Addressing systematic errors in axial distance measurements in single-emitter localization microscopy.

Optics Express
Petar N Petrov, W E Moerner

Abstract

Nanoscale localization of point emitters is critical to several methods in optical fluorescence microscopy, including single-molecule super-resolution imaging and tracking. While the precision of the localization procedure has been the topic of extensive study, localization accuracy has been less emphasized, in part due to the challenge of producing an experimental sample containing unperturbed point emitters at known three-dimensional positions in a relevant geometry. We report a new experimental system which reproduces a widely-adopted geometry in high-numerical aperture localization microscopy, in which molecules are situated in an aqueous medium above a glass coverslip imaged with an oil-immersion objective. We demonstrate a calibration procedure that enables measurement of the depth-dependent point spread function (PSF) for open aperture imaging as well as imaging with engineered PSFs with index mismatch. We reveal the complicated, depth-varying behavior of the focal plane position in this system and discuss the axial localization biases incurred by common approximations of this behavior. We compare our results to theoretical calculations.

References

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Citations

Mar 11, 2021·Journal of the Optical Society of America. A, Optics, Image Science, and Vision·Oumeng Zhang, Matthew D Lew
Mar 24, 2021·Light, Science & Applications·Luzhe HuangAydogan Ozcan
Oct 10, 2020·Journal of the American Chemical Society·Leonhard Möckl, W E Moerner
Jul 3, 2021·Nano Letters·Nadav OpatovskiYoav Shechtman

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