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Abstract

Single-molecule localization microscopy achieves nanometer spatial resolution by localizing single fluorophores separated in space and time. A major challenge of single-molecule localization microscopy is the long acquisition time, leading to low throughput, as well as to a poor temporal resolution that limits its use to visualize the dynamics of cellular structures in live cells. Another challenge is photobleaching, which reduces information density over time and limits throughput and the available observation time in live-cell applications. To address both challenges, we combine two concepts: first, we integrate the neural network DeepSTORM to predict super-resolution images from high-density imaging data, which increases acquisition speed. Second, we employ a direct protein label, HaloTag7, in combination with exchangeable ligands (xHTLs), for fluorescence labeling. This labeling method bypasses photobleaching by providing a constant signal over time and is compatible with live-cell imaging. The combination of both a neural network and a weak-affinity protein label reduced the acquisition time up to ∼25-fold. Furthermore, we demonstrate live-cell imaging with increased temporal resolution, and capture the dynamics of the endoplasmic reticulum over extended time without signal loss.