Extended super-resolution three-dimensional mechanical probing in living cells.

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New perspective of mechanobiology is currently emerging across multiple disciplines in the physical and biomedical research fields. In contrast to conventional beliefs, recent evidence indicates that cells regulate their cell mechanics not only downstream of signalling events triggered by external stimuli, but that cells employ a diversity of feedback mechanisms of their cytoskeleton enabling them to dynamically adjust cell mechanics to meet physiological needs. Consequently, this provides a previously unforeseen picture wherein cells actively exert and resist biomechanical force to tune their mechanobiology, and thus facilitate their function. Quantifying cellular forces has therefore become an important mission across multiple disciplines at the interface of bioengineering and biomedical sciences. The overall goal of this project is the development of a new-generation force probing methodology to enable physiological mechanical probing in living cells at unprecedented accuracy and resolution. To engineer this technology, we will combine a new technique of state-of-the-art 3D high-speed total-internal-reflection (TIRF) and cutting-edge super-resolution structural-illumination (SIM) microscopy coupled with extended mechanical force TFM probing to create a novel breakthrough technology for mechanical probing in living cells. We will demonstrate the power of the new methodology by quantifying mechanical force production in a variety of adherent cells and activating immune T cells. We anticipate that our research might lead to the replacement of conventional TFM measurements with major implications for the understanding of the cellular mechanobiology. Ultimately, we therefore aim to commercialise the 3D eTIRF-SIM-TFM method.