Excimer-forming lipids in membrane research

Pyrenedecanoic acid and pyrene lecithin are optical probes well suited to investigate lipid bilayer membranes. The method is based on the determination of the formation of excited dimers or excimers. The rate of excimer formation yields information on the dynamic molecular properties of artificial as well as of natural membranes. This article will review applications of the excimer-forming probes.Pyrene lipid probes are used to determine the coefficient of the lateral diffusion in fluid lipid membranes. Results in artificial membranes are comparable to the values obtained in erythrocyte membranes.Moreover, the excimer formation rate is a very sensitive measure of changes in membrane fluidity. Membrane fluidity is an important regulator of membrane functional proteins. For example, there is a correlation between membrane fluidity and enzyme activities of the adenylate cyclase system.The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transversal mobility, that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. Again, artificial as well as natural membranes can be investigated by this technique.Another important area of investigation in membrane research is the interaction between lipids and proteins. Lipids, in the presence of a protein, show a different dynamic behavior from free lipids. Because of changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, our methods could also be applied to the examination of phase separation phenomena and to lipid-protein interactions.     

Protein-dependent Reduction of the Pyrene Excimer Formation in Membranes

The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk lipid. The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene enrichment in this area of the lipid bilayer. Received: 29 December 1996/Revised: 15 May 1996     

Kinetics of partly diffusion controlled reactions. VII — Pyrene excimer formation in erythrocyte membranes

Intermolecular excimer forming systems such as pyrene have been proposed as probes for the microviscosity measurement of model and biological membranes. However, our experimental study of pyrene excimer formation in erythrocyte membranes and the theoretical calculations show that it is quite difficult to get the diffusion coefficient of pyrene in membranes and therefore the microviscosity of its environment.     

Minor effects of bulk viscosity on lipid translational diffusion measured by the excimer formation technique

We have investigated the effect of bulk viscosity on lipid translational diffusion using the excimer formation technique. In contrast to a study by Vaz et al. (1987), performed with the fluorescence recovery after photobleaching technique, we observed only a minor decrease of less than a factor of two for pyrene labelled phosphatidylcholine in glycerinated phosphatidylcholine bilayer membranes compared to an aqueous dispersion. Even the diffusion of pyrene labelled gangliosides with an oligosaccharide head-group that protrudes from the membrane surface is not strongly restricted by the increased bulk viscosity. We conclude that the viscosity of the fluid bounding the lipid bilayers is of minor importance for the diffusion of membrane lipids.Abbreviations DPPC 1-2 dipalmitoyl-sn-glycero-3-phosphocholine - DSPC 1-2 distearoyl-sn-glycero-3-phosphocholine - PyPC 1-acyl-2-[10(-1-pyrene)decanoyl]-sn-glycero-3-phosphocholine - PyG_M1 N-12-(1-pyrene)dodecanoyl-lyso G_M1 - PyG_M2 N-12-(1-pyrene)dodecanoyl-lyso G_M2 - PyG_M3 N-12-(1-pyrene) dodecanoyl-lyso G_M3 - I_M fluorescence intensity of the monomeric pyrene probe - I_D fluorescence intensity of the excimer     

The lack of relationship between fluorescence polarization and lateral diffusion in biological membranes

An investigation has been carried out of the relationship between changes in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and concomittant changes in the lateral diffusion of proteins and lipid probes in membranes. Plasma membranes from lymphocytes and a CH1 mouse lymphoma line were treated with up to 70 mol% (relative to the total membrane phospholipid) of oleic or linoleic fatty acids. Under these conditions the fluorescence polarization of DPH decreased by between 8 and 15% which, in the framework of the microviscosity approach, suggests a membrane fluidity change of between 20 and 50%. The lateral diffusion coefficients of surface immunoglobin and the lipid probes 3,3′-dioctadecylindocarbocyanine and pyrene were also measured in these membranes using the fluorescence photobleaching recovery technique and the rate of pyrene excimer formation. The diffusion rates were found to be unaffected by the presence of free fatty acids. Hence despite large ‘microviscosity’ changes as reported by depolarization of DPH fluorescence, lateral diffusion coefficients are essentially unchanged. This finding is consistent with the idea that perturbing agents such as free fatty acids do not cause a general fluidization of the membrane but act locally to alter, for example, protein function. It is also consistent with the suggestion that lateral mobility of membrane proteins is not modulated by the lipid viscosity.     

On measuring biomembrane microviscosity using pyrene luminescence in aerobic conditions

The movement of pyrene in a lipid bilayer is shown to occur not only in the lateral but also transmembrane direction. Within the excited state lifetime, the pyrene monomer elevates from the depth to the polar region of the membrane and emits a luminescence photon. The excimer does not exhibit any marked transmembrane movement. The luminescence quenching efficiency of monomers and excimers depends on the depth of penetration of the quencher into the membrane. In the lipid bilayer, pyrene luminescence is strongly quenched by oxygen. The binding of pyrene to membrane proteins protects it from quenching. It has been concluded that the widely used estimations of membrane viscosity from pyrene luminescence intensity are incorrect.     

Use of pyrene as a luminescence indicator of the viscosity of model and biological membranes

The pyrene movement in a lipid bilayer has been shown to occur not only in the lateral but also transmembrane direction. Within the excited state lifetime the pyrene monomer elevates from the depth to the polar regions of the membrane and emits a luminescence photon. The excimer does not exhibit any marked transmembrane movement while luminescing from the hydrophobic regions. The luminescence quenching efficiency of monomers and excimers depends on the depth of quencher penetration into the membrane. In the lipid bilayer the pyrene luminescence is strongly quenched by molecular oxygen. The pyrene binding to membrane proteins protects it from quenching. A conclusion has been made that the carrying out estimations of membrane viscosity from pyrene luminescence require considerable correction.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

Evidence that pyrene excimer formation in membranes is not diffusion-controlled

Kinetic and steady-state measurements of pyrene fluorescence in a variety of model membranes are evaluated in terms of the theory of collisional excimer formation. In the region of 10(-3)-0.1 M pyrene, molecular fluorescence decay in membranes is biphasic and the two component lifetimes do not depend on the pyrene concentration. The lifetime data are consistent with the rate constant for collisional excimer formation being of the order 10(6) M-1 X s-1 or less. The concentration dependence of the component amplitudes is inconsistent with the theory of collisional excimer formation and suggests that pyrene exists in two forms in membranes: a slowly diffusing monomeric form and an aggregated form. The component of molecular fluorescence decay associated with aggregated pyrene is highly correlated with steady-state excimer fluorescence, suggesting that excimer fluorescence in membranes arises from aggregated pyrene in which excimers are formed by a static rather than a collisional mechanism. It is suggested that the concentration dependence of excimer to molecular fluorescence intensity ratios in membranes is related to the equilibrium constant for exchange between monomeric and aggregated pyrene forms rather than to the collisional excimer formation rate constant.     

Cholesterol dynamics in membranes.

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.     

Dipyrenylphosphatidylcholines as membrane fluidity probes. Relationship between intramolecular and intermolecular excimer formation rates.

In the intramolecular excimeric membrane probe, dipyrenylphosphatidylcholine (dipyn PC), pyrene moieties are linked to the terminal carbons of the two acyl chains, each of which contains n carbons. We show here how the probe intramolecular excimer production rate, K, may be determined from the excimer/monomer intensity ratio, rl, by making use of the fluorescence titrations of the related monopyrenyl probe, pyn PC, analyzed according to the milling crowd model. rl and the rate K of dipy10 PC in four model membrane systems were measured over a wide temperature range and both parameters are shown to be sensitive functions of the lateral fluidity of the host matrix. A model for relating the intramolecular and intermolecular excimer formation rates is proposed according to which both processes are limited by the reorientational rate of the pyrene moiety. Above the fluid-gel transition temperature, Tc, the diffusion rate (f) of the monopyrenyl probe (pyn PC) is accordingly related to K by: pE approximately K/(K + 1/2f + tau -1M), where pE is the probability of excimer formation between nearest neighbor pyn PC probes, and tau M is the monomer lifetime. Values of pE derived in this way are found to be consistent with pE values derived from the milling crowd analysis of fluorescence yield titration experiments. K for dipy10 PC in DMPC multibilayers ranges from 0.21 x 10(7) s-1 at 10 degrees C in the gel phase, to 5.7 x 10(7) s-1 at 60 degrees C in the fluid phase, whereas the lateral diffusion coefficient, D, for py10 PC in the same bilayers ranged from 8 to 34 microns2 s-1, when calculated with D = fL2/4, L being the average lipid-lipid spacing of the host membrane. Above Tc and at the same reduced temperature, (T - Tc)/Tc, both f for py10 PC, and K for dipy10 PC were found to have relative magnitudes in the order: DPPC greater than DMPC greater than POPC greater than DOPC. This and the similarity of the activation energies for f and K suggest that the rotation of the the pyrene moiety is the rate-limiting step for both the lateral mobility of py10 PC and intramolecular excimer formation in dipy10 PC.     

Effect of sealing on the incorporation of pyrene in goat erythrocyte ghosts — A fluorescence study

The incorporation of pyrene within the membrane interior of goat erythrocyte ghost has been estimated from its fluorescence spectrum. The excimer to monomer fluorescence intensity ratio of embedded pyrene is a function of the fluidity of its environment and the magnitude of its incorporation. Our study shows that this ratio is considerably less (30%) in a pre-sealed ghost than in the non-sealed ghost revealing that the site of incorporation of the probe is indeed the hydrophobic interior of the membrane; as in the later case, the probe has access to the membrane interior from both sides of the membrane. Our study on kinetics of molecular exchange indicates a very fast (of the order of seconds) transfer rate of pyrene from probed to unprobed erythrocyte ghosts through the aqueous phase rather than actual fusion of the membranes.     

Interactions of pyrene derivatives with lipid bilayers and with (Ca2+-Mg2+)-ATPase

The intensities of fluorescence emission for pyrene and a number of its derivatives increase on binding to lipid bilayers and to the (Ca2+-Mg2+)-ATPase purified from rabbit muscle sarcoplasmic reticulum. The effect is particularly marked for the less water-soluble derivatives. Changes in intensity for monomer and excimer emission as a function of lipid concentration can be fitted to a simple model to obtain binding parameters. The number of binding sites per lipid is 0.2-0.4. For the ATPase system, at least two classes of sites are necessary to fit the data, one corresponding to the lipid component and one to sites on the ATPase. Excimer emission from the postulated sites on the ATPase is less marked than that from lipid. Pyrene-dodecanoic acid and pyreneundecyltrimethylammonium bromide, which bind to a large number of sites on the ATPase, cause marked inhibition of ATPase activity at high concentration. Pyrene and a number of water-soluble derivatives cause stimulation of the ATPase reconstituted with dimyristoleoylphosphatidylcholine and little inhibition and bind to a small number of sites on the ATPase. It is concluded that excimer emission from pyrene derivatives in systems containing proteins cannot be used to obtain reliable information about rates of diffusion in the lipid component of the membrane.     

The lateral fluidity of erythrocyte membranes. Temperature and pressure dependence

The pressure and temperature dependence of the lateral and rotational fluidity of erythrocyte membranes was investigated by inserting the excimeric membrane probe 1'-pyrenedodecanoic acid (PDA) into the membranes of intact cells and measuring the probe excimer formation rate and the steady-state polarization of the monomer at pressures up to 2000 atm (2 kbar). At that pressure the lateral diffusivity of PDA was found to decrease by a factor of 10 and its emission anisotropy by a factor of 5 at 22 degrees C. At atmospheric pressure, the local lateral diffusion coefficient of PDA at 2 and 33 degrees C is 1.5 and 4.3 x 10(-8) cm2 s-1, respectively. The activation energy for probe translation was found to decrease from 6 to 3 kcal M-1 in going from atmospheric pressure to 2 kbar, while the entropy decreased by approx. 15 cal M-1 K-1, indicating greater lipid order at the high pressure. The experimental data are consistent with a 'free-area' model for the membrane, analogous to the free-volume model for nonassociated liquids. The lateral diffusivity of PDA was found to be proportional to the free membrane area and linear extrapolation to zero diffusivity indicates that at atmospheric pressure, the fractional free area of the erythrocyte membrane is 6%.     

Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains

Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.     

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes. A fluorescence probe study

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles (out greater than in) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47 degrees C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.     

Thermotropic behavior of liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and their mixtures

The thermotropic properties of multilamellar liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and several mixtures of these two lipids were studied with the application of excimer--forming optical probe pyrene and microcalorimetry. It was discovered that when the proportion of the egg yolk lecithin in the lipid mixture was raised the temperature of the main phase transition reduced. For all this, independent of the lipid mixture composition when the temperature was raised, apparently, polarity of pyrene microenvironment in the liposomes bilayers decreased. On the basis of the analysis of solidus and liquidus curves obtained from calorimetric studies of the lipid mixtures and bend points of Arrhenius anamorphose obtained during the pyrene excimer formation measurements some conclusions were made about the role of unmodified and hydrogenized egg yolk lecithin cluster formation in the determination of thermotropic properties of the liposomes from the above two lipids mixtures. High temperature phase transition discovered for the egg yolk lecithin while measuring the pyrene excimer formation is proposed to be closely connected with temperature-dependent changes in the organization of phospholipid heads on the interphase bilayer/H2O solution.     

Lateral and transversal diffusion and phase transitions in erythrocyte membranes. An excimer fluorescence study

The lateral diffusion of the excimer-forming probe pyrene decanoic acid has been determined in erythrocyte membranes and in vesicles of the lipid extracts. The random walk of the probe molecules is characterized by their jump frequency, v_j, within the lipid matrix. At T = 35°C a value of v_j = 1.6 · 10³ s^?1 is found in erythrocyte membranes. A somewhat slower mobility is determined in vesicles prepared from lipid extracts of the erythrocyte membrane. Depending on structure and charge of the lipids we obtain jump frequencies between 0.8 · 10⁸ s^?1 and 1.5 · 10⁸ s^?1 at T = 35°C. The results are compared with jump frequencies yielded in model membranes.The mobility of molecules perpendicular to the membrane surface (transversal diffusion) is investigated. Erythrocyte ghosts doped with pyrene phosphatidylcholine were mixed with undoped ghosts in order to study the exchange kinetics of the probe molecule. A fast transfer between the outer layers of the ghost cells ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{min at}\text{T = 37°}\text{C}$) is found. The exchange process between the inner and the outer layer of one erythrocyte ghost (flip-flop process) following this fast transfer occurs with a half-life time value of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 100}\text{min at}\text{T = 37°}\text{C}$.The application of excimer-forming probes presumes a fluid state of the membrane. Therefore we investigated the phase transition behaviour using the excimer technique. Beside a thermotropic phase transition at T = 23°C and T = 33°C we observed an additional fluidity change at T = 38°C in erythrocyte ghosts. This transition is attached to a separation of the boundary lipid layer from the intrinsic proteins. No lipid phase transition is observed in liposomes from isolated extracts of the erythrocyte membrane with our methods.     

Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy

We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (~7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is ~8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.