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Title: Developing Raman sorting and imaging techniques to characterise and differentiate unculturable microbial single cells
Author: Li, Mengqiu
ISNI:       0000 0004 2728 498X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2012
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Unculturable microorganisms are a major challenge facing researchers in environmental microbiology, and Raman spectroscopy has emerged as a novel technique in this field as it is able to analyse single microbial cells without cultivation. It has been combined with stable isotope probing (SIP) to identify the ecological functions of unculturable microorganisms. However, Raman signal from microorganisms is usually weak and taking single-cell Raman spectra is timing consuming. The interpretation of microorganisms' Raman spectra is also difficult due to their complexity. This thesis aims to improve Raman spectroscopic techniques for environmental microbiology research. Resonance Raman (RR) spectroscopy and surface enhanced Raman scattering (SERS) were employed to enhance single-cell Raman spectra. RR spectroscopy was combined with SIP to rapidly image natural photosynthetic microorganisms and reveal their CO2 fixation activities at single-cell level. Single photosynthetic microorganisms with only 10 % difference in their 13C content can be rapidly differentiated using RR spectroscopy in a non-destructive manner. Two potentially suitable methods to synthesise SERS-active nanoparticles for labelling the surface of microorganisms were investigated. These two methods both labelled single microorganisms to a satisfactory level as shown by electron microscopy images, but more work is needed to study the resultant SERS spectra. A novel quantitative spectral marker, the thymine Raman band, was identified during the investigation of using Raman spectroscopy to track carbon flow in a model food chain. This new spectral marker shows intriguingly different isotopic shift behaviour from the well documented phenylalanine Raman band. This difference was studied and brought to light a previously omitted aspect of Raman spectroscopy: the isotopic shift of Raman bands may reveal the biochemical pathways. With enhanced Raman signal, high-throughput Raman activated cell sorting (RACS) became possible and this thesis proves the concept that the combination of RR spectroscopy and microfluidic devices can rapidly profile photosynthetic microbial communities and potentially sort cells based on their in situ activities. This thesis also extended the Raman-SIP method to nitrogen and found that single-cell Raman spectra can quantify 15N-uptake in single bacterium using multivariate analysis. The studies included in this thesis revolve around the application of Raman spectroscopy in environmental microbiology. They strengthened and expanded the existing Raman-SIP method and opened the door to the development of important new techniques such as high-throughput RACS systems.
Supervisor: Huang, Wei Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available