Fluorescence imaging-induced phospholipid oxidation drives membrane phase separation by shifting miscibility boundaries
Fluorescence microscopy is a widely used tool for visualizing membrane organization in model and cellular systems; however, photoexcitation during imaging can generate reactive oxygen species, thereby altering membrane structure. Here, we show that fluorescence imaging actively drives membrane phase separation through photo-induced phospholipid oxidation. Using giant unilamellar vesicles, we observed that illumination of initially homogeneous membranes triggers the emergence of coexisting liquid-ordered and liquid-disordered domains. Chemical analysis by thin layer chromatography and mass spectrometry revealed the formation of a complex mixture of oxidized lipid species following excitation, characterized by stepwise oxygen additions to unsaturated phospholipids and multiple degradation products. These modifications increase lipid polarity and disrupt acyl chain packing, reducing favorable interactions with saturated phospholipids. As a result, membranes near the miscibility boundary become prone to demixing upon illumination, indicating that photo-induced lipid oxidation effectively shifts the miscibility boundary. Consistent with this model, light-induced phase separation is highly sensitive to membrane composition, with maximal effects observed near the liquid-liquid miscibility boundary. Electroformation substrate further modulated this effect, suggesting that pre-existing oxidative conditions sensitize membranes to subsequent photo-induced changes. Together, these findings demonstrate that excitation light can introduce artifacts in membrane phase behaviour. More broadly, subtle chemical modifications of phospholipids can shift membrane miscibility and reorganize membrane structure.