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Here we present a study of single processive myosin motors based on the combination of optical tweezers and fluorescence imaging techniques. Ultrafast force-clamp spectroscopy [1] is applied to study the dependence of the chemo-mechanical properties of myosin on load and on ATP concentration. On the other hand, single molecule localization through FIONA (Fluorescence Imaging with One Nanometer Accuracy) [2, 3] is applied to in vitro motility assay to measure parameters such as the runlength, the velocity and the step size of single myosin motors, labelled with Quantum Dots.

Recently, tremendous improvements have been achieved in the precision of localization of single fluorescent molecules, allowing localization and tracking of biomolecules at the nm level. Since the behaviour of proteins and biological molecules is tightly influenced by the cell's environment, a growing number of microscopy techniques are moving from in vitro to live cell experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). To satisfy these requirements we developed an automated routine that allow 3D tracking of single fluorescent molecules in living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.