Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy
Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy
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DOI：
10.1088/2050-6120/3/1/014006
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发表时间：
2015-03-01
影响因子：
3.2
通讯作者：
Gaus, Katharina
中科院分区：
文献类型：
--
作者：
Pandzic, Elvis;Rossy, Jeremie;Gaus, Katharina
测量质膜中的蛋白质动力学可以提供对受体信号和其他细胞功能的机制的深入了解。为了在单个分子水平上量化整个细胞表面的蛋白质动力学，已经发展了复杂的方法，如单粒子跟踪(SPT)、光激活定位显微镜(Palm)和基于涨落的分析。然而，分析高密度下具有间歇激发和低信噪比的荧光粒子的分子动力学仍然是一个挑战。我们通过将时空图像相关光谱(STICS)分析应用于光激活(PA)显微镜时间序列来克服这一问题。为了确定这种方法在什么成像条件下是有效的，我们模拟了均匀环境中扩散粒子的PA图像，以及不同的光活化、可逆闪烁和不可逆光漂白速率。此外，我们模拟了具有高粒子密度的数据，这些数据填充了经常干扰STICS和基于波动的分析的移动对象(如粘连和小泡)。我们在实验测量中证明，融合有PAGFP的表皮生长因子受体(EGFR)在活COS-7细胞中的扩散系数可以在质膜上测定，并揭示了野生型和突变型Lck在活化T细胞中的时间依赖扩散图的差异。综上所述，我们开发了一种新的基于图像相关光谱的活细胞光激活显微镜数据分析方法，以量化单个蛋白质的时空动力学。
Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.