双光子激发荧光显微是一种非线性光学显微技术, 它结合了激光扫描共聚焦显微镜和双光子激发技术, 具有高时空分辨率、高信噪比和固有的三维层析分辨能力等优点。介绍了双光子显微镜的软硬件组成和技术参数, 样品制备方法, 双光子图像采集的常用操作规程, 日常维护和仪器管理等方面。旨在为双光子仪器的使用者与管理者提供参考, 使之更好地服务于教学与科研。

Two photon excited fluorescence(TPEF) microscopy is a nonlinear optical microscopy technique, which combines laser scanning confocal microscopy and two-photon excitation technique. The main advantages of TPEF include high temporal and spatial resolutions, high signal-to-noise ratio and inherent three-dimensional sectioning. To provide good references for the users and managers of TPEF microscopy, the Olympus inverted two-photon microscopy (IX81-FV1000MPE) configuration, software and hardware composition, technical parameters, sample preparation requirements, common operating procedures, as well as the routine maintenance and management are introduced in this article. This study can be very useful for the improvement of the equipment efficiency for both teaching and scientific research in universities.