Lu Sijia, Chen Ji-Yao, Zhang Yu, Ma Jiong, Wang Pei-Nan, Peng Qian
Fudan University, Department of Physics, 200433, Shanghai, China.
J Biomed Opt. 2008 Mar-Apr;13(2):024014. doi: 10.1117/1.2907316.
Photodynamic therapy (PDT) involves a combination of a lesion-localizing photosensitizer with light and has been established as a new modality for some medical indications. Much evidence has shown the correlation between subcellular localization of a photosensitizer with its photodynamic efficiency. However, the fluorescence of most photosensitizers in cells is weak and easily photobleached. We compare the effect of single-photon excitation (SPE) with that of two-photon excitation (TPE) on fluorescence detection of protoporphyrin IX (PpIX), a potent photosensitizer, in the PLC hepatoma cells in vitro. By using laser scanning confocal fluorescence microscopy, both fluorescence images and spectra of intracellular PpIX are studied with SPE of 405- and 488-nm lasers, and TPE of 800-nm femtosecond laser. The 405-nm laser is more efficient at exciting PpIX fluorescence than the 488-nm laser, but causes a considerable photobleaching of the PpIX fluorescence and induces weak autofluorescence signals of native flavins in the cells as well. The 800-nm TPE is found to significantly improve the quality of PpIX fluorescence images with negligible PpIX photobleaching and minimized endogenous autofluorescence, indicating the potential of 800-nm TPE for studying cellular localization of porphyrin photosensitizers for PDT.
光动力疗法（PDT）是将一种可定位病变的光敏剂与光相结合，已被确立为用于某些医学适应症的一种新方法。大量证据表明，光敏剂的亚细胞定位与其光动力效率之间存在关联。然而，大多数光敏剂在细胞内的荧光较弱且容易发生光漂白。我们比较了单光子激发（SPE）和双光子激发（TPE）对体外培养的PLC肝癌细胞中强效光敏剂原卟啉IX（PpIX）荧光检测的效果。通过使用激光扫描共聚焦荧光显微镜，利用405纳米和488纳米激光的单光子激发以及800纳米飞秒激光的双光子激发，研究了细胞内PpIX的荧光图像和光谱。405纳米激光在激发PpIX荧光方面比488纳米激光更有效，但会导致PpIX荧光发生相当程度的光漂白，同时也会诱导细胞内天然黄素产生微弱的自发荧光信号。研究发现，800纳米双光子激发能显著提高PpIX荧光图像的质量，PpIX光漂白可忽略不计，内源性自发荧光也降至最低，这表明800纳米双光子激发在研究用于光动力疗法的卟啉类光敏剂的细胞定位方面具有潜力。
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