Predicting resolution and image quality in RESOLFT and other point scanning microscopes

The performance of fluorescence microscopy and nanoscopy is often discussed by the effective point spread function and the optical transfer function. However, due to the complexity of the fluorophore properties such as photobleaching or other forms of photoswitching, which introduce a variance in photon emission, it is not trivial to choose optimal imaging parameters and to predict the spatial resolution. In this paper, we analytically derive a theoretical framework for estimating the achievable resolution of a microscope depending on parameters such as photoswitching, labeling densities, exposure time and sampling. We developed a numerical simulation software to analyze the impact of reversibly switchable probes in RESOLFT imaging.