Ellenberg J, Lippincott-Schwartz J, Presley J F
National Institute for Child Health and Human Development, NIH, Bethesda, MD, USA.
Biotechniques. 1998 Nov;25(5):838-42, 844-6. doi: 10.2144/98255bt01.
The Aequorea victoria green fluorescent protein (GFP) is widely recognized as a powerful tool in cell biology, serving as a vital reporter for monitoring localization and dynamics of intracellular proteins and organelles over time. GFP variants with shifted spectral characteristics have been described and offer enormous potential for double-labeling experiments and protein-protein interaction studies. However, most GFP variant combinations are not suitable for double-label, time-lapse imaging experiments because of either extremely rapid photobleaching of blue-shifted GFP variants or crossover of their excitation and emission spectra, which must then be computer corrected. Here, we describe the successful use of two photostable spectral GFP variants, W7 and 10C, in dual-color, time-lapse imaging of fusion proteins in living cells using either wide-field or confocal microscopy. W7 and 10C were highly photostable during repetitive long-term imaging and were cleanly separated by their different excitation spectra alone with negligible crossover of fluorescence. We present time-lapse image sequences of COS-7 cells co-expressing both a marker of the Golgi complex (galactosyl transferase) fused to W7 and a marker of the nuclear envelope (lamin-B receptor) fused to 10C. To our knowledge, these image sequences provide the first simultaneous visualization of Golgi and nuclear envelope membranes in living cells.
维多利亚多管水母绿色荧光蛋白（GFP）在细胞生物学中被广泛认为是一种强大的工具，作为一种重要的报告分子，用于监测细胞内蛋白质和细胞器随时间的定位和动态变化。已经描述了具有不同光谱特征的GFP变体，它们在双标记实验和蛋白质-蛋白质相互作用研究中具有巨大潜力。然而，大多数GFP变体组合不适合用于双标记、延时成像实验，这是因为蓝移GFP变体的光漂白速度极快，或者它们的激发光谱和发射光谱存在交叉，之后必须进行计算机校正。在此，我们描述了两种光稳定的光谱GFP变体W7和10C在活细胞中融合蛋白的双色延时成像中的成功应用，该成像使用宽场显微镜或共聚焦显微镜。在重复的长期成像过程中，W7和10C具有高度的光稳定性，仅通过它们不同的激发光谱就能清晰分离，荧光交叉可忽略不计。我们展示了共表达与W7融合的高尔基体复合物标记物（半乳糖基转移酶）和与10C融合的核膜标记物（核纤层蛋白B受体）的COS-7细胞的延时图像序列。据我们所知，这些图像序列首次实现了对活细胞中高尔基体和核膜的同时可视化。
