4D Confocal Imaging of Endogenous Proteins Tagged by CRISPR/Cas9 Genome Editing

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The application is to finance a significant and very urgent upgrade of our multi-channel Leica SP5-II scanning confocal microscope, an essential and key instruments in the programs of each of the 8 applicants. The fact that this is the third consecutive application to NSERC for this upgrade, our 2016 application having missed funding by only one place, attests to its urgency and to the lack of any other funding sources. The applicants’ research groups use a broad range of biological systems, Xenopus, Drosophila and mouse, and human, mouse and insect cell and stem cell culture to elucidate fundamental mechanisms in Cellular and Molecular Biology. All use cutting-edge technologies in Genomics, Bioinformatics and Proteomics, CRISPR/Cas9 Genome Editing, and especially extensive High Resolution Live-, Fixed-Cell and Tissue Subcellular Imaging. Our strong research grouping, therefore, encompasses a very wide range of expertise, and hence provides a truly exceptional training environment. The requested upgrade is to provide a critically important increase in sensitivity to the SP5-II confocal microscope and to provide near Super-Resolution imaging. CRISPR/Cas9 gene editing has provided us with a simple and efficient means to insert in-phase epitope and fluorescent gene tags into the genes of complex animal genomes. Our groups have successfully established this technology in order to study endogenous proteins under their normal cellular control and at truly physiological abundance. However, the imaging of tagged proteins at natural abundance requires highly sensitive imaging systems. Our Leica SP5-II point scanning confocal microscope presently lacks the sensitivity to allow us to take advantage of endogenous protein tagging, whether epitope or fluorescent. Further, our research involves imaging subcellular detail and we are continuously pushing the limits of optical confocal microscopy. Both the need for enhanced sensitivity and for enhanced resolution can be fulfilled by the cost effective addition to the SP5-II of photo-counting HyD detectors, HyVolution™ deconvolution and an adapted immersion objective. With its standard and resonance modes, our Leica SP5-II instrument provides all the advantages of a scanning microscope, e.g. true confocal resolution and the availability of techniques such as FRET and FRAP, with the extremely low photo-toxicity of spinning disk systems and is fully equipped for live-cell studies. It allows simultaneous monitoring of 4 or 5 freely selectable fluorescence channels in all imaging modes. The SP5-II is also optically and mechanically equal to the best systems available. With the installation of Hybrid (HyD) detectors, a task-adapted objective and improved deconvolution software the SP5-II will fulfill our pressing needs for increased sensitivity and for near Super-Resolution imaging.