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

SUBMITTER: Hur KH 

PROVIDER: S-EPMC4018325 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature