High performance live cell imaging unit

数字技术的最新进展与荧光显微镜相结合，可以观察到活细胞内发生的事件。传统上，荧光显微镜由激发标本的光源适当照明和捕获荧光发射的光传感器组成，但由于缺乏捕获活细胞中细胞组分动态相互作用的适当技术，其使用受到限制。然而，在过去的几年里，荧光显微镜与高速数码相机相结合，使科学家能够获得令人着迷的数据，以测试涉及实时细胞功能的新假设。本提案中要求的设备包含这种技术，并将允许从活细胞中高速获取荧光图像，而不会造成光损伤。所要求的电子倍增电荷耦合器件相机（EMCCD）， ImagEM (Hamamatsu)，可以在非常低的光水平下获得高分辨率图像，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，我们需要Volocity软件包（Improvision），它提供了一套完整的工具，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CO2浓度是细胞生理学的基本变量，必须保持恒定，以便在整个实验期间在显微镜阶段创造适当的实验条件。出于这个原因，我们要求购买Chalmide TC，一种二氧化碳/温度控制器设备。这种特殊的设备组合将包括一个独特的活细胞成像单元（LCIU），它将交替地与已经位于UTSC神经生物学中心的应激设施中的epifluorescent， TIRF或扫描共聚焦显微镜相结合。目前在UTSC校园还没有类似的系统，而且这种设备的组合在加拿大也相当罕见。

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.