Practical two‐photon‐absorption cross sections and spectra of eosin and hematoxylin

Abstract We experimentally investigate the two‐photon absorption cross sections and spectra of eosin and hematoxylin for applications in nonlinear microscopy. The experiments are carried out on pure samples of the two dyes in DI‐water solvent with different concentrations, in the typical range employed in standard staining procedures. Nonlinear fluorescence is excited by a line‐shaped beam emitted by a Ti:Sapphire mode‐locked laser in the wavelength range from 740 to 880 nm and is detected through a microscope setup. The two‐photon absorption spectral response is systematically analyzed and discussed. Finally, the staining is applied on biological tissue samples, which are imaged by two‐photon microscopy. Our results show that the employed dyes are fully suitable for applications in nonlinear imaging.