Offsetting ROS-Mediated Arrest of Endothelial Fenestration Dynamics Permits Long-Term Optical Super-Resolution Imaging Validated by AFM.

Advances in cell biology create the demand for developing methods capable of resolving the structure and dynamics of subcellular organelles in living cells, which are beyond the reach of classical microscopy. Live-cell super-resolution fluorescence imaging provides this capability; however, in practice, its application is limited by phototoxicity, which perturbs cellular features and interferes with natural mechanisms of biological processes, providing a biased interpretation. Liver Sinusoidal Endothelial Cells (LSECs), with their nanoscale fenestrations that are physiologically critical and highly dynamic structures in the native state, represent a particularly demanding system for fluorescence-based microscopy. Here, we identify that photoactivation-generated reactive oxygen species (ROS) are the principal cause of fenestration arrest in fluorescence microscopy. By implementing three-dimensional super-resolution structured illumination microscopy (3D SR-SIM), we systematically evaluate a range of fluorophores and ROS scavengers to optimize imaging conditions. By combining BioTracker staining, carbon dioxide-independent medium supplemented with N-acetylcysteine (NAC), we preserved fenestration dynamics without altering the number/size of fenestrations. Complementary atomic force microscopy (AFM) validated that the combination of light and dye exposure impairs fenestration dynamics through ROS, in the absence of antioxidant supplementation. Additionally, AFM provides insights into the cells' nanomechanical changes upon illumination. Our findings confirm the mechanism underlying imaging-induced artifacts in LSECs observed in the literature and provide a broadly applicable framework for extending live-cell super-resolution microscopy of living cells.