IMAGE-FORMATION AND DATA-ACQUISITION IN A STAGE SCANNING 4PI CONFOCALFLUORESCENCE MICROSCOPE

We describe the three-dimensional (3-D) image formation and data acqui sition in a stage scanning 4Pi confocal fluorescence microscope with t he use of two-photon excitation. The 3-D point-spread functions of the 4Pi confocal and regular confocal microscope are measured and compare d. Particular emphasis is given to the data acquisition procedure. 4Pi confocal microscopy results in a point-spread function that is 4 time s sharper than that of a regular confocal microscope, ultimately leadi ng to superior 3-D imaging of translucent fluorescent specimens. For a two-photon excitation wavelength of approximately 800 nm, we obtain a n axial resolution of 140 nm. (C) 1997 Optical Society of America.