Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, United States.
Elife. 2018 Jan 11;7:e32418. doi: 10.7554/eLife.32418.
Two-color fluorescence co-localization in 3D (three-dimension) has the potential to achieve accurate measurements at the nanometer length scale. Here, we optimized a 3D fluorescence co-localization method that uses mean values for chromatic aberration correction to yield the mean separation with ~10 nm accuracy between green and red fluorescently labeled protein epitopes within single human kinetochores. Accuracy depended critically on achieving small standard deviations in fluorescence centroid determination, chromatic aberration across the measurement field, and coverslip thickness. Computer simulations showed that large standard deviations in these parameters significantly increase 3D measurements from their true values. Our 3D results show that at metaphase, the protein linkage between CENP-A within the inner kinetochore and the microtubule-binding domain of the Ndc80 complex within the outer kinetochore is on average ~90 nm. The Ndc80 complex appears fully extended at metaphase and exhibits the same subunit structure in vivo as found in vitro by crystallography.
双色荧光共定位在 3D（三维）中具有实现纳米级精确测量的潜力。在这里，我们优化了一种 3D 荧光共定位方法，该方法使用平均值进行色差校正，以实现绿色和红色荧光标记的蛋白质表位在单个人类动粒内的平均分离，精度约为 10nm。准确性取决于在荧光质心确定、测量场中的色差和盖玻片厚度方面实现小的标准偏差。计算机模拟表明，这些参数的大标准偏差会大大增加 3D 测量值与其真实值之间的偏差。我们的 3D 结果表明，在中期，内动粒内的 CENP-A 与外动粒内的微管结合域之间的蛋白质连接平均约为 90nm。Ndc80 复合物在中期完全伸展，并表现出与体外结晶学发现的相同的亚基结构。
