Department of Physics, Ben-Gurion University of the Negev, Beer Sheva, Israel.
Phys Biol. 2022 Apr 4;19(3). doi: 10.1088/1478-3975/ac5e0f.
Quantifying the absolute protein number using the ratio between the variance and the mean of the protein Fluorescence intensity is a straightforward method for microscopy imaging. Recently, this method has been expanded to fluorescence decaying processes due to photobleaching with binomial distribution. The article examines the method proposed and shows how it can be adapted to the case of variance in the initial number of proteins between the cells. The article shows that the method can be improved by the implementation of the information processing of each frame independently from other frames. By doing so, the variance in determining the protein number can be reduced. In addition, the article examines the management of unwanted noises in the measurement, offers a solution for the shot noise and background noise, examines the expected error caused by the decay constant inaccuracy, and analyzes the expected difficulties in conducting a practical experiment, which includes a non-exponential decay and variance in the photobleaching rate of the cells. The method can be applied to any superposition ofdiscrete decaying processes. However, the evaluation of expected errors in quantification is essential for early planning of the experimental conditions and evaluation of the error.
使用荧光强度的方差与均值之比来定量绝对蛋白质数量是一种用于显微镜成像的简单方法。最近，由于光漂白的二项式分布，该方法已扩展到荧光衰减过程。本文研究了所提出的方法，并展示了如何将其应用于细胞间初始蛋白质数量方差的情况。文章表明，通过独立于其他帧对每一帧的信息进行独立处理，可以改进该方法。通过这样做，可以减少确定蛋白质数量的方差。此外，本文还研究了测量中无用噪声的管理问题，为散粒噪声和背景噪声提供了一种解决方案，检查了由于衰减常数不准确而导致的预期误差，并分析了在进行实际实验时可能遇到的困难，包括非指数衰减和细胞光漂白率的方差。该方法可应用于任何离散衰减过程的叠加。然而，对定量预期误差的评估对于早期规划实验条件和评估误差至关重要。
