Metal dication-ligand sandwich complexes have attracted intense attention recently for their widely use in catalysis, biochemistry and material science. The experimental techniques developed by our group have allowed forming, confining, cooling and investigating a wide range of metal dication complexes in the gas phase. In this thesis, the ultraviolet photofragmentation studies of Pb(II), Cu(II) and Ca(II) sandwich complexes with aromatic ligands are performed using a hybrid quadrupole ion trap instrument, followed by DFT/TDDFT calculations. The experimental results indicate that the complexes are capable of yielding structured, sometimes conformation resolved, UV spectra. The spectra of metal dication-benzene complexes exhibit features in the wavelength range 220-270 nm and a big raise as the wavelength decreases. The lead dication-bis(toluene) complex spectrum shows some well-resolved features arising from different conformers. The theory suggests that all of these complexes have excitations including significant contributions from the metal-based orbital. The adiabatic TDDFT methodology is able to give reasonable agreement between the calculated excitations and the experimental spectra for the close-shell complexes. But for some open-shell complexes, the calculated excitations are spin contaminated, which need to be discarded or corrected in the future. The degree of spin contamination for selected excitations is qualified by calculating the < S2 > values. For lead and calcium open-shell complexes, most of the excitations that can match the experimental features can be trusted. However, for the copper open-shell complex, only three states are ~90% doublet in their character which are responsible for some excitations that can match the spectra. Challenges such as developing the theory to describe the open-shell system and refining the experimental techniques to improve the resolution of the spectra, still remain.

The focus of this study is the potential use of FTIR imaging as a tool for objective automated histopathology. The Thesis also reports the use of multivariate statistical techniques to analyse the FTIR imaging data. These include Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), Multivariate Curve Resolution (MCR) and Fuzzy C-Means Clustering (FCM). The development of a new PCA-FCM Clustering hybrid that can automatically detect the optimum clustering structure is also reported. Chapter 1 provides a brief introduction to the use of vibrational spectroscopy to characterise biomolecules in tissues and cells for medical diagnosis. Chapter 2 details the basic histology of a lymph node before proceeding to present imaging results gained from the analysis of both healthy and diseased lymph node tissue sections. The ability of each multivariate technique to discriminate different tissue types is discussed. In addition, the spectral features that are characteristic for each tissue type are reported. The development and application of a new PCA-FCM Clustering algorithm that can automatically determine the best clustering structure is also described in full. The results indicate that cellular abnormality provides changes to both the protein and nucleic acid vibrations. However, similar spectral profiles were identified for highly proliferating cells that were contained within reactive germinal centres of the lymph node. Chapter 3 provides a short introduction to the histology of the cervlx before presenting imaging results that were gained from the analysis of both healthy and diseased cervical tissue sections. The ability of each multivariate technique to discriminate different tissue types is discussed. In addition, the spectral features that are characteristic for each tissue type are described in detail. Novel imaging experiments upon exfoliated cervical cells are also presented. It would appear that cellular abnormality in cervical tissues and cells affects both the protein and nucleic acid features of the spectra. Glycogen and glycoprotein contributions that are prevalent in healthy tissues are also absent. Chapter 4 details sample preparation methods, the instrumentation and procedures used for data acquisition, and the subsequent data processing and multivariate techniques applied to analyse the collected spectral datasets.

The thesis describes infrared spectra recorded during the adsorption of water, acetone, acetic acid and hexifluaroacetone onto oxidized and reduced rutile, and the development of a technique for recording the infrared spectrum of a solid immersed in a liquid. Bands observed on the hydroxylated rutile surface have been assigned to hydroxyl groups on the (110) plane and water IrDlecules adsorbed onto strong and weak Lewis sites on all exposed planes. The hydroxyl groups exist as isolated or hydrogen bonded groups on surface titanium ions or as hydrogen ions on bridging oxygen ions. Reduction of the rutile surface considerably decreased the amount of rmlecular water adsorbed on the hydroxylated surface. The adsorption of acetone onto the hydroxylated surface took place in three consecutive stages, the first involved acetone molecules Lewis bonding to weak sites, the second resulted in the formation of mesityl oxide on strong surface sites and occurred with stage one in the absence of surface water molecules. In the third stage acetate molecules were formed as a result of the decomposition of mesityl oxide. Adsorption of acetic acid onto rutile resulted in the formation of water and arpeaxeme of bands due to acetate groups and Lewis-bonded co lexes on the weak sites. Hexafluoroacetone reacted with surface hydroxyls to produce a salt of the gem-diol hexifluoropropane-2,2-dio1, which decomposed on the removal of water to form trifluoroacetate species. An infrared cell has been developed enabling solid discs to be treated and inmiersed in a solution under inert conditions. The cell, of path length 0.7cm, has been used to study the adsorption of ether, from a solution in carbon tetrachloride, onto silica. Designs of variable path length cells for use Hexafluoroacetone reacted with surface hydroxyls to produce a salt of the gem-diol hexifluoropropane-2,2-dio1, which decomposed on the removal of water to form trifluoroacetate species. An infrared cell has been developed enabling solid discs to be treated and inmiersed in a solution under inert conditions. The cell, of path length 0.7cm, has been used to study the adsorption of ether, from a solution in carbon tetrachloride, onto silica. Designs of variable path length cells for use unier vacuum are included.

In order to develop safe and effective topical and transdermal formulations to treat either local skin disorders or for systemic drug delivery, it is first imperative to assess skin permeation using a reliable and comprehensive analysis method. The assessment of drug permeation into/across the skin is traditionally accomplished using Franz diffusion cells with subsequent analysis by conventional chromatographic methods such as HPLC and more recently using advanced imaging techniques. In this context, time of flight-secondary ion mass spectrometry (ToF-SIMS) offers distinctive advantages in mapping drugs within skin with high sensitivity and chemical specificity without the need for fluorescent tags or radiolabels. The work in this thesis uses the combination of conventional and advanced methods to evaluate drug permeation into the skin. This approach provides complementary and detailed information regarding the permeated mass, the permeation depth and the spatial distribution and localisation of drugs within skin. As ToF-SIMS does not produce quantitative results, due to the matrix effects, a novel high throughput method was successfully developed to quantify ToF-SIMS data. This method involved the homogenisation of skin tissue followed by microarray printing of this skin homogenate with known concentrations of active pharmaceutical ingredients, specifically imiquimod and chlorhexidine. The subsequent analysis by ToF-SIMS of the resulting array allowed the generation of a calibration curve that can be used in the quantification of the unknown drug concentration in the tape strips. This work has demonstrated the potential of a method to quantify ToF-SIMS data of drugs within skin. Imiquimod is an immune modulator drug approved by the FDA for the treatment of superficial basal cell carcinoma (BCC) but not the nodular lesions. An assessment of imiquimod permeation from commercially available Aldara™ cream into ex vivo porcine skin was carried out using the complementary approach of HPLC and ToF-SIMS analysis. This work represents the most detailed assessment to date of the true extent of permeation of imiquimod from Aldara™ cream as previous studies analysed the permeation of Aldara™ cream showed a limitation in the analytical methodology employed (i.e. analysis by HPLC only). The results showed that imiquimod does permeate into the stratum corneum but is very limited in the deeper skin cell layers. In addition, the ToF-SIMS ion images of Aldara™ cream tape strips illustrated a non-uniform distribution of imiquimod within skin which may result in a decreased efficacy of the cream to uniformly treat whole BCC lesions giving rise to the likelihood of tumour recurrence. This offers previously unobserved insights about the spatial distribution of imiquimod delivered from Aldara™ cream. As other studies have reported that Aldara™ cream has some limitations in the treatment of nodular BCC lesions due to the cream’s inability to deliver imiquimod into the deeper more invasive nodular lesions, an enhancement of imiquimod permeation is thought to be useful to overcome these limitations. Therefore, an attempt to improve delivery of imiquimod into the deeper skin layers using microemulsions and microneedles was investigated. Imiquimod microemulsions were formulated, characterised and then tested for skin permeation enhancement. However, the assessment of imiquimod permeation from the formulated microemulsions alone and with microneedle pre-treatment using HPLC and ToF-SIMS demonstrated a limited ability of the microemulsions to improve delivery of imiquimod over Aldara™ cream. In contrast, Aldara™ cream with microneedle pre-treatment using a derma stamp electric pen showed improved delivery of imiquimod into the skin. This work is believed to be the first attempt to enhance imiquimod delivery using microemulsions and microneedles. Utilising the high sensitivity offered by the ToF-SIMS instrument in the analysis of individual tape strips, an in vivo and ex vivo comparison of chlorhexidine permeation into the stratum corneum was performed using commercial products currently used in hospitals within the UK for skin antisepsis. A comparison was carried out using the tape stripping technique with subsequent analysis of an individual tape strip by ToF-SIMS. The results showed that HiBiSCRUB® 4% produces a higher concentration of chlorhexidine in the upper stratum corneum layers than other products. This work demonstrated the first known application of ToF-SIMS to compare the in vivo skin permeation of commercially available chlorhexidine products and provides the foundation for the potential application of ToF-SIMS in assessing bioequivalence of topical products.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has proven its ability to characterise (in)organic surfaces, and is increasingly used for the characterisation of biological samples such as single cells. By combining ion imaging and molecular depth profiling it is possible to render 3D chemical images, which provides a novel, label-free way to investigate biological systems. Major challenges lie, however, in the development of data analysis tools and protocols that preserve the cell morphology. Here, we develop and employ such tools and protocols for the investigation of neuronal networks. One of the reasons 3D ToF-SIMS imaging of cells is underused is the lack of powerful data analysis tools as 3D ToF-SIMS measurements generate very large data sets. To address this issue, we developed a method that allows the application of principal component analysis (PCA) to be expanded to large 3D images making 3D ToF-SIMS image processing of whole, intact cells and cellular networks with multivariate analysis now accessible on a routine basis. Using this method, we are able to separate cellular material from the substrate and can then correct z-offsets due to the cells' topography resulting in a more accurate surface heightmap. The method also facilitates differentiation between cellular components such as lipids and amino acids allowing the cell membrane, the cytoplasm and the extracellular matrix (ECM) to be easily distinguished from one another. These developments permit us to investigate the intracellular localisation of specific native and non-native compounds label-free, not just in single cells but also in larger cellular networks. The visualisation of the cellular uptake of non-native compounds, namely fluorescent dyes, in primary rat cortical neurons and the chemical differentiation between cell types, namely primary rat cortical neurons and retinal pigment epithelium (RPE) cells, are presented as applications. Even though the dyes have distinct fragment ions in the high mass range, it was not possible to detect the fluorophores by 3D ToF-SIMS imaging of freeze-dried cells. However, it was possible to detect distinct differences in the kind of ions detected for freeze-dried primary rat cortical neurons and RPE cells albeit in the low mass range. To obtain meaningful results, however, it is paramount that sample preparation does not induce significant physical or chemical changes. We present the first comprehensive comparison between large 3D ToF-SIMS images of freeze-dried and frozen-hydrated cells using PCA to facilitate the data analysis of these large data sets. A higher degree of colocalisation of the K+ signal with cell regions is observed for frozen-hydrated cells, which indicates a lower degree of membrane damage and migration of diffusible chemical species. Frozen-hydrated cell samples are therefore considered to best reflect the native cell state, but freeze-dried cell samples allow far easier sample handling. The mass spectrum of frozen-hydrated cellular material also has increased ion intensities for higher-mass fragments, which is an additional advantage, because the poor signal-to-noise ratio of molecular species with m/z > 200 is a major bottleneck in the advancement of ToF-SIMS imaging as a diagnostic tool.

Absorption and emission spectra from the lowest energy transition in BODIPY have been simulated in the gas and water phase using a quantum mechanics/molecular mechanics approach, with DFT and the maximum overlap method (MOM). A post-SCF spin-purification to MOM yields transition energies in agreement with experimental data. Spectral bands were simulated using structures from ab initio molecular dynamics simulations, in which the solvent water molecules are treated classically and DFT is used for BODIPY. The resulting spectra are consistent with experimental data, and demonstrate how absorption and emission spectra in solution can be simulated using a quantum mechanical treatment of the solute. The electronic structure and photoinduced electron transfer (PET) processes in a fluorescent K+ sensor have been studied using DFT and TDDFT to rationalise its function. Absorption and emission energies of the fluorophore-localised intense excitation are more accurately described using MOM than TDDFT. Analysis of molecular orbital energies from DFT calculations in different phases cannot account for the sensors function. It is necessary to consider the relative energies of the electronic states. The inclusion of implicit solvent lowers the energy of the charge transfer state making a reductive PET possible in the absence of K+, while no such process is possible when the sensor is bound to K+. Binding within the ethene–argon and formaldehyde–methane complexes in ground and electronically excited states is studied with equations of motion coupled-cluster theory (EOM-CCSD), MP2 theory and dispersion-corrected DFT (DFT-D). MP2/MOM potential energy curves are in good agreement with EOM-CCSD calculations for the Rydberg and valence states studied. B3LYP-D3 calculations are in agreement with EOM-CCSD for ground and valence excited states, however for Rydberg states significant deviation is observed for a variety of DFT-D methods. Varying D2 dispersion parameters results in closer agreement with EOM-CCSD for Rydberg states.

This Thesis utilises vibrational spectroscopy in combination with multivariate and two-dimensional analytical techniques to probe the interactions of a biopolymer in water-based systems. Chapter 1: This Chapter gives an introduction to the Thesis and briefly outlines the experimental tech- niques used to study biopolymeric systems before covering the theory and implementation of the multivariate and two-dimensional methods used. A brief introduction to carrageenan, the biopolymer of interest, is then given. Chapter 2: The general experimental procedures are discussed together with the development of a new low-temperature ATR system, which allows very precise control and variation of sample tem- perature. The latter is key to many of the measurements and analyses reported in this thesis. The ATR system is stable to 0.01 C over a temperature range of -30 to 80 C. Many of the tech- niques used within this thesis rely on the Matlab environment. Analysis methods that are not commercially available have been programmed as part of my work. The theoretical background is discussed and the scripts for these functions are included in the Appendix. Chapter 3: Three commercially available carrageenans, k-, i- and l-carrageenan are studied with infrared spectroscopy. The carrageenan solutions are cooled from ca. 80 to 10 C. k- and i-carrageenan undergo a gelation transition during the cooling and this is investigated with a variety of analysis methods. The gelation transition can be monitored with FTIR allowing insight into the struc- tural rearrangement of the biopolymer as a function of temperature. The spectral transitions are probed with multivariate (PCA & MCR) and two-dimensional (2DCOS, MW2D & PCMW2D) techniques. Structural rearrangement for k- and i-carrageenan is observed, with various sulfate based modes showing the most intense changes to temperature. As cooled from 80 to 10 C, k- and i-carrageenan showed sulfate vibrational modes changing predominantly before vibrations associated with the backbone of the polyelectrolyte (C-O-C), indicating a sequential order to the molecular rearrangement occurring during the gelation transition. Chapter 4: Investigation of a 2 % i-carrageenan in H2O is frozen and probed with infrared spectroscopy using a modified low-temperature ATR accessory. The sample is cooled past it’s freezing point and is then subjected to short term, constant temperature storage. Changes in structure of the water and carrageenan are observed as a function of time post-freezing. Analysis by multivariate, two-dimensional and band fitting routines is applied, allowing the post freezing spectral perturbations to be monitored. Several spectral changes within the fingerprint region occur at a different rate, these have been proposed as vibrations associated with the backbone and pro- truding groups of i-carrageenan showing di↵erent processes in response to being frozen. Initially post-freezing, large changes in the O-H stretch region for H2O are observed, before subsiding and and followed by changes in the structure of carrageenan. These effects suggests an interaction between i-carrageenan and ice. Chapter 5: The use of a confocal Raman microscope, installed at Unilever’s Colworth facility has been used to investigate i-carrageenan in frozen systems. A gradient temperature stage allows formation of ice-fronts and a variety of these type of systems are probed. Changes in the concentration of carrageenan are seen dependant on the movement of the ice-front. Progressing an ice front into a gelled (unfrozen) section of the sample results in a large increase in carrageenan concentration at the ice-front. This indicates that the slow growth of the ice crystal is excluding the carrageenan and causing a freeze-concentration effect at the ice-front.

The development of optical multidimensional spectroscopic techniques has opened up new possibilities for the study of biological processes. Ultrafast two-dimensional ultraviolet spectroscopy experiments have determined the rates of tryptophan → heme electron transfer and excitation energy transfer for the two tryptophan residues in myoglobin. Here we show that accurate prediction of these rates can be achieved using Marcus theory in conjunction with time dependent density functional theory (TDDFT). Key intermediate residues between the donor and acceptor are identified, and in particular the residues Val68 and Ile75 play a critical role in calculations of the electron coupling matrix elements. Our calculations demonstrate how small changes in structure can have a large effect on the rates, and show that the different rates of electron transfer are dictated by the distance between the heme and tryptophan residues, while for excitation energy transfer the orientation of the tryptophan residues relative to the heme is important. The absorption and fluorescence spectroscopy of a series of porphyrin based systems have been studied. The range of systems has been selected in order to investigate the influence of both the electronic and geometric configuration on the photophysical properties. The origin of the bathochromic shift in the absorption and fluorescence spectra of substituted porphyrins is attributed to both steric distortions of the ring and electronic effects of the substituents. Three DFT based approaches have been used to model and calculate these properties. The approach using the maximum overlap method (MOM) predicted the largest discrepancy from Excited States of Porphyrins experimental results whilst TDDFT calculated shifts within 0.05 eV of experimental values. Finally a third method labelled as a ‘hybrid’ approach has been used, where the MOM is employed to optimise excited state geometries and single point TDDFT calculations are used to evaluate the vertical excitation energies. This approach improves on the excitation energies predicted by the MOM but does not improve on the values that a full TDDFT calculation produced. However, this ‘hybrid’ approach is computationally less demanding. There is distinct trade-off between accuracy and feasibility of calculations, where this ‘hybrid’ method of MOM and TDDFT becomes beneficial and useful. High resolution spectra of both free base and metallocentred complexes of porphyrin are calculated for the Q band region. Calculations on the vibronic structure of porphyrin are performed using DFT and TDDFT. Both Franck–Condon (FC) and Herzberg–Teller (HT) approaches have been used to predict the frequency and intensity of vibronic bands in the simulated absorption spectra with respect to the S0 → S1 electronic transition as the summation of contributions from both schemes characterise the electronic transitions and provide a high resolution description of the Q-band. Chapter five shows that the first electronic transition into the singlet excited state is vibronically active in the Qx region of the absorption spectrum, in good agreement with experimental data. The HT scheme appears to have more significant contributions and provides more insight into resolving the vibrationally active area of the absorption spectra. HT contributions to the electronic transition dipole moments are essential to assign the weak vibrational transitions and reproduce the experimental spectral profile. In order to provide a detailed account of the vibronic structure it is necessary to assign the vibrational transitions using both HT and FC schemes. The importance of theoretical calculations are highlighted here and can help the general understanding of the photophysical properties of porphyrins.