Quantifying cytoskeletal organization from optical microscopy data

The actin cytoskeleton plays a pivotal role in a broad range of physiological processes including directing cell shape and subcellular organization, determining cell mechanical properties, and sensing and transducing mechanical forces. The versatility of the actin cytoskeleton arises from the ability of actin filaments to assemble into higher order structures through their interaction with a vast set of regulatory proteins. Actin filaments assemble into bundles, meshes, and networks, where different combinations of these structures fulfill specific functional roles. Analyzing the organization and abundance of different actin structures from optical microscopy data provides a valuable metric for assessing cell physiological function and changes associated with disease. However, quantitative measurements of the size, abundance, orientation, and distribution of different types of actin structure remains challenging both from an experimental and image analysis perspective. In this review, we summarize image analysis methods for extracting quantitative values that can be used for characterizing the organization of actin structures and provide selected examples. We summarize the potential sample types and metric reported with different approaches as a guide for selecting an image analysis strategy.