Application of Time-Resolved Fluorescence Microscopy for Enhancing the Selectivity of Fluorogenic Dyes of the Arylidene–Imidazolone Series toward the Endoplasmic Reticulum.

Objective: A number of previously synthesized fluorogenic arylidene-imidazolones, which predominantly stain the endoplasmic reticulum (ER) of living cells, were studied by time-resolved fluorescence microscopy. It was suggested that the use of fluorescence microscopy of this type can enhance the selectivity of ER staining. Methods: The lifetimes of arylidene-imidazolone compounds in set of solvents with different polarity were measured with time-correlated single photon counting spectroscopy. Live HeLa Kyoto cells were stained with studied dyes and analyzed with time-resolved fluorescence microscopy. Results and discussion: It was found that most of the studied compounds show bi- or triexponential fluorescence decay patterns in solutions. In live cell culture dye (I) showed a monoexponential decay pattern, while dyes (II–IV) were better fitted by a biexponential function. Dyes (I), (III), and (IV) stained both ER and adiposomes, while dye (II) stained only ER. Conclusions: It is shown that under FLIM conditions discriminative filtering of cellular organelles stained with studied fluorogenic dyes is possible and applicable if the difference of mean amplitude-weighted lifetime is more than 0.1 ns, thus increasing the selectivity of ER staining in live cells. [ABSTRACT FROM AUTHOR]