Fig. 3: Performance of globLoc on 4Pi-SMLM data.

a Localization precision for 4Pi-SMLM imaging of fluorescent beads. A single 40 nm red fluorescence bead (F8793, Invitrogen) was imaged at z positions from −500 nm to 500 nm with 20 nm step size and 37 frames were collected at each z position. The estimated photons/objective is 464 ± 40 (mean ± s.t.d) and the background photons/pixel is 0.47 ± 0.04 (mean ± s.t.d). The localization precision is calculated as the standard deviation. Theoretical estimated precision (\(\sqrt{{{\mbox{CRLB}}}}\)) in z increases when unlinking the photon and background in four phase channels. b Comparison of globLoc on 4Pi-SMLM data under different parameter sharing schemes and compared to the state-of-the art analysis (photometry)¹¹, on the example of Nup96-AF647. b2–3 Top-view and side-view reconstructions of selected NPCs at different z positions as indicated in b1 (all linked: globLoc with x, y, z, photons and background photons linked; x,y,z, \(\varphi\) linked: globLoc with x, y and z and the phase linked). Representative results are shown from 3 experiments for (b). Scale bars 10 µm 1 µm (b1), 100 nm (b2-3).