Extended Data Fig. 5: Flowchart for me4Pi-SMLM reconstruction.

Refer to the Methods section for a detailed description. Three sequential sub-images were acquired, corresponding to phase-shifted illumination patterns. 2D astigmatic Gaussian fitting was used to determine lateral positions (x, y) and PSF widths, which were then used to estimate coarse axial positions. Intensities across the three sub-images were analyzed to extract interferometric phase information. After phase-drift correction, a ridge-finding algorithm was used for axial phase unwrapping to convert phase data into precise axial positions. A 3D drift-correction algorithm compensated for both sample drift and residual system drift. Molecules that did not exhibit modulation across all three frames were excluded based on modulation depth (Step 1), followed by further exclusion based on photon count and localization precision (Step 2). Final reconstructions used the precise z positions for me4Pi-SMLM, whereas coarse z positions were used for the 3D-SMLM comparison.