B. BIOMEDICAL MULTIPHOTON MICROSCOPY

This imaging equipment has been explained in detail in our previous work [18]. All the multiphoton images were captured via an microscope (LSM 510, Zeiss, Germany) which was inverted and equipped with a femtosecond Ti:sapphire laser (Mira 900-F, Coherent, Inc., USA). A Zeiss Plan Apochromat oil-immersion objective lens (63×, NA=1.4) was applied to acquire high-resolution images, and the laser power at the focal plane of the objective is about 10mW. In this study, TPAF signal (a wavelength range of 430–716 nm) and SHG signal (a wavelength range from 389 to 419 nm) were collected respectively by a META detector using 810 nm wavelength laser. For this imaging system, a lateral resolution of 0.3μm could be achieved, and it takes 1.8μs for collecting each pixel and therefore the scanning speed is 2 frames per second (512 × 512 pixels/135μm×135μm). We can acquire large-area MPM image through automatically assembling an array of images with 512 × 512 pixels via ZEN imaging software.