Imaging Membrane Potential via Second Harmonic Generation

了解大脑是如何工作的是尚未解决的重大科学挑战之一。为了了解神经元网络如何处理信息，我们需要一种方法来映射神经元中的电压变化，具有高灵敏度，高空间分辨率和高时间分辨率。微电极是目前测量膜电位的主要方法;它们具有优异的灵敏度和时间分辨率，但空间分辨率非常有限。光学显微镜有可能彻底改变这一领域，允许非侵入性，实时，高分辨率的电压成像沿着个别神经元，或神经元组，在他们的原生网络。与电极相比，光学探针的巨大优势在于能够同时在许多神经元上绘制电位。目前，用于膜电位的最有效的光学探针是荧光钙指示剂，其通过Ca 2+浓度间接测量膜电位。然而，钙浓度的变化不能准确地反映电压瞬变，并且不能提供关于电压波形的信息。荧光电压敏感染料在30年前就被开发出来用于这一应用，但在大多数情况下，它们的响应很弱，并被背景荧光掩盖。近年来，二次谐波成像（SHG）技术成为一种新的成像技术。二次谐波产生于非对称环境中的可极化分子。在神经元质膜中取向的推挽发色团产生对局部电场敏感的高对比度信号。卟啉的高极化率和强的光学跃迁使它们成为工程高效SHG电压敏感探针的优秀候选者。此外，SHG是一种散射效应，它不需要激发态的人口，所以它应该是可能的设计SHG染料，这是免费的光漂白和光诱导degradation. We的第一个研究卟啉为基础的电压probes导致的染料，表现出强烈的SHG和生物膜具有高亲和力，允许观察强SHG信号从离体神经切片。本项目的目的是在这些初步结果的基础上，创造一系列新的电压敏感卟啉染料用于研究神经元网络，并探索该技术用于脑中膜电位成像的范围。该跨学科合作项目结合了新探针分子的合成，用于筛选电压敏感染料的新膜技术的开发，多光子显微镜和测试新的探针化合物，在体外细胞培养和离体神经元网络。

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.