Fluorescence anisotropy structured illumination microscopy for quantitative super-resolved mapping of cell microenvironment and cytoskeletal dynamics

The crowded intracellular milieu shapes organelle architecture and dynamics, yet nanoscale heterogeneity in its physicochemical properties remains difficult to visualize with conventional fluorescence anisotropy (FA) imaging. Here, we develop fluorescence anisotropy structured illumination microscopy (FA-SIM), which employs orthogonal-polarization structured illumination with dual-angle detection to achieve [~]100-nm resolution and quantitative FA retrieval with 0.56% relative error, representing over 20-fold higher accuracy than conventional FA imaging. With low phototoxicity, FA-SIM enables dual-color, hour-long quantitative super-resolution imaging in cells. Using viscosity standards, defined nanoparticles and small-molecule drug binding assays, we validate FA-SIM as a quantitative reporter of rotational mobility and molecular interactions. In cells, FA-SIM resolves nanoscale crowding heterogeneity, correlates anisotropy landscapes with condensate dynamics, and uncovers a radial crowding gradient across the microtubule network and mitotic spindle. Long-term dual-color imaging further resolves coordinated actin-microtubule remodeling and associated microenvironmental changes. By enabling quantitative, super-resolved mapping of intracellular physical properties in living systems, FA-SIM provides a powerful platform for investigating the physical regulation of cellular organization and dynamics in health and disease.