Enhanced supercritical angle localization microscopy through point spread function modeling.

Single-molecule localization microscopy (SMLM) enables precise spatial localization of single molecules in cellular structures. A phenomenon called supercritical angle fluorescence (SAF) is utilized in SMLM (SAF-SMLM) to estimate the axial positions of single fluorophores. It is based on the fact that SAF intensity is highly sensitive to the fluorophore-coverslip distance. Conventional SAF-SMLM methods typically involve splitting the fluorescence emission into supercritical and undercritical components, which requires a complicated two-channel system and can lead to reduced light efficiency. In this work, we introduce a simplified approach to traditional SAF-SMLM by directly detecting all fluorescence into a single channel. Through simulations, we found that by accurately modeling the point spread function (PSF) with SAF, a single-channel system achieves better localization precision than two-channel-based SAF-SMLM systems. Furthermore, we developed a stage-tilt correction algorithm, incorporating stage tilt in the PSF model, to improve axial precision over the entire field of view. We applied our method experimentally by imaging F-actin filaments in HeLa cells. We demonstrate that our method efficiently exploits the information from SAF and achieves enhanced axial localization precision and accuracy compared to traditional SMLM localization methods for single-channel systems.