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Fluorescence emission after two-photon absorption of coumarins and xanthenes in an alcoholic solution was measured in the tuning range of a femtosecond-pulsed titanium-sapphire laser (750-840 nm). Xanthenes, which have a low one-photon absorption in the near UV, show a higher fluorescence signal after two-photon absorption than the UV-excitable coumarins. When fluxes of 10(28) photons/(cm(2) s) are used, the two-photon absorption cross sections for xanthenes are 1 order of magnitude higher than the two-photon absorption cross sections of the coumarins. Absolute cross sections have been estimated for three coumarins and three xanthenes. For the xanthenes a significant wavelength-dependent departure from the expected fluorescence intensity square law was observed. The coumarins follow the square-law dependence. The consequences of the findings are discussed for analytic and diagnostic methods. An especially important result is that the resolution in two-photon microscopy of xanthenes is worse than expected.
Fischer et al. (1995) studied this question.