Formation of hemoglobin photoproduct is responsible for two-photon and single photon-excited fluorescence of red blood cells

Two-photon excited fluorescence of red blood cells (RBC) has been reported to be applicable for their assessment in vitro and in vivo. The corresponding fluorescence emission was ascribed to hemoglobin (Hb), however, as Hb is essentially non-fluorescent at single-photon excitation, the mechanism of two-photon excited fluorescence of RBC remains debatable. Here we show that a fluorescent photoproduct, characterized by an ultrafast decay of excitation, is formed after irradiation of Hb with femtosecond laser pulses with ca. 8 · 10⁻⁵ quantum yield, and that it is also fluorescent at single-photon excitation. The formation of a similar photoproduct was also shown for Hb continuous wave irradiation with blue light with ca. 10⁻⁵ formation quantum yield. The kinetics of the Hb photoproduct formation and its spectral properties were investigated. The obtained results clarify the processes responsible for RBC fluorescence observed in two-photon microscopy experiments. (letter)