High performance live cell imaging unit

数字技术的最新进展与荧光显微镜相结合，使人们能够观察活细胞内发生的事件。传统上，荧光显微镜由激发样本的适当光源和捕捉荧光发射的光传感器组成，但由于缺乏适当的技术来捕捉活细胞中细胞成分的动态相互作用，荧光显微镜的使用受到了限制。然而，在过去的几年里，荧光显微镜与高速数码相机结合在一起，使科学家能够获得引人入胜的数据，以测试涉及实时细胞功能的新假设。这项提议中要求的设备包括这种技术，并将允许从活细胞中高速获取荧光图像，而不会造成光损害。所需的电子倍增器电荷耦合器件相机(EMCCD)Imagem(Hamamatsu)可以在非常低的光线水平下获取高分辨率图像，使其成为高性能活细胞成像的理想选择。为了控制相机和显微镜并处理采集到的数据，我们要求使用Volocity软件包(IMPROFILE)，该软件包提供一整套工具，用于3D图像采集、恢复、物体测量和跟踪。活细胞成像还需要维持细胞持续存活的最佳环境条件。温度和二氧化碳浓度是细胞生理学的基本变量，必须保持恒定，以便在整个实验过程中在显微镜阶段创造适当的实验条件。为此，我们请求购买查莱姆TC，这是一种二氧化碳/温度控制设备。这一特定的设备组合将包括一个独特的活细胞成像单元(LCIU)，该单元将与UTSC应激设施神经生物学中心内已安装的荧光、TIRF或扫描共焦显微镜交替耦合。UTSC校园目前还没有类似的系统，事实上，这种设备的组合在加拿大相当罕见。

Recent advances in digital technology coupled with fluorescent microscopy allow the observation of events as they take place inside living cells. Traditionally, fluorescent microscopes consist of a source appropriate illumination that excites specimens and a light sensor that captures fluorescent emissions but its use has been limited by the lack of appropriate  technology to capture the dynamic interplay of cellular components in living cells.  However, during the last few years, fluorescent microscopes have been coupled with high-speed digital cameras allowing scientists to acquire fascinating data to test new hypotheses involving real-time cellular function. The equipment requested in this proposal encompasses such technology and will allow high-speed acquisition of fluorescent images from live cells without causing photodamage. The requested electron multiplier charge-coupled device camera (EMCCD), ImagEM (Hamamatsu), can acquire high resolution images at very low levels of light, making it ideal for high-performance live cell imaging. To control the camera and microscope and to process the acquired data, we are requesting the Volocity software package (Improvision), which provides a full suite of tools for 3D image acquisition, restoration, object measurement and tracking. Live cell imaging also requires the maintenance of optimal environmental conditions for the continued cell survival. Temperature and CO2 concentrations are fundamental variables for cell physiology, and must be kept constant in order to create proper experimental conditions at the microscope stage throughout the duration of the experiment. For this reason we are requesting the purchase of the Chalmide TC, a CO2/ Temperature controller device. This specific combination of equipment will comprise a unique Live Cell Imaging Unit (LCIU) that will be alternatively coupled to either an epifluorescent, a TIRF or a scanning confocal microscope already located within the Centre for Neurobiology of Stress facility at UTSC. An equivalent system does not currently exist at the UTSC campus and indeed this combination of apparatus is quite rare in Canada.