Precise Photo-Manipulation and Rapid Imaging in Developing Organisms

该提案旨在将最新的靶向照明系统（FRAP/FLIP/光活化单元）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）相结合。SDCM目前配备了激光烧蚀系统和两个EM-CCD相机，可以同时进行双色成像。该提案还旨在将EE-CCD相机与两台Hamamatsu 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对驱动苍蝇，蠕虫和小鼠胚胎和神经元发育的关键因素进行同步多通道成像。结合激光消融和FRAP单元将允许精确操作多个荧光标记的目标，从而能够定量测量蛋白质-蛋白质相互作用和动力学，以及活细胞中光遗传学实验的靶向光活化。 此次升级包括一个Mosaic Digital Diaphragm System，能够对多个感兴趣点/区域的多个荧光探针进行靶向照明。它是光漂白（FRAP/FLIP）和光活化/光遗传学实验不可或缺的工具。光漂白实验将用于量化非肌肉蝇细胞（Schoeck实验室）中肌球蛋白的相互作用特性和小鼠小脑深部核（DCP）脑回路的关键元素（Watt实验室），并研究果蝇（Lasko实验室）中生殖系决定簇的定位动力学。Mosaic系统的光激活特征将使光遗传学实验能够理解小鼠DCP（Watt Lab）中的突触整合和C. elegans神经系统对刺激的反应（Hendricks实验室）。sCMOS相机能够以几乎三倍的分辨率和四倍的成像速度同时进行多色成像，从而最大限度地利用Mosaic照片处理工具。高速和高分辨率成像将有利于所有实验，特别是在整个C. elegans神经系统（Hendricks实验室）和真实的时间中多个中心体组分的同时成像（Roy实验室）。 本申请中提到的5个NSERC支持的实验室目前培训了18名研究生和博士后，他们都将使用该系统。这些研究人员将接受CIAN工作人员的专业培训。该设备还将提供给数百名其他CIEN用户。

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.