Single-molecule orientation measurements with a quadrated pupil

Abstract

This Letter presents a means of measuring the dipole orientation of a fluorescent, orientationally fixed single molecule, which uses a specially designed phase mask, termed a “quadrated pupil,” conjugate to the back focal plane of a conventional wide-field microscope. The method leverages the spatial anisotropy of the far-field emission pattern of a dipole emitter and makes this anisotropy amenable to quantitative analysis at the image plane. In comparison to older 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 and optical aberrations. Precision of 1°–5° is achieved in proof-of-concept experiments for both azimuthal ($\phi$) and polar ($\theta$) angles without defocusing. Since the phase mask is implemented on a liquid-crystal spatial light modulator that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into clear aperture imaging studies.

© 2013 Optical Society of America

Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements

Adam S. Backer, Maurice Y. Lee, and W. E. Moerner
Optica 3(6) 659-666 (2016)

Single-molecule orientations determined by direct emission pattern imaging

M. Andreas Lieb, James M. Zavislan, and Lukas Novotny
J. Opt. Soc. Am. B 21(6) 1210-1215 (2004)

Engineering the collected field for single-molecule orientation determination

Zbigniew Sikorski and Lloyd M. Davis
Opt. Express 16(6) 3660-3673 (2008)

Orientation imaging of single molecules by wide-field epifluorescence microscopy

Martin Böhmer and Jörg Enderlein
J. Opt. Soc. Am. B 20(3) 554-559 (2003)

Three dimensional single molecule localization using a phase retrieved pupil function

Sheng Liu, Emil B. Kromann, Wesley D. Krueger, Joerg Bewersdorf, and Keith A. Lidke
Opt. Express 21(24) 29462-29487 (2013)