A LARGE FOV PILLAR FORCE TRACKING SETUP WITH SELF CALIBRATION AND MINIMAL OVERSAMPLING

Xiaochun XU1, Felix Margadant1, Xian HU1,3, Bo YANG1, and Michael Sheetz1,2 1MechanoBiology Institute, National University of Singapore, Singapore 117411 2Biological Sciences, 713 Fairchild Center, Colombia University, New York 10027, USA 3Department of Biosciences, UiO Oslo, Blindernveien 31, 0371 Oslo, Norway emails: mbifmm@nus.edu.sg, xuxiaochun27@gmail.com KEY WORDS: Actin, Cell Spreading, Force Sensing, Living Cells, Localization Microscopy, MechanoBiology, Myosin Step, Oversampling, Pillar Tracking ABSTRACT: We show results from mouse embryonic fibroblast spreading assays at high frame rates in order to follow contractile forces and fast and faint myosin steps occurring in the force sensing period in focal adhesions during spreading on rigid or semi-rigid substrates. Protein observation was performed in conventional widefield microscopy at about one recording a second but pillar deflections were tracked in transmitted far red monochromatic light with near parallel illumination and up to 600 frames per second. Narrow spectral light and forgoing Kohler illumination made the pillar shape more homogenous across the field of view and hence easier and more robust to track. It also enables a proper shape detection without supersampling which allows for a sixfold area of observation over centroid localization methods. High refractive index pillar coats reduce the optical footprint of the pillar structure and reduce crosstalk between neighboring pillars. For even larger fields of view, the barrel distortion of the optics needs to be compensated if absolute deflection distances are needed, rather than just relative changes. FIGURES EQUATIONS AND REFERENCES

Figure 1: time-to-color coded 10x magnified traces (blue is the past and red is the present) of a sea-of-pillars partially covered by early spreading cells. The observation window here is 5 minutes @ 150 frames per second (left). An instant of time with 10x magnified deflection vectors is shown on the right)\. Note the opposite force vectors of contractile pairs in the right micrograph and the force free field outside of the spreading cell. [1] M. Gupta et al. “Adaptive rheology and ordering of cell cytoskeleton govern matrix rigidity sensing”. Nat. Commun. (2015) [2] B. Yang et al, “Mechanosensing Controlled Directly by Tyrosine Kinases”, Nano Lett. 16(9):5951-61 (2016) [3] H. Wolfenson et al. “Tropomyosin Controls Sarcomere-like Contractions for Rigidity Sensing and Suppressing Growth on Soft Matrices”. Nature Cell Biology, 18, 33–42 (2015)