Polarization effects in 4Pi confocal microscopy studied with water-immersion lenses

We studied the effect of electric field orientation on the point-spread function (PSF) of a 4Pi microscope. We show that in a standard 4Pi arrangement the orientation of the field can be used for changing between constructive- and destructive-mode 4Pi microscopy. The effect is counteracted by introduction of a phase shift of pi into one of the half-arms. This compensation is compulsory during illumination with unpolarized or circularly polarized light. By performing our experiments with 1.2-N.A. water-immersion lenses, we demonstrate that water immersion is suitable for 4Pi confocal microscopy. At a two-photon excitation wavelength of 1064 nm, the water 4Pi confocal PSF features an axial lobe of 40% above and below the focal plane, which, by linear filtering, can be unambiguously removed. The measured axial full width at half-maximum of the PSF is 240 nm. This is 4.3 times narrower than its single-lens confocal counterpart. The 4Pi confocal microscope sets a new resolution benchmark in three-dimensional imaging of watery samples.