A fluorescence-free real-time three-dimensional (3D) super-localization method for the analysis of 3D structure of organelles (e.g.,
mitochondria-associated endoplasm reticulum [mito-ER] contacts) in live single cells under physiological conditions was developed
with dual-wavelength enhanced dark-field microscopy. The method was applied to live single cells under physiological conditions to
analyze the complex 3D mito-ER contact region by choosing an optimum nanotag with distinct scattering properties. Combining
dual-view with enhanced dark-field microscopy provided concurrent images of different scattering wavelengths of nanotag-labeled
mitochondria and ER. The reconstructed super-localized images resolved controversy over the distance between the intracellular
organelles at functional contacts. The distance between mitochondria and ER was measured to be 45 nm, which was ~ 50% greater
than in a previous report using electron microscopic tomography, and was a better fit for the likely features of these structures. These
results indicate that this method was a reliable and convenient approach for investigating the 3D structure of organelles, such as mitoER contacts in live single cells, and provided accurate information under physiological conditions.