Multiplexed measurement of molecular interactions using hyper-spectral imaging and multi-parametric detection

使用高光谱成像和多参数检测对分子相互作用进行多重测量

• 批准号: EP/F044011/1
• 负责人: Alessandro Esposito
• 金额: $ 43.59万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FF044011%2F1
• 关键词: Multiplexed; measurement; molecular; interactions; using

A large number of molecules cooperate in an intricate network of interactions for the maintenance of the structural integrity, the metabolism and the function of the living cell. The spatio-temporal localization of each molecule and their propensity to interact with each other regulate the complexity of life. Among modern techniques, fluorescence microscopy is one of the most important and productive allowing to see structures and molecules in their natural biochemical environment and to probe molecular dynamics and interactions.The challenge for engineering and physics in optical microscopy is to provide tools that could offer the highest spatio-temporal resolution with the capability to decode the complex network of molecular interactions by the development of technologies and methods that, at the same time, may provide cost-effective and user-friendly instruments that could be of widespread use in the biomedical community.This project focuses on the development of a novel architecture for a spectrograph that will permit to characterize fluorescence emission (excitation and emission spectra, fluorescence anisotropy and fluorescence lifetime) in a quantitative and efficient manner. By the exploitation of Foerster resonance energy transfer and other photophysical phenomena, it is indeed possible to probe molecular interactions. The system that will be developed will offer the opportunity to image complex biochemical events with high spatial resolution retaining a comparatively high temporal resolution (1 minute) owing to the use of parallel acquisition. Typical systems make use of multiple detectors at the detriment of simplicity of use and costs or sequential acquisitions that require several minutes for the acquisition of one (multi-parametric) image. The novel architecture will offer parallel acquisition with a single detector and, by the use of a novel solid-state detector (time-gated single-photon avalanche photodiodes) and a supercontinuum light source, will provide excellent versatility of use at comparatively low costs.These instruments and the related methods of analysis will be essential for the understanding of molecular mechanisms of crucial importance for both human physiology and pathologies such as neurodegenerative diseases. In fact, neurodegenerative diseases like Alzheimer's disease and Parkinson's disease are posing an increasing economical and social threat for the modern aging society. Therefore, the developed system will be used to investigate a molecular pathway that involves the cooperation between alpha-synuclein and the protein 14-3-3. Alpha-synuclein aggregates are the main hallmark of Parkinson's disease. Furthermore, mutations in the alpha-synuclein gene are responsible for a genetic variant of Parkinson's disease. 14-3-3s exhibit a degree of homology with alpha-synuclein and both proteins share common substrates. Are 14-3-3s and alpha-synuclein concurring in the regulation of common substrates such as the protein kinase C? Which is the role of this signalling pathway in neurodegeneration? Fortunately, technologies and methods such as the one proposed in this project will provide an answer to these and other intriguing questions in the near future.

大量的分子在一个复杂的相互作用网络中合作，以维持活细胞的结构完整性、代谢和功能。每个分子的时空定位及其相互作用的倾向调节着生命的复杂性。在现代技术中，荧光显微镜是最重要和最有效的技术之一，它允许在自然生化环境中观察结构和分子，并探测分子动力学和相互作用。光学显微镜工程和物理学面临的挑战是提供能够提供最高时空分辨率的工具，并能够通过开发技术和方法解码复杂的分子相互作用网络，同时可能提供具有成本效益和用户友好的仪器，可以在生物医学界广泛使用。该项目的重点是开发一种新的光谱仪结构，以定量和有效的方式表征荧光发射（激发和发射光谱，荧光各向异性和荧光寿命）。利用福斯特共振能量转移和其他光物理现象，确实有可能探测分子间的相互作用。将开发的系统将提供以高空间分辨率成像复杂生化事件的机会，由于使用并行采集，可以保持相对较高的时间分辨率（1分钟）。典型的系统使用多个检测器，损害了使用的简单性和成本，或者需要几分钟才能获取一个（多参数）图像的顺序采集。新架构将提供单一探测器的并行采集，并且通过使用新型固态探测器（时间门控单光子雪崩光电二极管）和超连续光源，将以相对较低的成本提供出色的多功能性。这些仪器和相关的分析方法对于理解对人类生理和病理（如神经退行性疾病）至关重要的分子机制至关重要。事实上，像阿尔茨海默病和帕金森病这样的神经退行性疾病正在对现代老龄化社会造成越来越大的经济和社会威胁。因此，开发的系统将用于研究α -突触核蛋白与14-3-3蛋白之间合作的分子途径。α -突触核蛋白聚集体是帕金森病的主要标志。此外，α -突触核蛋白基因的突变是帕金森病遗传变异的原因。14-3-3 -3与α -突触核蛋白具有一定程度的同源性，两者具有共同的底物。14-3-3和α -突触核蛋白是否同时调控常见底物，如蛋白激酶C？这个信号通路在神经退行性变中的作用是什么？幸运的是，像这个项目中提出的技术和方法将在不久的将来为这些和其他有趣的问题提供答案。

项目成果

Enhancing biochemical resolution by hyper-dimensional imaging microscopy

通过超维成像显微镜提高生化分辨率

• DOI: 10.1101/431452
• 发表时间: 2018
• 作者: Esposito A