Abstract
Quantifying small, rapidly progressing three-dimensional forces generated by cells remains a major challenge towards a more complete understanding of mechanobiology. Traction force microscopy is one of the most broadly applied force probing technologies but ascertaining three-dimensional information typically necessitates slow, multi-frame z-stack acquisition with limited sensitivity. Here, by performing traction force microscopy using fast single-frame astigmatic imaging coupled with total internal reflection fluorescence microscopy we improve the temporal resolution of three-dimensional mechanical force quantification up to 10-fold compared to its related super-resolution modalities. 2.5D astigmatic traction force microscopy (aTFM) thus enables live-cell force measurements approaching physiological sensitivity.
Highlights
Quantifying small, rapidly progressing three-dimensional forces generated by cells remains a major challenge towards a more complete understanding of mechanobiology
With the intent of combining Traction force microscopy (TFM) and astigmatic imaging, we introduced a cylindrical lens into the light path of the previously developed super-resolution total internal reflection fluorescence microscopy (TIRF)-SIM platform
Quantifying mechanical force at physiological sensitivity in living cells is vital towards understanding the functional significance of mechanobiology
Summary
Quantifying small, rapidly progressing three-dimensional forces generated by cells remains a major challenge towards a more complete understanding of mechanobiology. Recent evidence indicates that cells modulate their function downstream of signalling events triggered by external biochemical stimuli, but that cells employ a diversity of biomechanical mechanisms enabling them to dynamically adjust their mechanics to meet physiological needs[4,5,6] This forms a previously underappreciated picture wherein cells actively exert and resist biomechanical forces to tune their mechanobiology and facilitate their function[7,8]. TFM allows force measurements whereby the cell can deform the underlying substrate in all spatial dimensions, mimicking biological interactions This is in contrast to other force quantification modalities such as fluorescent-based force sensors, which while offering single-molecule sensitivity, are not able to capture the full range of biomechanical cellular interactions, for example it currently remains challenging to interpret pushing forces exerted by the cell using DNA-force sensors[17,18]. The spatial sampling density is constrained by the finite axial and lateral spatial resolution of the optical fluorescence microscope, as the finite size of the point spread function (PSF) resulting from each marker bead imposes an upper limit on the spatial density of beads that can be reliably imaged and tracked within the elastic gel[19]
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