Confocal theta fluorescence microscopy with annular apertures.

In a confocal theta fluorescence microscope, two objective lenses with circular apertures are used, one to illuminate the sample and the other to detect the emitted light at an angle to the illumination axis. We show that annular illumination and detection apertures lead to a reduction in the extent of the point-spread function. A spatial resolution improved by more than 50% can be achieved with a central obstruction blocking the inner 80% of the diameter. For the limit of a very narrow annular aperture and a numerical aperture of 0.75, the volume at half-maximum of the point-spread function is reduced from 15to5 aL. Amixed setup with anannular illumination aperture and a circular detection aperture is also considered.