Detection of liposome membrane viscosity perturbations with ratiometric molecular rotors

► Introduction of an engineered, self-calibrating, fluorescent viscosity sensor. ► Demonstration of its efficacy in viscosity gradients and in liposomes. ► The sensor allows quantitative measurement of membrane fluidity changes. ► Due to the ratiometric nature, the sensor allows to compensate for local dye concentration and fluid turbidity. ► We propose the sensor as a low-cost alternative to lifetime measurements for steady-state instrumentation and epifluorescent microscopes.