Tracking Subtle Membrane Disruptions with a Tethered Photoacid

Fluorescent dyes are common tools for tracking biological membranes. In this work, we introduce a new ratiometric fluorescent probe for membranes based on a surface-sensitive photoacid. As all other probes, it lights up within membranes, and furthermore, it has different ratios of fluorescent peaks for different membranes. The main unique feature of the probe is its ability to sense events taking place in the membrane far away from its actual location. This is due to the photoacidity of the probe, and the associated kinetics of its excited-state proton release and capture that are highly sensitive to the lateral proton diffusion on the surface of the membrane. We show here that minor disruptions to the membrane integrity, induced by the Triton X-100 surfactant at low concentration (surfactant:lipid ratio of 0.02), can be sensed by our probe using steady-state fluorescence, while other probes are invisible to them. Time-resolved fluorescence reveals that the surfactant insertion affects the lateral proton diffusion and accordingly the measured proton recombination of the photoacid.