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Probing the mechanical properties of phospholipid membranes is a fundamental characterization step for biomimetic membrane systems as well as for living cellular systems. A common method relies on the analysis of thermal membrane fluctuations, which has been implemented in video flicker spectroscopy. Here we present a new optical method that directly measures the dynamics of membrane fluctuations with nm and μs resolution, thus providing access to the bending modulus κ and the membrane tension σ for measurement times of 10 s. Our method allows the observation of bilayer membrane fluctuations of liposomes and the calculation of the power spectral density in yet unreported regimes of frequencies >50 Hz and amplitudes <10 nm. The recorded data are in agreement with the Helfrich membrane theory over 4 decades of frequencies (0.1 Hz–1000 Hz). However, we find a systematic overestimation of the buffer viscosity, which can not be simply explained by measurement errors, but unveils an effect that is not explained by the classical theory for membrane dynamics, and hence suggests that new physics must be developed in the observed frequency and amplitude range. The experimental method is easily reproducible on any optical tweezer setup equipped with a quadrant photodiode position detection sensor.
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