Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy
Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy
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DOI：
10.1016/s1046-2023(02)00305-5
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发表时间：
2003-02-01
期刊：
影响因子：
4.8
通讯作者：
Molloy, JE
中科院分区：
文献类型：
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作者：
Mashanov, GI;Tacon, D;Molloy, JE
在过去的10年里，激光和探测器技术的进步使得在水溶液中可以看到单个荧光团。在这里，我们描述了基于全内反射荧光显微镜（TIRFM）的方法，我们已经开发出研究活体哺乳动物细胞内单个蛋白质分子的行为。我们用培养的成肌细胞瞬时转染编码目标蛋白的DNA质粒与绿色荧光蛋白（GFP）融合。通过对照稀释GFP的共聚焦图像定量表达水平，然后使用TIRFM检测含有1-100 nM GFP的细胞。用完全内反射的氩离子激光束照射玻璃-水界面的浅区域（50100nm深），产生倏逝场。单个gfp标记的蛋白质进入消失场后，出现单个的、衍射有限的光点，可以从背景荧光中清楚地分辨出来。结合在基细胞膜上的分子在位置上保持固定数秒，而那些在细胞质中自由扩散的分子在几毫秒内消失。我们开发了自动检测和跟踪方法来识别和表征录制视频序列中的单个分子的行为。这使我们能够测量光漂白动力学和膜结合分子的横向扩散。(C) 2003 Elsevier Science（美国）版权所有。
Over the past 10 years, advances in laser and detector technologies have enabled single fluorophores to be visualized in aqueous solution. Here, we describe methods based on total internal reflection fluorescence microscopy (TIRFM) that we have developed to study the behavior of individual protein molecules within living mammalian cells. We have used cultured myoblasts that were transiently transfected with DNA plasmids encoding a target protein fused to green fluorescent protein (GFP). Expression levels were quantified from confocal images of control dilutions of GFP and cells with 1-100 nM GFP were then examined using TIRFM. An evanescent field was produced by a totally internally reflected, argon ion laser beam that illuminated a shallow region (50100 nm deep) at the glass-water interface. Individual GFP-tagged proteins that entered the evanescent field appeared as individual, diffraction-limited spots of light, which were clearly resolved from background fluorescence. Molecules that bound to the basal cell membrane remained fixed in position for many seconds, whereas those diffusing freely in the cytoplasm disappeared within a few milliseconds. We developed automated detection and tracking methods to recognize and characterize the behavior of single molecules in recorded video sequences. This enabled us to measure the kinetics of photobleaching and lateral diffusion of membrane-bound molecules. (C) 2003 Elsevier Science (USA). All rights reserved.