Precise Photo-Manipulation and Rapid Imaging in Developing Organisms

本提案旨在将最新的靶向照明系统（FRAP/FLIP/光激活单元）与细胞成像和分析网络（CIAN）中现有的旋转圆盘共聚焦显微镜（SDCM）结合起来。SDCM目前配备了一个激光烧蚀系统和两个EM-CCD相机，可以同时进行双色成像。本提案还旨在将EE-CCD相机与两台滨松sCMOS数码相机交换，以显着提高成像速度和分辨率。升级后的系统将能够以以前无法达到的速度和分辨率同时对驱动果蝇、蠕虫和小鼠胚胎和神经元发育的关键因素进行多通道成像。联合激光消融和FRAP单元将允许精确操作多个荧光标记的目标，实现蛋白质-蛋白质相互作用和动力学的定量测量，以及在活细胞中进行光遗传学实验的靶向光激活。

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.