title: Quantitative measurement of brightness from living cells in the presence of photodepletion.
abstract: The brightness of fluorescently labeled proteins provides an excellent marker for identifying protein interactions in living cells. Quantitative interpretation of brightness, however, hinges on a detailed understanding of the processes that affect the signal fluctuation of the fluorescent label. Here, we focus on the cumulative influence of photobleaching on brightness measurements in cells. Photobleaching within the finite volume of the cell leads to a depletion of the population of fluorescently labeled proteins with time. The process of photodepletion reduces the fluorescence signal which biases the analysis of brightness data. Our data show that even small reductions in the signal can introduce significant bias into the analysis of the data. We develop a model that quantifies the bias and introduce an analysis method that accurately determines brightness in the presence of photodepletion as verified by experiments with mammalian and yeast cells. In addition, photodepletion experiments with the fluorescent protein EGFP reveal the presence of a photoconversion process, which leads to a marked decrease in the brightness of the EGFP protein. We also identify conditions where the effect of EGFP's photoconversion on brightness experiments can be safely ignored.
authors.affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Kwang-Ho
authors.initials: KH
authors.lastname: Hur
authors.affiliation: Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Macdonald
authors.affiliation: Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Serkan
authors.initials: S
authors.lastname: Berk
authors.affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: C Isaac
authors.initials: CI
authors.lastname: Angert
authors.affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Joachim D
authors.initials: JD
authors.lastname: Mueller
mesh_terms: D000818:Animals; D016477:Artifacts; D002460:Cell Line; D002470:Cell Survival; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D038761:Photobleaching; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000166:cytology*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins