Abstract：The blue shift of a two-photon excitation absorption peak allows the application of a single wavelength to the simultaneous excitation of several fluorochromes with disparate emission characteristics. An output at 730nm of a mode-locked femtosecond Ti-sapphire laser was used to excite four different commonly used fluorochromes, namely Hoechst 33342, Fluo-4, PI and Indo-1, and characteristic fluorescence images were obtained using (455±15)nm, (540±15)nm, (580±16)nm and (500±15)nm filters respectively. An approach to exciting with a single wavelength, staining with two different fluorochromes, and collecting fluorescence in two separate channels was employed to study mouse preimplantation embryos by 3D and 4D real-time imaging. Combined with the merits of two-photon excitation fluorescence imaging, such as better penetration, less photon-damage, and higher signal-to-noise ratio, this approach was supposed to be a novel multi-parameter investigating tool for mouse preimplantation embryos development study.
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