Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy
Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy
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DOI：
10.1529/biophysj.105.072322
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发表时间：
2006-01-01
影响因子：
3.4
通讯作者：
Wiseman, PW
中科院分区：
文献类型：
--
作者：
Kolin, DL;Costantino, S;Wiseman, PW
我们提出了一个广泛的调查的准确性和精度的时间图像相关光谱（TICS）。使用模拟的激光扫描显微镜图像时间序列，我们调查的时空采样，粒子密度，噪声，采样频率，和荧光团的光漂白的恢复的输运系数和数密度的TICS的效果。我们发现，恢复的输运系数通常是有限的空间采样，而准确的数密度的测量受到限制的背景噪声的图像序列。我们还表明，荧光团的光漂白导致一致的高估的扩散系数和流速，和严重低估的数密度。我们推导出一个漂白校正方程，消除这两个偏见时，用于拟合时间自相关函数，而不增加拟合参数的数量。最后，我们的图像与EGFP/α-辅肌动蛋白的CHO细胞的基底膜，使用双光子显微镜，并分析了一个子区域，这一系列使用TICS和应用漂白校正。我们表明，光漂白校正可以简单地通过使用从时间序列的平均图像强度来确定，我们使用的模拟提供良好的估计与TICS测量的数密度和输运系数的准确性和精度。
We present an extensive investigation of the accuracy and precision of temporal image correlation spectroscopy (TICS). Using simulations of laser scanning microscopy image time series, we investigate the effect of spatiotemporal sampling, particle density, noise, sampling frequency, and photobleaching of fluorophores on the recovery of transport coefficients and number densities by TICS. We show that the recovery of transport coefficients is usually limited by spatial sampling, while the measurement of accurate number densities is restricted by background noise in an image series. We also demonstrate that photobleaching of the fluorophore causes a consistent overestimation of diffusion coefficients and flow rates, and a severe underestimation of number densities. We derive a bleaching correction equation that removes both of these biases when used to fit temporal autocorrelation functions, without increasing the number of fit parameters. Finally, we image the basal membrane of a CHO cell with EGFP/ alpha-actinin, using two-photon microscopy, and analyze a subregion of this series using TICS and apply the bleaching correction. We show that the photobleaching correction can be determined simply by using the average image intensities from the time series, and we use the simulations to provide good estimates of the accuracy and precision of the number density and transport coefficients measured with TICS.