Engineering of illumination and collection field profiles for single-molecule orientational imaging

Recently, spatial light modulators (SLMs) have been used to generate polarization-engineered laser beams, such as radially polarized doughnut modes, which may provide advantages for excitation of fluorophore dipoles in single-molecule (SM) spectroscopy. Here we investigate the additional use of SLMs for spatially-dependent transformation of the collected fluorescence field with a goal to improve the fidelity of three-dimensional molecular orientation determination. Numerical calculations of a high numerical aperture single-molecule confocal microscope are presented in which a SLM is placed in the back focal plane of the objective. The coherently imaged fluorescence undergoes spatially-dependent phase and polarization transformation by the SLM, before it passes to a polarization beamsplitter, and is subsequently focused onto two pinholes and single-photon avalanche photodiodes. We calculate the electric vector field in the back focal plane of the objective using the Weyl representation and taking into account the forbidden light emitted at angles above the critical angle of the cover glass-immersion fluid interface. The calculated electric field is then subject to the spatially-dependent polarization change implemented by SLM. We numerically study the effects of polarization control on the microscope sensitivity to molecule orientation. We also analyze the combined use of the intensity and polarization information in the back focal plane of the SM microscope for single-molecule orientation determination