The multi-photon microscope comprises a repetition rate tuner that lowers an optical pulse train emitted from a pulsed laser to a repetition rate for time-gated detection, a scanner that scans the optical pulse train transmitted from the repetition rate tuner in x-axis and y-axis directions, an objective lens that irradiates an optical signal scanned by the scanner to the sample and acquires a fluorescence signal emitted from the excited fluorescent material, a photodetector that photoelectrically converts the fluorescence signal acquired by the objective lens, an amplifier that converts a current signal output from the photodetector into a voltage signal, a digitizer that samples the voltage signal output from the amplifier, and a computing device that separates sampling data output from the digitizer with a detection window set in time domain, and generates an image based on the sampling data separated by the detection window.