Localization microscopy vastly improves the resolution achieved by fluorescence microscopy by fitting the positions of individual fluorophores. We examine the reconstructions produced by different fitting algorithms for instances of fixed pattern noise—systematic tendencies to alter estimated emitter positions according to their subpixel location in a way that does not reflect the ground truth structure. We show that while not readily visible at standard empirical signal strengths, fixed pattern noise can occur when performing sub-pixel fitting, and that its degree varies according to the algorithm used and the relative size of the pixels compared to the point spread function. For pixel sizes in the range 80–170 nm, this results in variations in accuracy of the order of 2–4 nm—comparatively small for many applications, but non-negligible in scenarios where very high accuracy is sought.
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