Ruthenium(II) Complex Enantiomers as Cellular Probes for Diastereomeric Interactions in Confocal and Fluorescence Lifetime Imaging Microscopy

Abstract

Ruthenium dipyridophenazine (dppz) complexes are sensitive luminescent probes for hydrophobic environments. Here, we apply multiple-frequency fluorescence lifetime imaging microscopy (FLIM) to Δ and Λ enantiomers of lipophilic ruthenium dppz complexes in live and fixed cells, and their different lifetime staining patterns are related to conventional intensity-based microscopy. Excited state lifetimes of the enantiomers determined from FLIM measurements correspond well with spectroscopically measured emission decay curves in pure microenvironments of DNA, phospholipid membrane or a model protein. We show that FLIM can be applied to monitor the long-lived excited states of ruthenium complex enantiomers and, combined with confocal microscopy, give new insight into their biomolecular binding and reveal differences in the microenvironment probed by the complexes.

Figure 1

Representative transmission (upper) and emission confocal laser scanning (middle) images of the intracellular distribution of Δ- and Λ-D4 in fixed CHO-K1 cells. The lower panel shows intensity profile plots resolving intracellular differences in emission intensity of the two enantiomers. The black horizontal bars indicate the location of the nucleus. Laser intensity and photo multiplier gain are the same in both images, and intensities are thus directly comparable.

Figure 2

False color fluorescence lifetime images (left), emission intensity images (middle) and transmission images (right) of Δ- and Λ-D4 in fixed CHO-K1 cells showing the enantiospecific difference in apparent intracellular lifetime as well as their different lifetime distributions. FLIM color bars: 200–360 ns.

Figure 3

False color fluorescence lifetime images of membrane bound Δ- and Λ-D4 in live CHO-K1 cells before (left) and after (middle) photoactivated uptake. The fluorescence intensity images (right) correspond to the lifetime images after uptake. FLIM color bars 75–250 ns.

Chart 1

Molecular structure of Δ- and Λ-D4. Ruthenium are shown in orange, nitrogen in purple and oxygen in cyan. Hydrogens are omitted for clarity.