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 可以让人追踪活体中特定分子的行为 cell. 通过这项技术，一种已知蛋白质的基因被拼接 用GFP基因，然后在活细胞中表达产物， 融合蛋白。 在有利条件下，融合蛋白 功能正常，其位置可以随着时间的推移在 活细胞的低亮度荧光成像。 但龙 长期GFP成像研究是棘手的，因为，像大多数荧光 它会被光漂白，而且随着时间的推移， 激发GFP所需的光对细胞可能是有毒的。 因此，长期以来， 术语研究低光级相机必须耦合到高光级相机。 高质量的荧光成像平台， 可以最小化和关闭对样本的照射。 也是 重要的是，该系统允许样本也被光学地 以落射荧光模式切片，并长时间随访 在非荧光成像模式下的周期。 由于BMIRR有许多 GFP成像所需的LM组件，我们花了1997年的一部分时间， 组装GFP成像站供内部和外部使用。 为 这一点，我们修改了DIC（De Senarmont）和荧光（尼康 Quadfluor）部分，我们的定量宽场荧光 显微镜工作站 这台工作站有一个大的格式冷却 CCD（PXL 14000 Photometrics Ltd.）用于图像采集的、电子的 用于延时成像的快门，以及用于收集 Z系列（Ludl Inc.）。相机和其他电子设备 由运行ISEE软件（Inovision）的SGI工作站控制 Corp）。 这台显微镜和工作站是在1996年购买的 通过纽约州的资助，我们升级了显微镜， 配备Quadfluor荧光系统和Omega荧光滤光片 立方体。 后者被设计为一个合作， 资源和欧米茄，并尽量减少内部反射，同时提供 增强的信噪比。 通过这些修改，我们可以 现在获得高分辨率和高对比度的图像， 标记结构的曝光时间非常短（200 ms）， 我们可以对GFP标记的细胞进行延时研究的时间长度 细胞 我们还为平台配置了一个气缸， 简单的电子设备（达林顿），使DIC分析仪可以 从荧光光路交替地插入和移除 使用ISEE软件。 这不仅能够实现最大荧光 信号到达相机，但它也允许DIC成像 显像

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.