Time-resolved fluorescence imaging of methylene blue reveals heterogeneous polarity in living cells

The paper presents time-resolved fluorescence analysis of methylene blue (MB) in solutions and in living cells in vitro. The analysis of MB fluorescence lifetime in solutions of different pH, viscosity and polarity revealed its independence on acidity and viscosity and linear rise with decreasing polarity. Moreover, MB binding to albumin and DNA did not affect its fluorescence lifetime. The obtained dependence of MB fluorescence lifetime on the Lippert-Mataga polarity parameter enabled analysis of polarity distributions in living cells. Fluorescence-lifetime images of MB fluorescence in cancerous HeLa and pseudo-normal bEnd.3 cells provided clear double-exponential signals, which were suggested to be due to diversity of polarity in different cell compartments. The longer fluorescence lifetime and its contribution were shown to differ in cells of different lines, that allowed us to suggest that polarity of low-polar structures and their amount differ in cells of these lines. In cells of both lines the fluorescence lifetimes in nuclei were shorter than those in cytoplasm. The combined analysis of fluorescence lifetimes and phasor plot coordinates allowed for segmentation of the intracellular area to regions of different polarity corresponding to nuclei and cytoplasm with the accuracy of about 90%, and to reveal differences in cells of the two lines.