Single-molecule experiments have changed the way we explore the physical world, yet data analysis remains time-consuming and prone to human bias. Here, we introduce Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software suite powered by deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, especially from single-molecule Förster Resonance Energy Transfer (smFRET) experiments. Deep-LASI automatically sorts recorded traces, determines FRET correction factors and classifies the state transitions of dynamic traces all in ~20-100 ms per trajectory. We benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data.
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