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Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations in biological samples, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the diffraction-limited three-dimensional distribution of the orientations and positions of ensembles of fluorescent dipoles that label biological structures. We share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model the distributions based on the polarization-dependent efficiency of excitation and detection of emitted fluorescence, using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labeled giant unilamellar vesicles, fast-scarlet-labeled cellulose in xylem cells, and phalloidin-labeled actin in U2OS cells. Additionally, we observe phalloidin-labeled actin in mouse fibroblasts grown on grids of labeled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales. 

Specificity of cellular responses to distinct cues from the ECM requires precise and sensitive decoding of physical information. However, how known mechanisms of mechanosensing like force-dependent catch bonds and conformational changes in FA proteins can confer that this sensitivity is not known. Using polarization microscopy and computational modeling, we identify dynamic changes in an orientational order of FA proteins as a molecular organizational mechanism that can fine-tune cell sensitivity to the ECM. We find that αV integrins and F-actin show precise changes in the orientational order in an ECM-mediated integrin activation-dependent manner. These changes are sensitive to ECM density and are regulated independent of myosin-II activity though contractility can enhance this sensitivity. A molecular-clutch model demonstrates that the orientational order of integrin–ECM binding coupled to directional catch bonds can capture cellular responses to changes in ECM density. This mechanism also captures decoupling of ECM density sensing from stiffness sensing thus elucidating specificity. Taken together, our results suggest relative geometric organization of FA molecules as an important molecular architectural feature and regulator of mechanotransduction.