Provided are a two-photon fluorescence imaging method and system, and an image processing device, which are applicable to the technical field of two-photon fluorescence. The method comprises: performing wavefront shaping on two incident pulsed light beams in sequence to obtain two light beams, light fields of which at a focal point of an objective lens are gradient light fields, and making light intensity distributions of the gradient light fields of the two light beams be opposite; and then scanning a sample to be tested through the two light beams sequentially to obtain two images, and performing three-dimensional data reconstruction according to the two images to obtain a three-dimensional depth image of the sample to be tested. According to the method, the imaging speed is high, the resolution is high, and the algorithm is simple, and the present application is easily implemented; furthermore, since the imaging speed is high, light damage and a photobleaching effect on the sample to be tested are effectively reduced, and the present application is especially applicable to performing three-dimensional imaging on embryonic development samples and neural activity samples.
TWO-PHOTON FLUORESCENCE IMAGING METHOD AND SYSTEM, AND IMAGE PROCESSING DEVICE