Combined LAPS and SICM for multimodal live cell imaging

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Epithelial and endothelial tissues line the cavities or surfaces of organs such as the eyes, lungs, gastrointestinal and urinary tract or blood vessels. Impaired function of these tissues is linked to many health issues including blindness, atherosclerosis and diabetes. The cells in these tissues are strongly polarised and display different properties on the apical and basal sides. This polarised nature is crucial for their function, and events on one side directly affect the other side. Hence, there is a great need for tools capable of investigating apical and basal sides simultaneously. Apart from environmental changes through nutritional stimuli or oxidative stress, cells often respond to localised factors like that of broken off photoreceptor outer segments. This may elicit response only in a single cell, but also affect neighbouring cells via changed secretion. In this project, a novel instrument will be developed that will revolutionise our ability to monitor cellular processes, and cell communication in polarised cells by simultaneously imaging cells apically and basally providing simultaneous information about apical cell morphology and basal ion concentrations and electrical signals such as cell surface charge and impedance. An instrument will be built that, for the first time, combines two electrochemical imaging techniques that have been used previously independently to measure cell responses. Light-addressable potentiometric sensors (LAPS) are based on the photocurrent measurements at electrolyte-insulator-semiconductor (EIS) structures. LAPS is sensitive to surface charges, ion concentrations and impedance. If cells are cultured on LAPS substrates, all these parameters can be measured on the substrate facing, basal, side of the cells. Scanning Ion Conductance Microscopy (SICM) measures ion currents through a nanopipette scanning the surface of the cell. SICM has been used to image detailed cell topography on the outer (apical) side of cells and to induce changes in the microenvironment by releasing reagents locally to a single cell. By combining LAPS and SICM, we can carry out functional electrochemical imaging simultaneously both apically and basally. The release of reagents locally on the apical side while continuously imaging ion concentrations and cell impedance basally will provide us with new insights into the transport mechanisms through epithelial and endothelial tissues with unprecedented detail and contribute to the understanding of physiological processes and disease mechanisms. By chemically modifying the insulator surface in the EIS structure, a change of surface charge can specifically be induced by different ionic species resulting in quantitative concentration dependent signals in LAPS. In this project, we will specifically measure pH, calcium, zinc and sodium ions. The instrument will be validated with polymer patterns to ensure proper synchronisation of LAPS and SICM and will then be further characterised using two cell models. (i) Retinal pigment epithelial (RPE) cells have been used as a cell model for the investigation of the mechanisms of age related macular degeneration (AMD) - the most prevalent cause of blindness in the elderly where basal changes in zinc, calcium and pH are implicated in deposit formation. We will stimulate RPE cells by apical release of ions and reagents inducing oxidative stress using the SICM nanopipette and simultaneously image ion concentrations and impedance changes at the basal side to gain more information about the mechanism of the formation of local deposits, which are the hallmark of AMD. (ii) The vascular endothelium lines the luminal surface of blood vessels performing a barrier function between circulating blood and the rest of the vessel wall. The new instrument will be used to study in detail the mechanism of vascular permeability for sodium and calcium ions, which is increased in diseases such as atherosclerosis, diabetes and renal diseases.