B CUBE-Center of Innovation Competence, Technische Universität Dresden, Dresden, Germany.
Biophys J. 2011 Jun 8;100(11):2820-8. doi: 10.1016/j.bpj.2011.04.023.
Recent developments in image processing have greatly advanced our understanding of biomolecular processes in vitro and in vivo. In particular, using Gaussian models to fit the intensity profiles of nanometer-sized objects have enabled their two-dimensional localization with a precision in the one-nanometer range. Here, we present an algorithm to precisely localize curved filaments whose structures are characterized by subresolution diameters and micrometer lengths. Using surface-immobilized microtubules, fluorescently labeled with rhodamine, we demonstrate positional precisions of ∼2 nm when determining the filament centerline and ∼9 nm when localizing the filament tips. Combined with state-of-the-art single particle tracking we apply the algorithm 1), to motor-proteins stepping on immobilized microtubules, 2), to depolymerizing microtubules, and 3), to microtubules gliding over motor-coated surfaces.
图像处理的最新进展极大地促进了我们对体外和体内生物分子过程的理解。特别是，使用高斯模型来拟合纳米级物体的强度分布，可以将其二维定位精度提高到纳米级。在这里，我们提出了一种算法，可以精确地定位具有亚分辨率直径和微米长度的弯曲细丝的结构。使用表面固定的微管，用罗丹明标记荧光，我们在确定纤维中心线时的位置精度约为 2nm，在定位纤维尖端时的位置精度约为 9nm。结合最先进的单颗粒跟踪技术，我们将该算法应用于 1）固定在微管上的运动蛋白的步进，2）微管的解聚，3）微管在运动蛋白覆盖的表面上的滑行。
