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 of n_0 discrete decaying processes. However, the evaluation of expected errors in quantification is essential for early planning of the experimental conditions and evaluation of the error.
Quantifying protein number using the ratio between the variance and the mean of the protein distribution is a straightforward calibration method in the experimental conditions for microscopy imaging. Recently the model has been expanded to decaying processes with binomial distribution. In this paper, we examine the model proposed, and show how the algorithm can be adapted to the case of variance in the initial number of proteins between cells. We propose improving the algorithm so that the information processing of each frame is done independently from other frames. By doing so, the variance in the process of determining the protein number can be reduced. In addition, we examine the handling of unwanted noises in the measurement, offer a solution for shot noise and background noise, and examine the expected error caused in calculating the decay constant. We also analyze the expected difficulties in conducting a practical experiment, which includes non-exponential decay, and variance in the decay constants of the cells. These methods can be applied to any superposition of n_0 discrete decaying processes. However, the evaluation of expected errors in quantification is essential for early planning of the experimental conditions, and for the evaluation of the error.
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