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 ofn0discrete decaying processes. However, the evaluation of expected errors in quantification is essential for early planning of the experimental conditions and evaluation of the error.

Stochastic nucleoid segregation dynamics as a source of the phenotypic variability in E. coli.

Segregation of the replicating chromosome from a single to two nucleoid bodies is one of the major processes in growing bacterial cells. The segregation dynamics is tuned by intricate interactions with other cellular processes such as growth and division, ensuring flexibility in a changing environment. We hypothesize that the internal stochasticity of the segregation process may be the source of cell-to-cell phenotypic variability, in addition to the well-established gene expression noise and uneven partitioning of low copy number components. We compare dividing cell lineages with filamentous cells, where the lack of the diffusion barriers is expected to reduce the impact of other factors on the variability of nucleoid segregation dynamics. The nucleoid segregation was monitored using time-lapse microscopy in live E. coli cells grown in linear grooves. The main characteristics of the segregation process, namely, the synchrony of partitioning, rates of separation, and final positions, as well as the variability of these characteristics, were determined for dividing and filamentous lineages growing under the same conditions. Indeed, the gene expression noise was considerably homogenized along filaments as determined from the distribution of CFP and YFP stochastically expressed from the chromosome. We find that 1) the synchrony of nucleoid partitioning is progressively decreasing during consecutive cell cycles, but to a significantly lesser degree in filamentous than in dividing cells; 2) the mean partitioning rate of nucleoids is essentially the same in dividing and filamentous cells, displaying a substantial variability in both; and 3) nucleoids segregate to the same distances in dividing and filamentous cells. Variability in distances is increasing during successive cell cycles, but to a much lesser extent in filamentous cells. Our findings indicate that the variability of the chromosome segregation dynamics is reduced upon removal of boundaries between nucleoids, whereas the remaining variability is essentially inherent to the nucleoid itself.