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Optical microscopy methods such as calcium and voltage imaging enable fast activity readout of large neuronal populations using light. However, the lack of corresponding advances in online algorithms has slowed progress in retrieving information about neural activity during or shortly after an experiment. This gap not only prevents the execution of real-time closed-loop experiments, but also hampers fast experiment-analysis-theory turnover for high-throughput imaging modalities. Reliable extraction of neural activity from fluorescence imaging frames at speeds compatible with indicator dynamics and imaging modalities poses a challenge. We therefore developed FIOLA, a framework for fluorescence imaging online analysis that extracts neuronal activity from calcium and voltage imaging movies at speeds one order of magnitude faster than state-of-the-art methods. FIOLA exploits algorithms optimized for parallel processing on GPUs and CPUs. We demonstrate reliable and scalable performance of FIOLA on both simulated and real calcium and voltage imaging datasets. Finally, we present an online experimental scenario to provide guidance in setting FIOLA parameters and to highlight the trade-offs of our approach.