The aim of this study was to examine the characteristics of skeletal muscle fibers in tail tendons, explore the content of intrinsic components at different depths and ascertain the optimum excitation wavelength, which will help to establish a relationship between diagnosis and therapy and the tendon injury. A multiphoton microscopic imaging system was used to achieve the images and spectra via an imaging mode and a Lambda mode, respectively. This work demonstrates that the skeletal muscle fibers of the tail tendon are in good order. Second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals originating from certain intrinsic components are varied with depth, and the SHG/TPEF intensity ratios are varied at different excitation wavelengths. Below 800 nm is the optimum for cell TPEF, while above 800 nm is the optimum for SHG. With the development of imaging techniques, a nonlinear optical imaging system will be helpful to represent the functional behaviors of tissue related to tendon injury.

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