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是如何处理信息的，我们需要一种高灵敏度、高空间分辨率和高时间分辨率的映射神经元电压变化的方法。微电极是目前测量膜电位的主要方法；它们具有良好的灵敏度和时间分辨率，但空间分辨率非常有限。光学显微镜有可能使这一领域发生革命性的变化，因为它允许对自然网络中单个神经元或神经元组的电压进行非侵入性、实时、高分辨率的成像。与电极相比，光学探针的巨大优势是能够一次映射多个神经元的电位。目前，最有效的膜电位光学探针是荧光钙指示剂，它通过钙离子的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，也不能提供有关电压波形的信息。荧光电压敏感染料是30年前为这一应用而开发的，但在大多数情况下，它们的响应很弱，并且被背景荧光遮挡。它们还存在严重的光不稳定和光毒性问题。最近，二次谐波成像(SHG)已经成为一种强有力的替代方案。倍频产生于非对称环境中的可极化分子。定位于神经元质膜的推挽式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烈的光学跃迁使其成为工程高效倍频电压敏感探测器的极佳候选者。此外，倍频是一种散射效应，它不需要激发态的布居，因此设计没有光漂白和光诱导降解的倍频染料是可能的。我们首次研究了基于卟啉的电压探针，导致了染料表现出强烈的倍频效应，并对生物膜具有高亲和力，使得从体外神经元切片观察到强的倍频信号成为可能。这项提议的目的是在这些初步结果的基础上，创造一系列新的基于卟啉的电压敏感染料用于研究神经元网络，并探索这项技术在脑部成像膜电位的范围。这一跨学科的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、多光子显微镜以及在体外细胞培养和体外神经元网络中对新探针化合物的测试。

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.