双光子激发显微镜中的光漂白
Photobleaching in two-photon excitation microscopy.
作者信息
Patterson G H, Piston D W
机构信息
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615, USA.
出版信息
Biophys J. 2000 Apr;78(4):2159-62. doi: 10.1016/S0006-3495(00)76762-2.
The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.
激光扫描显微镜中双光子激发的强度平方依赖性将激发限制在焦平面，并导致厚样品中的光漂白减少。然而，这些实验中使用的高光子通量可能会在焦体积内引发高阶光子相互作用。在单光子和双光子激发下，研究了薄荧光样品（约1微米）的荧光强度和光漂白速率对激发功率的依赖性。正如预期的那样，荧光素单光子激发的激发功率与荧光强度和光漂白速率的对数-对数图以约1的斜率增加。荧光强度与双光子激发功率的类似图以约2的斜率增加。然而，双光子光漂白速率以大于或等于3的斜率增加，表明存在高阶光子相互作用。对Indo-1、NADH和氨基香豆素进行的类似实验产生了类似的结果，并表明这种高阶光漂白在双光子激发显微镜中很常见。因此，使用多光子激发显微镜研究薄样品可能会受到光漂白增加的限制。
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