Abstract
Out-of-plane fluctuations, also known as stochastic displacements, of biological membranes play a crucial role in regulating many essential life processes within cells and organelles. Despite the availability of various methods for quantifying membrane dynamics, accurately quantifying complex membrane systems with rapid and tiny fluctuations, such as mitochondria, remains a challenge. In this work, we present a methodology that combines metal/graphene-induced energy transfer (MIET/GIET) with fluorescence correlation spectroscopy (FCS) to quantify out-of-plane fluctuations of membranes with simultaneous spatiotemporal resolution of approximately one nanometer and one microsecond. To validate the technique and spatiotemporal resolution, we measure bending undulations of model membranes. Furthermore, we demonstrate the versatility and applicability of MIET/GIET-FCS for studying diverse membrane systems, including the widely studied fluctuating membrane system of human red blood cells, as well as two unexplored membrane systems with tiny fluctuations, a pore-spanning membrane, and mitochondrial inner/outer membranes.