublish
SO  - Environ Microbiol. 2007 Aug;9(8):1878-89. doi: 10.1111/j.1462-2920.2007.01352.x.