Depth estimation in fluorescence microscopy using degree of polarization

Accurate axial depth estimation in fluorescence microscopy remains challenging in non-interferometric systems due to weak signals and strong background noise. Here, we investigate the depth-dependent behavior of the degree of polarization (DOP) and demonstrate its applicability for axial localization under low signal-to-noise ratio conditions. By formulating a noise-aware model based on the cross-spectral density matrix that explicitly incorporates unpolarized background fluorescence and detector noise, we explain the experimentally observed degradation of DOP with defocus. Polarization-resolved fluorescence experiments show that the measured DOP exhibits a maximum near the geometrical focus and gradually decays toward zero at larger defocus distances. Based on a calibrated DOP-defocus relationship, reliable depth estimation of fluorescent beads is achieved without interferometric or phase-sensitive measurements. Furthermore, combining DOP information from multiple axial planes improves robustness and enables accurate depth reconstruction of multi-layer samples, showing improved depth-estimation accuracy over the representative comparison methods under the tested conditions.