Measurement-based estimation of global pupil functions in 3D localization microscopy

We report the use of a phase retrieval procedure based on maximum likelihood estimation (MLE) to produce an improved, experimentally calibrated model of a point spread function (PSF) for use in three-dimensional (3D) localization microscopy experiments. The method estimates a global pupil phase function (which includes both the PSF and system aberrations) over the full axial range from a simple calibration scan. The pupil function is used to refine the PSF model and hence enable superior localizations from experimental data. To demonstrate the utility of the procedure, we apply it to experimental data acquired with a microscope employing a tetrapod PSF with a 6 μm axial range. The phase-retrieved model demonstrates significant improvements in both accuracy and precision of 3D localizations relative to the model based on scalar diffraction theory. The localization precision of the phase-retrieved model is shown to be near the limits imposed by estimation theory, and the reproducibility of the procedure is characterized and discussed. Code which performs the phase retrieval algorithm is provided.