CONFOCAL THETA-FLUORESCENCE MICROSCOPY USING 2-PHOTON ABSORPTION AND ANNULAR APERTURES

In a confocal theta fluorescence microscope, a second objective lens p laced orthogonally to the illumination objective lens is used for dete ction. The addition of annular apertures to such an arrangement leads to a reduction of the volume at half maximum of the point spread funct ion. We show that when the sample is illuminated in a two-photon absor ption mode a three-dimensional resolution improvement by more than 60% can be achieved in the limit of a narrow annular aperture. The volume at half maximum of a confocal theta microscope with a numerical apert ure of 0.75 and an illumination wavelength of 800 nm is then reduced f rom 12.3 to 4.6 attoliters. An important advantage of the two-photon a bsorption mode is that no diffraction side lobes are present when an a nnular illumination aperture is used.