Breast cancer is the most common cancer in women. It is a highly heterogeneous disease in terms of histology, therapeutic response and patient outcomes. Early and accurate detection of breast cancer is crucial as the patient prognosis varies greatly depending on the diagnosis of the disease. Nonetheless current breast cancer classification methods fail to precisely sub-classify the disease, resulting in potential inadequate therapeutic management of patients and subsequent poor clinical outcomes. Substantial effort is therefore put in cancer research to develop methods and find new biomarkers efficiently identifying and characterizing breast tumor cells. Moreover it is now well-recognized that the intensive cross-talk between cancer cells and their microenvironment (including non-tumor cells) highly influences cancer progression. Recently, a growing body of clinical evidence reported the prognostic and predictive value associated with the presence of tumor infiltrating lymphocytes (TILs) in the microenvironment of breast tumors. Although the evaluation of TILs would be of great value for the management of patients and the development of new immunotherapies, it is currently not assessed in routine practice. Furthermore Fourier transform infrared (FTIR) imaging has shown its usefulness to study a panel of human cancers. Infrared (IR) spectroscopy coupled to microscopy provides images composed of multiple spectra reflecting the biochemical composition and subtle modifications within biological samples. IR imaging therefore provides useful information to improve breast cancer identification and characterization. The ultimate aim of this thesis is to improve breast cancer diagnosis using FTIR imaging to better identify and characterize cancer cells and the tumor microenvironment of breast cancers. In a first step we carried out a feasibility study aiming at evaluating the impact of the sample fixation process on IR spectra. While spectra were undeniably influenced by this biochemical alteration, our results indicated that closely-related cell types were influenced similarly and could still be discriminated on the basis of their spectral features. We then demonstrated the capability of IR imaging to discriminate a tumor from a normal tissue environment based on the spectral features of tumor cells and the surrounding extracellular matrix. A particular focus was placed on the identification of lymphocyte spectral signatures of cells isolated from blood or present within secondary lymphoid organs such as tonsils. Our results revealed that IR imaging was sensitive enough to discriminate lymphocyte subpopulations and to identify a particular spectral signature that we assigned to lymphocyte activation. Finally we highlighted the potential value of IR imaging as complementary tool to identify and characterize TILs in breast tumor samples. Altogether, our results suggest that IR imaging provides interesting and reliable information to improve breast cancer characterization and to assess the immune microenvironment of breast tumors.<p>/<p>Le cancer du sein est le carcinome le plus fréquent chez la femme. C’est une maladie très hétérogène du point de vue histologique, de la réponse thérapeutique et de l’évolution clinique. Une détection rapide et précise de la maladie est cruciale, un diagnostic du cancer du sein dès les premiers stades de la maladie permet une meilleure prise en charge du patient et est directement associé à un meilleur pronostic. Néanmoins la classification actuelle des cancers du sein ne permet souvent pas de caractériser la maladie de manière précise, ce qui donne lieu à la mise en place de traitements moins ciblés et une évolution clinique peu favorable. Pour remédier à cela, des efforts conséquents sont réalisés en recherche, dans le but de mettre au point des méthodes capables d’identifier et de caractériser les cellules tumorales. De plus il est actuellement reconnu que le micro-environnement tumoral (composé des cellules non-tumorales) influence fortement la progression du cancer. Récemment de nombreuses études ont montré que la présence de lymphocytes au niveau des tumeurs mammaires (TILs) était corrélée à un meilleur facteur pronostic et prédictif. Bien que l’évaluation des TILs soit de grande importance dans le cadre des immunothérapies, cet élément n’est actuellement pas pris en compte dans les analyses de routine. Par ailleurs, l’imagerie infrarouge par transformée de Fourier (FTIR) a démontré son utilité dans l’étude de plusieurs cancers humains. La spectroscopie infrarouge (IR) couplée à la microscopie fourni des images composées de multiples spectres qui reflètent la composition biochimique et les modifications dans les échantillons biologiques. De ce fait l’imagerie infrarouge procure des informations utiles pour améliorer l’identification et la caractérisation du cancer du sein. L’objectif général de cette thèse est d’améliorer le diagnostic du cancer du sein par imagerie FTIR pour mieux identifier et caractériser les cellules cancéreuses et le micro-environnement tumoral des tumeurs mammaires. Dans un premier temps nous avons effectué une étude de faisabilité afin d’évaluer l’impact du protocole de fixation des tissus sur les spectres IR. Bien que les spectres soient indéniablement influencés par cette altération biochimique, nos résultats indiquent que des types cellulaires proches sont influencés de manière similaire et peuvent donc être discriminés sur base de leurs caractéristiques spectrales. Nous avons ensuite démontré la capacité de l’imagerie IR de distinguer un environnement tumoral d’un environnement normal sur base des particularités spectrales des cellules tumorales et de la matrice extracellulaire. Une attention particulière a ensuite été portée afin d’identifier des signatures spectrales de cellules immunitaires du sang et au sein d’organes lymphoïdes secondaires, tels que les amygdales. Nos résultats ont révélé que l’imagerie IR permet d'identifier une signature spectrale particulière, que nous avons associée à une stimulation lymphocytaire. Finalement nous avons mis en évidence l’utilité de l’imagerie IR en tant qu’outil complémentaire pour identifier et caractériser les TILs dans les échantillons tumoraux mammaires. De manière générale, nos résultats suggèrent que l’imagerie IR fournit des informations intéressantes et fiables pour améliorer la caractérisation et l’évaluation du micro-environnement immunitaire dans les tumeurs mammaires. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

There is an urgent need to develop reliable and cost-saving methods to select pre-clinically new drug candidates with original mechanism for cancer therapy. Previous results have shown that IR spectra of cancer cells exposed to various drugs provided a global signature of all the metabolic changes induced by the treatments. In this thesis, we attempted to develop a selection criterion – based on FTIR spectroscopy – for potential antitumor compounds according to their mechanism of action. <p>In chapter III, it was demonstrated that spectral variations in IR spectra of cancer cells induced by a treatment can be correlated to the mechanism of the drug. Human prostate cancer PC-3 cells were exposed to 7 well-described anticancer drugs belonging to 3 distinct classes. Each class is characterized by a unique mode of action. Drugs known to induce similar types of metabolic disturbances appear to cluster when spectrum shapes are analyzed. Chapter IV generalized the results obtained on PC-3 cells with six other cell lines. We showed that the spectral signatures of drug effects are mainly independent of the cell line. Chapter V indicated that, while the cell cycle phase influence IR spectra of cells, the drug spectral signature was dominated by global metabolic modifications and not much by the cell cycle perturbations due to this drug. <p>Chapter VI and VII focused on lipids. While the precise identification of particular molecules is particularly complex with IR spectroscopy, we attempted to extract more precise information and to assign spectral variations to specific changes in lipids. IR spectra of lipids contain very interesting details on their nature and structure. We achieved to build a tool which quantifies five major lipid classes in complex mixtures such as total lipid cell extracts. However, based on this tool, the treatments used do not induce any variation in the lipid cell composition (for five classes).<p>Finally, in chapter VIII, we applied the method developed previously on a new potential class of anticancer molecules: the polyphenols. A global method was particularly interesting as the development of therapy using these compounds is hampered by the complexity of the multiple anticarcinogenic mechanisms of these molecules. We have noticed the similarities and discrepancies among 3 very close synthetic molecules and the observations were coherent with previous biological data. We also compared them with 3 natural molecules already in clinical phase for treatment of various cancers.<p>In conclusion, we developed an objective classifier for potential anticancer drugs based on their global effects on cancer cells. Applied to a larger scale, this method could constitute a first step in the screening method to select drugs with original mode of action.<p>There is an urgent need to develop reliable and cost-saving methods to select pre-clinically new drug candidates with original mechanism for cancer therapy. Previous results have shown that IR spectra of cancer cells exposed to various drugs provided a global signature of all the metabolic changes induced by the treatments. In this thesis, we attempted to develop a selection criterion – based on FTIR spectroscopy – for potential antitumor compounds according to their mechanism of action. <p>In chapter III, it was demonstrated that spectral variations in IR spectra of cancer cells induced by a treatment can be correlated to the mechanism of the drug. Human prostate cancer PC-3 cells were exposed to 7 well-described anticancer drugs belonging to 3 distinct classes. Each class is characterized by a unique mode of action. Drugs known to induce similar types of metabolic disturbances appear to cluster when spectrum shapes are analyzed. Chapter IV generalized the results obtained on PC-3 cells with six other cell lines. We showed that the spectral signatures of drug effects are mainly independent of the cell line. Chapter V indicated that, while the cell cycle phase influence IR spectra of cells, the drug spectral signature was dominated by global metabolic modifications and not much by the cell cycle perturbations due to this drug. <p>Chapter VI and VII focused on lipids. While the precise identification of particular molecules is particularly complex with IR spectroscopy, we attempted to extract more precise information and to assign spectral variations to specific changes in lipids. IR spectra of lipids contain very interesting details on their nature and structure. We achieved to build a tool which quantifies five major lipid classes in complex mixtures such as total lipid cell extracts. However, based on this tool, the treatments used do not induce any variation in the lipid cell composition (for five classes).<p>Finally, in chapter VIII, we applied the method developed previously on a new potential class of anticancer molecules: the polyphenols. A global method was particularly interesting as the development of therapy using these compounds is hampered by the complexity of the multiple anticarcinogenic mechanisms of these molecules. We have noticed the similarities and discrepancies among 3 very close synthetic molecules and the observations were coherent with previous biological data. We also compared them with 3 natural molecules already in clinical phase for treatment of various cancers.<p>In conclusion, we developed an objective classifier for potential anticancer drugs based on their global effects on cancer cells. Applied to a larger scale, this method could constitute a first step in the screening method to select drugs with original mode of action.<p><p>Afin d’améliorer les thérapies contre le cancer, il devient actuellement cruciale de développer une méthode pour améliorer la sélection préclinique de nouvelles molécules, potentiellement anticancéreuses. Des publications précédentes ont mis en évidence que les spectres infrarouges de cellules cancéreuses exposées à différents agents thérapeutiques fournissent une empreinte globale de l’ensemble des changements métaboliques induit par ce médicament. Dans cette thèse, nous proposons d’utiliser la spectroscopie infrarouge pour mettre au point un critère de sélection basé sur le mode d’action des agents anticancéreux. Plusieurs aspects de la technique ont été investigués. Nous avons d’abord démontré la possibilité d’utiliser les spectres infrarouges de cellules cancéreuses de prostate PC-3 traitées avec 7 drogues pour classer ces molécules selon leur mode d’action. Nous avons ensuite reproduit les résultats obtenus sur PC-3 avec 6 autres lignées cellulaires et montré que la signature spectrale obtenue était largement indépendante de la lignée. Par la suite, nous avons étudié si l’effet sur le cycle cellulaire induit par de nombreuses molécules anticancéreuses, pouvait expliquer certains changements spectraux observés suite au traitement. Nous avons pu montrer que la majorité des variations spectrales n’étaient pas liées à une perturbation du cycle cellulaire. Nous nous sommes ensuite concentrés sur une classe de molécules en particulier: les lipides. Après avoir mis en évidence l’ensemble des informations contenues dans un spectre infrarouge de lipides, nous avons mis au point un modèle permettant de quantifier 5 classes de lipides dans des mélanges complexes tels que des extraits lipidiques provenant de cellules. Néanmoins, aucune variation du contenu en ces 5 classes de lipides n’a été observée pour les traitements utilisés dans cette étude. Enfin, nous avons appliqué la méthode mise au point dans cette thèse à une classe de molécules prometteuses :les polyphénols. Une approche globale s’avère particulièrement intéressante pour ces composés étant donné qu’ils présentent des mécanismes anticancéreux multiples et complexes. Nous avons comparé 3 molécules naturelles en phase clinique pour le traitement de certains cancers et 3 molécules synthétiques présentant une structure très proche. Par notre méthode, nous avons mis en évidence certaines similarités et différences de ces 6 molécules en termes d’effets globaux sur les cellules. En conclusions, nous avons développé un outil objectif de classification pour les molécules anticancéreuses potentielles, basée sur le mécanisme global des composés. Appliquée à plus large échelle, cette méthode pourrait constituer une première étape permettant de sélectionner les molécules avec un mode d’action original. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

We investigate the spectra of asteroids at near- and mid-infrared wavelengths. In 2010 and 2011 we reported the detection of 3 ?m and 3.2-3.6 ?m signatures on (24) Themis and (65) Cybele indicative of water-ice and complex organics [1] [2] [3]. We further probed other primitive asteroids in the Cybele dynamical group and Themis family, finding diversity in the shape of their 3 ?m [4] [5] [6] and 10 ?m spectral features [4]. These differences indicated mineralogical and compositional variations within these asteroid populations. Also in the mid-infrared region we studied a larger population of asteroids belonging to the Bus C, D, and S taxanomic classes to understand the relationship between any mineralogy and hydration inferred in the visible and near- infrared with the shape, strength, and slope of the 10 ?m emission. We have discovered that at least 3 of the main Bus taxanomic groups (Cs, Ds, and Ss as defined by their visible spectra) clearly cluster into 3 statistically distinct groups based on their 8-13 ?m spectra. Additionally we have attempted to simulate in a laboratory the possible organic compounds we have detected on two asteroids, using various mixtures containing aromatic and aliphatic hydrocarbons. We find that asteroid (24) Themis and (65) Cybele have ?CH2/?CH3 and NCH2/NCH3 ratios similar to our 3- methylpentane, propane, and hexane residues, suggesting that the organics on these asteroids may be short chained and/or highly branched. The ?CH2/?CH3 and NCH2/NCH3 for asteroid(24)Themis are most consistent with the DISM, and some carbonaceous chondrites. The band centers of the C-H stretch absorptions indicate that both asteroids may have aliphatic carriers chemically bonded to electronegative groups (i.e. aromatics), and some that are not. We also detect a 3.45 ?m feature in the spectra of both asteroids that is present in several dense molecular clouds. Our results suggest an interstellar origin for the organics on (24) Themis, and likely (65) Cybele. The differences in the organics of Themis and Cybele are likely related to variations in thermal processing, irradiation and/or formation region in the solar nebula.

Call number: LD2668 .T4 1986 C64 / Master of Science / Chemical Engineering

Master of Science / Department of Kinesiology / Thomas J. Barstow / Near infrared spectroscopy is currently routinely used to assess tissue (muscle) oxygenation at rest and during exercise. While most investigators assume that hemoglobin ([Hb]) is the major contributor to the responses seen during exercise, the relative contribution of myoglobin ([Mb]) to the NIRS signals remains controversial. PURPOSE: a) To calculate the range of light absorbing potential (LAP) of hemoglobin and myoglobin in mammalian skeletal muscle at rest based on analysis of published chemical and morphometric data in humans and other mammals (Part 1), and b) use the information in a) to interpret changes in total [Hb+Mb] from NIRS during exercise (Part 2). METHODS: Part 1: Information was retrieved from five published studies with regard to capillary density (#caps/mm2) and [Mb] in skeletal muscle of human, horse and rat. Preference was given to studies in which both measurements were provided for the same muscles. [Hb] in skeletal muscle was estimated as a function of capillary density, [Hb] in systemic blood, and the ratio of capillary-to-systemic hematocrit at rest and during exercise. Part 2: Changes in total [Hb] + [Mb] (as t[Hb+Mb]) from published NIRS data obtained from human subjects performing cycling or knee extension exercise were interpreted in the context of the results of Part 1. RESULTS: Part 1: Individual group mean values for skeletal muscle [Mb] in the literature ranged from 0.25-0.67 mM in human samples, with a similar range for muscles of the rat hindlimb; horse limb muscles tended to be higher (up to 1.0 mM). Capillary densities ranged from ~200 to 600 caps/mm2 in human and rat muscles, and up to 800 caps/mm2 in horse muscle. Assuming a resting capillary hematocrit of 22% and 4 fold greater LAP for each mole [Hb] vs [Mb], the resulting estimation of capillary [Hb] ranged from ~0.03 to 0.09 mM in human and rat muscles, and up to ~0.13 mM in horse muscles. The results suggest that [Mb] could contribute ~50-70% of the total LAP at rest in human skeletal muscle. Part 2: With exercise, total heme by NIRS can increase ≥ 30% in individual human subjects. Assuming this increase reflects only increased [Hb], this fits well with the observed increase in capillary hematocrit with exercise. CONCLUSIONS: 1) In skeletal muscle at rest, [Mb] is likely to be at least as significant a light absorbing heme as is [Hb] in most mammalian muscles, including the human leg. 2) Observed increases in t[Hb+Mb] with NIRS during exercise can be explained by an increase in capillary hematocrit, even in the presence of significant [Mb].

Master of Science / Department of Diagnostic Medicine/Pathobiology / Larry C. Hollis / Mark F. Spire / Two studies were undertaken to evaluate the use of Near Infrared Spectroscopy (NIRS) to determine arterial oxygen saturation (StO[subscript]2) in cattle with naturally-occurring Undifferentiated Bovine Respiratory Disease (UBRD) and experimentally-induced UBRD utilizing Mannheimia haemolytica. The first study was a natural infection model utilizing 679 beef heifers weighing approximately 227 kg (500 pounds) originating from a southeastern U.S. salebarn. Heifers were evaluated for UBRD upon feedlot arrival, at revaccination, at day 35 on feed, at re-implant time, and two weeks prior to shipment for slaughter. Animals deemed to have UBRD were treated for UBRD and data was collected for 5 days following treatment, while a comparable healthy cohort was also evaluated at the time of treatment. There was a trend for NIRS to be able to predict the incidence of subsequent UBRD when cattle were evaluated on arrival (p=0.0552). However, the ability to detect UBRD in clinically ill cattle was not significantly different (p>0.1690) when compared to healthy cohorts in this model. When carcass characteristics were evaluated at each time point, NIRS StO[subscript]2 values were able to differentiate between yield grades of animals with UBRD and healthy cohorts when evaluated at revaccination, day 35, re-implant, and pre-shipping (p<0.0199). NIRS tended to be able to differentiate yield grades at initial processing (p=0.0513). StO[subscript]2 was not a predictor of quality grade at any time point (p>0.1023), nor was there any correlation between lung lesions at slaughter and StO[subscript]2 (p>0.2292). The second study involved 12 head of 181 kg (400 pound) heifers which were subjected to an experimental challenge model of Mannheimia haemolytica. Animals were evaluated daily and StO[subscript]2 readings recorded 12 hours pre-inoculation, at inoculation, 6, 12 and 24 hours post inoculation and daily for the next 12 days. While NIRS could not definitively differentiate healthy cohort cattle from challenge cattle (p>0.0713), there were trends toward challenge cattle having lower StO[subscript]2 values than healthy controls. The authors conclude that while these studies did not provide conclusive evidence of the ability of NIRS to detect UBRD, further studies with a machine that is specifically calibrated and designed for use with cattle should be performed.

Thesis (MEng)--Stellenbosch University, 2015. / AFRIKAANSE OPSOMMING: Raadpleeg die volteks vir opsomming, asseblief. / ENGLISH ABSTRACT: Please refer to full text for abstract

Applications of laser absorption spectroscopy for trace gas detection are many and diverse, ranging from the environmental and atmospheric to the medical and industrial. The aim of creating a spectrometer which combines high sensitivities and selectivities (in order to measure small amounts of absorbers or species that are only weakly absorbing, in a complex background matrix) with a wide spectral coverage (to allow broadband absorbers or multi-component samples to be studied) can be realised by implementing three separate concepts: the exploitation of the strong, fundamental transitions of the mid-infrared; the use of sensitive spectroscopic techniques; and the selection of a widely tunable laser source. In this thesis, these ideas are investigated individually and in combination in order to achieve such a goal. Laser spectroscopic techniques based on optical cavities are used to build a high resolution spectrometer covering a large spectral range capable of selectively detecting low levels of gaseous compounds of interest, especially those of medical or environmental significance. Work in both the near- and mid-infrared is presented, including much of the initial, developmental work which was conducted in the former region. The thesis begins with an overview of both narrowband and broadband near-infrared radiation sources, with a particular emphasis on commonly available diode lasers (DLs). A novel laser source, the digital supermode distributed Bragg reector (DS-DBR) laser, is introduced as a useful laser source for spectroscopy, combining the usual benefits of telecom DLs with a wide tunability (1563 – 1613 nm). The laser can be operated in an internal or external ramping mode, allowing the output wavelength to be scanned or stepped across a desired region. The observation of mode-hopping during the application of the scanning methodology is examined and rationalised. The ability of the DS-DBR laser to perform high resolution spectroscopy over its entire spectral coverage is demonstrated by recording spectra of carbon dioxide (CO₂) over this range, covering transitions from two of the four Fermi resonance components of the 3ν₁ + ν₃ combination band. The results of conducting wavelength modulation spectroscopy on CO₂ are also reported. A system developed for performing cavity ring-down spectroscopy (CRDS), capable of the real-time retrieval of ring-down times (RDTs), is presented and discussed. The outcomes of initial tests performed with a conventional DL at 1557 nm, to study a calibrated mixture of CO₂ in air at various pressures, are given. In addition, the results of combining this system with the DS-DBR laser are discussed. The bandwidth of the DS-DBR laser was found to be larger than that of a standard DFB DL, resulting in the presence of noisy cavity modes. Despite this, the acquisition of reproducible RDTs is demonstrated, with single wavelength studies of an evacuated cavity at 1605.5 nm yielding a RDT of 24.54 ± 0.04 µs and Allan variance calculations signalling an attainable minimum detectable absorption coefficient, α_min, of 2.8 x 10^-10 cm^-1 over 20 s. The ability to perform CRDS across the whole DSDBR laser wavelength range without the need for cavity re-alignment is illustrated, and studies conducted on CO₂ in air, calibrated mixtures and breath are reported. Investigations are also described into the accurate determination of the ¹³C/¹²C ratio in exhaled CO₂ undertaken using CRDS and cavity enhanced absorption spectroscopy (CEAS) on CO₂ isotopologues, an approach which can be utilised as a diagnostic aid in determining Helicobacter pylori infection. The focus of the thesis then moves to the mid-infrared, to describe quasi phase matching difference frequency generation (QPM-DFG) and its use to generate laser light at 3 µm by optically mixing near-infrared DLs. The theory behind this non-linear optical interaction is outlined, and the construction of a free-space QPM-DFG system using periodically poled lithium niobate is detailed and characterised. This DL-based QPM-DFG arrangement has been coupled with the CRDS system developed to create a mid-infrared CRD spectrometer. The results of single wavelength studies indicate RDTs of ~ 6 µs and an achievable αmin of 2.9 x 10^-9 cm^-1 over 44 s for an evacuated cavity. Spectroscopic investigations carried out on methane (CH₄), acetone and deuterium are documented; for the latter species, Dicke narrowing of the electric quadrupole ν(1←0) Q(2) transition at 2987.29 cm^-1 is observed and the integrated absorption cross-section for the same transition measured as 2.29 ± 0.03 x 10^-27 cm²cm^-1molec^-1. The results of modifications made to the system, namely the use of a more powerful Nd:YAG laser as the pump radiation source, as well as a faster detector combined with a variable amplifier, are presented; these include the observation of an improved optimal α_min of 6.4 x 10^-10 cm^-1 over 151 s for an empty cavity. Finally, work utilising the DS-DBR laser as one of the near-infrared sources for the QPM-DFG set-up is presented. This configuration generates radiation covering a wide mid-infrared range (3130 – 3330 nm) and has been used to perform direct absorption and wavelength modulation spectroscopy on ro-vibrational transitions within the fundamental ν₃ (F₂) band of CH₄. The spectrum of methanethiol (CH₃SH) over this region has also been investigated, with preliminary studies identifying a feature at 3040 cm^-1 as a potential indicator for monitoring this biomarker in breath. The results of coupling this mid-infrared radiation with an optical cavity to perform CEAS combined with phase sensitive detection are subsequently reported. Studies were conducted on calibrated CH₄ mixtures and ambient air to examine two transitions of the fundamental ν₃ (F₂) band of CH₄ in order to characterise the system: effective path lengths of ~ 700 m and α_min of 6.2 x 10^-8 cm^-1 over 8 s were found. The ^RQ₄ CH₃SH absorption feature at 3040 cm^-1 was also further studied with this system using prepared samples of CH₃SH in N₂ at different concentrations, yielding a CH₃SH detection limit of 2.4 ppm at 19 Torr. The potential of such a cavity-based, DS-DBR sourced, QPM-DFG mid-infrared spectrometer for trace gas sensing having thus been demonstrated, possible improvements that could be implemented to increase the sensitivity of the system are then discussed.

Cleaning validation of pharmaceutical manufacturing equipment is required by legislation. Generally, wet chemical techniques are employed using swabbing and/or rinse sampling methods. These are generally either selective and time consuming, or less selective and give results in a shorter period. The infrared reflection absorption spectroscopy (IRRAS) technique explored here attempts to deliver accurate, selective surface contamination information in real time to complement current methods and reduce down-time. The IRRAS instrument used in this research is a Fourier transform infrared (FTIR) spectrometer coupled by an IR fibre-optic cable to a grazing-angle sampling head with a fixed incidence angle of 80°. The introduced flexibility permits collection of in situ spectra from contaminated surfaces. Calibration models are developed using the multivariate, linear partial least squares (PLS) statistical method. The research focuses on sodium dodecyl sulfate (SDS), a model cleaning agent, on metal (aluminium and stainless steel) and dielectric (glass, EPDM and silicone) surfaces. The effects of surface finish are investigated for SDS on stainless steel. Calibrations for SDS and paracetamol in the presence of each other on glass surfaces are examined, as well as a common industrial cleaner (P3 cosa® PUR80) on polished stainless steel. For the calibration sets in this thesis, RMSECV values were < 0.41 µg cm⁻², corresponding to conservative surface residues detection limits of better than ~0.86 µg cm⁻². However, RMSECV values depend on the calibration loading range, and the detection limits were typically ~0.2 µg cm⁻² for loading ranges 0-2.5 µg cm⁻². These are below visual detection limits, generally taken to be 1-4 µg cm⁻², depending on the analyte and substrate. This shows that IRRAS is a viable method for the real-time detection and quantification of surface contamination by surfactants and active pharmaceutical ingredients on metals and dielectrics.

The objective of this thesis is to describe the development, construction and use of a new tool for optical coherent transient spectroscopy. The new tool is a frequency-switched CO₂ laser. A highly stable laser design was modified to include an intra cavity electro-optic modulator, which al lows the output of the laser to be frequency-switched. The frequency modulated output is used in spectroscopic experiments whose goals are the determination of decay rates for infrared moIecuIar transitions. The use of a frequency-switched laser is the most prom i sing means of making such measurements on nonpoIar molecules. The use of an electro-optic crystal inside a laser cavity introduces a number of fundamental problems which must be overcome before the instrument can be used to make useful spectroscopic measurements. These problems are brought about by the need for a stable laser amplitude and frequency output. The development of a novel stabilization technique to overcome these problems is documented in this thesis. Also included in this thesis is a description of the microcomputer and associated electronics necessary to integrate the laser into an experimental apparatus capable of performing signal averaging and background subtraction on raw time resolved data. The final chapters of this work describe experiments and results of measurements of the scattering cross sect ions of a nonpolar molecule with rare gas perturbers. The nonpolar molecule is SF₆ and the rare gas collision partners are Helium, Argon, and Xenon. The results indicate that the scattering cross section for state changing collisions displays a mass dependance predicted by classical collision theory. However, the measured cross sections for elastic velocity-changing collisions appears to be mass independent, which is at variance with theory.