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Keywords:

  • fluorescence microscopy;
  • fluorophores;
  • localization precision;
  • single-molecule studies;
  • superresolution imaging

Abstract

Single-molecule localization-based superresolution imaging is complicated by emission from multiple emitters overlapping at the detector. The potential for overlapping emitters is even greater for 3D imaging than for 2D imaging due to the large effective “volume” of the 3D point spread function. Overlapping emission can be accounted for in the estimation model, recovering the ability to localize the emitters, but with the caveat that the localization precision has a dependence on the amount of overlap from other emitters. Whether a particular 3D imaging modality has a significant advantage in facilitating the position estimation of overlapping emitters is investigated. The variants of two commonly used and easily implemented imaging modalities for 3D single-molecule imaging are compared: astigmatic imaging; dual focal plane imaging; and the combination of the two approaches, dual focal plane imaging with astigmatism. The Cramér–Rao lower bound is used to quantify the multiemitter estimation performance by calculating the theoretical best localization precision under a multiemitter estimation model. The performance of these 3D modalities is investigated under a wide range of conditions including various distributions of collected photons per emitter, background counts, pixel sizes, and camera readout noise values. Differences between modalities are small and it is therefore concluded that multiemitter fitting performance should not be a primary factor in selecting between these modalities.