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Chapter 13 : Raman-Fluorescence In Situ Hybridization

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Raman-Fluorescence In Situ Hybridization, Page 1 of 2

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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% C. Raman spectroscopy has also been combined with optical trapping and manipulation to achieve cell sorting of isotopically labeled cells.

Citation: Read D, Whiteley A. 2011. Raman-Fluorescence In Situ Hybridization, p 279-294. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch13

Key Concept Ranking

Microbial Ecology
0.73556197
16s rRNA Sequencing
0.46080306
Raman Spectroscopy
0.40555432
0.73556197
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Figures

Image of FIGURE 1
FIGURE 1

Jablonski diagram. A Jablonski diagram illustrates the transitions between the electronic states of a molecule and how these relate to infrared (IR) absorption, Rayleigh scattering, and Stokes and anti-Stokes Raman scattering.

Citation: Read D, Whiteley A. 2011. Raman-Fluorescence In Situ Hybridization, p 279-294. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch13
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Image of FIGURE 2
FIGURE 2

The main features of a Raman microspectrometer. Transmitted light is used to visualize microbial cells on a Raman-inert slide. Light from a monochromatic laser source is directed down the microscope objective, and Raman scattered light is collected back through the same objective. Unwanted light frequencies are filtered with a notch filter, before the light beam is diffracted using a grating and dispersed onto a cooled CCD camera. This signal can be used to create a Raman spectrum or a Raman chemical image. ( .)

Citation: Read D, Whiteley A. 2011. Raman-Fluorescence In Situ Hybridization, p 279-294. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch13
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Image of FIGURE 3
FIGURE 3

Raman spectra of microbial cells. Raman spectra collected from a nonpigmented bacterial cell () and a pigmented bacterial cell (unidentified environmental isolate) using a LabRAM HR800 UV confocal Raman microscope (Horiba Scientific) with an excitation wavelength of 532 nm.

Citation: Read D, Whiteley A. 2011. Raman-Fluorescence In Situ Hybridization, p 279-294. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch13
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Image of FIGURE 4
FIGURE 4

Raman spectra of labeled and unlabeled compounds. Raman spectra of unlabeled (solid line) and labeled [U-C] (dashed line) glucose collected on a LabRAM HR800 UV confocal Raman microscope (Horiba Scientific) with an excitation wavelength of 532 nm.

Citation: Read D, Whiteley A. 2011. Raman-Fluorescence In Situ Hybridization, p 279-294. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch13
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Image of FIGURE 5
FIGURE 5

Isotope labeling series. Raman spectra collected from cultured in media containing 0, 50, or 100% [U-C]glucose as the sole carbon source. Figures are offset for clarity. Raman spectra collected on a LabRAM HR800 UV confocal Raman microscope (Horiba Scientific) with an excitation wavelength of 532 nm. Major peak shifts are highlighted in gray.

Citation: Read D, Whiteley A. 2011. Raman-Fluorescence In Situ Hybridization, p 279-294. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch13
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Image of FIGURE 6
FIGURE 6

Outline of the major steps involved in Raman-FISH. The stable isotope is added to the environmental sample, either in a microcosm (shown) or in the field. Microbes are permeabilized and labeled with fluorescently tagged FISH probes, and epifluorescence microscopy is used to identify cells of interest. Once identified, Raman optics are switched to Raman mode to analyze uptake of stable isotope at the single-cell level. .

Citation: Read D, Whiteley A. 2011. Raman-Fluorescence In Situ Hybridization, p 279-294. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch13
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