A lightweight deep learning framework for fast, real-time super-resolution fluctuation imaging

Capturing dynamic cellular processes in live cells requires fast imaging with very high resolution beyond the diffraction limit. Fluctuation-based super-resolution techniques overcome this limit by exploiting correlations in fluorescence blinking, but they typically require hundreds of frames and computationally intensive post-processing, prohibiting real-time imaging of fast cellular events. Recent deep learning approaches aim to increase temporal resolution; however, many rely on extensive pre-processing or large, complex models that increase training costs and inference latency, preventing real-time deployment. To address this, we employ a lightweight recurrent neural network model that integrates sequential low-resolution frames to extract spatio-temporally correlated signals. It significantly improves temporal resolution by reducing the required number of frames down to as few as 8 frames while doubling the spatial resolution in an inference time under 30 ms. Furthermore, gentle imaging conditions are essential for extracting reliable biologically relevant information, especially in long-term experiments. Our method is suitable for live-cell imaging under extreme signal/noise ratio conditions, allowing imaging under very low laser intensities to prevent photodamage. By combining simulation-based training with an efficient network architecture, we introduce real-time super-resolution fluctuation imaging (RESURF), a deep-learning-based real-time super-resolution fluctuation imaging framework. We demonstrate that RESURF generalizes across different biological structures and can be readily adapted to various microscope setups using a small data set for transfer learning. The accompanying data set, comprising simulations and experiments across multiple subcellular structures and labeling strategies, establishes a benchmarking platform for fluctuation-based super-resolution techniques. RESURF offers a practical, low-latency deep-learning framework for high-throughput imaging and real-time, smart live-cell super-resolution imaging.