Biological systems can be quantitatively explored using single-molecule manipulation techniques such as optical or magnetic tweezers or atomic force microscopy. Though a plethora of discoveries have been accomplished using single-molecule manipulation techniques in vitro, such investigations constantly face the criticism that conditions are too far from being physiologically relevant. Technical achievements now allow scientists to take the next step: to use single-molecule manipulation techniques quantitatively in vivo. Considerable progress has been accomplished in this realm; for example, the interaction between a protein and the membrane of a living cell has been probed, the mechanical properties of individual proteins central for cellular adhesion have been measured and even the action of molecular motors in living cells has been quantified. Here, we review the progress of in vivo single-molecule manipulation with a focus on the special challenges posed by in vivo conditions and how these can be overcome.
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