Petrásek Zdenek, Schwille Petra
Biophysics Group, Biotechnologisches Zentrum, Technische Universität Dresden, Tatzberg 47-51, 01307 Dresden, Germany.
Chemphyschem. 2008 Jan 11;9(1):147-58. doi: 10.1002/cphc.200700579.
Two-photon excitation in fluorescence correlation spectroscopy (FCS) is often preferred to one-photon excitation because of reduced bulk photobleaching and photodamage, and deeper penetration into scattering media, such as thick biological specimens. Two-photon FCS, however, suffers from lower signal-to-noise ratios which are directly related to the lower molecular brightness achieved. We compare standard FCS with a fixed measurement volume with scanning FCS, where the measurement volume is scanned along a circular path. The experimental results show that photobleaching is the dominant cause of the effects observed at the high excitation powers necessary for good signal-to-noise ratios. Theoretical calculations assuming a nonuniform excitation intensity profile, and using the concept of generalized volume contrast, provide an explanation for the photobleaching effects commonly observed in two-photon FCS at high excitation intensities, without having to assume optical saturation. Scanning alleviates these effects by spreading the photobleaching dose over a larger area, thereby reducing the depletion of fluorescent molecules in the measurement volume. These results, which facilitate understanding of the photobleaching in FCS and of the positive effects of scanning, are particularly important in studies involving the autocorrelation amplitude g(0), such as concentration measurements or binding studies using fluorescence cross-correlation between two labeled species.
在荧光相关光谱学（FCS）中，双光子激发通常比单光子激发更受青睐，这是因为它能减少整体光漂白和光损伤，并且能更深入地穿透散射介质，比如厚的生物样本。然而，双光子FCS存在较低的信噪比，这与所实现的较低分子亮度直接相关。我们将具有固定测量体积的标准FCS与扫描FCS进行比较，在扫描FCS中，测量体积沿着圆形路径进行扫描。实验结果表明，在获得良好信噪比所需的高激发功率下，光漂白是观察到的这些效应的主要原因。假设激发强度分布不均匀并使用广义体积对比度概念进行的理论计算，为双光子FCS在高激发强度下通常观察到的光漂白效应提供了解释，而无需假设光学饱和。扫描通过将光漂白剂量分散在更大的区域来减轻这些效应，从而减少测量体积中荧光分子的消耗。这些结果有助于理解FCS中的光漂白以及扫描的积极作用，在涉及自相关幅度g(0)的研究中尤为重要，例如浓度测量或使用两种标记物种之间的荧光交叉相关进行的结合研究。
