Tauer Ulrike
Leica Microsystems Heidelberg GmbH, Mannheim, Germany.
Exp Physiol. 2002 Nov;87(6):709-14. doi: 10.1113/eph8702464.
Multiphoton microscopy is based on the simultaneous absorption of two photons emitted by a pulsed infrared laser source. In this technique, the excitation is restricted to a very small focus and thus results in optical sectioning a priori without the need of a confocal aperture. Multiphoton microscopy was introduced in live cell imaging as an alternative to confocal microscopy due to its superior qualities, such as the deep penetration depth, the reduced photodamage and the lack of out-of-focus bleaching. However, during the past years, examinations revealed severe limitations to the initial expectations. In the focal plane, photodamage and photobleaching can be worse than in single photon microscopy. However, studies showed that with low excitation intensity and by special technical adaptations photodamage could be avoided successfully. For functional biological imaging multiphoton excitation provides an excellent tool such as the release of caged compounds in a diffraction-limited volume combined with multiphoton or confocal imaging.
多光子显微镜基于脉冲红外激光源发射的两个光子的同时吸收。在这项技术中，激发被限制在一个非常小的焦点上，因此无需共焦孔径就能先验地实现光学切片。由于其诸如深穿透深度、减少的光损伤和无离焦漂白等优越特性，多光子显微镜作为共聚焦显微镜的替代技术被引入活细胞成像。然而，在过去几年中，研究发现其存在严重局限，与最初的预期不符。在焦平面上，光损伤和光漂白可能比单光子显微镜更严重。不过，研究表明，通过低激发强度和特殊的技术调整，可以成功避免光损伤。对于功能性生物成像，多光子激发提供了一个出色的工具，例如在衍射极限体积内结合多光子或共聚焦成像释放笼形化合物。
