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Cell stability and motility depends on a complex dynamic cytoplasmic scaffolding called the cytoskeleton. It is composed of actin filaments, intermediate filaments and microtubules, and interacts with neighbouring cells and the extracellular matrix via specialized adhesion sites - multimolecular complexes responsible for the transmission of mechanical force and regulatory signals. The dynamic behaviour of these subcellular structures in living cells can be analysed by fluorescence microscopy yielding series of 2D or 3D images. Towards a quantitative analysis, we present methods for the segmentation and motion estimation of cytoskeletal filaments as well as for the tracking of adhesion sites, allowing the quantification of cytoskeletal dynamics under different conditions.