Ultrashort laser pulses interaction with hemoglobin and label-free imaging of erythrocytes

Label-free imaging of erythrocytes using single-photon excitation microscopy (e.g. confocal) is practically impossible because the hemoglobin (Hb) fluorescence is hardly detectable. On the other hand, two photon excited fluorescence (TPEF), of a newly formed photoproduct upon the interaction of ultrashort laser pulses with Hb, was used in number of studies for label-free erythrocytes imaging. We present the photophysical properties of the formed photoproduct and its possible applications. We created fluorescent photoproduct patterns on the thin Hb film using femtosecond laser writing. The photoproduct is characterized using different spectroscopic and imaging techniques. Spectroscopic properties of Protoporphyrin IX (PpIX) are considered as well. Moreover, we treated Hb solution with hydrogen peroxide to test the similarity in optical response between chemically and laser induced degradation of Hb. Photo stability of Hb photoproduct was sustained in the time range of several months. Based on the photoproduct formation that can be controlled spatiotemporally, photo-labelling of individual RBCs and tracing of their movement in the whole blood was successfully performed.