In this thesis we investigated the ability of two-photon multifocal microscopy for single-molecule microscopy in live cells and organisms. Two-photon excitation combined with multifocal scanning... Show moreIn this thesis we investigated the ability of two-photon multifocal microscopy for single-molecule microscopy in live cells and organisms. Two-photon excitation combined with multifocal scanning has the potential to achieve, high (temporal) resolution imaging at a low background. Splitting the laser beam into multiple beamlets reduced laser power to all but eliminate photobleaching. The low background combined with the fast scan speeds and absence of photobleaching allowed us to measure single-particles and single-molecules in live zebrafish embryo's for long time periods. To the best of our knowledge, it is the first time that single-molecule molecules have been observed in a wide-field two-photon microscope. Show less
Surface charge plays a fundamental role in determining the fate of a nanoparticle, and any encapsulated contents, in vivo. Herein, we describe, and visualise in real time, light-triggered switching... Show moreSurface charge plays a fundamental role in determining the fate of a nanoparticle, and any encapsulated contents, in vivo. Herein, we describe, and visualise in real time, light-triggered switching of liposome surface charge, from neutral to cationic, in situ and in vivo (embryonic zebrafish). Prior to light activation, intravenously administered liposomes, composed of just two lipid reagents, freely circulate and successfully evade innate immune cells present in the fish. Upon in situ irradiation and surface charge switching, however, liposomes rapidly adsorb to, and are taken up by, endothelial cells and/or are phagocytosed by blood resident macrophages. Coupling complete external control of nanoparticle targeting together with the intracellular delivery of encapsulated (and membrane impermeable) cargos, these compositionally simple liposomes are proof that advanced nanoparticle function in vivo does not require increased design complexity but rather a thorough understanding of the fundamental nano-bio interactions involved. Surface charge plays an important role in determining nanoparticle fate in vivo. Here the authors report on the development of a light triggered charge switching liposome and demonstrate light triggered liposome targeting, uptake and payload delivery in a zebrafish model. Show less
Surface charge plays a fundamental role in determining the fate of a nanoparticle, and any encapsulated contents, in vivo. Herein, we describe, and visualise in real time, light-triggered switching... Show moreSurface charge plays a fundamental role in determining the fate of a nanoparticle, and any encapsulated contents, in vivo. Herein, we describe, and visualise in real time, light-triggered switching of liposome surface charge, from neutral to cationic, in situ and in vivo (embryonic zebrafish). Prior to light activation, intravenously administered liposomes, composed of just two lipid reagents, freely circulate and successfully evade innate immune cells present in the fish. Upon in situ irradiation and surface charge switching, however, liposomes rapidly adsorb to, and are taken up by, endothelial cells and/or are phagocytosed by blood resident macrophages. Coupling complete external control of nanoparticle targeting together with the intracellular delivery of encapsulated (and membrane impermeable) cargos, these compositionally simple liposomes are proof that advanced nanoparticle function in vivo does not require increased design complexity but rather a thorough understanding of the fundamental nano-bio interactions involved. Show less
