Confocal theta fluorescence microscopy using two-photon absorption and annular apertures

Confocal theta fluorescence microscopy using two-photon absorption and annular apertures. In a confocal theta fluorescence microscope, a second objective lens placed orthogonally to the illumination objective lens is used for detection. The addition of annular apertures to such an arrangement leads to a reduction of the volume at half maximum of the point spread function. We show that when the sample is illuminated in a two-photon absorption mode a threedimensional 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 aperture of 0.75 and an illumination wavelength of 800 nm is then reduced from 12.3 .to 4.6 attoliters. An important advantage of the two-photon absorption mode is that no diffraction side lobes are present when an annular illumination aperture is used.