Single-molecule orientation measurements with a quadrated pupil

Adam S. Backer, Mikael P. Backlund, Matthew D. Lew, Alexander R. Diezmann, Steffen J. Sahl, W. E. Moerner

Research output: Chapter in Book/Report/Conference proceedingConference contribution


We present a means of measuring the dipole orientation of a fluorescent, rotationally fixed single molecule (SM), using a specially designed phase mask, termed a "quadrated pupil," conjugate to the back focal plane of a conventional widefield microscope. In comparison to image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies, defocus, and optical aberrations. Precision on the order of 1°-5° is achieved in proofof- concept experiments for both azimuthal (φ) and polar (θ) angles. Since the phase mask is implemented on a liquid-crystal spatial light modulator (SLM) that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into conventional imaging studies.

Original languageEnglish (US)
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VII
ISBN (Print)9780819498632
StatePublished - 2014
Externally publishedYes
EventSingle Molecule Spectroscopy and Superresolution Imaging VII - San Francisco, CA, United States
Duration: Feb 1 2014Feb 2 2014

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


OtherSingle Molecule Spectroscopy and Superresolution Imaging VII
Country/TerritoryUnited States
CitySan Francisco, CA


  • Fluorescence microscopy
  • Optical Fourier processing
  • Single-molecule microscopy
  • Super-resolution

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging


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