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Updated: Jun 6, 2026

Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy
Published on: November 11, 2025
Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy
Johann Engelhardt1, Jan Keller, Patrick Hoyer
1German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. shell@gwdg.de
Abstract:
We investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-field superresolution microscopy based on stochastic single molecule switching. If tilt angle and defocus are unknown, the localization contains systematic errors up to about ±125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about ±32 nm, restricting the attainable resolution accordingly.
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