Within cells, functional changes in mitochondrial metabolic state are associated with alterations in organelle mobility, shape, and configuration of the mitochondrial matrix. Fluorescence correlation spectroscopy (FCS) is a technique that measures intensity fluctuations caused by single fluorescent molecules moving through a small detection volume. By mathematically correlating these fluctuations, information can be obtained about the concentration and rate of diffusion of the fluorescent molecules. Here we present an FCS-based approach for determining the mobility of enhanced yellow fluorescent protein (mitoEYFP) in the mitochondrial matrix of primary human skin fibroblasts. This protocol allows simultaneous quantification of intramatrix EYFP concentration and its diffusion constant, as well as the fraction of moving mitochondria and their velocity.
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