Fig. 2.

Optical setup. Sample is mounted on a piezo stage and imaged with an oil-immersion objective lens (100x, NA 1.4). The image formed by the tube lens is relayed via a 4f system to the final image plane at the EMCCD camera. A phase mask is placed conjugate to the back focal plane. Inset shows phase patterns for the different PSFs used in radians (top) and examples of scaled images acquired at different focal positions (columns) from beads on the glass coverslip. Focal positions are offset by 500 nm (OA, astigmatism) or 750 nm (DH). Pixel size of the PSF images is 160 nm at the sample plane.