Combined LAPS and SICM for multimodal live cell imaging

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.

UK expertise in electrochemical cell imaging (academic researchers and companies):
The proposed instrumentation has new capabilities such as simultaneous apical and basal functional electrochemical imaging in in-vitro cell models. This will be shared with the scientific community (see above). The development of new measurement capabilities will increase the competitiveness of UK science in this field of research. The planned conference presentations and publications will enable us to demonstrate the benefits of the new technique to the wider research community. Research groups who wish to adopt this technique will be invited to training workshops.
We will pursue KTP opportunities to transfer our knowledge to relevant companies.
The PDRA employed for this project will develop new expertise and gain a highly multidisciplinary research profile by receiving training in a variety of areas including electrochemistry, cell biology, device fabrication and basic chemical synthesis and by taking the lead in the presentation and dissemination of results. This will result in a highly trained scientist with broad multidisciplinary skills, which will be very attractive to prospective employers in both academia and industry and ultimately contribute to the UK economy.
Understanding of disease mechanisms (academic researchers & pharmaceutical companies):
The instrument will provide new insights into transport processes in in-vitro cell models, which cannot be gained with any other electrochemical or electrophysiological techniques currently available. In the long-term, this could aid the development of organ on-a-chip devices, which can reduce the need for animal models (the 3 Rs). The information gained with the new instrument will improve the understanding of disease mechanisms and can therefore lead to new treatments that benefit the health of the population in the UK and worldwide. More specifically, one of the models used for the validation of the instrument is looking into the mechanism of age related macular degeneration (AMD) by studying the transport of ions through the retinal pigment epithelium (RPE), which is essential to understanding how sub-RPE material is deposited at the RPE/choroid interface - the site of initial degeneration in AMD. The information gained may lead to new treatments to prevent, forestall, or reverse the effects of the disease. The endothelial cell model used for the validation of the new instrument aims to elucidate ion transport mechanisms through the vascular endothelium, which are important to the regulation of vascular permeability that is affected in diseases such as atherosclerosis, diabetes and renal dysfunction.
The instrument can also be applied to drug screening in blood-brain barrier (BBB) models. The BBB is less permeable than other types of endothelium and is believed to be one of the reasons for the higher failure rate of brain disorder treatments compared to other disease areas. The new instrument will be able to map transport pathways of conventional therapeutic molecules and novel nanoscale materials for drug delivery (nanomedicine) in BBB models. This information would help to identify compounds, which may fail in the later stages of clinical trials and thus reduce the cost of drug development. To facilitate the transfer of knowledge to pharmaceutical industry, we will utilise Industrial Advisory Boards within our school which include GSK, Pfizer, and Johnson & Johnson.
Inspiring school children:
The outreach activities proposed in this project are closely aligned with the aims and objectives of QMUL's "Centre of The Cell" (http://www.centreofthecell.org/). The PDRA will use this link to ensure outreach activity is delivered and will develop materials for exhibitions and web-based materials. Participation in open days delivered by the Centre of Cell will inspire school children to become the next generation scientists at the interface of the biological and physical sciences.