Single-molecule orientation measurements with a quadrated pupil

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 (φ) and polar (θ) 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.