Adherent cells sense the physical properties of their environment via focal adhesions. Improved understanding of how cells sense and response to their physical surroundings is aided by quantitative evaluation of focal adhesion size, number, orientation, and distribution in conjunction with the morphology of single cells and the corresponding nuclei. We developed a fast, user-friendly and automated image analysis algorithm capable of capturing and characterizing these individual components with a high level of accuracy. We demonstrate the robustness and applicability of the algorithm by quantifying morphological changes in response to a variety of environmental changes as well as manipulations of cellular components of mechanotransductions. Finally, as a proof-of-concept we use our algorithm to quantify the effect of Rho-associated kinase inhibitor Y-27632 on focal adhesion maturation. We show that a decrease in cell contractility leads to a decrease in focal adhesion size and aspect ratio.
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