Rustom A, Gerlich D, Rudolf R, Heinemann C, Eils R, Gerdes H H
University of Heidelberg, Germany.
Biotechniques. 2000 Apr;28(4):722-8, 730. doi: 10.2144/00284rr01.
The generation of spectral mutants of the green fluorescent protein (GFP) set the stage for multiple-color imaging in living cells. However, the use of this technique has been limited by a spectral overlap of the available GFP mutants and/or by insufficient resolution in both time and space. Using a new setup for dual-color imaging, we demonstrate here the visualization of small, fast moving vesicular structures with a high time resolution. Two GFP-fusion proteins were generated: human chromogranin B, a secretory granule matrix protein, and phogrin, a secretory granule membrane protein. They were tagged with enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), respectively. Both fusion proteins were cotransfected in Vero cells, a cell line from green monkey kidney. EYFP and ECFP were excited sequentially at high time rates using a monochromator. Charged coupled device (CCD)-based image acquisition resulted in 5-8 dual-color images per second, with a resolution sufficient to detect transport vesicles in mammalian cells. Under these conditions, a fully automated time-resolved analysis of the movement of color-coded objects was achieved. The development of specialized software permitted the analysis of the extent of colocalization between the two differentially labeled sets of cellular structures over time. This technical advance will provide an important tool to study the dynamic interactions of subcellular structures in living cells.
绿色荧光蛋白（GFP）光谱突变体的产生为活细胞中的多色成像奠定了基础。然而，该技术的应用受到可用GFP突变体光谱重叠和/或时间与空间分辨率不足的限制。在这里，我们使用一种新的双色成像装置，展示了以高时间分辨率对小的、快速移动的囊泡结构进行可视化。我们构建了两种GFP融合蛋白：人嗜铬粒蛋白B，一种分泌颗粒基质蛋白，以及嗜铬素，一种分泌颗粒膜蛋白。它们分别用增强型黄色荧光蛋白（EYFP）和增强型青色荧光蛋白（ECFP）进行标记。这两种融合蛋白共转染到非洲绿猴肾细胞系Vero细胞中。使用单色仪以高时间频率依次激发EYFP和ECFP。基于电荷耦合器件（CCD）的图像采集每秒可得到5 - 8张双色图像，其分辨率足以检测哺乳动物细胞中的运输囊泡。在这些条件下，实现了对颜色编码物体运动的全自动时间分辨分析。专门软件的开发使得能够分析两组不同标记的细胞结构之间随时间的共定位程度。这一技术进步将为研究活细胞中亚细胞结构的动态相互作用提供一个重要工具。
