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Throughout the past few decades, science has progressed towards the ability to probe many extremely fast processes and a large amount of research has been aimed at the area of few-femtosecond pulse generation. This thesis describes the generation of coherent broadband radiation through two-colour pumping of molecular hydrogen confined to a unidirectional ring cavity, and the subsequent synthesis of high peak power and few-femtosecond pulses. A set of normalised semi-classical field equations are derived in Bloch form describing the process of ultra-broadband multi-frequency Raman generation or UMRG, and a 3-wave gain suppression analysis is derived from a subset of the plane wave UMRG field equations which describes gain suppression within the ring cavity in terms of both medium and cavity parameters. The gain suppression analysis is further generalised to include finite levels of linear two-photon frequency detuning of the pump beams. Simulations of the plane wave ultra-broadband multi-frequency Raman (UMRG) equations show that a broad frequency spectrum of mutually coherent sideband can be generated. The inverse Fourier transform of spectra generated in this way yields a train of high power near Fourier limited pulses in the time domain which can range from a few-femtoseconds in duration to tens of attoseconds with repetition rates equal to the Raman transition frequency. Pulses synthesised in this way are limited only by the level of medium dispersion, the reflection bandwidth of the chosen coupling mirror and the chosen Raman medium. Simulations of the transverse UMRG equations within the ring cavity geometry have shown ring cavity enhanced UMRG to be resilient to transverse effects such as finite beam width, beam diffraction and the transverse beam separation of the applied pump beams.

Part I. A number of modifications were made to the large spheroidal field B-spectrometer and a small lens spectrometer was attachedto it for 3-3 coincidence measurements. The method of ion ejection from a capillary tube for preparing thin sources was developed and used for making Ce144 sources. An attemptwas made to verify the existence of a 166 keV excited statein Pr 144 suggested by some investigators by observing the predicted Y-Y cascade, i.e. 33 keV Y-ray followed by a 133 keV Y-ray. No evidence of B-B coincidences between the K133 and L133 conversion lines was found. Part II. The trajectories of electrons in a spheroidal field were found by integrating the equations of motion using the electronic computer 'Mercury' of the University of London. The focusing properties of a B-spectrometer using this field were investigated and an attempt was made to find an analytical form for the field intensity which would describe the field in the large spectrometer.

Coherent Antistokes Raman Scattering (CARS) microscopy has emerged in the last decade as a powerful multiphoton microscopy technique to rapidly image lipid droplets (LDs) label-free with intrinsic three-dimensional spatial resolution in cells. In this thesis I investigate and compare the ability of hyperspectral CARS and dual-frequency/differential CARS (D-CARS) to enable the chemical specificity required to distinguish lipids of different chemical composition. In hyperspectral CARS a series of spatially-resolved images are acquired over a frequency range thus proving high chemical specificity. In D-CARS two vibrational frequencies are simultaneously excited and probed, and the resulting sum and difference CARS intensities are detected by a fast and efficient single photomultiplier. This results in a higher image speed than hyperspectral CARS and in an improved image contrast against the nonresonant CARS background with a straightforward data analysis. D-CARS and hyperspectral CARS techniques were applied to LDs in model and cellular systems. In model systems made by agarose gel, droplets of pure lipids with different degree of unsaturation (number of carbon-carbon double bonds in the fatty acyl chain) were used as test sample to compare Raman spectra with CARS spectra, and measure D-CARS images at specific chemically-selective wavenumbers. Building from this knowledge, cytosolic droplets induced by loading fatty acids to the culture media of human adipose-derived stem cells (ADSCs) were distinguished in composition both in fixed cells and in living cells during differentiation into adipocytes. Furthermore, the application of a in-house developed Hyperspectral Image Analysis (HIA) software on hyperspectral data provided spatial distributions and absolute concentrations for the chemical components of the investigated specimens. In particular quantitative information was extracted about the concentration of pure neutral lipid components within cytosolic LDs, and changes over time were inferred in living ADSCs according to the type of pure fatty acid added to the culture media.

Analysis has been made of the amplitudes of the second and third harmonics when pumping a discrete frequency to the Josephson-vortex photonic crystal within the THz range of the electromagnetic spectrum. The results of numerical simulations show that there are certain resonance frequencies for these harmonics where the amplitudes are strongly enhanced. The frequencies at which these resonances occur can be tuned by an applied magnetic field and tilting the material with respect to the incident radiation. For the second harmonic it has been possible to describe these resonances analytically with a resonance approximation which displays good agreement with numerical simulations at and near the resonances. A similar perturbative method has been used to simulate the nonlinear mixing of two discrete THz frequencies in the JV photonic crystal, producing resonances for harmonics at the sum and the difference of these two input frequencies. This method can allow a high degree of control over the harmonic frequencies produced.

Traditionally, many advances in the field of biology have been driven by optical microscopy based techniques which reveal morphological information about the samples under study [1, 2, 3, 4]. The scope of the applications of these methods is limited due to the lack of contrast from most biological materials (cells and tissues) which are transparent to visible light. The introduction of extraneous materials (such as fluorescent quantum dots or other fluorescent proteins/labels) with affinity towards certain sub-cellular components which are then imaged, has emerged as a popular and powerful method to image biological materials. Fluorescence microscopy using visible wavelengths in its simplest application includes the identification and imaging of interesting features of a sample which are fluorescently labelled in a structurally/ chemically specific way. In more recent developments, fluorescence lifetimes have been imaged. Fluorescence based techniques can also be applied to track protein dynamics or drug delivery in live samples. Despite the benefits of imaging samples with labels, and a host of associated applications, the issues of photobleaching and induced phototoxicity remain. Another very important aspect which gains relevance in live sample imaging is that the system dynamics may be influenced by the introduction of fluorescent labels. Spectroscopy techniques, which rely on the material resonances are chemically specific, sensitive, and if combined with microscopy, bridge the gap between non-invasive imaging and fluorescence microscopy. Instead of an extraneous label, the contrast generated originates from molecular transitions of the chemical species in the sample. Coherent Raman scattering (CRS) based techniques rely on the chemical contrast generated due to molecular vibrations and have been applied to biology [5]. One CRS technique, stimulated Raman scattering (SRS) has been used to distinguish between the macromolecular constituents of cells [6, 7] and tissues [8]. Additionally, quantitative hyperspectral SRS has been demonstrated in polymer and lipid mixtures [6]. Another type of CRS, coherent anti-Stokes Raman scattering (CARS) [9] was reported in 1965, nearly half a decade before SRS. However, due to difficulties in implementation, CARS was not readily put to application. Since its revival in 1999 [10], CARS has emerged as a label-free, chemically specific microscopy technique and has been applied to image various biological materials [11, 12]. In addition to the studies of lipid rich samples [13, 14], spectral differences between the cytosol and the nucleus have been reported using CARS microscopy [15, 16]. However, none of the previous works in this field present full 3D hyperspectral data, or make quantitative volumetric estimates of the various chemical components present in the sample. With respect to biomedical/biochemical application based studies, literature suffers from a paucity of examples investigating the effects of drugs on biological materials using CARS microscopy. This project aims to overcome these shortcomings. In this work, CARS microscopy is applied to single cells (osteosarcoma, U-2OS which are lipid poor due to their functional profile) with volumetric quantitative analysis to determine the absolute masses of the component species. For the first time in our knowledge, full 3D hyperspectral data has been acquired and analyzed. Correlative fluorescence imaging to ascertain the origin of various components of the cells as imaged with CARS was also performed. Furthermore, reports of no observed (with CARS) correlation in protein content in the intranuclear region with the mitotic stage in cells one publication [16] have been disproved, shown in this thesis. Osteosarcoma is a rare type of cancer, most commonly diagnosed in children and adolescents. However, due to its rarity, it is not well researched. The usual line of treatment includes surgery followed by chemotherapy, of which Taxol (microtubule stabilizer) and ICRF-193 (topoisomerase II poison) form an important part. The effects of these drugs on cells are often investigated in literature using a range of techniques, of which, the most non-invasive one is chemically non-specific optical microscopy [17, 18]. Among the chemically specific methods used to perform such studies, flow cytometry [19, 20, 21] is one of the most commonly employed; and the most invasive methods, also in widespread use, are Western blotting and gel electrophoresis [22]. This means that in the best case scenario, we can perform optical microscopy on the cells with no chemical specificity or sensitivity, or sacrifice non-invasiveness for chemical information. Identifying a need for label-free methods to study the effects of these drugs, we applied CARS microscopy to study the effects of Taxol and ICRF-193 on U-2OS cells. This was done to determine whether CARS microscopy is suitable for population phenotyping and profiling the effects of anticancer drugs over a period of time, following treatment. Also for the first time, in this project, CARS microscopy has been demonstrated with chemical specificity and sensitivity on structurally and functionally multicellular 3D assemblies, organoids. In the past, other groups have reported studies on organoids, their metabolism and drug interactions using fluorescence microscopy [23, 24, 25], with the already mentioned shortcomings and pitfalls of photodamage and photobleaching. This thesis is structured into five chapters. The required background is given in the first two chapters. The first chapter introduces optical spectroscopy with emphasis on CARS, including a discussion of the theory and the various implementations of CARS microscopy. The second chapter contains the biology background in cells and cell division requisite for this project. An overview of the current state of the art in imaging techniques is also given. The set-up and analysis techniques used to acquire and analyze the data presented in this work are described in the third chapter, along with a characterization of the effects of the imaging optics and the sample’s refractive index on the analysis method and the quantitative calculations, using polystyrene and polymethymethacrylate beads of different sizes. The next two chapters describe the applications of CARS microscopy to fixed U- 2OS cells and organoids. In chapter four, the results of CARS imaging and spectral analysis of U-2OS cells correlated with two-photon fluorescence are shown. Furthermore, applications of CARS to study the effects of two kinds of anti-cancer drugs i.e, Taxol and ICRF-193 on U-2OS cells are demonstrated. Additionally, a side project not related to CARS microscopy, but presenting a simple method to quantitatively investigate the number of eGFP molecules attached to cyc-B, across the cell cycle is also described in this chapter. Chapter five demonstrates the suitability of CARS microscopy to image higher levels of biological organization, specifically organoids which are miniature lab grown 3D models of organs. The summary and outlook of this project are given in the last chapter, which is followed by the Appendices including additional detailed information referenced in the thesis. All 3D data are available as videos in the data DOI related to this thesis.

This thesis presents the theoretical modelling and commissioning of the laser station at the newly constructed IGISOL 4 facility. The experimental techniques and apparatus of the facility, focusing on a line dedicated to laser spectroscopy, are described, followed by details on simulations of the beam-line. First spectroscopic data has been acquired at IGISOL 4. The isotope shifts and hyperfine structure of stable and radioactive molybdenum isotopes were acquired using collinear laser spectroscopy. The spectroscopy of this element was chosen in order to compare the results with those previously acquired at IGISOL 3. The isotope shifts and hyperfine dipole constants calculated were found to agree and the hyperfine structure of the 4d4(5D)5s 6D1/2 to 4d4(5D)5p 6F1/2 transition in 107Mo has been observed for the first time. A new electrostatic ion trap (ConeTrap) has been designed for trapping and optical pumping of ions in high vacuum using realistic ion optical simulations. The ConeTrap has been designed with an extended central section in order to facilitate the optical pumping of contained ions. The trap uses asymmetric voltages to achieve greater extraction efficiency and an acceptance time of ~10 us. Fast high-voltage switching circuitry has been created, with fall and rise times of <240 ns and <700 ns respectively, for dynamic trapping of ion bunches. The IGISOL ConeTrap has been designed and built for axial and radial optical pumping, however several other possible applications are also presented. These applications, to be deployed at the IGISOL 4, include using the ConeTrap as a potential energy elevator, a "high-density'' ConeTrap for two-photon spectroscopy, a "dual-stability'' ConeTrap that can provide pure, doubly-ionised bunches, a ConeTrap designed for radiation detection for spectroscopy and lifetime measurements and a combination of the ConeTrap and the current IGISOL light collection region for collinear laser spectroscopy of trapped ions.

This thesis is focused on the development of Raman micro-spectroscopy for label free imaging and discrimination of stem cells. The thesis is divided into six chapters. Chapter 1 gives an overview of the existing techniques used for molecular analysis of cells, with emphasis on methods that allow non-invasive label-free imaging. A literature review of the main relevant applications of Raman micro-spectroscopy for imaging cells was also included. Chapter 2 discusses the basic theoretical principles of Raman scattering and design of Raman micro-spectrometers. The practical aspects related to the design of an optimised Raman micro-spectrometer are presented in Chapter 3 along with experimental characterisation of its performance. The chapter concludes with examples of Raman spectral maps of endothelial cells. Chapter 4 and Chapter 5 present experimental results obtained by Raman micro-spectroscopy for molecular analysis of live neural and mesenchymal stem cells. In these investigations Raman spectroscopy was used to identify, image and quantify spectral markers for label-free discrimination between glial cells and their neural progeny. The potential of Raman micro-spectroscopy to measure timecourse molecular changes of individual bone nodules was demonstrated in Chapter 5. Future work and final conclusion are discussed in Chapter six of this thesis.

Given their unique structural properties, quantum rings (QRs) structures have recently been of particular interest for investigating quantum interference which is called the neutral charged exciton optical Aharonov-Bohm (AB) effect. A delocalized wavefunction around the rim is a prerequisite for the AB effect, but asymmetry and anisotropy seem to have been overlooked in the spectroscopy of QRs. In this thesis, the presence of a localized state in a single GaAs QR is presented.

Raman spectroscopy is a powerful tool in oncological imaging. Optical biopsies in which an accurate diagnosis of the tumour areas is spectroscopically performed are especially interesting for application to skin cancer treatments. In the first part of this dissertation a study on automated Raman spectral imaging allowed accurate diagnosis and delineation of the borders of a common type of skin cancer, basal cell carcinoma (BCC). Automated detection and imaging of BCC in skin sections excised during surgery was performed by combining Raman micro-spectroscopy with supervised multivariate mathematical algorithms based on linear discriminant analysis (LDA). The model allowed 90±9% sensitivity and 85±9% specificity in BCC detection. Raman spectral images based on the LDA model were created and compared with the gold-standard of the conventional histopathological diagnoses resulting in excellent agreement. Additional studies on the ability of the model in discriminating between BCC and hair follicles produced accurate diagnoses. In this thesis instrumental implementation and design of a Raman spectral imaging prototype aiming to reduce the acquisition time required to build the Raman spectral images was developed. High sensitivity variants of Raman spectroscopy such as surface enhanced Raman spectroscopy (SERS) are known to enable optical detection down to single molecules and can be applied to thin supported lipid research. The combination of SERS with a complementary topographic technique simultaneously synchronised adds to the chemical information the morphology of the sample surface. In the second part of this thesis simultaneous atomic force microscopy (AFM) and SERS characterisation of thin (≈15-20 nm) supported films of arachidic acid and cationic phospholipids on sapphire/silver substrates was successfully achieved. Supports were fabricated with nanosphere lithographic procedures and allowed enhancement of the weak Raman signals from the amphiphilic films by a maximum factor of ×10^8.