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CARS has already been used for the rapid profiling of microorganisms at the subcellular level Okuno et al. Another technique which has found surprisingly limited use in the field of microbial ecology is TERS Mariani et al. Indeed, TERS has already been used for the analysis and classification of viral strains Hermann et al. FT-IR Imaging While Raman spectroscopy relies on irradiating a sample with a monochromatic laser beam, Fourier-transform infrared FT-IR spectroscopy is based on measuring the absorption of polychromatic infrared light.
The functional groups in a given molecule are identified according to their vibrational modes at different IR frequencies for detailed information, see Skoog et al. Raman analyses depend on a shift in the polarizability of a molecule, whereas FT-IR measurements depend on changes in the dipole moment.
Indeed, Raman-active vibrational modes often exhibit weak IR signals and vice versa with symmetric and asymmetric moieties producing strong Raman and IR spectral bands, respectively , and the two methods provide complementary information on the molecular composition of microbial cells Lu et al. Infrared imaging could, therefore, provide insights into microbial physiology in samples that are difficult to analyze using Raman spectroscopy alone. Indeed, high-speed imaging of large centimeter-scale sample areas can be achieved using a focal plane array FPA detector that enables the simultaneous acquisition of tens of thousands of IR spectra Dorling and Baker, Chemical mapping by reflectance FT-IR microspectroscopy has also been used to characterize bacteria on opaque steel surfaces, without a need for destructive sampling Ojeda et al.
Even so, several ways to overcome these challenges have been developed.
For example, synchrotron radiation sources have enabled FT-IR measurements at the micron scale Nasse et al. While synchrotron-FT-IR analyses require dedicated facilities, advances in the development of high-magnification optics have made it possible to perform FPA-based infrared imaging at a spatial resolution comparable with Raman instruments, even without access to a synchrotron beamline Findlay et al.
Where required, techniques for nano-scale infrared imaging have been developed Reddy et al. Crucially for the in situ analysis of microbial activities, there is evidence that FT-IR spectroscopy is compatible with SIP and can be used to track the cellular uptake of stable-isotope-labeled carbon 13C and nitrogen 15N compounds Muhamadali et al. FT-IR microspectropy can detect differences in the spectra of water and heavy water D2O , due to absorbance peaks corresponding to O—H and O—D bending modes occurring at different wavenumber regions Miller et al.
While we are unaware of studies that have combined D2O labeling with FT-IR spectroscopy to monitor the activities of individual microbial cells, this has recently been achieved using Raman spectroscopy Berry et al. Further to the studies discussed above, Muhamadali et al.
Of these techniques, infrared spectroscopy was found to provide the most consistent results for the entire sample set in terms of spectral quality and reproducibility , which led the authors to suggest that FT-IR analyses could be particularly useful for characterizing mixed cultures also see Wenning et al. Indeed, FT-IR microspectroscopy has already been used to quantify compare the abundances of bacteria and archaea within subsurface aquifer samples, based on domain-specific CH3:CH2 absorbance ratios Igisu et al.
Recommendations and Outlook The spectroscopic imaging of microbial cells in physically and chemically complex samples involves diverse analytical challenges.
While addressing these will often require sample-specific optimization steps such as identifying an appropriate laser wavelength; Edwards et al. Based on the case studies discussed in this Mini Review, it is possible to identify several general guidelines for achieving this Table 1.
The suggestions provided in Table 1 additionally highlight the promising role that live-cell FT-IR imaging could play in environmental microbiological research, further to Raman measurements which have traditionally been more common in this field.
The future development of vibrational spectroscopy instrumentation and analytical methods may serve to further enhance the cross-compatibility of Raman and FT-IR techniques e.
Experimental goals associated with the Raman and FT-IR imaging of single microbial cells in complex biological samples. Additionally to considering the benefits and pitfalls inherent to Raman vs.
FT-IR measurements, experiments focusing on the imaging of single cells in complex habitats can be expected to profit from combining these techniques with other analytical approaches Figure 1. Synchrotron-FT-IR microspectroscopy has been paired with synchrotron ultraviolet microspectroscopy and time-of-flight-secondary ion mass spectrometry ToF-SIMS for the analysis of human liver tissue, with each technique yielding unique information on the chemical composition of the sample Petit et al.
Jarmin, and J. Miranda Small vs. Large vs.
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Small Business Kim, J. Ling International Journal of Management, 29 1 , International Journal of Economics and Finance, 4 5 , Minniti, M. European Journal of Development Research, 22 3 , Journal of Business and Enterprise Development, 2, Menon, V. International Journal of Business and Management, 7 1 , Misango, S. ACase in Kenya. International Review of Management and Business Research, 2 1 , Nagesh, P.
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Abstract Resonance Raman spectra of Fe—S proteins are sensitive to the cluster type, structure and symmetry. Furthermore, bands that originate from bridging and terminal Fe—S vibrations in the 2Fe—2S, 3Fe—4S and 4Fe—4S clusters can be sensitively distinguished in the spectra, as well as the type of non-cysteinyl coordinating ligands, if present. For these reasons, resonance Raman spectroscopy has been playing an exceptionally active role in the studies of Fe—S proteins of diverse structures and functions.
We provide here a concise overview of the structural information that can be obtained from resonance Raman spectroscopy on Fe—S clusters, and in parallel, refer to their thermodynamic properties e. We demonstrate how the knowledge gained over the past several decades on simple clusters nowadays enables studies of complex structures that include Fe—S clusters coupled to other centers and transient processes that involve cluster inter-conversion, biogenesis, disassembly and catalysis.
Keywords: Resonance Raman spectroscopy, Iron-sulfur proteins, Reduction potential Introduction Resonance Raman RR spectroscopy is a powerful tool for identification and characterization of the metal active site and elucidation of structure—function relationship in metalloproteins and metalloenzymes, including hemic, iron—sulfur, diiron and copper proteins [ 1 , 2 ].
When the wavelength of the excitation laser coincides with that of an allowed electronic transition of the protein chromophore, the intensities of certain Raman bands become selectively enhanced by several orders of magnitude.