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

大量的分子在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方法之一，可以看到自然生物化学环境中的结构和分子，并探测分子动力学和相互作用。光学显微镜中的工程和物理挑战是提供能够提供最高空间分辨率的工具。时间分辨率，通过开发技术和方法来解码分子相互作用的复杂网络的能力，同时，该项目的重点是开发一种新型光谱仪结构，这种光谱仪将允许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）。通过利用Foerster共振能量转移和其他物理现象，确实有可能探测分子间的相互作用。将开发的系统将提供机会，以高空间分辨率对复杂的生化事件成像，由于使用并行采集，同时保留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，这损害了使用的简单性和成本，或者顺序采集需要几分钟来采集一个（多参数）图像。新的架构将提供并行采集与一个单一的探测器，并通过使用一个新的固态探测器（时间选通单光子雪崩光电二极管）和超连续谱光源，这些仪器和相关的分析方法对于理解对人类生理学和病理学都至关重要的分子机制是必不可少的例如神经退行性疾病。事实上，阿尔茨海默病和帕金森病等神经退行性疾病正在对现代老龄化社会构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涉及α-突触核蛋白和蛋白质14-3-3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。14-3-3s表现出与α-突触核蛋白的一定程度的同源性，并且两种蛋白质共享共同的底物。14-3-3s和α-突触核蛋白在调节蛋白激酶C等常见底物中是否同时存在？这种信号通路在神经退行性变中的作用是什么？幸运的是，本项目中提出的技术和方法将在不久的将来为这些问题和其他有趣的问题提供答案。

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.