Single molecules are the building blocks of life. Probing single molecules in action in living cells, a formidable task until now, promotes a basic understanding of the biophysical mechanism of living matter. Here, we advanced single-molecule imaging as a comprehensive tool for quantitative measurements in vivo. Using bacterial gene expression as a model system, we monitored intracellular dynamics with high spatiotemporal precision and established analytical models to comprehend the workings of life.
To probe gene regulation dynamics, we studied both how and how fast a single transcription factor molecule searches for its binding site on DNA. Through direct observation of diffusion and binding of the classic lac repressor in vivo, we characterized its search time, as well as the kinetics during the search process. Quantitative agreement between the search time and the search mechanism was met with the aid of analytical modeling of the reaction-diffusion dynamics in a crowded cell.
To probe fluctuations subsequent to gene regulation, we profiled the variation and co-variation of gene expression products in individual E. coli cells. Using fluorescence in situ hybridization, we established simultaneous detection and digital counting of mRNA and protein. A complete lack of correlation between these two products of the same gene was observed in single cells, shedding light on the source of gene expression noise.
To put gene expression in the spatial context, we mapped out the subcellular organization of bacterial gene expression machineries. Capitalizing on the single-molecule based super-resolution imaging techniques, we determined the localization of global transcription factors, RNA polyermases, RNases, and ribosomes with nanometer resolution. Distinct patterns were observed for molecular complexes responsible for gene activation or silencing, suggesting a novel mechanism of gene regulation.
Beyond bacterial gene expression, we further developed single-molecule methods for higher eukaryotic cells. With a combination of two-photon excited fluorescence and sensitive detection, we imaged, for the first time, the binding of single Klf4 transcription factors in a mouse embryonic stem cell. Advancing these techniques and analytical modeling will open up new possibilities in quantitative biology.