1.

Introduction

Detailed monitoring of a physiological process in living cells is one of the main goals in biological and medical sciences. To this aim, laser scanning confocal fluorescence microscopy (LSCFM) offers higher resolution and sensitivity than conventional fluorescence microscopy and allows 3-D analysis. Among commercial dyes, few fluorophores are characterized as mitochondrial stains, most of them being potentiometric. 2,5-Bis[1-(4- $N$ -methylpyridinium)ethen-2-yl)]- $N$ -methylpyrrole ditriflate (PEPEP) (see Fig. 1 ) is a representative chromophore of a large family of heterocyclic fluorescent dyes that shows fluorescence emission even in aqueous media, an important prerequisite for cellular fluorescence microscopy.^{1, 2} In vitro interaction studies with biomolecules show that PEPEP has DNA affinity.³ In view of possible applications of PEPEP as a fluorescent marker in living cells, we checked its cytotoxicity and intracellular localization in different cell cultures such as human umbilical vein endothelial cells (HUVEC), human lymphoblast U937, immortalized monkey kidney COS7, and yeast Saccharomyces cerevisiae.

Fig. 1

Molecular structure of PEPEP.

2.

Materials and Methods

3.

Results and Discussion

PEPEP cytotoxicity has been evaluated using the assay based on MTT reduction via mitochondrial dehydrogenase activity of living cells. Up to $48\mathrm{h}$ of incubation with $5\text{-}\mu \mathrm{M}$ PEPEP, the growth of U937 (Fig. 2 ) and HUVEC is not affected, showing that the new dye does not compromise cell viability and can be used as nucleic acid probe in living cells.

Fig. 2

PEPEP viability test. U937 were treated with dye solution $\left({10}^{-5}\mathrm{M}\right)$ for 24 and $48\mathrm{h}$ , followed by $4\mathrm{h}$ of MTT reduction at $37°\mathrm{C}$ . Absorbance values were determined at $570\mathrm{nm}$ . Absorbance is directly related to viable cell numbers.

The intracellular localization of PEPEP in different cell lines was investigated by LSCFM using the MRC-600 confocal microscope with argon laser excitation at $488\mathrm{nm}$ and photon counting detection. The high sensitivity of this detection allowed us to employ a low power laser excitation $\left(0.1\mathrm{mW}\right)$ at the entry of the optical head,^{4, 5} an essential condition for the study of active mitochondria—known to be damaged by light^{6, 7, 8, 9}—in living cells.

Cells incubated with PEPEP at a final concentration of $5\mu \mathrm{M}$ for $15\text{to}30\min$ were found to be permeable to the dye. We report the intracellular distribution of PEPEP in HUVEC for short [Fig. 3a ] and long [Fig. 3b] incubation times. In both cases, the compound was found to localize selectively in mitochondria, with an intense fluorescent signal and a higher accumulation at $24\mathrm{h}$ .

Fig. 3

Fluorescence images of mitochondrial localization of PEPEP in HUVEC. Cells were incubated with: (a) $5\text{-}\mu \mathrm{M}$ PEPEP for $30\min$ ; (b) $5\text{-}\mu \mathrm{M}$ PEPEP for $24\mathrm{h}$ ; (c) $1\text{-}\mu \mathrm{M}$ R123 for $10\min$ ; and (d) $10\text{-}\mu \mathrm{M}$ DASPMI for $15\min$ .

To estimate the efficiency of PEPEP in staining mitochondria, we present for comparison the HUVEC intracellular localization of two well known potentiometric vital probes, R123 [Fig. 3c] and DASPMI [Fig. 3d], whose mitochondria affinity is well documented in the literature.^{10, 11}

To validate PEPEP as a mitochondrial probe, we studied its intracellular distribution in different cell cultures. In addition to HUVEC, we considered U937 (Fig. 4 ) and COS7 (Fig. 5 ) as model systems for physiological and pathological mammalian cultures, and yeast Saccharomyces cerevisiae for its relevance in biotechnology and bioengineering (Fig. 6 ). In particular, we performed colocalization experiments of PEPEP and R123 in HUVEC and U937. Colocalization of the two probes is evident in the merged images reported in Fig. 4, and strongly supports the mitochondrial specificity of PEPEP. In addition, colocalization levels in the two images for HUVEC and U937 cells were evaluated by using the colocalization test plug-in for image J (Wright Cell Imaging Facility, Toronto, Canada). The Pearson correlation coefficient ranges at about 95% for HUVEC and 80% for U937 cells.

Fig. 4

Fluorescence images of mitochondrial colocalization of PEPEP and R123 in HUVEC and U937. Cells were incubated in the presence of $5\text{-}\mu \mathrm{M}$ PEPEP for $20\min$ , then $1\text{-}\mu \mathrm{M}$ R123 was added for $10\min$ . HUVEC cells: (a) red channel, PEPEP fluorescence; (b) green channel, R123 fluorescence; and (c) merged image. U937 cells: (d) red channel, PEPEP fluorescence; (e) green channel, R123 fluorescence; and (f) merged image (color online only).

Fig. 5

Images of mitochondrial localization of PEPEP in COS7 cells in (a) the absence and (b) the presence of $2\text{-}\mu \mathrm{M}$ FCCP for $5\text{to}10\min$ .

Fig. 6

Fluorescence images of mitochondrial localization of PEPEP in Saccharomyces cerevisiae cells. Cells were incubated in the presence of $5\text{-}\mu \mathrm{M}$ PEPEP for $30\min$ .

Stimulated by these results, we investigated whether the mitochondrial uptake of this new probe was driven by the organelle membrane potential or by its high DNA affinity. We studied the fluorescence distribution of PEPEP in COS7 cells treated with a membrane-potential uncoupler, the protonophor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The images of stained cells in the absence and in the presence of $2\text{-}\mu \mathrm{M}$ FCCP are reported in Figs. 5a and 5b, respectively. Unambiguously, the dye fluorescence intensity and localization are not appreciably affected by the uncoupler, suggesting that mtDNA is the target of PEPEP, as expected taking into account its affinity for DNA in solution.³ We confirmed that no nuclear localization was observed in viable cells, in agreement with what was already reported for other nucleic acid probes in living intact cells.^{4, 8, 9, 12} The impact of alterations in mitochondrial membrane potential $\left(\Delta \Psi m\right)$ and oxidant stress on PEPEP staining in mitochondria with eventual relocalization of the dye will be the object of future investigations.

In conclusion, our results indicate that PEPEP enters easily into living cells, is not cytotoxic and localizes in mitochondria with a nonpotentiometric mechanism. All of these data unequivocally prove that PEPEP is a very efficient dye for fluorescence confocal microscopy, and a valuable alternative to the most frequently used mitochondrial stains.