STED and parallelized RESOLFT optical nanoscopy of the tubular endoplasmic reticulum and its mitochondrial contacts in neuronal cells

Abstract

The classic view of organelle cell biology is undergoing a constant revision fueled by the new insights unraveled by fluorescence nanoscopy, which enable sensitive, faster and gentler observation of specific proteins in situ. The endoplasmic reticulum (ER) is one of the most challenging structure to capture due the rapid and constant restructuring of fine sheets and tubules across the full 3D cell volume. Here we apply STED and parallelized 2D and 3D RESOLFT live imaging to uncover the tubular ER organization in the fine processes of neuronal cells with focus on mitochondria-ER contacts, which recently gained medical attention due to their role in neurodegeneration. Multi-color STED nanoscopy enables the simultaneous visualization of small transversal ER tubules crossing and constricting mitochondria all along axons and dendrites.Parallelized RESOLFT allows for dynamic studies of multiple contact sites within seconds and minutes with prolonged time-lapse imaging at ~50 nm spatial resolution. When operated in 3D super resolution mode it enables a new isotropic visualization of such contacts extending our understanding of the three-dimensional architecture of these packed structures in axons and dendrites.

Keywords

• Endoplasmic reticulum
• Mitochondria-ER contacts
• Super resolution microscopy
• RESOLFT
• STED