Abstract: Because of the complexities and varieties of biological tissues, and the influences of sample preparation, the backward SHG and TPEF of different tissues are different. In this thesis, rat-tails were used as experiment objects, there are 40 slices including transverse and longitudinal, HE-dyed and undyed. Excited by femtosecond laser, the changes of backward SHG and TPEF versus sample preparation, excitation wavelength, excitation power, penetration depth and so on were observed by Two-photon Laser Scanning Confocal Microscope(TPLSCM), effect factors of backward SHG and TPEF and differences of both were discussed and compared, and related comments were also presented.