Supplementary Figure 5: Astigmatism calibration curves.

The astigmatism calibration curves for all nine beads at different depths (a subset was shown in Fig. 1). The widths of the PSFs are measured and combined into a single calibration curve using the metric, \(\omega _x^3/\omega _y - \omega _y^3/\omega _x\). Calibration curves without AO correction using the metric (A) or the measured widths (D). The slope of the calibration curves (A) decreases at greater depths reducing the range and sensitivity of the axial localizations. The insets with the green (orange) outline show the PSF 360 nm below (above) the focal plane of the 45.4 µm (92.0 µm) deep bead. Removing aberrations improves the calibration curves (B, E). Up to ~10 µm, the calibration curves nearly overlap. The magnitude of astigmatism can be increased based on the imaging depth to ensure that the calibration curves overlap (C, F). PSF modulation is enforced to match the calibration curve on the coverslip. Scale bar is 500 nm.