Quantitative Brightness Analysis of Fluorescence Intensity Fluctuations in E. Coli.

ABSTRACT: The brightness measured by fluorescence fluctuation spectroscopy specifies the average stoichiometry of a labeled protein in a sample. Here we extended brightness analysis, which has been mainly applied in eukaryotic cells, to prokaryotic cells with E. coli serving as a model system. The small size of the E. coli cell introduces unique challenges for applying brightness analysis that are addressed in this work. Photobleaching leads to a depletion of fluorophores and a reduction of the brightness of protein complexes. In addition, the E. coli cell and the point spread function of the instrument only partially overlap, which influences intensity fluctuations. To address these challenges we developed MSQ analysis, which is based on the mean Q-value of segmented photon count data, and combined

SUBMITTER: Hur KH 

PROVIDER: S-EPMC4476568 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature