A water-soluble, long fluorescent lifetime DNA probe for real-time dynamic visualization of mitosis in live cells and applicability for FLIM/time-gated imaging

Abstract

Long-lived and photostable fluorescent probes for real-time monitoring of cell division, particularly for dynamic visualization of mitosis and interphase, are rare. We have developed a water-soluble diazaoxatriangulenium cation-based fluorescent probe, Nuc-DAOTA⁺, that meets these criteria. Nuc-DAOTA⁺ has a long fluorescence lifetime (∼20 ns) and exclusively targets the nucleus for specific DNA binding in live cells, essential for real-time dynamic imaging of cell-division, including mitosis and interphase. Addition of dsDNA (0–50 μg mL⁻¹) to Nuc-DAOTA⁺ results in a red shift (∼10 nm) of the absorption and a blue shift (∼5 nm) of the fluorescence maxima, along with an intensity increase in both. A very good linear correlation (R² = 0.999) from the plot of fluorescence intensity versus concentration of dsDNA (up to 30 μg mL⁻¹) resulted in a detection limit of 0.7 μg mL⁻¹. The Benesi–Hildebrand plot was used to calculate the binding constant between the Nuc-DAOTA⁺ and DNA, which was found to be 1.6 × 10⁴ M⁻¹ and the mechanism of binding interactions was investigated using CD spectroscopy. The long fluorescence lifetime and excellent biocompatibility of Nuc-DAOTA⁺ enabled its use for real-time dynamic imaging of mitotic phases and interphase in live CHO (Chinese hamster ovary) cells by using confocal microscopy. In addition, the Nuc-DAOTA⁺ exhibited high photostability during photo-bleaching experiments and was successfully applied for fluorescence lifetime imaging microscopy (FLIM) and time gated imaging of the mouse embryonic fibroblast 3T3 cell line.