Super-resolution Light-Addressable Potentiometric Sensors (LAPS)

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In-vitro cell models have been used very successfully to investigate disease mechanisms and the efficacy and toxicity of drugs. They have the potential to eventually abolish the need for animal experiments. The investigation of complex biological processes in cell culture requires sophisticated measurement tools, and there is currently a lack of tools capable of providing quantitative chemical information on the surface-attached (basal) side of living cells. In this project, a novel instrument will be developed that can revolutionise our ability to understand ion fluxes and pH changes in living cells by quantitatively measuring the concentrations of ions, electrical cell-signals and the transport properties of living cells in the surface attachment area with nanoscale lateral resolution. The instrument proposed is based on light-addressable potentiometric sensors (LAPS). In this technique, light is modulated and then focused onto an electrolyte/ insulator/ semiconductor (EIS) structure. The light excites a local photocurrent, which depends on the local surface charge of the insulator and the local impedance of anything in contact with the insulator surface. Using thin semiconductor layers in silicon-on-sapphire (SOS) and a femtosecond, near-infrared laser for photocurrent excitation, a resolution of 800 nm was achieved, which allowed measurement of single cells, but was not sufficient for reliable sub-cellular resolution.Super-resolution microscopy or nanoscopy (Nobel Prize in Chemistry 2014) has brought fluorescence microscopy into the nanodimension of live-cell imaging beyond the diffraction limit, which provided a new level of detail of living cells in bioimaging research. In this project, the principle of Stimulated Emission Depletion (STED) nanoscopy will be applied to LAPS to obtain a novel instrument capable of imaging ion concentrations and cell impedance at the nanoscale. As in STED, we propose to use two laser beams. However, in contrast to STED, we will use these two beams to excite and inhibit photocurrent in EIS structures instead of fluorescence. We will illuminate the LAPS substrates with one focused, modulated light beam for photocurrent excitation and with a doughnut shaped light beam of high, constant intensity of the same wavelength for inhibition of the photocurrent outside the central focus of the modulated beam. As in STED, this is expected to result in a resolution well below the diffraction limit of less than 50 nm and will allow resolution of subcellular features. In contrast to the femtosecond laser technology employed previously for high-resolution LAPS, we envisage a sixteen-fold improvement in the resolution (without incurring any of the disadvantages of STED such as photobleaching or the necessity of specialised fluorescent dyes) while reducing the cost of the optical setup tenfold.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 this project, we will specifically measure pH on the basal (surface facing) side of cells. The instrument will be validated with polymer patterns to obtain quantitative information about the resolution that can be achieved and will then be further characterised using two cell models. (i) Yeast cells will be immobilised on the pH sensitive surfaces using agarose gel. The change of pH under and around a single yeast cell in the presence of glucose will be monitored using the new measurement system. (ii) 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. We will image pH and impedance changes at the basal side of the retinal pigment epithelium to gain more information about the mechanism of the formation of local deposits, which are the hallmark of AMD.