AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.
Live-Cell Super-Resolution Imaging of Endogenous Ligand-Activated Protein Dimers by Combining Upaint and Single Molecule FRET
