LOW LIGHT QUANTITATIVE SYS: LONG TIME LAPSE GFP & DIC IMAGING OF LIVING CELLS

绿色荧光蛋白(GFP)标记的最新进展 允许一个人跟踪活着的选定分子的行为 手机。利用这项技术，一种已知蛋白质的基因被拼接 然后该产物在活细胞中表达为 一种融合蛋白。在有利的条件下，融合蛋白 函数正常运行，并且其位置可以随时间在 通过微光荧光成像获得活细胞。然而，Long 术语GFP成像研究是棘手的，因为像大多数荧光物质一样 探测它的光漂白，也是因为随着时间的推移，蓝色的水平 激发绿色荧光蛋白所需的光对细胞可能是有毒的。因此，在很长一段时间里 学期研究微光摄像机必须与高光摄像机相连接 高质量的荧光成像平台上使用的光 照亮样本可以最小化和关闭。它也是 重要的是，该系统允许样品也是光学的 以表观荧光模式切片，ALS o长时间跟随 在非荧光成像模式下的经期。因为BMIRR有很多 在GFP成像所需的LM组件中，我们花了1997年的部分时间 组装GFP成像站，供内部和外部使用。为 我们对DIC(德塞纳蒙特)和荧光(尼康)进行了修改 Quadfluor)部分我们的定量广场荧光 显微镜工作站。这台工作站有一台大幅面冷却系统 Ccd(PXL 14000 Photoetrics Ltd.)对于图像采集，电子， 用于延时成像的快门和用于采集的步进电机 Z系列(Ludl Inc.)。摄像机和其他电子设备是 由运行ISEE软件(Inovision)的SGI工作站控制 公司)。这台显微镜和工作站完全是在1996年购买的 通过纽约州的资助，我们对显微镜进行了升级 带有四氟荧光系统和欧米茄荧光滤光片 立方体。后者被设计为 资源和欧米茄，并最大限度地减少内部反射，同时提供 增强的信噪比。有了这些改装，我们可以 现在获得极暗的高分辨率和高对比度的图像 具有非常短(200毫秒)曝光的标记结构--这增加了 我们可以进行GFP标记的时间推移研究的时间长度 细胞。我们还为平台配置了一个气动气缸和 简单的电子设备(达林顿)，以便DIC分析仪可以 交替地插入和从荧光光路移除 使用ISEE软件。这不仅实现了最大程度的荧光 信号到达摄像机，但它也允许由DIC成像 成像。

The recent advent of green fluorescent protein (GFP)-labeling allows one to follow the behavior of selected molecules in the living cell. With this technology the gene for a known protein is spliced with a GFP gene, and the product is then expressed in a living cell as a fusion protein. Under favorable conditions the fusion protein functions normally and its position can be followed over time in the living cell by low light level fluorescence imaging. However, long term GFP imaging studies are tricky because, like most fluorescent probes it photobleaches, and also because over time the levels of blue light needed to excite GFP can be toxic to the cell. Thus, for long term studies a low light level camera must be coupled to a high quality fluorescent imaging platform on which the light used to illuminate the specimen can be minimized and shuttered. It is also important that the system allow the specimen to also be optically sectioned in the epi-fluorescent mode, and als o followed for long periods in a non-fluorescent imaging mode. Since the BMIRR had many of the LM components necessary for GFP imaging, we spent part of 1997 assembling a GFP imaging station for internal and external use. For this we modified the DIC (De Senarmont) and fluorescence (Nikon Quadfluor) portions of our quantitative wide-field fluorescent microscope workstation. This workstation has a large format cooled CCD (PXL 14000 Photometrics Ltd.) for image acquisition, electronic shutters for time-lapse imaging, and a stepper motor for collecting Z-series (Ludl Inc.). The camera and other electronic devices are controlled by a SGI workstation running ISEE software (Inovision Corp). This microscope and workstation was purchased in 1996 entirely through NY State funding We upgraded the microscope by equipping it with a Quadfluor fluorescence system and Omega fluorescence filter cubes. The latter were designed as a collaboration between the Resource and Omega, and minimize internal reflections while providing an enhanced signal to noise ratio. With these modification's we can now obtain high resolution and high contrast images of extremely dimly labeled structures with very short (200 ms) exposures--which increases the length of time we can conduct time-lapse studies on GFP labeled cells. We also configured the platform with a pneumatic cylinder and simple electronic device (Darlinton) so that the DIC analyzer could be alternatively inserted and removed from the fluorescence light path using the ISEE software. This not only enables a maximum fluorescence signal to reach the camera, but it allows to also be imaged by DIC imaging.