Maximizing the field of view and accuracy in 3D Single Molecule Localization Microscopy.
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Peer-reviewed
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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.
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1094-4087
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Engineering and Physical Sciences Research Council (EP/M003663/1)
Engineering and Physical Sciences Research Council (EP/L015455/1)