Non linear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume

Abstract

It is shown that two-photon absorption confines the illumination volume and present quantitative evidence that an additional confocal arrangement of the detector further improves the resolution by 48%. The axial resolution in a confocal fluorescence microscope using two-photon absorption with infra-red light is comparable to that achievable with ultra-violet light half the wavelength. An important advantage of two-photon microscopy over single-photon microscopy is that absorption is almost confined to the observed volume. This means no photodamage is caused outside the observed volume.