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Fluorescence microscopy is a photon-limited imaging modality that allows the study of subcellular objects and processes with high specificity. The best possible accuracy (standard deviation) with which an object of interest can be localized when imaged using a fluorescence microscope is typically calculated using the Cramér-Rao lower bound, that is, the inverse of the Fisher information. However, the current approach for the calculation of the best possible localization accuracy relies on an analytical expression for the image of the object. This can pose practical challenges since it is often difficult to find appropriate analytical models for the images of general objects. In this study, we instead develop an approach that directly uses an experimentally collected image set to calculate the best possible localization accuracy for a general subcellular object. In this approach, we fit splines, i.e. smoothly connected piecewise polynomials, to the experimentally collected image set to provide a continuous model of the object, which can then be used for the calculation of the best possible localization accuracy. Due to its practical importance, we investigate in detail the application of the proposed approach in single molecule fluorescence microscopy. In this case, the object of interest is a point source and, therefore, the acquired image set pertains to an experimental point spread function.

Determination of localization accuracy based on experimentally acquired image sets: applications to single molecule microscopy