Use of fluorescent dyes as molecular probes for the study of multidrug resistance

Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.     

Pairs of fluorescent dyes as probes of DNA and chromosomes.

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.     

Intracellular distribution of lipophilic fluorescent probes in mammalian cells

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and $\text{2-p-}\text{toluidinyl-6-naphthalene}$ sulfonate (TNS)) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

Intracellular distribution of lipophilic fluorescent probes in mammalian cells.

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and 2-p-toluidinyl-6-naphthalene sulfonate (TNS) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

Nonsymmetrical polymethine dyes as fluorescent probes for the study of microviscosity of membrane phospholipid bilayers

Nonsymmetrical polymethine dyes are shown to be applied as a new approach in the studies of phospholipid membrane microviscosity. The method requires determination of the intensity ratio for the long-wave length (Ig) and short-wave length (Ik) bands of fluorescence spectra in the region of 730-770 nm at exitation 600 nm. It allows determination of microviscosity by comparative measurements of the fluorescence parameter Ig/Ik in the model medium of the known viscosity (glycerol) and the object under study. Microviscosity in egg phosphatidylcholine vesicules (liposomes) is found to be 0.6-1.2 P. It depends on the surface curvature (size of vesicle), cholesterol content and temperature. It the studies of dimiristoylphosphatidyl choline liposomes the changes in microviscosity at the phase transition temperature are shown to be from 4.5 to 1.1 P. The transition temperature is 24.5 degrees C, the transition range being 2.2 degrees C. The results of this work demonstrate the advantages of the suggested approach to study mobility in phospholipid membranes and confirm it to be promising to study natural membranes and whole cells.     

Carbocyanine dyes used as fluorescent triplet probes for measuring slow rotational diffusion of lipids in membranes

A method for measuring the slow rotational diffusion of lipids or lipid domains in membranes has been developed. It covers the time range from 20 microseconds-5 ms, and has a greater than 5 x 10(4) molecules of probe. The method uses acyl-substituted carbocyanine dyes as fluorescent triplet probes and a laser-microscope combination for excitation and measurement of the triplet state. Rotation rates in dimyristoylphosphatidylcholine vesicles were sensitive to the liquid-to-gel transition. Slow rotations with relaxation times of about 100 microseconds were detected at the transition temperature region.     

Molecular probes for fluorescent sensing of metal ions in non-mammalian organisms

While metal ions play an important role in the proper functioning of all life, many questions remain unanswered about exactly how different metals contribute to health and disease. The development of fluorescent probes, which respond to metals, has allowed greater understanding of the cellular location, concentration and speciation of metals in living systems, giving a new appreciation of their function. While the focus of studies using these fluorescent tools has largely been on mammalian organisms, there has been relatively little application of these powerful tools to other organisms. In this review, we highlight recent examples of molecular fluorophores, which have been applied to sensing metals in non-mammalian organisms.     

Triazole-containing BODIPY dyes as novel fluorescent probes for soluble oligomers of amyloid Aβ1-42 peptide

A straightforward functionalization of BODIPY dyes via incorporation of a triazole moiety produced fluorescent dyes that were capable of distinguishing between secondary structure conformations of soluble oligomeric species of Aβ1-42 peptide. Small concentrations of the dyes, relative to Aβ1-42, provided up to an 8-fold and 35-fold fluorescence increase in the presence of the unordered and ordered, β-sheet-rich conformations of soluble Aβ1-42 oligomers, respectively. These triazole-containing dyes could prove to be useful probes for monitoring amyloid conformational transitions in vitro.     

Visualization and sensing of intermolecular interactions with two-color fluorescent probes

We developed a new generic fluorescence sensing technology based on the change of relative intensities between two well-separated emission bands of the novel functional 3-hydroxychromone (3HC) dyes. A greatly enhanced self-calibrating wavelength-ratiometric response is obtained to all major types of non-covalent interactions that can be used in sensing--to polarity, hydrogen bonding ability and to local electrostatic fields. This technology may find a broad range of applications--from homogeneous assays in solutions to microarrays, microfluidic devices, nanosensors and whole cell imaging systems. It allows transforming micelles or phospholipid vesicles into nanosensor devices. In cellular research a high sensitivity to membrane potentials can be obtained and the membrane changes during apoptosis detected.     

Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments

The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.     

Studies of monomeric and homodimeric oxazolo[4,5-b]pyridinium cyanine dyes as fluorescent probes for nucleic acids visualization

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.     

Trimethine cyanine dyes as fluorescent probes for amyloid fibrils: The effect of N,N′-substituents

The effect of various N,N′-substituents in the molecule of benzothiazole trimethine cyanine dye on its ability to sense the amyloid aggregates of protein was studied. The dyes are low fluorescent when free and in the presence of monomeric proteins, but their emission intensity sharply increases in complexes with aggregated insulin and lysozyme, with the fluorescence quantum yield reaching up to 0.42.     

Fluorescent dyes as probes to study lipid-binding proteins

We studied the equilibrium binding of two hydrophobic fluorescent dyes, ANS and bisANS, to four members of a family of intracellular lipid-binding proteins: IFABP, CRABP I, CRABP II, and ILBP. The spectral and binding parameters for the probes bound to the proteins were determined. Typically, there was a single binding site on each protein for the ligands. However, IFABP cooperatively bound a second bisANS molecule in the binding pocket. Comparative analysis of affinities and spectral characteristics for the two probes allowed us to examine the contributions of electrostatic and hydrophobic interactions to the binding process, and to address some aspects of the internal structure of the studied proteins.     

Evaluation of delocalized lipophilic cationic dyes as delivery vehicles for photosensitizers to mitochondria

Mitochondria are attractive targets in photodynamic therapy. Two conjugates: TPP–Rh (a porphyrin–rhodamine B conjugate) and TPP–AO (a porphyrin–acridine orange conjugate), each possessing a single delocalized lipophilic cation, were designed and synthesized as photosensitizers. Their ability to target the mitochondria for photodynamic therapy was evaluated. The conjugates were synthesized by conjugating a monohydroxy porphyrin (TPP-OH) to rhodamine B (Rh B) and acridine orange base (AO), respectively, via a saturated hydrocarbon linker. To evaluate the efficiency of the conjugates as photosensitizers, their photophysical properties and in vitro photodynamic activities were studied in comparison to those of TPP-OH. Although fluorescence energy transfer (FRET) was observed in the conjugates, they were capable of generating singlet oxygen at rates comparable to TPP-OH. Biologically, exciting results were observed with TPP–Rh, which showed a much higher phototoxicity [IC₅₀, 3.95 μM: irradiation of 400–850 nm light (3 mW cm⁻²) for 1 h] than either TPP-OH or Rh B (both, IC₅₀, >20 μM) without significant dark toxicity at 20 μM. This improved photodynamic activity might be due to a greater cellular uptake and preferential localization in mitochondria. The cellular uptake of TPP–Rh was 8 and 14 times greater than TPP-OH and Rh B, respectively. In addition, fluorescence imaging studies suggest that TPP–Rh localized more in mitochondria than TPP-OH. On the other hand, TPP–AO showed some dark toxicity at 10 μM and stained both mitochondria and nucleus. Our study suggests that conjugation of photosensitizers to Rh might provide two benefits, higher cellular uptake and mitochondrial localization, which are two important subjects in photodynamic therapy.     

Interaction of cyanine dyes with nucleic acids. XII.beta-substituted carbocyanines as possible fluorescent probes for nucleic acids detection.

Results of investigations of fluorescent properties of a beta-substituted carbocyanine and its complexes with nucleic acids in comparison with those for the unsubstituted dye are presented. Carbocyanine substituted in polymethine chain has shown promising properties for use as a fluorescent probe in homogeneous systems of nucleic acids detection.     

New fluorescent cholesterol analogs as membrane probes.

New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.     

Metal complexes as optical probes for DNA sensing and imaging

Transition and lanthanide metal complexes have rich photophysical properties that can be used for cellular imaging, biosensing and phototherapy. One of the applications of such luminescent compounds is the detection and visualisation of nucleic acids. In this brief review, we survey the recent literature on the use of luminescent metal complexes (including Re^I, Ru^II, Os^II, Ir^III, Pt^II, Eu^III and Tb^III) as DNA optical probes, including examples of compounds that bind selectively to non-duplex DNA topologies such as quadruplex, i-motif and DNA mismatches. We discuss the applications of metal-based luminescent complexes in cellular imaging, including time-resolved microscopy and super-resolution techniques. Their applications in biosensing and phototherapy are briefly mentioned in the relevant sections.     

Quantum dots versus organic dyes as fluorescent labels

Suitable labels are at the core of Luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technology is the emerging development of quantum dots (QDs)--inorganic nanocrystals with unique optical and chemical properties but complicated surface chemistry--as in vitro and in vivo fluorophores. Here we compare and evaluate the differences in physicochemical properties of common fluorescent labels, focusing on traditional organic dyes and QDs. Our aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels.     

Natural fruit extracts as non-toxic fluorescent dyes for staining fungal chlamydospores

Currently, most synthetic dyes utilized for fungal fluorescent staining are toxic, carcinogenic, or harmful to animals, humans, and the environment. This study proposes non-toxic extracts of fruits from the genera Rhamnus, Ribes, Sambucus, Viburnum, Sorbus and Beta as simple, safe, and ecological alternatives to chemical fluorescent dye for efficient staining of Fusarium chlamydospore cells using, as test strains, five different pathogenic Fusarium species.