飞秒激光双光子荧光显微系统的构建与应用

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

摘要:

为进行双光子荧光显微成像研究,搭建了一套飞秒激光光源双光子荧光显微成像系统．对超短脉冲锁模激光器的成像优势、双光子激励饱和功率及系统分辨率进行了理论推导,利用飞秒激光器、显微镜、数据采集设备与控制装置及扫描控制软件搭建了显微成像系统,并对Rhodamine B样品进行双光子荧光显微成像实验．结果表明:相同条件下,超短脉冲锁模激光器的双光子荧光产率为连续光输出激光器的10⁵倍;采用UPLSAPO60XO型物镜时,双光子激励饱和功率为50 mW,理论横向和轴向分辨率为303 nm与727 nm;该系统具有显微成像能力,且实际横向分辨率小于3 μm．

Abstract:

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.