From Pixels to Patterns: A Multidimensional Framework to Decode Cytoskeletal Organization

The cytoskeleton is a dynamic filamentous network that supports essential cellular processes, from shape maintenance to cell division and migration. Advances in microscopy and computational image analysis now enable visualization and quantification of its organization with increasing precision, either as a whole structure or at an individual filament level. However, existing approaches to describe cytoskeletal architecture remain constrained by network complexity and heterogeneity in imaging and processing methods. In most studies, analysis focuses on a single organizational parameter, providing valuable but limited insights into cytoskeletal behavior and thus lacking an integrated perspective. Herein, we have compiled and analyzed the quantitative metrics reported for cytoskeletal characterization in 2-dimensional microscopy images, and organized them into a structured framework that synthesizes current methodologies. This pipeline addresses 8 complementary aspects, including morphology, orientation, quantity, compactness/density, bundling/thickness, connectivity, complexity, and interaction with cellular organelles, with each aspect representing a distinct dimension of filament structure. By classifying descriptors from diverse studies into a coherent conceptual framework, this review unveils a foundation for systematic and comparable analyses of cytoskeletal organization. Further, it can guide future investigations, supporting a more consistent interpretation of cytoskeletal organization across biological systems and experimental contexts.