双光子显微镜在生物医学中的应用及其进展

光学显微镜的发展历史是一段不断提高显微镜的分辨率和对比度的历史。双光子显微镜是近30年来非线性显微镜的研究发展的代表。它在分辨率上与共聚焦显微镜相当，但在成像的层析穿透深度上有显著提高，并且大大减少了光毒性与光漂白。由于生物细胞组织中富有各种自家荧光源，因此双光子显微镜被广泛应用于皮肤组织甚至癌组织以及细胞的成像。基于共聚焦扫描显微镜的双光子显微镜可以很容易的与二次谐波显微镜组合，对皮肤组织中的重要成分胶原纤维进行成像。双光子显微镜还可以结合其他非线性光学现象对组织以及细胞进行成像，显示其强大的生命力。将来随着携带方便且廉价的双光子显微镜的出现，双光子显微镜有望在临床医学上发挥其有效的作用。

The Applications of Two-photon Microscopy in Biomedicine and Its Development

The history of the development of the light microscopy was a history of constant improvement of the resolution and the contrast of the microscopy. Two photon microscopy represented the development of nonlinear microscopy in the recent 30 years. The resolution limit of two photon microscopy was close to that of confoeal microscopy, whereas with nsing two photon microscopy the imaging contrast and depth of intact sectioning had been improved obviously. Furthermore phototoxicity and photobleaching had been reduced in large extent. Because there were many kinds of autofluorescent fluorophores in tissue and cell, two photon microscopy had been widely applied in imaging of skin tissue, even cancerous tissue and cell. Based on laser scanning confocal microscopy, two photon microscopy can be combined easily with second harmonic generation microscopy, so the important component of skin tissue including collagen fiber can be imaged simultaneously. Besides, two photon microscopy can also couple with other nonlinear optical phenomenon for imaging tissues and living cells, which implies its prospective development in future. Following by the appearance of portable and low-cost two photon devices, we expect the effective use of the two photon microscopy in the clinical medicine.