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.

Optimisation of computational, biological and physical methods to study the cellular entry of nanoparticles

Multiscale Synthetic Biology

Live cell imaging of the movement of various membrane-bounded organelle cargos has enhanced our understanding of their function. Eukaryotic cells utilize microtubules and two classes of microtubule-based motor proteins, cytoplasmic dynein and members of the kinesin family, to deliver a variety of membrane-bounded organelles and other cargos to their appropriate locations. In order to better understand the functions and regulation of cytoplasmic dynein, we developed a method to study its location and motility in living cells. The technique takes advantage of the long thin axons of cultured hippocampal neurons. We use calcium phosphate to transfect fluorescent-tagged dynein intermediate chain (IC) subunits (DYNC1I) into cultured neurons. When the ICs are expressed at low levels, they are effective probes for the location of the cytoplasmic dynein complex in axons when living cells are imaged with fluorescence microscopy. The fluorescent subunit probes can be used to identify specific cargos of dynein complexes with different IC isoforms as well as the kinetic properties of cytoplasmic dynein.

Live cell imaging of cytoplasmic dynein movement in transfected embryonic rat neurons.

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Live cell imaging of cytoplasmic dynein movement in transfected embryonic rat neurons.