Precise Photo-Manipulation and Rapid Imaging in Developing Organisms

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This proposal aims to combine the latest targeted illumination system (FRAP/FLIP/photo-activation unit) with an existing spinning disc confocal microscope (SDCM) in the Cell Imaging and Analysis Network (CIAN). The SDCM is currently equipped with a laser ablation system and two EM-CCD cameras that allow simultaneous dual-color imaging. This proposal also aims to exchange the EE-CCD cameras with two Hamamatsu sCMOS digital cameras to significantly increase imaging speed and resolution. The upgraded system will be capable of simultaneous multi-channel imaging of key factors driving embryonic and neuronal development in flies, worms and mice at previously unattainable speed and resolution. The combined laser ablation and FRAP unit will allow for precise manipulations of multiple fluorescently tagged targets, enabling quantitative measurements of protein-protein interactions and dynamics as well as targeted photo-activation for optogenetic experiments in living cells. The upgrade includes a Mosaic Digital Diaphragm System capable of targeted illumination of multiple fluorescent probes in multiple points/regions of interest. It is an indispensible tool for photo-bleaching (FRAP/FLIP) and photo-activation/optogenetic experiments. Photo-bleaching experiments will be used to quantify the interaction properties of myosin in non-muscle fly cells (Schoeck Lab) and of key elements of the deep cerebellar nuclei (DCP) brain circuit of mice (Watt Lab) and to study the localization dynamics of germ line determinants in Drosophila (Lasko Lab). The photo-activation feature of the Mosaic system will enable optogenetic experiments aimed to understand synaptic integration in the mouse DCP (Watt Lab) and network properties of the C. elegans nervous system in response to stimuli (Hendricks Lab). The sCMOS cameras enable simultaneous multi-color imaging at almost triple the resolution and four times the imaging speed thus allowing to maximally exploit the Mosaic photo-manipulation tool. High speed and resolution imaging will benefit all experiments, in particular fast volumetric calcium imaging in the entire C. elegans nervous system (Hendricks Lab) and the simultaneous imaging of multiple centrosome components in real time (Roy Lab). The 5 NSERC supported laboratories that are named in this application presently train 18 graduate students and postdocs, all of whom will use the system. These researchers will receive professional training from the CIAN staff. The equipment will also be available to hundreds of other CIAN users.