Periodontal disease is one of the main chronic infectious diseases of oral cavity and the principal cause of tooth loss in humans. The main cause of periodontal disease is the bacteria in calculus or plaque to cause the gum inflamed. Therefore detection and removal of subgingival calculus remains important to all periodontal therapy. However, subgingival calculus within the periodontal pocket remains a complicated and potentially delicate structure to be detected and treated with current dental armamentaria, namely dental x-rays and the dental probe. In this study, an innovative subgingival calculus detection system employing autofluorescence imaging has been developed. Feasibility of such a system on the detection of subgingival calculus is investigated in vitro. Autofluorescence imaging is based on two-photon time correlated fluorescence technique by a femtosecond pulsed Ti-sapphire laser.