Fluorescence spatial anisotropy of emission dipoles in an orthogonal imaging system

In this study, we explore the relatively unexplored spatially anisotropic fluorescence emission induced by rotationally polarized excitation light in orthogonal imaging systems , a phenomenon that is particularly pronounced in orthogonal setups compared to coaxial configurations. Despite its significance, this aspect has been largely neglected, given the prevalent use of coaxial Fluorescence Polarization Microscope (FPM) setups. Our research endeavors to bridge this gap by formulating a physical model to investigate the polarization-dependent emission of dipoles and the corresponding fluorescence signal within an orthogonal imaging configuration. Additionally, we introduce the Fluorescence Spatial Anisotropy Index (FSAI) to quantify fluorescence spatial anisotropy. • Explored the fluorescence spatial anisotropy of dipoles excited by rotationally polarized light, a phenomenon relatively unexplored in orthogonal setups. • Developed a physical model investigating fluorescence spatial anisotropy of dipoles in orthogonal setups. • Introduced the Fluorescence Spatial Anisotropy Index (FSAI) for quantitative measurements.