Rhodamine 123 (R302; FluoroPure Grade, R22420; Figure 12.22) is a cell-permeant, cationic, fluorescent dye that is readily sequestered by active mitochondria without inducing cytotoxic effects.56 Uptake and equilibration of rhodamine 123 is rapid (a few minutes) compared with dyes such as DASPMI (4-Di-1-ASP, D288), which may take 30 minutes or longer.14 Viewed through a fluorescein longpass optical filter (Table 23.12), fluorescence of the mitochondria of cells stained by rhodamine 123 appears yellow-green. Viewed through a tetramethylrhodamine longpass optical filter, however, these same mitochondria appear red. Unlike the lipophilic rhodamine and carbocyanine dyes, rhodamine 123 apparently does not stain the endoplasmic reticulum.
Rhodamine 123 has been used with a variety of cell types such as presynaptic nerve terminals,57 live bacteria,58,59 plants 60,61 and human spermatozoa.62 Using flow cytometry, researchers employed rhodamine 123 to sort respiratory-deficient yeast cells 63,64 and to isolate those lymphocytes that are responsive to mitogen stimulation.65 Rhodamine 123 has also been used to study:
Apoptosis 52,66
Axoplasmic transport of mitochondria 67
Bacterial viability and vitality 58
Mitochondrial enzymatic activities 68,69
Mitochondrial transmembrane potential and other membrane activities 15,60,70–73
Multidrug resistance 74–81 (Section 15.6)
Mycobacterial drug susceptibility 82,83
Oocyte maturation 84
Although rhodamine 123 is usually not retained by cells when they are washed, a variety of human carcinoma cell lines (but not sarcomas or leukemic cells) retain the dye for unusually long periods 85 (>24 hours), making rhodamine 123 a potential anticancer agent for photodynamic therapy.86–91 Rhodamine 123 is known to be preferentially taken up and retained by mitochondria of carcinoma cells 92 and to inhibit their proliferation;93,94 cardiac muscle cells also retain rhodamine 123 for days.95
Other mitochondrion-selective dyes include tetramethylrosamine (T639, Figure 12.23), whose fluorescence contrasts well with that of fluorescein for multicolor applications, and rhodamine 6G 89,96–98 (R634, Figure 12.24), which has an absorption maximum between that of rhodamine 123 and tetramethylrosamine. Tetramethylrosamine and rhodamine 6G have both been used to examine the efficiency of P-glycoprotein–mediated exclusion from multidrug-resistant cells 74 (Section 15.6). Rhodamine 6G has been employed to study microvascular reperfusion injury 99 and the stimulation and inhibition of F1-ATPase from the thermophilic bacterium PS3.100
At low concentrations, certain lipophilic rhodamine dyes selectively stain mitochondria in live cells.101 Molecular Probes' researchers have observed that low concentrations of the hexyl ester of rhodamine B (R 6, R648MP) accumulate selectively in mitochondria (Figure 12.25) and appear to be relatively nontoxic. We have included this probe in our Yeast Mitochondrial Stain Sampler Kit (Y7530, see below for description). At higher concentrations, rhodamine B hexyl ester and rhodamine 6G stain the endoplasmic reticulum of animal cells 101 (Section 12.4).
The accumulation of tetramethylrhodamine methyl and ethyl esters (TMRM, T668; TMRE, T669) in mitochondria and the endoplasmic reticulum has also been shown to be driven by their membrane potential 102,103 (Section 22.3). Moreover, because of their reduced hydrophobic character, these probes exhibit potential-independent binding to cells that is 10 to 20 times lower than that seen with rhodamine 6G.104 Tetramethylrhodamine ethyl ester has been described as one of the best fluorescent dyes for dynamic and in situ quantitative measurements — better than rhodamine 123 — because it is rapidly and reversibly taken up by live cells.105–107 TMRM and TMRE have been used to measure mitochondrial depolarization related to cytosolic Ca2+ transients 108 and to image time-dependent mitochondrial membrane potentials.106 A high-throughput assay utilizes TMRE and our low-affinity Ca2+ indicator fluo-5N AM (F14204, Section 19.3) to screen inhibitors of the opening of the mitochondrial transition pore.109 Researchers have also taken advantage of the red shift exhibited by TMRM, TMRE and rhodamine 123 upon membrane potential–driven mitochondrial uptake to develop a ratiometric method for quantitating membrane potential.70
Inside live cells, the colorless dihydrorhodamines and dihydrotetramethylrosamine are oxidized to fluorescent products that stain mitochondria.110 However, the oxidation may occur in organelles other than the mitochondria. Dihydrorhodamine 123 (D632, D23806; Figure 12.26) reacts with hydrogen peroxide in the presence of peroxidases,111 iron or cytochrome c 112 to form rhodamine 123. This reduced rhodamine has been used to monitor reactive oxygen intermediates in rat mast cells 113 and to measure hydrogen peroxide in endothelial cells.112Dihydrorhodamine 6G (D633, Figure 12.27) is another reduced rhodamine that has been shown to be taken up and oxidized by live cells.114–116 Chloromethyl derivatives of reduced rosamines (MitoTracker Orange CM-H2TMRos, M7511; MitoTracker Red CM-H2XRos, M7513), which can be fixed in cells by aldehyde-based fixatives, have been described above. The acetoxymethyl (AM) ester of dihydrorhod-2, which is prepared by chemical reduction of the calcium indicator rhod-2 AM (R1244, R1245MP; Section 19.3) has been extensively used to measure the relatively slow changes in intramitochondrial Ca2+ (Figure 19.33, Figure 19.39).
Most carbocyanine dyes with short (C1–C6) alkyl chains (Section 22.3) stain mitochondria of live cells when used at low concentrations (~0.5 μM or ~0.1 μg/mL); those with pentyl or hexyl substituents also stain the endoplasmic reticulum when used at higher concentrations (~5–50 μM or ~1–10 μg/mL). DiOC6(3) (D273) stains mitochondria in live yeast 21,117–119and other eukaryotic cells,98,120 as well as sarcoplasmic reticulum in beating heart cells.121 It has also been used to demonstrate mitochondria moving along microtubules.23 Photolysis of mitochondrion- or endoplasmic reticulum–bound DiOC6(3) specifically destroys the microtubules of cells without affecting actin stress fibers, producing a highly localized inhibition of intracellular organelle motility.122 We have included DiIC1(5) and DiOC2(3) in two of our MitoProbe Assay Kits for flow cytometry (M34151, M34150; Section 22.3). Several other potential-sensitive carbocyanine probes described in Section 22.3 also stain mitochondria in live cultured cells.98
The carbocyanine DiOC7(3) (D378), which exhibits spectra similar to those of fluorescein, is a versatile dye that has been reported to be a sensitive probe for mitochondria in plant cells.123Its other uses include:
Distinguishing cycling and noncycling fibroblasts 124 and viable and nonviable bacteria 125
Following the reorganization of the endoplasmic reticulum during fertilization in the ascidian egg 126
Identifying functional vasculature in murine tumors 127,128
Studying multidrug resistance 129 (Section 15.6)
Visualizing the detailed morphology of neurites of Alzheimer's disease neurons 130
The styryl dyes DASPMI (4-Di-1-ASP, D288) and DASPEI (D426) can be used to stain mitochondria in live cells.14 These dyes have large fluorescence Stokes shifts and are taken up relatively slowly as a function of membrane potential. The kinetics of mitochondrial staining with styrylpyridinium dyes has been investigated using the concentration jump method.131DASPMI and DASPEI have been shown to be useful for:
Determining the distribution of mitochondria in yeast mutants 63
Long-term imaging of live mammalian nerve cells and their connections 132–134
Monitoring the metabolic state of Pneumocystis carinii mitochondria 31
Screening aberrant mitochondrial distribution and morphology in yeast 135