3D super-localization of intracellular organelle contacts at live single cell by dual-wavelength synchronized fluorescence-free imaging.

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 mito-ER contacts in live single cells, and provided accurate information under physiological conditions. Graphical abstract Fluorescence-free enhanced dark-field 3D super-resolution microscopy (3D SRM) method, with dual-wavelength simultaneous imaging (DWSI) for 3D analysis of mitochondria-endoplasmic reticulum (Mito-ER) at their functional contact site.