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Imaging Membrane Potential via Second Harmonic Generation

通过二次谐波产生膜电位成像

基本信息

批准号：
EP/H018565/1
负责人：
Harry Anderson
金额：
$ 100.56万
依托单位：
University of Oxford
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2010
资助国家：
英国
起止时间：
2010 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=EP%2FH018565%2F1
关键词：
Imaging Membrane Potential via Second

项目摘要

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.

理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，我们需要一种高灵敏度、高空间分辨率和高时间分辨率的神经元电压变化映射方法。微电极是目前测量膜电位的主要方法；它们具有出色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，因为它允许对单个神经元或神经元群在其原生网络内的电压进行非侵入性、实时、高分辨率成像。与电极相比，光学探针的巨大优势在于它能够同时绘制多个神经元的电位图。目前，最有效的膜电位光学探针是荧光钙指示剂，它通过Ca2+浓度间接测量膜电位。然而，钙浓度的变化不能准确反映电压瞬变，也不能提供电压波形的信息。荧光电压敏感染料是在30年前开发的，但在大多数情况下，它们的反应很弱，被背景荧光遮挡。它们也存在严重的光不稳定性和光毒性问题。最近，二次谐波成像（SHG）已成为一种强大的替代方法。SHG产生于不对称环境中的极化分子。推拉发色团定位于神经元质膜，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，它不需要激发态的填充，因此应该有可能设计出不发生光漂白和光致降解的SHG染料。我们对基于卟啉的电压探针的首次研究导致染料表现出强烈的SHG，并且对生物膜具有高亲和力，可以观察到来自离体神经元切片的强SHG信号。本提案的目的是在这些初步结果的基础上，为研究神经元网络创造一系列新的电压敏感卟啉染料，并探索该技术在脑膜电位成像中的应用范围。这个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，多光子显微镜和新探针化合物的测试，体外细胞培养和离体神经网络。