SIM imaging of bacterial membrane dynamics and lipid peroxidation during photodynamic inactivation with a dual-functional activatable probe

Photodynamic inactivation (PDI) has emerged as a promising antibacterial strategy that mitigates the risk of bacterial resistance. However, the precise morphological dynamics and mechanisms underlying bacterial cell death during PDI remain insufficiently understood. In this study, we developed a dual-functional activatable probe, RDP, which integrated rhodamine B as a fluorophore with moderate brightness and fatty chains for selective bacterial membrane localization. The probe employed an aggregation-disaggregation mechanism to achieve both fluorescence activation and PDI functionality. Using super-resolution fluorescence imaging, we unveiled the selective rupture of bacterial membranes at specific sites during PDI, followed by membrane contraction and internalization, ultimately leading to the formation of lipid-enriched droplets within the bacteria. Further mechanistic investigations confirmed that this membrane rupture was driven by lipid peroxidation. Statistical analysis of bacterial morphological changes during PDI revealed that membrane rupture predominantly occurred at the septum during cell division, whereas in other growth phases, rupture sites were mainly localized at the poles. These findings provide critical insights into the role of selective membrane rupture in bacterial growth and viability, paving the way for the rational design of targeted and highly efficient antibacterial agents.