Figure 1.

Emission path of DHPSF setup and empirically-determined localization precision of the DHPSF as a function of detected photons. (a) The DHPSF is implemented by the addition of a 4f system comprised of two lenses (L1 & L2) into the emission path of a fluorescence microscope with a DHPSF PM placed in the Fourier-transfer plane of the 4f system (center of the two lenses). The first plane of the 4f system is placed in the image plane of the microscope, relaying the emission signal onto an EMCCD placed a distance 4f away. Scale bars are 500 nm. (b) Measured lateral (xy) and axial (z) localization precision compared to a 2D fluorescence microscope as a function of detected photons with example DHPSFs. The highlighted region represents typical experimentally determined detected photon numbers when imaging fluorescent proteins and organic dyes. Localization precision was determined by imaging immobilized fluorescent beads.