QUANTIFYING PHOTOINDUCED MEMBRANE DAMAGE IN GIANT UNILAMELLAR VESICLES: FROM LIPID COMPOSITION TO IMAGE-BASED ANALYSIS

Understanding how membranes respond to oxidative and photoinduced stress is central to a wide range of biological and biophysical processes, from cell damage to the action of photosensitizing agents. In this study, we investigate photodamage to giant unilamellar vesicle (GUV) membranes induced by photoirradiation of methylene blue in solution. GUVs composed of different lipid mixtures, including POPC and POPC–cholesterol membranes, are used to assess how lipid composition modulates membrane susceptibility to photoinduced damage. Using optical videomicroscopy, we follow the evolution of membrane perturbations over time, focusing on morphological changes such as vesicle deformation, fluctuations, contrast loss, and membrane destabilization. These observables provide direct and quantitative signatures of membrane integrity loss, allowing us to track the progression of damage under controlled irradiation conditions. To quantitatively describe these processes, we developed a computational image analysis pipeline for automated detection and tracking of GUV contours. This approach enables measurements of vesicle area, shape variations, and time-dependent morphological descriptors, as well as the quantification of contrast decay as a proxy for membrane permeability and structural disruption. By comparing vesicles with distinct lipid compositions, we evaluate how membrane organization influences the kinetics and extent of photodamage. The results indicate that the interplay between methylene blue photoactivation and membrane composition governs the pathways of vesicle destabilization, highlighting the role of the lipid environment in modulating oxidative membrane processes. Beyond the specific system studied here, this experimental–computational approach provides a controlled benchmark for the development of quantitative analysis tools. The image-based pipeline is readily extendable to other membrane–agent interactions, including those involving membrane-active molecules and proteins, establishing a versatile platform for studying membrane dynamics under external perturbations.