Two-photon absorption cross-sections of eosin and hematoxylin

We present a detailed investigation of the two-photon absorption cross-sections and spectra of eosin and hematoxylin to be applied in nonlinear microscopy. In our configuration the two-photon fluorescence is excited by a mode-locked Ti:Sapphire laser tunable in the wavelength range from 740 to 880nm in a novel microscope setup exploiting a line-shaped illumination beam. Each dye is diluted at different concentrations in deionized water solution. The emission of nonlinear fluorescence is demonstrated by the quadratic dependence of the fluorescence intensity upon the excitation intensity. While the nonlinear emission of eosin increases for increasing concentration, the emission of hematoxylin decreases suggesting that concentration quenching phenomena arise. The feasibility of two-photon microscopy exploiting the two dyes is finally confirmed by carrying out imaging of biological tissues. Our results demonstrate that eosin and hematoxylin, which are usually used together for staining samples in white-light and one-photon-fluorescence microscopy, exhibit an excellent nonlinear fluorescence response. Indeed, the images of biological samples are obtained from already prepared specimens.