Physical Sciences and Mathematics Commons^™

Absolute Quantification Of Gene Expression In Individual Bacterial Cells Using Two-Photon Fluctuation Microscopy, Matthew L. Ferguson, Dominique Le Coq, Matthieu Jules, Stéphane Aymerich, Nathalie Declerck, Catherine A. Royer

Matthew L. Ferguson

Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We …