Pandžić Elvis, Rossy Jérémie, Gaus Katharina
ARC Centre for Advanced Molecular Imaging, Australian Centre for NanoMedicine University of New South Wales Australia, Sydney, NSW, Australia.
Lowy Cancer Research Centre, Centre for Vascular Research Level 3, Kensington, NSW, Australia.
Methods Appl Fluoresc. 2015 Mar 9;3(1):014006. doi: 10.1088/2050-6120/3/1/014006.
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.
测量质膜中的蛋白质动力学可以深入了解受体信号传导机制和其他细胞功能。为了在整个细胞表面的单分子水平上量化蛋白质动力学，已经开发了诸如单粒子追踪（SPT）、光激活定位显微镜（PALM）和基于涨落的分析等复杂方法。然而，分析在高密度下存在的间歇性激发和低信噪比的荧光颗粒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）显微镜时间序列克服了这个问题。为了确定该方法在哪些成像条件下有效，我们模拟了均匀环境中扩散颗粒的PA图像，并改变了光激活、可逆闪烁和不可逆光漂白速率。此外，我们模拟了具有高密度颗粒的数据，这些颗粒构成了经常干扰STICS和基于涨落分析的移动物体（如黏附物和囊泡）。我们在实验测量中证明，在活COS-7细胞中，与PAGFP融合的表皮生长因子受体（EGFR）的扩散系数可以在质膜中测定，并揭示了活化T细胞中野生型和突变型Lck之间时间依赖性扩散图谱的差异。总之，我们基于图像相关光谱开发了一种用于活细胞光激活显微镜数据的新分析方法，以量化单个蛋白质的时空动力学。
