Imaging Membrane Potential via Second Harmonic Generation

Understanding how the brain works is one of the great unsolved scientific challenges. In order to learn how neuronal networks process information, we need a way of mapping the voltage changes in neurons, with high sensitivity, high spatial resolution and high temporal resolution. Microelectrodes are currently the primary method for measuring membrane potentials; they give excellent sensitivity and temporal resolution, but very limited spatial resolution. Optical microscopy has the potential to revolutionise this field by allowing the non-invasive, real-time, high resolution imaging of voltages along individual neurons, or groups of neurons, within their native networks. The huge advantage of optical probes, compared to electrodes, is the ability to map potential across many neurones at once.At present, the most effective optical probes for membrane potential are fluorescent calcium indicators, which measure membrane potential indirectly, via the concentration of Ca2+. However changes in calcium concentration do not accurately reflect voltage transients, and provide no information on the voltage waveform. Fluorescent voltage-sensitive dyes were developed 30 years ago for this application, but in most cases their response is weak and obscured by background fluorescence. They also have severe problems of photo-instability and photo-toxicity.Recently, second harmonic generation (SHG) imaging has emerged as a powerful alternative. SHG arises from polarisable molecules in asymmetric environments. Push-pull chromophores orientated in the neuronal plasma membrane generate a high contrast signal that is sensitive to the local electric field. The high polarisability and intense optical transitions of porphyrins make them excellent candidates for engineering efficient SHG voltage-sensitive probes. Furthermore, SHG is a scattering effect and it does not require the population of excited-states, so it should be possible to design SHG dyes which are free from photobleaching and photo-induced degradation.Our first studies on porphyrin-based voltage probes led to dyes which exhibit strong SHG and have high affinities for biological membranes, allowing observation of strong SHG signals from ex vivo neuronal slices. The purpose of this proposal is to build on these initial results, to create a new series of voltage-sensitive porphyrin-based dyes for studying neuronal networks, and to explore the scope of this technology for imaging membrane potential in the brain.This collaborative interdisciplinary project combines synthesis of new probe molecules, development of new membrane technology for screening voltage sensitive dyes, multiphoton microscopy and testing of new probe compounds, within vitro cell cultures and ex vivo neuronal networks.

1) Generation of Important New Knowledge, New Methodology and New Materials. The main impacts of this project will be through the generation of new knowledge with relevance to four different areas: (a) better understanding of the design and synthesis of dyes for biological and non-biological applications, (b) new methods for testing voltage-sensitive dyes, and (c) new dyes for imaging membrane potential via SHG. [see Academic Beneficiaries section and Impact Plan]. 2) Economic Impact. This project aims to develop a new tool for fundamental research. The main impact will be an advance in basic understanding. If the new dyes that we develop become widely used by electrophysiologists, the project could make a significant contribution towards understanding the function of the brain and other organs, but the economic impact would be modest. For example the dyes might be used by 250 research groups around the world; each group might spend 2k per year on these compounds, corresponding to a total market of only about 500k p.a. We will seek to commercialise these dyes by license agreements with companies such as Invitrogen or GE Healthcare. Greater economic impact will ensue if it turns out that our dyes can be used for the optical-screening of drug candidates, by evaluating their effect on membrane potential (for example in tissue culture cells). Debilitating medical conditions such as epilepsy and cardiac dysrhythmia are known as channelopathies because they are associated with disorder in ion channels, leading to poorly regulated membrane potential. A rapid optical screening technique for drug candidates for these diseases would have high market value to pharmaceutical research companies. Who will benefit? Oxford University, the inventors of the technology, and UK biotechnology industry. How will they benefit? Financially. How will we ensure impact? All results and know-how with potential for commercial exploitation will be treated confidentially, patented and exploited in conjunction with the Oxford University intellectual property company Isis Innovation Limited. 3) Training Impact. The interdisciplinary and collaborative nature of this project will contribute towards an excellent training environment for two PhD students and two PDRAs. These researchers will also be exposed to a wide range of other current research projects in the Anderson, Bayley, Barford, Wilson and Paulsen labs. We will train scientists with highly transferable skills at the Chemistry-Biology-Engineering interface. 4) Track Record Relating to Impact. All the investigators involved with this project have strong track records for realising the potential impact of their research, as demonstrated primarily by highly cited publications in top international journals). All the investigators are also proactive in the commercial exploitation of their research. For example, Bayley founded a spin-out company (Oxford Nanopore Technologies) to exploit the potential of his stochastic sensing technology. Wilson has published 12 patents, several of which are under licence to commercial enterprises.