Technical Advancement and Biomedical Application of Multiphoton Excitation Microscopy

Fluorescence microscopic techniques are widely used in studies for biology and medicine as a powerful tool to visualize morphology and distribution of target molecules in cells and tissues. Laser scanning confocal microscopy enables to obtain optical sectioned high contrast images of biological samples with fluorescent labeling. Multiphoton excitation microscopy is a promising technique for intravital imaging in both basic biology and clinical medicine. Multiphoton excitation is a nonlinear optical phenomenon based on simultaneous absorption of two or more photons by irradiation of ultra-short pulse laser. In particular, nonlinear optical imaging technique has make it possible to analyze deep portions of tissues. Furthermore, second harmonic generation, a nonlinear optical process, is also useful for label-free imaging of collagen in biological specimen. Here we performed intravital imaging to reveal the mechanism of chemotherapy induced alopecia, one of the side effect in anticancer drugs, using multiphoton excitation microscopy in mice. In the result, we observed alterations in the vascular structure and the hair papillae of subcutaneous tissue treated with cyclophosphamide. The technical development in multiphoton excitation microscopy implies a practical tool not only for bioimaging analysis but also for future clinical applications.