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

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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.