We conducted a systematic study on two-photon excited fluorescence (TPEF) of hemoglobin using the near transform-limited and Gaussian-shaped femtosecond pulse sources. We found that the two-photon action cross section of hemoglobin drops over 2 orders of magnitude in the wavelength range from 550 to , while the spectral and temporal characteristics of hemoglobin TPEF are insensitive to the change of excitation wavelength. In particular, our new findings showed that the hemoglobin fluorescence could be excited with sufficient efficiency using a conventional Ti:sapphire laser tuned at the wavelength close to . With the employment of a time-resolved detection method, we demonstrated that the TPEF signals of hemoglobin excited by a Ti:sapphire laser could be clearly differentiated from other nonlinear signals presented within the living biological tissues, indicating that a standard TPEF microscope can become a routine tool for in vivo label-free microangiography imaging.
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