Wide field video-rate two-photon imaging by using spinning disk beam scanner

The microscope technology with wider view field, deeper penetration depth, higher spatial resolution and higher imaging speed are required to investigate the intercellular dynamics or interactions of molecules and organs in cells or a tissue in more detail. The two-photon microscope with a near infrared (NIR) femtosecond laser is one of the technique to improve the penetration depth and spatial resolution. However, the video-rate or high-speed imaging with wide view field is difficult to perform with the conventional two-photon microscope. Because point-to-point scanning method is used in conventional one, so it’s difficult to achieve video-rate imaging. In this study, we developed a two-photon microscope with spinning disk beam scanner and femtosecond NIR fiber laser with around 10 W average power for the microscope system to achieve above requirements. The laser is consisted of an oscillator based on mode-locked Yb fiber laser, a two-stage pre-amplifier, a main amplifier based on a Yb-doped photonic crystal fiber (PCF), and a pulse compressor with a pair of gratings. The laser generates a beam with maximally 10 W average power, 300 fs pulse width and 72 MHz repetition rate. And the beam incident to a spinning beam scanner (Yokogawa Electric) optimized for two-photon imaging. By using this system, we achieved to obtain the 3D images with over 1mm-penetration depth and video-rate image with 350 x 350 um view field from the root of Arabidopsis thaliana.