Characterization and Optimization of the Biplane Distance in Three-Dimensional Single-Molecule Localization Microscopy

Biplane single-molecule localization microscopy (SMLM) enables three-dimensional (3D) super-resolution imaging by extracting the axial position of fluorophores from a pair of emission patterns detected at two axially separated planes. The separation between these two imaging planes, termed the biplane distance, is a key parameter that determines axial localization precision, yet a systematic investigation of its optimal selection remains lacking. Here, we calculate the theoretical localization precision across a range of biplane distances and identify an optimal value, construct a tunable biplane detection module and experimentally evaluate axial localization precision at three different biplane distances using both fluorescent beads and biological specimens. Experimental results confirm the theoretical predictions and provide a practical framework for optimizing the biplane distance in 3D-SMLM systems.