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

We studied the effect of electric field orientation on the point-spread fun ction (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 introd uction of a phase shift of a into one of the half-arms. This compensation i s compulsory during illumination with unpolarized or circularly polarized l ight. By performing our experiments with 1.2-N.A, water-immersion lenses, w e demonstrate that water immersion is suitable for 4Pi confocal microscopy. At a two-photon excitation wavelength of 1064 nm, the water 4Pi confocal P SF features an axial lobe of 40% above and below the focal plane, which, by linear filtering, can be unambiguously removed. The measured axial full wi dth at half-maximum of the PSF is 240 nm. This is 4.3 times narrower than i ts single-lens confocal counterpart. The 4Pi confocal microscope sets a new resolution benchmark in three-dimensional imaging of watery samples. (C) 2 000 Optical Society of America.