Raman-Fluorescence in Situ Hybridization

Abstract

As with many other molecular microbiology methods, the family of stable isotope probing (SIP) techniques based on the analysis of isotope- labeled nucleic acids, phospholipid fatty acids (PLFAs), or proteins are frequently performed at the level of the community. Single-cell studies encompass a range of techniques, including bacterial bioreporters, flow cytometry, fluorescence in situ hybridization (FISH) in combination with microautoradiography (MAR), nanoscale secondary ion mass spectrometry (nanoSIMS), and Raman spectroscopy. In general, FISH procedures are almost identical to standard epifluorescence microscopic procedures. The ability to analyze microbial communities at the level of the single cell, rather than at the level of the population or community, is one of the main advantages of Raman, and the area where it differs from conventional SIP techniques. Conventional SIP techniques tend to focus on isotope incorporation into one specific biomolecule, such as PLFAs, DNA, RNA, or proteins. Raman has a significant advantage in that it is looking at isotope incorporation by all these biomolecules simultaneously, depending of course on the exact constitution and quality of the collected Raman spectra. Raman is a sensitive technique, with isotope incorporation being detected in microbes cultured in growth media containing as low as 10% 13C. Raman spectroscopy has also been combined with optical trapping and manipulation to achieve cell sorting of isotopically labeled cells.