Sample datasets of microscopy images were used to illustrate the analysis and are provided as Supplementary Material . In particular, cultured fibroblasts transfected with GFP-actin, fibroblasts fixed and stained for filamentous actin (F-actin, phalloidin), fixed cell-derived matrices immunostained for actin (before decellularisation) and fibronectin (after decellularisation), and second harmonic generation imaging of tissue samples were utilised. Fixed actin images were acquired at different magnifications using either single cells or cells at confluence, to allow for evaluation of the length scale of alignment. Y-27632 treated cells were incubated in the indicated concentration of the drug for 30 minutes and then fixed and stained with phalloidin.
Fixed samples were imaged using a Zeiss LSM 880 equipped with a 40x NA 1.3 Plan-Apochromat oil objective or 63x NA 1.4 Plan-Apochromat oil objective. Decellularised matrices were imaged using a Zeiss LSM 880 equipped with 63x NA 1.4 Plan-Apochromat oil. For second harmonic generation imaging, tissue sections were imaged using a Zeiss LSM 7MP equipped with a 20x NA 1.0 water immersion objective. Y-27632 treated cells were imaged on a 3i Marianas Imaging System consisting of a Zeiss Axio Observer 7 inverted microscope attached to a Yokogawa W1 Confocal Spinning Disk using a 63x NA 1.4 Plan-Apochromat oil objective.
Fixed samples were imaged using a Zeiss LSM 880 equipped with a 40x NA 1.3 Plan-Apochromat oil objective or 63x NA 1.4 Plan-Apochromat oil objective. Decellularised matrices were imaged using a Zeiss LSM 880 equipped with 63x NA 1.4 Plan-Apochromat oil. For second harmonic generation imaging, tissue sections were imaged using a Zeiss LSM 7MP equipped with a 20x NA 1.0 water immersion objective. Y-27632 treated cells were imaged on a 3i Marianas Imaging System consisting of a Zeiss Axio Observer 7 inverted microscope attached to a Yokogawa W1 Confocal Spinning Disk using a 63x NA 1.4 Plan-Apochromat oil objective.