Raman FISH

  • D. S. Read
  • W. E. Huang
  • A. S. Whiteley
Reference work entry


Linking both identity and function within natural microbial communities has long been seen as one of the ultimate goals for microbial ecologists. The phenotypic data obtained from single-cell Raman microspectroscopy can be combined with the phylogenetic resolution of fluorescence in situ hybridization (FISH) to provide a powerful tool for investigating the structure and function of natural microbial communities (Raman-FISH). This chapter provides information about the use of isotopically labeled substrates to track isotope incorporation and hence specific metabolic processes within bacterial functional groups, through the use of Raman-FISH. A general overview of the equipment and techniques necessary for conducting Raman-FISH analysis is followed by a case study highlighting the utility of this technique for discovering the bacterial communities responsible for naphthalene degradation in groundwater microcosms.


Fluorescence in situ hybridization Raman spectroscopy stable isotopes 


  1. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in-situ detection of individual microbial-cells without cultivation. Microbiological reviews 59: 143–169.Google Scholar
  2. Buijtels P, Willemse-Erix HFM, Petit PLC, Endtz HP, Puppels GJ, Verbrugh HA, Belkum A, van Soolingen D, Maquelin K (2008) Rapid identification of mycobacteria by Raman spectroscopy. J Clin Microbiol 46: 961–965.PubMedCrossRefGoogle Scholar
  3. Cheng JX, Pautot S, Weitz DA, Xie XS (2003) Ordering of water molecules between phospholipid bilayers visualized by coherent anti-stokes Raman scattering microscopy. Proc Natl Acad Sci USA 100: 9826–9830.PubMedCrossRefGoogle Scholar
  4. Cheung HY, Cui JX, Sun SQ (1999) Real-time monitoring of Bacillus subtilis endospore components by attenuated total reflection Fourier-transform infrared spectroscopy during germination. Microbiology-UK 145: 1043–1048.CrossRefGoogle Scholar
  5. Choo-Smith LP, Maquelin K, Vreeswijk T, Bruining HA, Puppels GJ, Thi NAG, Kirschner C, Naumann D, Ami D, Villa AM, Orsini F, Doglia SM, Lamfarraj H, Sockalingum GD, Manfait M, Allouch P, Endtz HP (2001) Investigating microbial (micro)colony heterogeneity by vibrational spectroscopy. Appl Environ Microbiol 67: 1461–1469.PubMedCrossRefGoogle Scholar
  6. Culha M, Stokes D, Allain LR, Vo-Dinh T (2003) Surface-enhanced Raman scattering substrate based on a self- assembled monolayer for use in gene diagnostics. Anal Chem 75: 6196–6201.PubMedCrossRefGoogle Scholar
  7. Daims H, Stoecker K, Wagner M (2005) Fluorescence in situ hybridization for the detection of prokaryotes. In Advanced Methods in Molecular Microbial Ecology. AM Osborn, CJ Smith (eds.). Bios-Garland, UK: Abingden, pp. 213–239.Google Scholar
  8. Ellis DI, Broadhurst D, Kell DB, Rowland JJ, Goodacre R (2002) Rapid and quantitative detection of the microbial spoilage of meat by Fourier transform infrared spectroscopy and machine learning. Appl Environ Microbiol 68: 2822–2828.PubMedCrossRefGoogle Scholar
  9. De Gelder J, De Gussem K, Vandenabeele P, Moens L (2007) Reference database of Raman spectra of biological molecules. J Raman Spectros 38: 1133–1147.CrossRefGoogle Scholar
  10. Huang WE, Griffiths RI, Thompson IP, Bailey MJ, Whiteley AS (2004) Raman microscopic analysis of single microbial cells. Anal Chem 76: 4452–4458.PubMedCrossRefGoogle Scholar
  11. Huang WE, Stoecker K, Griffiths RI, Newbold L, Daims H, Whiteley AS, Wagner M (2007) Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. Environ Microbiol 9: 1878–1889.PubMedCrossRefGoogle Scholar
  12. Jarvis RM, Goodacre R (2008) Characterisation and identification of bacteria using SERS. Chem Soc Rev 37: 931–936.PubMedCrossRefGoogle Scholar
  13. Kirschner C, Maquelin K, Pina P, Thi NAN, Choo-Smith LP, Sockalingum GD, Sandt C, Ami D, Orsini F, Doglia SM, Allouch P, Mainfait M, Puppels GJ, Naumann D (2001) Classification and identification of enterococci: a comparative phenotypic, genotypic, and vibrational spectroscopic study. J Clin Microbiol 39: 1763–1770.PubMedCrossRefGoogle Scholar
  14. Lee N, Nielsen PH, Andreasen KH, Juretschko S, Nielsen JL, Schleifer KH, Wagner M (1999) Combination of fluorescent in situ hybridization and microautoradiography-a new tool for structure-function analyses in microbial ecology. Appl Environ Microbiol 65: 1289–1297.PubMedGoogle Scholar
  15. Li T, Wu T, Mazeas L, Toffin L, Guerquin-Kern J, Leblon G, Bouchez T (2008) Simultaneous analysis of microbial identity and function using NanoSIMS. Environ Microbiol 10: 580–588.PubMedCrossRefGoogle Scholar
  16. Lieber CA, Majumder SK, Ellis DL, Billheimer DD, Mahadevan-Jansen A (2008) In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy. Lasers Surg Med 40: 461–467.PubMedCrossRefGoogle Scholar
  17. Matsui H, Pan S (2001) Distribution of DNA in cationic liposome complexes probed by Raman microscopy. Langmuir 17: 571–573.CrossRefGoogle Scholar
  18. Maquelin K, Choo-Smith LP, Vreeswijk T, Endtz HP, Smith B, Bennett R, Bruining HA, Puppels GJ (2000) Raman spectroscopic method for identification of clinically relevant microorganisms growing on solid culture medium. Anal Chem 72: 12–19.PubMedCrossRefGoogle Scholar
  19. Maquelin K, Choo-Smith LP, Endtz HP, Bruining HA, Puppels GJ (2002) Rapid identification of Candida species by confocal Raman micro spectroscopy. J Clin Microbiol 40: 594–600.PubMedCrossRefGoogle Scholar
  20. Maquelin K, Kirschner C, Choo-Smith LP, Ngo-Thi NA, Vreeswijk T, Stammler M, Endtz HP, Bruining HA, Naumann D, Puppels GJ (2003) Prospective study of the performance of vibrational spectroscopies for rapid identification of bacterial and fungal pathogens recovered from blood cultures. J Clin Microbiol 41: 324–329.PubMedCrossRefGoogle Scholar
  21. Movasaghi Z, Rehman S, Rehman IU (2007) Raman spectroscopy of biological tissues. Appl Spectros Rev 42: 493–541.CrossRefGoogle Scholar
  22. Naumann D (2001) FT-infrared and FT-Raman spectroscopy in biomedical research. Appl Spectros Rev 36: 239–298.CrossRefGoogle Scholar
  23. Petrich W (2001) Mid-infrared and Raman spectroscopy for medical diagnostics. Appl Spectros Rev 36: 181–237.CrossRefGoogle Scholar
  24. Petry R, Schmitt M, Popp J (2003) Raman Spectroscopy – A prospective tool in the life sciences. Chemphyschem 4: 14–30.PubMedCrossRefGoogle Scholar
  25. Potma EO, de Boeij WP, van Haastert PJM, Wiersma DA (2001) Real-time visualization of intracellular hydrodynamics in single living cells. Proc Natl Acad Sci USA 98: 1577–1582.PubMedCrossRefGoogle Scholar
  26. Schuster KC, Reese I, Urlaub E, Gapes JR, Lendl B (2000a) Multidimensional information on the chemical composition of single bacterial cells by confocal Raman microspectroscopy. Anal Chem 72: 5529–5534.PubMedCrossRefGoogle Scholar
  27. Schuster KC, Urlaub E, Gapes JR (2000b) Single-cell analysis of bacteria by Raman microscopy: spectral information on the chemical composition of cells and on the heterogeneity in a culture. J Microbiol Methods 42: 29–38.PubMedCrossRefGoogle Scholar
  28. Shafer-Peltier KE, Haka AS, Fitzmaurice M, Crowe J, Myles J, Dasari RR, Feld MS (2002) Raman microspectroscopic model of human breast tissue: implications for breast cancer diagnosis in vivo. J Raman Spectros 33: 552–563.CrossRefGoogle Scholar
  29. Tang HW, Mei EW, Chen GQ, Zeng Y (1999) Measurements of the DNA content in a breast tumor cell based on the Hadamard transform microscopic fluorescence image. Anal Sci 15: 113–119.CrossRefGoogle Scholar
  30. Wagner M, Nielsen PH, Loy A, Nielsen JL, Daims H (2006) Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays. Curr Opin Biotechnol 17: 83–91.PubMedCrossRefGoogle Scholar
  31. Whiteley AS, Thomson B, Lueders T, Manefield M (2007) RNA stable-isotope probing. Nat Protocols 2: 838–844.CrossRefGoogle Scholar
  32. Wu Q, Nelson WH, Elliot S, Sperry JF, Feld M, Dasari R, Manoharan R (2000) Intensities of E coli nucleic acid Raman spectra excited selectively from whole cells with 251-nm light. Anal Chem 72: 2981–2986.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  • D. S. Read
    • 1
  • W. E. Huang
    • 2
  • A. S. Whiteley
    • 1
  1. 1.Centre for Ecology and Hydrology OxfordOxfordUK
  2. 2.Kroto Research InstituteThe University of SheffieldSheffieldUK

Personalised recommendations