Award details

Super-resolution Light-Addressable Potentiometric Sensors (LAPS)

Reference	BB/P026788/1
Principal Investigator / Supervisor	Professor Steffi Krause
Co-Investigators / Co-Supervisors	Dr Jose Castrejon Pita, Professor Michael Watkinson, Dr Dewen Zhang
Institution	Queen Mary University of London
Department	School of Engineering & Materials Scienc
Funding type	Research
Value (£)	151,056
Status	Completed
Type	Research Grant
Start date	20/07/2017
End date	19/10/2018
Duration	15 months

Abstract

For the first time, we will deliver an instrument that is capable of potentiometrically imaging ion concentrations released basally by surface attached cells and electrical impedance with subcellular resolution (< 50 nm) without the need for fluorescent dyes. Light-addressable potentiometric sensors (LAPS) are based on photocurrent measurements at electrolyte/ insulator/ semiconductor field-effect structures and are capable of monitoring local ion concentrations, extracellular potentials and impedance with good lateral resolution. The best resolution of LAPS that has been reported is 800 nm and has been achieved by using organic monolayer modified silicon-on-sapphire substrates with two-photon excitation of photocurrents using a near infrared femtosecond laser. We now propose to use the optics developed for Stimulated Emission Depletion (STED) nanoscopy (the Nobel Prize in Chemistry 2014) to achieve nanoscale resolution for LAPS. A new instrument will be developed that allows illumination of the substrate with one focused, modulated light beam for photocurrent excitation and with a doughnut shaped light beam of high, constant intensity of the same wavelength, which will inhibit the photocurrent generation outside the central focus of the modulated laser beam. The imaging technology described in this proposal promises to achieve 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. To demonstrate the capabilities of the instrument, we will carry out validation experiments with polymer patterns obtained by micro-contact printing and photolithography, and we will study both, cell impedance and basal pH changes in retinal pigment epithelial cells and their connection with processes such as exocytosis and deposit formation in age related macular degeneration (AMD).

Summary

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 willuse 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.

Impact Summary

UK expertise in electrochemical cell imaging (academic researchers and companies): The proposed instrumentation has new capabilities such as nanoscale, quantitative potentiometric imaging of ion concentrations in the cell-attachment area, which will allow us to gain new insights into electrochemical cell imaging. This will be shared with the scientific community (see Academic beneficiaries). The development of new measurement capabilities will increase the competitiveness of UK science in this field of research. The planned conference presentation 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 also consider KTP opportunities to transfer our knowledge to relevant companies. Dr Dewen Zhang, the scientist employed for this project, will develop new expertise and establish himself as one of the leading researchers in the field. He aims to return to China and establish his own research group in his employment as Assistant Professor in the Institute of Materials at the China Academy of Engineering Physics. D Zhang and S Krause intend to set up strong collaborative links between their research groups, which will benefit both groups' international research profiles and ultimately 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 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, the model used for the validation of the instrument is looking into the mechanism of age related macular degeneration (AMD). The measurements proposed will look at nanoscale changes in pH at the retinal pigment epithelium (RPE)/choroid interface, which is essential to understanding how the sub-RPE material is deposited at the RPE/choroid interface - the site of initial degeneration in AMD. Enhanced understanding of biological processes in AMD may lead to new treatments to prevent, forestall, or reverse the effects of the disease and may also help to elucidate the role of different ions in other diseases including type 2 diabetes, pancreatic cancer, and Alzheimer's disease. Inspiring school children in cell biology and biomedical research: 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.

Committee	Not funded via Committee
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	Tools and Resources Development Fund (TRDF) [2006-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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