Biological Applications of A Multiplexed Confocal Fluorescence Lifetime Imaging Microscope

Abstract

Understanding dynamic molecular interactions taking place within living systems has been a crucial aspect of modern biological research. In this work, the development and execution of fixed sample and live cell imaging protocols are central to characterizing and validating the performance and capabilities of a new Multiplexed Multispectral Confocal Fluorescence Lifetime Imaging Microscope (FLIM) for visualizing and interpreting such interactions. This system presents a new approach that integrates FLIM with other advanced imaging modalities, involving multiplexed and multispectral imaging as well as confocal microscopy. With FLIM, simultaneous spatial and temporal information can be measured to provide comprehensive analyses of dynamic biological processes, transcending the limitations of conventional fluorescence microscopy. Its multiplexed and multispectral imaging capabilities then add another layer of benefits by facilitating a 30x30 scan encompassing 900 pixels that captures six spectral images across a spectral band range of 450 nm to 700 nm within 0.9 s from only 1ms exposure time. Furthermore, incorporating confocal microscopy enhances the system's ability to capture detailed structural and functional information within biological specimens with less noise by rejecting background fluorescence from other planes. Through this thesis, the applications of this multiplexed multispectral confocal FLIM system in various biological contexts, ranging from studying protein-protein interactions to monitoring intracellular signalling events, are explored. The experiments conducted use standard fluorescence samples and live cell models, serving as examples demonstrating the system's functionality and potential. While the scope of this research primarily encompasses straightforward cellular experiments, its implications extend far beyond, promising substantial contributions to imaging technologies and offering insights into the dynamic molecular processes governing cellular function and disease progression. As the multiplexed multispectral confocal FLIM system undergoes further optimization and application across diverse biological investigations, it holds the promise to significantly advance our understanding of disease pathogenesis, elevate medical diagnostics, and transform drug discovery.