Image formation and data acquisition in a stage scanning 4Pi confocal fluorescence microscope.

We describe the three-dimensional (3-D) image formation and data acquisition in a stage scanning 4Pi confocal fluorescence microscope with the use of two-photon excitation. The 3-D point-spread functions of the 4Pi confocal and regular confocal microscope are measured and compared. Particular emphasis is given to the data acquisition procedure. 4Pi confocal microscopy results in a point-spread function that is 4 times sharper than that of a regular confocal microscope, ultimately leading to superior 3-D imaging of translucent fluorescent specimens. For a two-photon excitation wavelength of approximately 800 nm, we obtain an axial resolution of 140 nm.