Quantifying 3D live-cell membrane dynamics using dynamic metal-induced energy transfer spectroscopy (dynaMIET).

The dynamic behavior of cellular membranes underpins essential biological processes, including signal transduction, intracellular trafficking, and mechanotransduction. However, simultaneously quantifying lateral molecular diffusion and vertical membrane fluctuations in live cells remains challenging. Here, we present dynamic metal-induced energy transfer spectroscopy (dynaMIET), which integrates metal-induced energy transfer with fluorescence correlation spectroscopy to resolve three-dimensional membrane dynamics with nanometer axial sensitivity and microsecond temporal resolution. dynaMIET enables concurrent measurement of lateral diffusion and vertical undulations within a single acquisition. We validate the method using simulations and model membranes and demonstrate its robustness in living cells, applying it to the plasma membrane, endoplasmic reticulum, and nuclear envelope. By capturing both molecular mobility and membrane fluctuations, dynaMIET provides a powerful, noninvasive tool for probing membrane mechanics and organization. This advance opens avenues for studying membrane-associated phenomena in health and disease, including cancer cell mechanics, protein-membrane interactions, and organelle dynamics.