Stiffening cells with light

Fluorescence microscopy is commonly used to observe living cells and organisms, but it can induce phototoxicity, which may lead to erroneous interpretations. The primary cause of phototoxic cell damage is the production of reactive oxygen species (ROS), which can crosslink intracellular proteins and nucleic acids. Using profile microindentation and atomic force microscopy, we demonstrate that excitation of various fluorescent probes causes a rapid dose-dependent increase in cell stiffness across multiple cell types. This photostiffening effect explains why T cells loaded with the Fluo-4 calcium probe stop protruding within seconds after light excitation. We show that upon fluorophore excitation, ROS production is correlated with increased stiffness, and we reproduce the effect by incubating cells with H₂O₂ or the photosensitizer pheophorbide a . This study underscores the importance of controls and proposes photostiffening as a method for quantifying phototoxicity.