Fluorescence imaging for monitoring the colocalization of two single molecules in living cells
Fluorescence imaging for monitoring the colocalization of two single molecules in living cells
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DOI：
10.1529/biophysj.104.048967
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发表时间：
2005-03-01
影响因子：
3.4
通讯作者：
Kusumi, A
中科院分区：
文献类型：
--
作者：
Koyama-Honda, I;Ritchie, K;Kusumi, A
活细胞中两个或多个分子的相互作用、结合和共定位是许多生物分子过程的重要方面，如果成功开发用于研究活细胞中这些过程的单分子技术，将成为非常强大的工具。在这里，我们开发了同步、双色、单色。荧光分子共定位成像，以定量检测两种单独分子的共定位。我们首先建立了一种方法，用于空间校正的两个完整的图像同步获得的两种不同的颜色，然后重叠它们的精度为13 nm。通过进一步评估位置确定的精确度以及信号/噪声和信号/背景比，我们发现对于GFP和Alexa 633，双色的两个单分子可以在细胞膜中共定位到64 - 100 nm内（68 - 90%可检测性）。在分子水平上的真正的共定位的可检测性和错误地包括偶然的方法的两个分子的共定位必须在不同的水平在每个实验中妥协，这取决于它的目的。该技术在培养的活细胞中成功地证明，监测与在质膜中扩散的GFP融合的E-钙粘蛋白的单分子与从外部添加到培养基中的与抗E-钙粘蛋白Fab缀合的Alexa 633的单分子的共定位。这项工作建立了监测两个单分子共定位的基准，这可以应用于分子相互作用的广泛研究，无论是在单分子水平还是在分子集合水平。
The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single. uorescent molecule colocalization imaging, to quantitatively detect the colocalization of two species of individual molecules. We first established a method for spatially correcting the two full images synchronously obtained in two different colors, and then for overlaying them with an accuracy of 13 nm. By further assessing the precision of the position determination, and the signal/noise and signal/ background ratios, we found that two single molecules in dual color can be colocalized to within 64 - 100 nm ( 68 - 90% detectability) in the membrane of cells for GFP and Alexa633. The detectability of true colocalization at the molecular level and the erroneous inclusion of incidental approaches of two molecules as colocalization have to be compromised at different levels in each experiment, depending on its purpose. This technique was successfully demonstrated in living cells in culture, monitoring colocalization of single molecules of E-cadherin fused with GFP diffusing in the plasma membrane with single molecules of Alexa633 conjugated to anti-E-cadherin Fab externally added to the culture medium. This work established a benchmark for monitoring the colocalization of two single molecules, which can be applied to wide ranges of studies for molecular interactions, both at the levels of single molecules and collections of molecules.