Tracking single particles and elongated filaments with nanometer precision

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.