School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, USA.
School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, USA; Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA; Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, USA.
Biophys J. 2023 Jan 3;122(1):241-253. doi: 10.1016/j.bpj.2022.10.027. Epub 2022 Oct 20.
The experimental autocorrelation function of fluorescence correlation spectroscopy calculated from finite-length data is a biased estimator of the theoretical correlation function. This study presents a new theoretical framework that explicitly accounts for the data length to allow for unbiased analysis of experimental autocorrelation functions. To validate our theory, we applied it to experiments and simulations of diffusion and characterized the accuracy and precision of the resulting parameter estimates. Because measurements in living cells are often affected by instabilities of the fluorescence signal, autocorrelation functions are typically calculated on segmented data to improve their robustness. Our reformulated theory extends the range of usable segment times down to timescales approaching the diffusion time. This flexibility confers unique advantages for live-cell data that contain intensity variations and instabilities. We describe several applications of short segmentation to analyze data contaminated with unwanted fluctuations, drifts, or spikes in the intensity that are not suited for conventional fluorescence correlation analysis. These results demonstrate the potential of our theoretical framework to significantly expand the experimental systems accessible to fluorescence correlation spectroscopy.
荧光相关光谱学的实验自相关函数是从有限长度数据计算得出的，它是理论相关函数的有偏估计。本研究提出了一个新的理论框架，明确考虑了数据长度，从而可以对实验自相关函数进行无偏分析。为了验证我们的理论，我们将其应用于扩散的实验和模拟中，并对得到的参数估计的准确性和精度进行了表征。由于活细胞中的测量通常受到荧光信号不稳定性的影响，因此通常会对分段数据进行自相关函数计算，以提高其稳健性。我们重新制定的理论将可用分段时间的范围扩展到接近扩散时间的时间尺度。这种灵活性为包含强度变化和不稳定性的活细胞数据带来了独特的优势。我们描述了几种短分段的应用，以分析因强度的不希望的波动、漂移或尖峰而受到污染的数据，这些数据不适合传统的荧光相关分析。这些结果表明，我们的理论框架有可能显著扩展可用于荧光相关光谱学的实验系统。
