The eukaryotic cytoskeleton consists of three different types of biopolymers - microtubules, actin filaments, and intermediate filaments - and provides cells with versatile mechanical properties, combining stability and flexibility. The unique molecular structure of intermediate filaments leads to high extensibility and stability under load. With high laser power dual optical tweezers, the mechanical properties of intermediate filaments may be investigated, while monitoring the extension with fluorescence microscopy. Here, we provide detailed protocols for the preparation of single vimentin intermediate filaments and general measurement protocols for (i) stretching experiments, (ii) repeated loading and relaxation cycles, and (iii) force-clamp experiments. We describe methods for the analysis of the experimental data in combination with computational modeling approaches.
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