The link between haemodynamics and cardiac tissue mechanics is an active area of research in developmental biology. Nevertheless, previous study of fluid-structure interaction in the developing heart was mostly confined to single projection blood flow measurements or computational fluid dynamics simulations using only the information of the heart wall structure. Hence, techniques capable of direct 3D + time resolved blood flow and heart wall motion are necessary to deepen the understanding of the cardiac function in the developing heart. This work presents an imaging system which combines selective plane illumination microscopy (SPIM), with optical gating techniques, and micro particle image velocimetry (uPIV). This combination (referred here as SPIM-uPIV) allowed non-invasively measuring blood flow in the developing zebrafish heart in a depth and time-resolved manner. Our system surpasses conventional uPIV measurement systems based on wide-field illumination which suffer from measurement errors due to volume illumination of the sample. The proposed SPIM-uPIV system was validated in a control microfluidics experiment where flow of fluorescent microspheres was measured in a 50 um diameter tube. Both qualitative and quantitative analysis was performed to compare our SPIM-uPIV against conventional brightfield-uPIV measurements. Furthermore, this work implements a different metric for “cross-correlation” which was empirically found to perform better than the traditional algorithm used in PIV analysis, when motion of large particles is measured. By implementing optical gating techniques to our analysis, 3D + time blood flow measurements in the beating hearts of 3, 4, and 5 day old zebrafish hearts were obtained. The recovered 3D + time velocity information enabled further investigation of the heart function such as the pumping efficiency which was obtained by calculating the flow rate through a section of a heart chamber. In summary, it is proposed that SPIM-uPIV system can be a useful tool for direct bloodflow measurements in transparent small-animal models. Such measurements would benefit the current knowledge of fluid-structure interaction phenomena in the developing heart, and could be used to validate previous work by other groups.

Bone marrow-derived mesenchymal and hematopoietic stem cells (MSCs and HSCs) have rapidly become the leading cells for consideration to aid tissue regeneration following injury. However, potential HSCs and MSCs migration to the injured tissue after infusion is impeded by cell trap within the upstream vessels, where physical and mechanical properties of cells play an important role. Microfluidic system has a potential to sort cells/particles based on their mechanical properties. It is hypothesized that such sorting system could be utilized to separate smaller and more deformable SCs from a cell population, infusion of which might be able to enhance the recruitment of the cells. The mechanical properties of murine HSCs were determined using micromanipulation and atomic force microscopy (AFM). Microfluidic devices were fabricated to separate sub-set of HSCs, followed by the infusion of the isolated cells into ischemia-reperfusion injured animals. HSCs as a whole became weaker and more deformable after pre-treatment with SDF-la and H 2 02, but HSC surface stiffened after the same pre-treating, accompanied by the expansion and polymerization ofF-actin interacting with the plasma membrane. A spiral microfluidic system with channel width 300m and height 40m was found to effectively isolate smaller and more deformable HSCs from a cell population, resulting in a significant increase of free flowing cells in vivo. This study comprehensively characterized cell mechanics at different levels using micromanipulation and AFM, determining mechanical markers of therapeutic cells. Most importantly, a simple cell sorting system was successfully developed to isolate target cells without introducing any chemical modification, and the possible underlying mechanism was discussed, which can be valuable to cellular therapy.

This thesis describes the improvements to the image replicating imaging spectrometer (IRIS) and the development of novel applications in the field of oximetry. IRIS is a snapshot multispectral device with a high transmission output and no need of inversion for data recovering, hence, with high signal-to-noise ratio (SNR). IRIS shows great versatility due to the possibility of choosing multiple contiguous or non-contiguous wavelengths inside its free spectral range. IRIS uses a set of waveplates and Wollaston prisms to demultiplex the spectral information of an object and replicate the image of such object in different wavelengths. The birefringent nature of IRIS means that different wavelengths are separated by the Wollaston prisms with different angles, introducing multiple images of the same object. In addition, the spectral transmission function shows multiple spectral sidelobes that contaminate each IRIS band with light belonging to other wavelengths. These issues can lower the performance of IRIS as a multispectral imaging device. In this thesis, these problems were assessed with the introduction of a filter plate array placed in the image plane of the optical system. This filter array is a set of narrow-band filters (Full Width Half Maximum (FWHM) =10 ± 2 nm ) that removes undesired wavelengths from each IRIS band. Since the spectral transmission of IRIS is replicated along the free spectral range, the filters can be designed to match any of the present spectral lobes in IRIS. The design and fabrication of a filter array enhance the performance of IRIS as a multispectral imaging device: it allows wavelength selection and improves spectral and spatial image quality. The design and manufacture of the corresponding filter holder and camera adapter were critical in terms of offering an easy filter-camera implementation. The filter plate allowed the removal of other dispersed wavelengths by the Wollaston prisms, improving image registration between the set of spectral images created by IRIS, and so, improving the quality of the registered spectral 3-D cube. The implemented improvements on IRIS allow high quality, calibration-free oximetry using eight different wavelengths optimised for oximetry. Two main experiments were performed: 1) Using an inverted microscopy interfaced with IRIS and a linear spectral unmixing technique, we measured the deoxygenation of single horse red blood cells (RBC) in vitro in real time. The oximetry was performed with a subcellular spatial resolution of 0.5 μ m , a temporal resolution of 30 Hz, and an accuracy (standard error of the mean) of ± 1.1% in oxygen saturation. 2) Eight-wavelength calibration-free retinal oximetry performed in nine healthy subjects demonstrated an increase in the stability of the oxygen saturation measurements along retinal vessels when compared with more traditional analysis methods such as two wavelengths oximetry. The stability was measured as the standard deviation along the retinal vessels of the nine subjects and was found to be ∼ 3% in oxygen saturation for eight-wavelengths oximetry and ∼ 5% in oxygen saturation for two-wavelengths oximetry. A modified physical model was used to improve the characterization of light propagation through the eye, retina, and blood vessels by applying a set of feasible physiological assumptions. This model was optimised by an algorithm which solves for the different variables involved in the retinal vessels transmissions in order to accurately calculate the oxygen saturation. The oximetry algorithm was applied in retinal vessels, in collaboration in vivo on rat spinal cord to assess hypoxia in inflammatory diseases such as multiple sclerosis and rheumatoid arthritis and on mice legs to assess hypoxia on autoimmune diseases. A third experiment using a microscope interfaced with IRIS was performed. The experiment aimed to replicate laminar flow conditions observed in retinal vessels and to calculate oxygen diffusion between adjacent streams of blood with different oxygen saturation. For this purpose a PDMS multichannel flow cell with cross sections of 40x100 μm was designed and fabricated allowing us to replicate conditions found in retinal blood vessels. Laminar flow was replicated but the experiment failed in calculating oxygen diffusion due to flaws in the experiment. The experiment with the results and recommendations on how to improve it can be found in Apendix B for future researchers.

The assessment of nanoparticle penetration through skin is of increasing importance not only to evaluate the toxicity associated with occupational or environmental exposure to nanoparticles, but also to design rules for the fabrication of new types of transdermal drug delivery or diagnostics approaches. While these have been the subject of much research, the lack of a systematic approach in the penetration experiments has created controversial results regarding whether nanoparticles do or not penetrate the skin. The aim of the research presented in the thesis is to investigate the penetration of gold nanoparticles through human and mouse skin, focusing on the effect of surface charge, morphology and specific functionalisation. To study this, a penetration protocol using organ culture was designed and tested to assure the maintenance of the skin integrity in the course of our experiments. Skin samples incubated with gold nanoparticles were characterized for penetration by NPs using inductively coupled plasma atomic emission spectroscopy, transmission electron microscopy, energy-dispersive Xray spectroscopy and photoluminescence microscopy. Furthermore, epithelial cell monolayers were exposed to the gold nanoparticles to evaluate the transport through the cellular barrier. Transmission electron microscopy, light microscopy and trans epithelial electric resistance were used to characterise the cell monolayers exposed to gold nanoparticles. Results obtained are important to enhance our understanding of the interaction of gold nanoparticles with skin, providing valuable information for the design of new nanoparticle-based transdermal delivery systems.

In recent years biomolecules have been used to infer specific functionality to nanomaterials. Advances in conjugation techniques have allowed for the development of a vast range of hybrid bio-nano materials. Their applications range from biosensing and targeted therapy to metamaterials. In particular the conjugation of nanomaterials to functional oligonucleotides has been a thriving area of scientific research. In this project the main aim was to explore the uses of gold nanoparticle-DNA conjugates for biomedical applications. Probes for the real-time intracellular detection of mRNA were synthesized. These probes showed great target specificity, excellent biocompatibility and good cellular uptake. Importantly, unlike free nucleic acids, they displayed no susceptibility to degradation by nuclease enzymes. The ability to detect mRNAs in a live cell, in real time has tremendous diagnostic applications. Furthermore, multifunctional probes were designed. In addition to live cell mRNA detection, we developed probes with the ability to deliver a cytotoxic drug. Utilizing their inherent high specificity for target mRNAs, we demonstrated that cell-type specific targeted drug delivery was possible. In the absence of the target mRNA, the drug remained tightly bound within the probe. With a view of developing advanced materials, capable of performing multiple roles simultaneously, we investigated the use of nanoparticle assemblies for biomedical applications. In order to create highly stable nanoconstructs, a novel tool for the programmed assembly of DNA-nanomaterials was demonstrated. The use of copper-free click chemistry resulted in nano-assemblies connected by ssDNA. The employment of this novel tool proved to produce assemblies with covalently linked particles. Moreover, it was shown that gold nanoparticle dimers displayed excellent stability with respect to a variety of conditions commonly met within a biological environment. Additionally, the formation of heterogeneous nanoassemblies was demonstrated. Dimers of optical and either semi-conductor or magnetic nanocrystals were assembled representing examples of multi-role probes with exciting potential for applications in biomedicine.

According to the data from government institutions (Energy Information Administration, 2012), the consumption of energy will show an average growth of 42.5% by 2035 from the heating, ventilation and air conditioning devices solely (Perez-Lombard et al., 2008). For this reason the interest in technologies that can reduce the consumption of energy for heating or cooling houses is growing. In particular, the glazing of houses could offer great potential for energy saving as these elements of the building envelopment still have a margin for improvement. Currently, the research is focusing on stopping the heat exchange through radiative transfer. The problems of the current technologies are associated with the high costs and the colour they give to windows. Technologies using nanoparticles have started to emerge and have shown promise as methods for absorbing the radiation which passes through the glazing. Thanks to the unique control over their size and shape dependent properties, their absorbance can be moved to lower energy and this can satisfy both the requirements of stopping the heat carrying radiation and still providing a good illumination. Through the wide range of nanoparticle materials, sizes and shapes, silver triangular nanoprisms are a promising candidate for further research due to their strong absorption in the near infrared region. As their synthesis and the control over their geometrical properties are challenging using conventional batch-based macro reactor systems, a novel microreactor system was developed in this study in order to continuously produce silver triangular nanoprisms and monitoring their optical properties by mean of integrated spectroscopy techniques. By using sol-gel chemistry, the particles were coated with a shell of SiO2 which can further be functionalised with various chemical functional groups such as thiol and allyl. Coated particles were then embedded in polymeric matrix (i.e. poly(methylmethacrylate), or PMMA) with covalent interactions between the polymer and the functional groups attached to the silica shell surface. Finally, the composite solutions were casted onto a glass-slide and the optical performance was evaluated using spectroscopic methodologies. Compared to similar composite materials, the systems herein reported offers several advantages, such as the low coloration in the visible spectrum and no risk of aggregation of the metal nuclei once they are dispersed in the polymer matrix. The use of a microreactor can also grant good control over high volumes of such colloids, opening to the possibility for a large scale production of such materials.

The colicin and lectin-like bacteriocins are a broad class of antimicrobial proteins produced by Gram-negative bacteria. They are generally narrow spectrum, killing or inhibiting the growth of closely related bacteria. Numerous Gram-negative bacteria that are important pathogens of both animals and plants produce and are susceptible to these bacteriocins. As such, these proteins represent an attractive alternative to traditional small molecule antibiotics for controlling bacterial infection. Very little is known about bacteriocins produced by Gram-negative plant pathogens and so the aim of this work was to discover novel bacteriocins active against globally important plant pathogens from the genera Pectobacterium and Pseudomonas. The bacteriocins discovered in this study were then structurally and functionally characterised and assessed for their ability to impart disease resistance when expressed in a model transgenic system. This study presents the discovery and characterisation of the bacteriocins syringacin M, syringacin L1 and pyocin L1 from the genus Pseudomonas, As well as the discovery and characterisation of the unusual ferredoxin containing pectocins from the genus Pectobacterium. Also presented is the discovery of a novel virulence related ferredoxin/iron-uptake system in Pectobacterium, which is parasitised by the pectocins for cell entry. Additionally, the transgenic expression of the bacteriocin putidacin L1 in both Arabidopsis thaliana and Nicotiana benthamiana was shown to provide these plants with resistance to infection by strains of the plant pathogen P. syringae.

Adaptive radiotherapy (ART) aims to adapt the treatment plan to account for inter-fraction anatomical variations, based on online acquired images. However, ART workflows are not –yet– routinely used in clinical practice, primarily due to the dramatic increase of the workload required and the inadequate understanding of optimal methods to maximise clinical benefit. This thesis reports on investigations of procedures for the automation of the ART process and the identification of optimal adaptation methodologies. Investigated auto-segmentation algorithms were found insufficient for an automated workflow, while a hybrid deformable image registration (DIR), incorporating both intensity based and feature-based components, revealed the most accurate and robust performance. An evaluation method was proposed for interfraction treatment monitoring through dose accumulation following DIR. The robustness of several treatment methods to observable anatomical changes were investigated, highlighting cases whereby substantial dosimetric consequences may arise. Offline ART workflows were explored, specifically investigating the effects of treatment monitoring frequency, adaptation method (simple re-plan or re-optimisation addressing cumulative dose), and adaptation timing. Contrary to simple re-planning, re-optimisation demonstrated its ability to compensate for under-/over-dose, however, non-uniform dose distributions and hot-spots may be generated. Therefore established planning techniques are applicable for re-planning while advanced approaches are required for treatment re-optimisation accounting for radiobiological consequences.

Magnetic Resonance Spectroscopic Imaging (MRSI) is a functional MRI technique allowing non-invasive biochemical mapping of the brain. MRSI is advantageous for characterising many neurological conditions; however, its clinical application is limited by lengthy scan time and low spatial resolution, which are intrinsically linked. This research investigated the potential of Compressed Sensing (CS) to speed-up MRSI or enhance spatial resolution. CS allows accelerated acquisition by reducing the data sampling requirements, whilst preserving image quality. The focus of this work was the effect of CS-MRSI at different acceleration factors upon spatial integrity. CS reconstruction software was developed and applied to retrospective MRSI data. Imaging test objects and software simulations were developed to assess MRSI spatial resolution via metabolite edge response measurements. CS-MRSI was also investigated in realistic scenarios using data from healthy volunteers and a child with Optic Pathway Glioma (OPG). The potential of CS-MRSI to enable high-resolution MRSI in feasible scan times was investigated using simulations of focal and infiltrative OPG. Results suggest that CS-MRSI can reduce scan duration by up to a factor of 5 whilst simultaneously eliminating ringing artefacts and increasing spatial resolution compared with conventionally filtered MRSI. Therefore, CS could greatly increase the clinical utility of MRSI.