MUFASA: a continuous-time stochastic framework for realistic fluorescence microscopy simulation

We present MUFASA ( M ulti-Protocol U nified F luorescence-based A dvanced S imulation A lgorithm), a physically grounded, continuous-time simulator of several super-resolution fluorescence microscopy protocols. By modeling fluorophore dynamics using continuous-time Markov chains, MUFASA’s simulation features yield realistic photon emission behavior across both Single Molecule Localization Microscopy (SMLM) and fluorescence fluctuation-based (FF-SRM) settings–independently of frame duration and sampling. The framework supports both individual emitters and structure-level simulations, incorporating photophysical transitions, photobleaching, and camera properties.To quantitatively compare simulations with real data, we introduce a novel validation metric based on the 1-Wasserstein distance between simulated and experimental photon-count distributions. Another functionality of MUFASA is the estimation of key photophysical parameters (e.g., molar extinction coefficient) and the suggestion of optimal light-source power ranges from fluctuation data.An intuitive Python-based graphical interface for real-time parameter tuning, visualization, and TIFF export is provided.Designed for biologists, physicists, microscopists, and computational imaging engineers, MUFASA offers a practical platform for microscopy experiment design, hypothesis testing and holds the potential of being used for the generation of realistic training data for data-driven methods across modalities.