Fig. 5.

Comparing off-focus imaging at a water/glass interface and in the water volume, for equal detected signal and background levels. (a) Graphical explanation of the compared measurement conditions. Imaging in the volume means that the coverslip is sufficiently far away from the emitter to prevent any near-field coupling effects. (b) Theoretically obtainable z-precisions for various distances of the emitter to the nominal focus point ($\delta$). A good off-focus range for volume imaging regarding z-localization is in between about $\delta$=150 and 400 nm. For surface-near imaging, an off-focus distance of $\Gamma$=600 nm is a good choice for axial localizations. There, the interface boosts the z-precision by almost up to a factor of 2. The images on the right show simulated molecule images for different values of $\delta$. The presence of the water/glass interface leads to clearly visible shape-changes of the PSF (signal=2000 photons, background level=100 photons per pixel, wavelength=670 nm, pixel size=115 nm, image size=15$\times$15 pixel, NA=1.49).