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

作者：Chakkarapani, Suresh Kumar; Zhang, Peng; Kang, Seong Ho^*

来源：Analytical and Bioanalytical Chemistry, 2018, 410(5): 1551-1560.

DOI：10.1007/s00216-017-0805-9

• Multispectral plasmon
• Concurrent imaging
• Gaussian fitting
• Super-localization
• Intracellular organelles

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 similar to 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.

• 出版日期2018-2