Variance reducing and noise correction in protein quantification by measuring fluctuations in fluorescence due to photobleaching.

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.