Abstract:Twophoton fluorescent probes have been widely studied for biomedical imaging and treatment. Understanding the excitation characteristics of this type of probes in biological tissue and knowing the compared advantages and disadvantages with singlephoton probes are useful for the probe selection and application. However, the actual measurement is difficult to reveal their inherent characteristics due to too many interference factors existing. In this study, following the selected typical parameters, imaging of the two types of excitation probes in tissue is simulated. It is found that, during irradiation with same power of the flat light beams, twophoton 〖JP〗probes have more rapid intensity attenuation and lower tissue penetration than single probes, due to their low excitation efficiency and high excitation threshold, as well as the twophoton absorption. In addition, the rapid attenuation along the depth can result in unevenness distribution of light. The unevenness affects the fluorescence of twophoton probes more when comparing to singlephoton probes. Simulation analysis further indicates that enhancing the twophoton power in biological tissue tolerance range can improve the fluorescence imaging of twophoton probes more; and increasing the irradiation area can weaken the inhomogeneity of lateral excitation for better imaging effect. The results provide the basic data for twophoton fluorescence excitation and imaging. The simulation method also provides a reference for the rapid study of the interaction between light and biological tissue.