For biological imaging, two-photon lifetime imaging was applied for melanin imaging (Krasieva; et al. J. Biomed. Opt. 18 (3), 031107). Furthermore, Yang et al. (J. Biomed. Opt. 18 (3), 031104) showed that cell diagnosis in mouse cochlea can be achieved with two-photon microscopy, and infection was studied in two and three dimensions with ultrashort pulse microscopy by Gibbs et al. (J. Biomed. Opt. 18 (3), 031111). In medical optics, Burke et al. (J. Biomed. Opt. 18 (3), 031106) studied matrix alterations due to the progression of breast tumors, and Zhuang and collaborators (J. Biomed. Opt. 18 (3), 031103) used micro-Raman spectroscopy in studying renal tumors. Moreover, Yasui et al. (J. Biomed. Opt. 18 (3), 031108) used 1250 nm as the light source to induce second harmonic generation from skin dermis to study skin aging. Finally, ultrasound and nonlinear optical imaging was combined to study the structure of the human cervix (Reusch et al. (J. Biomed. Opt. 18 (3), 031110)). The manuscripts described in this special section demonstrate the wide array of problems researchers can address with multiphoton technology. With continual technical development, one can expect additional biological and medical applications to be found in the future.