Microfluidic devices for 3D super-resolution imaging of single molecules in live cells

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Single molecule experiments in living cells are of interest for a variety of reasons: to be able to track macromolecules within living systems and quantify their role in the regulation of gene expression, to provide experimental insight into the mechanism of gene expression and regulation in living systems and to be able to provide insight into other stochastic mechanisms present at a molecular scale in living systems that may be missed in an ensemble measurement typically representative of conventional tools and interfaces. This proposal builds on initial proof-of-concept experiments and presents an approach combining microfluidics, nanoscale structures and optical detection to open up a range of options for integrating single-cell molecular imaging together with chemical analysis of small volumes of complex mixtures present in such systems. This unique convergence of technologies could enable new platforms for unique single-molecule studies in living systems. Such systems could combine stochastic measurements of protein molecules using super-resolution optical microscopy with the high-throughput analysis of nuclear structure to obtain truly novel insight into the 3D organisation of chromatin in live mammalian cells. The proposed tools will address the current limitations that exist in extending super-resolution imaging technology to mammalian systems by integrating appropriate trapping structures and optical light sheets within microfluidic devices to prevent auto-fluorescence that results in the presence of much larger nuclear structures found in these systems.