This thesis describes the development of instrumentation for a high resolution 0.78 mum laser diode spectrometer operated at room temperature. Single mode GaAlAs laser diodes are used with a tunability of 0.06 nm/°C. A computer controlled temperature scanning control system, which completely removes the need for precision instrumentation amplifiers and ultra-low-temperature coefficient components featured in previous designs, was developed. This system is capable not only of generating rapid temperature scans but maintaining a set point temperature to an accuracy of +/-2 mK in a range of 11 to 36 °C. A Michelson-type infrared wavemeter of 0.01 nm precision is used for coarse frequency calibration and the water vapour spectrum is used as an absolute wavelength standard. Fine frequency calibration is done using a Fabry-Perot etalon of 0.25 cm-1 free spectral range, which is also used to monitor the mode structure of the laser and record the occurrence of a mode hop during a scan. By modulating the injection current of the laser, relatively weak, overtone spectral features of molecules can be extracted from background by means of frequency modulation spectroscopy. More than fifty lines of the nu1+3nu3(a) and the nu1+2nu2+nu3+4v40(b) bands of acetylene in the 12666-12731 cm-1 region, have been measured with the above spectrometer to a precision of 0.003 cm-1, yielding much improved band centres and upper state molecular constants as; nu0(a) = 12675.668(7) cm-1, B'(a) = 1.15158(16) cm-l and D'(a) = 2.98(83)xl0-6 cm-1; nu0(b) = 12710.913(4) cm-1, B'(b) = 1.15794(18) cm-1 and D'(b) = 5(l)xl0-6 cm-1. A two-level crossing interaction treatment has been applied to both bands, giving reasonable explanations of closely split lines within them. An -resonance treatment is further included to analyse a global perturbation in the nu1+2nu2+nu3+4nu40 band.

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.

Raman and infrared (IR) spectroscopies provide detailed information about biological constituents such as lipids, proteins, carbohydrates and DNA/RNA, etc. Based on this, these techniques can be used to differentiate cells and tissues, as well as employed as a diagnostic tool for detecting post-exposure biochemical alterations in toxicity assessment due to the induced changes of chemical composition and structure reflected by their spectral properties. Over the past few decades, Raman and IR spectroscopies with the development of more sophisticated instruments can provide high-resolution spectral data from heterogeneous biological samples, which consisting of large amount of biochemical information, is complex. Therefore, computational analysis is employed to process and analyse the data for obtaining meaningful information and getting deeper insight into the wavenumbers-related biochemical alterations. Carbon-based nanoparticles (CNPs) are most widely used novel nanomaterials. With their widespread application, concerns emerge on their potential risk to the health of organism and human, and investigation on their possible toxicity is urgently required. This thesis is contributing to the toxicity assessment of CNPs by using spectroscopic techniques coupled with computational analysis. Findings from our projects indicated that this approach has the capability of detecting the CNPsinduced biochemical alterations both in vitro and in vivo, which implies that techniques involved in IR and Raman spectroscopy can provide a rapid and highly sensitive tool to detect minimal changes at the subcellular level.

There remains a continuing threat of terrorist/insurgent attacks on military/civilian personnel and key strategic infrastructures both within the UK mainland and in operational theatres. The development of a novel, innovative, low cost, field deployable bionanosensor, which will have the capability to detect low levels of explosive in a multiplexed fashion is required. The use of the specific interaction between small molecules and biological capture molecules such as antibodies coupled with the detection technique of surface enhanced Raman scattering (SERS) allows a ‘one shot’ analysis. This research makes use of antibody functionalised silver nanoparticles for the detection of the explosives TNT, RDX and PETN by surface enhanced Raman scattering (SERS). Commercially available antibodies specific for TNT and RDX have been modified to specifically orientate ‘flat’ on the surface of silver nanoparticles bringing the target close enough to the metal surface to allow an intrinsic SERS signal of the target molecule to be obtained. Quantitative detection of TNT and RDX explosives was achieved, with pM sensitivity demonstrated for RDX. Furthermore, TNT was detected in two different types of dirt, natural and synthetic dirt in order to mimic a more realistic matrix in which TNT would be found in the field. However, for the detection of PETN, it was required to develop a method to modify a PETN antibody in-house, to specifically orientate ‘flat’ on the nanoparticles surface similarly to the commercially available antibodies. This was achieved by using carbodiimide chemistry and the antibody was purified by cartridge centrifugation and HPLC. The PETN modified antibody was then functionalised onto silver nanoparticles and detection of PETN was achieved by SERS. In addition, PCA was used to allow multiplexed analysis based on unique Raman bands for the three different explosives which could be clearly identified in the SERS spectra. Finally, TNT was detected by using magnetic nanoparticles which were functionalised with a terminal amine group in combination with FITC modified TNT antibody functionalised silver nanoparticles. This assay was designed to allow for the formation of a Meisenheimer complex in the presence of TNT, between the amine functionalised magnetic nanoparticles and the TNT. Furthermore, the TNT antibody functionalised silver nanoparticles also binds to TNT, aggregating the nanoparticles. The magnetic nanoparticles were subsequently used to remove the nanoparticle assembly from the matrix, resulting in a concentrated sample on the magnet, resulting in an increase in SERS.

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HHG spectroscopy is a powerful tool for observing electronic structure along with electronic and nuclear dynamics with state of the art resolution on the attosecond (10⁻¹⁸ s = 1 as) timescale and angstrom (10⁻¹⁰ m = 1 Å). Thus far, HHG spectroscopy has been applied to small hydrocarbons such as ethylene and methane using an 800 nm drive laser field wherein dynamics can only be measured in a 0.9 - 1.6 fs time window. This PhD has two primary aims: 1) to extend the time window over which we can make measurement up to 4 fs using an 2 μm drive laser field and 2) to perform HHG spectroscopy on molecules which are in the liquid phase at room temperature. The molecules at the focus of this thesis are benzene and substituted benzenes. The design and development of an apparatus to generate a stable vapour jet from a thin continuous nozzle is presented. The completed and tested apparatus has demonstrated that stable and reproducible spectra can be acquired with no contamination between different samples as they are switched over. Comparison of methylated benzene to benzene harmonic spectra show good agreement with theoretical results on coupled electron-nuclear dynamics developed by our collaborators. We also compare HHG spectra acquired under similar generating conditions from deuterated and protonated benzene which enables us to track nuclear dynamics and to determine the origin of the ionized electron in benzene. HHG spectra from the halogenated benzenes, in particular fluorobenzene, seem to illustrate the contribution of more tightly bound orbitals to the HHG signal which is indicative of dynamical interferences.

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