To unravel dynamic processes underpinning key functions in cell biology, it is essential to develop imaging technologies able to track the movement of individual bio-nano-objects under physiologically relevant conditions, hence at high speed (ms) and in 3D. We demonstrate interferometric gated off-axis reflectometry (iGOR) which detects the back-scattered light of the structure of interest using an external off-axis reference, enabling label-free high-speed tracking of nanoparticles and suspended membranes in 3D volumes. Employing coherence time-gating by femtosecond pulses, the axial extension of the detected volume is controlled. We show tracking of single nanoparticles down to 10nm size freely diffusing in volume, which allows us to determine their geometrical and hydrodynamic radius as well as non-sphericity. We also show the spatiotemporal dynamics of suspended lipid bilayers, and the influence of lipid phase transitions on these dynamics with sub-nm thickness precision.
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