Abstract

Localization microscopy approaches with enhanced depth-of-field (EDoF) are commonly optimized using the Cramér–Rao bound (CRB) as a criterion. It is widely believed that the CRB can be attained in practice by using the maximum-likelihood estimator (MLE). This is, however, an approximation, of which we define in this paper the precise domain of validity. Exploring a wide range of settings and noise levels, we show that the MLE is efficient when the signal-to-noise ratio (SNR) is such that the localization standard deviation of a single molecule is less than 20 nm. Thus, our results provide an explicit and quantitative validity boundary for the use of the MLE in EDoF localization microscopy setups optimized with the CRB.

© 2021 Optical Society of America

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