In a 4Pi confocal fluorescence microscope two opposing microscope objective lenses were used to illuminate a fluorescent object from both sides and to collect the fluorescence emissions on both sides. Constructive interference of either the illumination wave fronts in the common focus or the detection wave fronts in the common detector pinhole resulted in an axial resolution approximately four times higher than that in a confocal fluorescence microscope. A precise 4Pi confocal fluorescence microscope that uses simultaneous illumination was built. The full width at half-maximum of the depth discrimination was determined experimentally to be approximately 110 nm when lenses with a numerical aperture of 1.4, an excitation of 633 nm, and detection of approximately 725 nm were used.
© 1992 Optical Society of America
Original Manuscript: April 15, 1992
Revised Manuscript: June 29, 1992
Manuscript Accepted: June 10, 1992
Published: December 1, 1992
Stefan Hell and Ernst H. K. Stelzer, "Properties of a 4Pi confocal fluorescence microscope," J. Opt. Soc. Am. A 9, 2159-2166 (1992)