A versatile reference-based TFM method that allows increased spatial resolution consists of coating the topmost gel layer with randomly distributed sub-diffraction-sized fluorescent marker beads. Despite the advantage of not having to record a reference frame of the gels relaxed state, the reference-free methods require specialist equipment and are also limited in their lateral spatial resolution. To accurately track the gel deformation, both reference and reference-free TFM methods are available, with the later made possible via a number of strategies, including the deposition of a structured pattern of quantum dot inks on the gel surface or two-photon laser scanning lithography 27, 28, 29, 30. ![]() To extract the mechanical force of the cell, the gel deformation is tracked with respect to the relaxed state 24. Both hydrogels and elastomers, such as polyacrylamide and polydimethylsiloxane (PDMS), respectively, are used in TFM experiments as elastic substrates with tunable stiffness and, in the case of PDMS, tunable refractive index allowing total internal reflection fluorescence (TIRF) microscopy on top of the substrate 24, 25, 26. The molecular properties of the gel surface and its bulk mechanical properties can be adjusted to provide physiological surface conditions by tuning the surface protein functionalisation and gels stiffness, respectively. In a TFM experiment, living cells are plated onto the topmost layer of a homogeneous, isotropic, elastic, thin (20–30 µm) gel substrate, deposited on a glass coverslip 13. Consequently, there is a pressing need to improve the sensitivity and biocompatibility of TFM to quantify these biomechanical forces of living cells. In addition, rapid force generation is vital for cell migration, for example, in the case of wound-healing processes 21, 22, 23. For example, in the context of immune cell activation, mechanical forces generated by immune cells have been implicated in their function, playing a role in antigen recognition and discrimination 18, 19, 20. Despite an urgent demand from the biological context, these limitations have thus far precluded TFM in its application to biological questions involving cellular force production exhibiting both sub-micron length-scales and sub-second time-scales 17. Nevertheless, the sensitivity and biocompatibility of TFM have remained dependent on the individually chosen imaging modality, highlighting the difficulty of overcoming the trade-off between spatial resolution, temporal resolution, and acquisition duration 14, 15, 16. Efforts aiming to approach physiological sensitivity have led to combinations of TFM and different optical microscopy modalities with extended spatial and temporal resolution 11, 12. Despite its importance and proven biological significance, quantifying the dynamic process of force production in living cells via TFM remains challenging due to technical constraints. Traction force microscopy (TFM) has become one of the most commonly applied force probing methodologies 10, 11, 12, primarily due to its adaptability in modelling biological and mechanical properties, ease of implementation, and reliance on widely available materials and fluorescence microscopy 13. Quantifying mechanical forces within the context of fast nanoscale dynamics of cortical actin architectures has therefore become an important step in understanding the mechanobiology of living cells. Cells are thus able to adjust their biomechanics in order to meet their physiological needs 5, 8, 9. ![]() The strength of the forces generated by cells evolve in time, as does a cell’s sensitivity to changes in the magnitude, frequency and duration of the surrounding biomechanical stimuli 4. The ability of cells to generate and respond to these mechanical cues are primarily processed by the actin cytoskeleton, a key determinant of cellular biomechanics, that comprises dynamic actin architectures undergoing continuous re-arrangements and turnover 3, 6, 7. New insights in the field of mechanobiology have revealed that cellular physiology is directly influenced by the mechanical properties and forces generated within the cellular environment 3, 4, 5. Although the biological effects of biomechanics have perhaps always been most evident in the context of the physical behaviour of cells, it has become apparent that mechanical forces can direct the function of cells in many biological contexts in health and disease. Mechanical forces guide the function of living cells 1, 2.
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