Photobleaching in two-photon excitation microscopy
Photobleaching in two-photon excitation microscopy
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DOI：
10.1016/s0006-3495(00)76762-2
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发表时间：
2000-04-01
影响因子：
3.4
通讯作者：
Piston, DW
中科院分区：
文献类型：
--
作者：
Patterson, GH;Piston, DW
在激光扫描显微镜中，双光子激发的强度平方依赖性限制了对焦平面的激发，并导致厚样品中光漂白的减少。然而，这些实验中使用的高光子通量可能会导致焦点体积内的高阶光子相互作用。在单光子和双光子激发下，研究了薄荧光样品（类似于1 μ m）的荧光强度和光漂白速率的激发功率依赖性。正如预期的那样，对于荧光素的单光子激发，激发功率与荧光强度和光漂白速率的对数-对数曲线以类似于1的斜率增加。荧光强度与双光子激发功率的类似曲线以类似于2的斜率增加。然而，双光子光漂白速率增加的斜率大于或等于3，表明存在更高阶的光子相互作用。对Indo-1、NADH和氨基香豆素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限制，增加光漂白。
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 (similar to 1 mu m) 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 similar to 1.A similar plot of the fluorescence intensity Versus two-photon excitation power increased with a slope of similar to 2. However, the two-photon photobleaching rate increased with a slope greater than or equal to 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.