Maximizing the field of view and accuracy in 3D Single Molecule Localization Microscopy

Optics Express
Sohaib Abdul RehmanKevin O'Holleran

Abstract

Super-resolution techniques that localize single molecules in three dimensions through point spread function (PSF) engineering are very sensitive to aberrations and optical alignment. Here we show how double-helix point spread function is affected by such mis-alignment and aberration. Specifically, we demonstrate through simulation and experiment how misplacement of phase masks in infinity corrected systems is a common source of significant loss of accuracy. We also describe an optimal alignment and calibration procedure to correct for these errors. In combination, these optimizations allow for a maximal field of view with high accuracy and precision. Though discussed with reference to double-helix point spread function (DHPSF), the optimization techniques are equally applicable to other engineered PSFs.

References

Aug 10, 2006·Nature Methods·Michael J RustXiaowei Zhuang
Aug 12, 2006·Science·Eric BetzigHarald F Hess
Jun 11, 2008·Optics Express·Sri Rama Prasanna Pavani, Rafael Piestun
Feb 13, 2009·Proceedings of the National Academy of Sciences of the United States of America·Sri Rama Prasanna PavaniW E Moerner
Jun 22, 2010·Proceedings - Society of Photo-Optical Instrumentation Engineers·Matthew D LewW E Moerner

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Citations

Jan 1, 2020·Proceedings of the IFAC World Congress·Nicholas A Vickers, Sean B Andersson

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