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Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization

By Yiming Li, Yu-Le Wu, Philipp Hoess, Markus Mund, Jonas Ries

Posted 21 Feb 2019
bioRxiv DOI: 10.1101/555730 (published DOI: 10.1364/boe.10.002708)

3D Single molecule localization microscopy relies on fitting of the individual molecules with a point spread function (PSF) model. The reconstructed images often show local squeezing or expansion in z. A common cause are depth-induced aberrations in conjunction with an imperfect PSF model calibrated from beads on a coverslip, resulting in a mismatch between measured PSF and real PSF. Here, we developed a strategy for accurate z-localization in which we use the imperfect PSF model for fitting, determine the fitting errors and correct for them in a post-processing step. We present an open-source software tool and a simple experimental calibration procedure that allow retrieving accurate z-positions in any PSF engineering approach or fitting modality, even at large imaging depths.

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