Fast and Long‐Term Super‐Resolution Imaging of Endoplasmic Reticulum Nano‐structural Dynamics in Living Cells Using a Neural Network

Stimulated emission depletion (STED) microscopy is a super‐resolution technique that surpasses the diffraction limit and has contributed to the study of dynamic processes in living cells. However, high laser intensities induce fluorophore photobleaching and sample phototoxicity, limiting the number of fluorescence images obtainable from a living cell. Herein, these challenges are addressed by using ultra‐low irradiation intensities and a neural network for image restoration, enabling extensive imaging of single living cells. The endoplasmic reticulum (ER) is chosen as the target structure due to its dynamic nature over short and long timescales. The reduced irradiation intensity combined with denoising permits continuous ER dynamics observation in living cells for up to 7 h with a temporal resolution of seconds. This allows for quantitative analysis of ER structural features over short (seconds) and long (hours) timescales within the same cell, and enabled fast 3D live‐cell STED microscopy. Overall, the combination of ultralow irradiation with image restoration enables comprehensive analysis of organelle dynamics over extended periods in living cells. Photobleaching and phototoxicity limit the observation time of living cells with super‐resolution fluorescence microscopy. Herein, the authors use low‐illumination microscopy and neural network assisted denoising to achieve fast and long‐term stimulated emission depletion microscopy in living cells. Imaging of the endoplasmic reticulum in live cells at sub‐100 nm spatial resolution and and ≈4 s time resolution over 7 h is demonstrated.