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This thesis focuses on current methods for nanoparticle characterisation and analysis of cellular interactions. The specific interaction between HeLa cells and 20 nm carboxylate-modified polystyrene nanoparticles is examined in detail, and the effects of serum proteins in corona formation is analysed. Fluorescence microscopy image acquisition is studied, with particular attention given to colocalisation analysis. An automated program is presented, making use of a novel de-noising algorithm which makes analysis of low signal-to-noise images possible without additional input. This is validated using computer generated images, and data acquired and analysed by hand. This program was used to analyse confocal microscopy images to quantify nanoparticle colocalisation with intracellular protein markers and membrane stains. A protocol is developed to enable use of total internal reflection fluorescence (TIRF) microscopy for the study of nanoparticle internalisation. Finally, innovative TIRF methods that permit identification of areas of local plasma membrane curvature and super-resolution analysis of fluorophore motion in Z are examined, and significant steps are made to combine these into a single imaging system. Thus, this project will develop computational analysis methods, novel biological assays, and physical enhancements to existing hardware with the aim of accelerating the characterisation of nanoparticle-cell interactions. |
