Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy
Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy
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DOI：
10.1016/s0006-3495(02)73990-8
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发表时间：
2002-10-01
影响因子：
3.4
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Hess, ST;Webb, WW
荧光相关光谱（FCS）可以在广泛的时间尺度（1秒）范围内提供丰富的生物和化学系统信息。然而，FCS观测体的数值模拟结合测量结果表明，在许多常见的测量条件下，三维高斯FCS观测体的标准假设并不是有效的近似值。因此，FCS自相关将包含重要的系统伪影，当使用大探测器孔径和孔径限制照明时，这些伪影在共聚焦光学中最为严重。这些光学伪影在荧光相关性中表现为明显的附加指数成分或具有显著（bbb30 %）振幅的扩散物质，这可能意味着外来动力学，将测量的扩散时间移动多达相似的80%，并导致轴向比发散。伪影可以最小化或几乎消除通过使用一个小的共聚焦探测器孔径，未填充物镜后孔径，或双光子激发。然而，使用比最佳值（类似于4.5光学单位）更小或更大的检测器孔径大大降低了每个分子的计数率和信噪比。因此，在单光子FCS中，存在优化信噪比和减少实验伪影之间的权衡。
Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (1 s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions. As a result, the FCS autocorrelation will contain significant, systematic artifacts that are most severe with confocal optics when using a large detector aperture and aperture-limited illumination. These optical artifacts manifest themselves in the fluorescence correlation as an apparent additional exponential component or diffusing species with significant (>30%) amplitude that can imply extraneous kinetics, shift the measured diffusion time by as much as similar to80%, and cause the axial ratio to diverge. Artifacts can be minimized or virtually eliminated by using a small confocal detector aperture, underfilled objective back-aperture, or two-photon excitation. However, using a detector aperture that is smaller or larger than the optimal value (similar to4.5 optical units) greatly reduces both the count rate per molecule and the signal-to-noise ratio. Thus, there is a tradeoff between optimizing signal-to-noise and reducing experimental artifacts in one-photon FCS.