Automated analysis of time-lapse imaging of nuclear translocation by retrospective strategy and its application to STAT1 in HeLa cells.

Cell-based image analysis of time-lapse imaging is mainly challenged by faint fluorescence and dim boundaries of cellular structures of interest. To resolve these bottlenecks, a novel method was developed based on "retrospective" analysis for cells undergoing minor morphological changes during time-lapse imaging. We fixed and stained the cells with a nuclear dye at the end of the experiment, and processed the time-lapse images using the binary masks obtained by segmenting the nuclear-stained image. This automated method also identifies cells that move during the time-lapse imaging, which is a factor that could influence the kinetics measured for target proteins that are present mostly in the cytoplasm. We then validated the method by measuring interferon gamma (IFNγ) induced signal transducers and activators of transcription 1 (STAT1) nuclear translocation in living HeLa cells. For the first time, automated large-scale analysis of nuclear translocation in living cells was achieved by our novel method. The responses of the cells to IFNγ exhibited a significant drift across the population, but common features of the responses led us to propose a three-stage model of STAT1 import. The simplicity and automation of this method should enable its application in a broad spectrum of time-lapse studies of nuclear-cytoplasmic translocation.