Method for live-cell super-resolution imaging of mitochondrial cristae and quantification of submitochondrial membrane potentials

Abstract The emergence of diffraction-unlimited live-cell imaging technologies has enabled the examination of mitochondrial form and function in unprecedented detail. We recently developed an approach for visualizing the inner mitochondrial membrane and determined that cristae membranes possess distinct mitochondrial membrane potentials, representing unique bioenergetic subdomains within the same organelle. Here, we outline a methodology for resolving cristae and inner boundary membranes using the LSM880 with Airyscan. Furthermore, we demonstrate how to analyze TMRE fluorescence intensity using the Nernst equation to calculate membrane potentials of individual cristae. Altogether, using these new techniques to study the electrochemical properties of the cristae can help to gain deeper insight into the still elusive nature of the mitochondrion.