Scanning two photon fluorescence microscopy with extended depth of field

We describe the design and implementation of a light efficient binary phase-only diffraction optical element which converts a laser beam into an annular ring with the loss of less than 25% of the overall power. The use of annular illumination permits us to obtain an increase in the axial extent of the point spread function of a microscope objective without compromising the lateral resolution. The light efficiency of the system permitted two photon fluorescence images with extended depth of field to be obtained. The combination of two such extended depth of field images obtained at different parallaxes allowed the generation of high resolution two photon stereo pairs.