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

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.

Biomedical sciences increasingly recognise the importance of mechanobiology for human health. Consequently to-date, most groups at the fore-front of their disciplines quantify mechanical force production in living cells on a daily basis - knowing that state-of-the-art force probing remains limited in its sensitivity. The central aim of the proposed project is to engineer and disseminate a new generation of mechanical force probing owning the technical potential to transform the landscape of the biomedical community in the years to come. Therefore, this project will have impact on academic biophysical and biomedical sciences, as well as on the private sectors, and thus ultimately on the public.

Being awarded an EPSRC New Investigator Award would allow us to explore and validate the methodology of 3D TIRF-SIM-TFM. Positive outcome of this project will enable our colleagues across multiple disciplines to effectively rebuild the system at a comparatively low cost, while in principle also allowing large scale manufacture to be performed. These are crucial considerations as, at present, our previously-developed extended force probing technology has attracted considerable interest (see also Case of support: Academic Beneficiaries). We are in the ideal position to further bring this approach to fruition, not only because of the commitment of our Research Co-investigator Dr. Huw Colin-York to this project with whom we established for-the-first-time the combination of force probing and super-resolution microscopy, but also because of the promising preliminary results using cutting-edge super-resolution microscopy. Investment in this new generation force probing technology is crucial because there are uncertainties regarding, for example, the effects of the fluorescence imaging on the sensitivity of the method, as well as the chemistry of the hydrogels, which are acting to limit the popularity of force techniques. Successful completion of the project will in turn boost the competitiveness of the method and open the door for the realistic commercial production of the methodology in collaboration with either established microscopy companies or supported by the local startup Innovation Initiative of the University of Oxford.

Academic Impacts

This research project will also impact on capacity building within the field of physical and biomedical sciences. The project involves the highly-skilled researchers from different disciplines, which will provide an excellent research environment for young researcher such as graduate or master students. The proposed research will expose them to an innovative and unique training programme involving the engineering of the new force probing technology, cell-biology, and the best cellular imaging and biophysical technology available to date.

Research of the University of Oxford will benefit in general from this project, since it represents another highly interdisciplinary research project, strengthening the links between different scientific disciplines and departments.

Societal impacts

Long term, we aim to inform industrial and academic research that target the mechanobiology of cells. Application, following the dissemination of the technical capabilities of the method, will contribute to the understanding of academics and the public and provide broad mechanistic insights underlying cellular events by which cells adjust their mechanobiology for instance during immune responses. To this end, we plan a number of complementary actions including seminars with academics and relevant companies active in the Oxford area and beyond. Towards the end of the project, commercial microscope companies such as JPK, Leica, or Zeiss could be contacted with concrete results to show a proof of concept arrangement of 3D TIRF-SIM-TFM.