Search

The world’s population is increasing rapidly and higher calorific diets are becoming more common; as a consequence the demand for grain is predicted to increase by more than 50% by 2050 without a significant increase in the available agricultural land. Maximising the productivity of the existing agricultural land is key to maintaining food security and agrochemicals continue to be a key enabler for the efficiency gains required. However, agrochemicals can be susceptible to significant losses and thus often require further chemical to be applied to compensate. Sources of such losses include spray drift, poor spray retention/capture by the target and poor penetration through the plant cuticle. The effectiveness of a crop protection agent depends not only on the intrinsic activity of the active ingredient (AI) but also on the physicochemical properties of the formulation. These properties can be modified by using formulation components, known as adjuvants, which can be used to help mitigate such losses. Adjuvants exert their effects by, for example, controlling droplet size and distribution through their effect on surface tension which can also improve penetration into leaves through the cuticle wax which coats the epidermis of leaves and acts as a protective barrier. However, characterising how they alter the movement of the AIs can be challenging. Optical techniques have shown promise in a multitude of scientifically related areas, such as in vivo tissue imaging, but none have yet been applied to aiding the agrochemical industry. By probing the interactions between leaf surface and agrochemical agent, with light, one is able to obtain a large amount of diagnostic information, non-invasively. Whereas techniques like Raman 3 spectroscopy are limited by long acquisition times, coherent Raman techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) are coherently driven and provide an enhanced signal, and also allow for video-rate imaging. In this thesis, I have applied this cutting-edge laser imaging technique as a novel analytical technique that allows the in situ analysis of agrochemicals in living plant tissues at a cellular level. In Chapters 4 through 7, multiple factors essential for a functional and efficient agrochemical were considered and experimented. In Chapter 4, a typical industry study highlights the need for innovative and rapid technologies in the agrochemical industry. The resulting chapters (5, 6, and 7) outline several ways in which Coherent Raman Scattering (CRS) techniques can improve the current capabilities of agrochemical testing. By utilising a model system, paraffin wax, a cheap and rapid protocol can provide accurate diffusion information and repeatable results. Chapters 6 and 7 use both this protocol to gain comparative data on several adjuvants and active ingredients in paraffin wax and in vivo, in a variety of plants. The ability to visualise agrochemical products on a leaf surface to reveal interactions between the materials of the product and with the leaf surface will enable a step change in the agrochemical design process, through determination of the spatial distribution of the materials and their roles within the applied products. It is hoped that the technology developed in this thesis could play a big role in the development of future agrochemical products that are tailored to maximise efficacy and minimise environmental impact.

Gold nanoparticles (AuNPs) are increasingly being used in biomedicine for enhancing drug delivery and facilitating imaging, as well as directly in applications such as cancer photothermal therapy. Despite this, very little is known about their potential impacts on the environment. Importantly, the effectiveness of AuNPs in such applications depends on a range of particle specifications that have been optimised during development to serve their specific purpose. The manipulations include their size and the addition of coatings, usually to enhance biocompatibility. How these different particle characteristics affect their bioavailability and biological effects on wildlife once released into the environment, has received very little research attention. Consequently, the main aims of this thesis were to investigate, principally via imaging methods, how the characteristics of size and surface coatings of gold nanoparticle (AuNP) affected bioavailability, tissue distribution and potential for toxicity using zebrafish (Danio rerio) as an experimental model. To undertake this work, a second major objective of the thesis work was to construct an imaging platform to trace the uptake and biodistribution of the gold nanoparticles. For this, I constructed an open source selective-plane illumination microscopy system (OpenSPIM) which allowed me to quantitatively trace fluorescently tagged AuNPs in exposed zebrafish embryos with relatively high-throughput. Applying SPIM through multiple angles, the fluorescence tag on the AuNPs was detectable throughout the zebrafish embryo body enabling concentration-response uptake analyses. Having built the OpenSPIM system from the ground up, careful method developments were undertaken to optimise the SPIM which included the imaging of multiple transgenic models of zebrafish such as the MPO:GFP model with fluorescent neutrophils, the elavl3:GCaMP6s utilised in chapter 6 to measure neural activity, and the casper model which was utilised in all uptake studies. Studies into the toxicity of the AuNPs were carried out with models for detecting assess oxidative stress responses in the 3EpRE:hsp70:mCherry model (using the Zeiss inverted microscope) and the pod::NTR-mCherry/l-fabp::VDBP-GFP model to assess for damage to the pronephric glomerular filtration barrier (using the EVOS fluorescent microscope). The OpenSPIM system was applied to image uptake and distribution of quantum dots (CdTe) in the casper model which indicated minimal uptake in the entire zebrafish embryo. Extensive oxidative stress was detected for exposures to aqueous levels of CdTe QDs down to 10 µg/L in the lateral line neuromasts, fionocytes and pronephros (undeveloped kidney) but there were no effects to the glomerular filtration barrier. Aqueous exposure to AuNPs illustrated a size selective uptake with the highest uptake for 40-80 nm AuNPs across a size range between 10 to 100 nm. This was principally found accumulating in the pronephros and gut with some fluorescence visualised in the heart. The effect of AuNP functionalisation on uptake in the casper model demonstrated varied levels of uptake depending on surface modification. Modifications of the AuNPs resulted in enhanced uptake levels for coatings with PEG and TNFα but not for NHS/PAMAM where there was decreased uptake. Depuration studies with all AuNP studies indicated that the AuNPs were cleared (but not completely) from the zebrafish embryos over a period of 4 days. No toxic effects were seen in any study with AuNP exposure in concentrations that exceed environmentally relevant doses (2 mg/L). This body of work illustrates the utility of SPIM for detecting the uptake and fate of selected NPs in exposed fish embryos. When combined with transgenic models that allow for functional analyses across specific tissue targets and effect mechanisms, thus providing a powerful tool for nanoparticle screening. Finer level details relating to the biological effects of NPs, however, require imaging methods that allow for sub-cellular resolution to analyse cell mechanics and how NPs influence cellular functions.

Oesophageal cancer, one of the most aggressive cancer types is considered the seventh most common cancer in terms of incidence and the sixth most common cause of cancer deaths worldwide due to late diagnosis. In the UK, the oesophageal cancer incidence rate has increased by approximately 10% since the 1990s. At present, histopathology is the gold standard method for the diagnosis of oesophageal cancer, which rely on biopsy collection using an endoscopy procedure followed by the histological sample's preparation. This method is invasive, time-consuming, and largely based on the pathologist's experience of diagnosis. Therefore, new diagnostic techniques are required to provide non-invasive methods for early and rapid diagnosis. Raman scattering has the potential to replace histopathology as the gold standard for diagnosis for a wide range of diseases. Raman scattering provides stain-free imaging with chemical-specificity derived from the intrinsic vibrational signatures of biomolecules. However, the low scattering cross-section severely limits the image acquisition speeds and like conventional histopathology, requires tissue sectioning to provide morphological imaging below the surface of tissue biopsies. Stimulated Raman scattering (SRS) has recently appeared as a powerful technique for (near)real-time Raman imaging in intact tissue samples. Thework in this thesis aimed to develop the stimulated Raman scattering (SRS) for rapid wavelength tuning and chemical imaging of clinical samples, such as cancer biopsies. This was achieved by making modification to a laser cavity to reduce the time of the wavelength tuning by approximately 35 times compared to the original cavity design. Furthermore, the cavity modification led to the spectra being separated efficiently and the wavelength tuning controlled by cavity length changes only. The improved design was applied to image frozen oesophageal tissues, which have four major pathology groups, normal, inflammation, columnar-lined (Barrett's) oesophagus (CLO) and low-grade dysplasia. A large area imaging was performed using the SRS technique at 2930 cm-1 for four different oesophageal tissues, which presented the morphological and structural information. However, histopathological diagnosis depends on the visualisation of the cell nucleus in the tissue. This component was not highlighted until the stimulated Raman histology approach was developed for small regions of interest in the CLO and the low-grade dysplasia sample, which required two different frequencies at 2840 cm-1 and 2930 cm-1. All SRS images were compared to haematoxlin and eosin (H&E) stained sections. Further comparisons were made between SRS and Raman imaging techniques, with SRS offering faster acquisition times and a higher spatial resolution. The spectral signature for the different pathological groups in the oesophageal tissues were explored in the high wavenumber (2800 - 2930 cm-1) region using hyperspectral SRS and compared with the spectra from the Raman. K-means clustering analysis was used to explore the morphochemical information using the CLO and low-grade dysplasia sections. Both techniques were able to demonstrate unique information such as the epithelial cells that form the oesophagus glands and surrounding connective tissue. It is concluded that SRS has the power to be one of the ideal imaging modalities to gather the molecular information in biological samples. However, it still needs more development due to the complexity of the system.