Super-resolution radial fluctuation microscope for 4D imaging of cells and cellular dynamics

Super-resolution radial fluctuation microscope for 4D imaging of cells and cellular dynamics Our 6 research groups use a state-of-the-art custom-built spinning-disk confocal microscopy originally funded by investments from both CFI and NSERC. This microscope is cable of both fixed and high-speed live-cell imaging that is central to our research programs, and is used to observe a wide-array of proteins (e.g. mitochondrial, DNA repair and RNA-binding factors), cellular processes (e.g. DNA repair, synapse formation, autophagy, host-pathogen interactions) and structures (e.g. nuclear subdomains, synapses, mitochondria, autophagosomes and stress granules) within several different organisms including protists, bacteria, viruses, and both human and zebrafish cells. When imaging fluorescent proteins and subcellular structures in living cells by light microscopy, the precise localization of protein complexes is limited by the resolution of conventional light microscopy (~200 nm in x/y). Super-resolution techniques developed in the last decade allow increased resolution in the range of 20-120 nm but often require high-intensity light illumination over long periods of time, which affects the viability of cells through photo-toxicity. In 2016, the research group of Dr. Ricardo Henriques (University College London (UCL)) developed a new live-cell imaging technique called S uper- R esolution R adial F luctuation ( SRRF ) microscopy, which readily achieves 60-120 nm resolution in live-cells with low levels of illumination on conventional light microscopes with modest software and camera upgrades. Currently, there are no super-resolution microscopes in Atlantic Canada. Therefore, we are requesting funds to support the upgrade of our existing spinning-disk system to enable SRRF live-cell super-resolution microscopy. This upgrade will enable our 6 research groups to more accurately localize and track the dynamics of biological molecules and cellular process in live cells in 4 dimensions (3D and over-time) uncovering new fundamental biology otherwise unresolvable by conventional light microscopy. The requested funds to upgrade the CFI/NSERC funded microscope for SRRF capability will result in a one-of-kind instrument in Canada, providing a unique training opportunity for highly qualified personnel to learn advanced live-cell super-resolution microscopy techniques. The requested equipment will also be accessible to other research groups on a collaborative basis, facilitating super-resolution live-cell imaging research not only at Dalhousie University but throughout Atlantic Canada.