Construction and application of femtosecond laser two-photon fluorescence microscopy system

By using a femtosecond laser as the excitation source, a two-photon fluorescence microscopy system was constructed to conduct some researches on two-photon microcsopy. Firstly, the advantage of introducing an ultrashort pulse mode-locked laser was verified, the definite expression of saturation power was calculated, and the resolution of two-photon fluorescence microscopy was deduced. Then based on the femtosecond laser, microscope, data acquisition device, control equipment and the software that we wrote, a two-photon fluorescence microscopy system was built. At last, the two-photon fluorescence microscopy system is applied into the microscopy study of Rhodamine B specimen. The results show that in the same condition, two-photon excitation productivity is as much as 10⁵ times with an ultrashort pulse mode-locked laser compared with a CW laser. By using the UPLSAPO60XO objective, the saturation power is 50 mW while the radial and the axial resolution reach 303 nm and 727 nm. The image ability of the system is proved by the results, and the actual radial resolution is less than 3 μm.