Highly sensitive detection of hybridization of oligonucleotides to specific sequences of nucleic acids by application of fluorescence resonance energy transfer

We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light.     

Potential charge transfer probe induced conformational changes of model plasma protein human serum albumin: Spectroscopic, molecular docking, and molecular dynamics simulation study

The nature of binding of specially designed charge transfer (CT) fluorophore at the hydrophobic protein interior of human serum albumin (HSA) has been explored by massive blue-shift (82 nm) of the polarity sensitive probe emission accompanying increase in emission intensity, fluorescence anisotropy, red edge excitation shift, and average fluorescence lifetimes. Thermal unfolding of the intramolecular CT probe bound HSA produces almost opposite spectral changes. The spectral responses of the molecule reveal that it can be used as an extrinsic fluorescent reporter for similar biological systems. Circular dichrosim spectra, molecular docking, and molecular dynamics simulation studies scrutinize this binding process and stability of the protein probe complex more closely.     

Some physiological and biochemical changes in marine eukaryotic red tide alga Heterosigma akashiwo during the alleviation from iron limitation

Some physiological and biochemical changes in the marine eukaryotic red tide alga Heterosigma akashiwo (Hada) were investigated during the alleviation from iron limitation. Chlorophyll a/carotenoid ratio increases as a result of iron alleviation. In vivo absorption spectra of iron-limited cells showed a chlorophyll (Chl) absorption peak at 630 nm, 2 nm blue-shifted from the normal position. Low-temperature fluorescence emission spectra of the cells have one prominent Chl emission peak at 685 nm. The cells showed a decrease in fluorescence yield from 685 nm band during alleviation from iron limitation. Low-temperature fluorescence excitation spectra and room-temperature fluorescence spectra indicated an efficient excitation energy transfer in the cells alleviated from iron limitation. Photosynthetic efficiency and carbohydrate content per cell increased after alleviation from iron limitation. Total protein decreased in iron-limited cells, while iron deficiency induced the appearance of specific soluble proteins (17 and 55 kDa).     

Excitation spectra and brightness optimization of two-photon excited probes

Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross section can be extracted from molecular brightness measurements at low excitation intensities, while peak molecular brightness (the maximum molecular brightness with increasing excitation intensity) is measured at higher intensities at which probe photophysical effects become significant. The spectral shape of these two parameters was similar across all dye families tested. Peak molecular brightness spectra, which can be obtained rapidly and with reduced experimental complexity, can thus serve as a first-order approximation to cross-section spectra in determining optimal wavelengths for two-photon excitation, while providing additional information pertaining to probe photostability. The data shown should assist in probe choice and experimental design for multiphoton microscopy studies. Further, we show that, by the addition of a passive pulse splitter, nonlinear bleaching can be reduced--resulting in an enhancement of the fluorescence signal in fluorescence correlation spectroscopy by a factor of two. This increase in fluorescence signal, together with the observed resemblance of action cross section and peak brightness spectra, suggests higher-order photobleaching pathways for two-photon excitation.     

荧光光谱法研究铈离子与二棕榈酰磷脂酰胆碱（DPPC）脂质体的相互作用

通过荧光光谱法研究了稀土元素Ｃｅ３＋与二棕榈酰磷脂酰胆碱（ＤＰＰＣ）脂质体的相互作用,结果表明,Ｃｅ３＋－ＤＰＰＣ体系的激发波长在２４７ｎｍ,２９４ｎｍ,发射波长在３４４ｎｍ,与水合Ｃｅ３＋的荧光光谱完全不同。这表明Ｃｅ３＋与ＤＰＰＣ形成了复合物,该复合物的荧光光谱同Ｃｅ３＋－ＤＨＰ复合物的荧光光谱一致。可以认为Ｃｅ３＋与ＤＰＰＣ分子上的磷酸基团相作用。     

荧光光谱法研究铈离子与二棕榈酰磷脂酰胆碱（DPPC）脂质体的相互作用

通过荧光光谱法研究了稀土Ｃｅ＾３＋与二棕榈酰磷脂酰胆碱（ＤＰＰＣ）脂质体的相互作用,结果表明,Ｃｅ＾３＋－ＤＰＰＣ体系的激发波长在２４７ｎｍ,２９４ｎｍ,发射波长在３４４ｎｍ,与水合Ｃｅ＾３＋的荧光光谱完全不同。这表明Ｃｅ＾３＋与ＤＰＰＣ形成了复合物,该复合物的荧光光谱同Ｃｅ＾３＋－ＤＨＰ复合物的荧光光谱一致,可以认为Ｃｅ＾３＋与ＤＰＰＣ分子上的磷酸基团相作用。     

Biophysical properties of ergosterol-enriched lipid rafts in yeast and tools for their study: characterization of ergosterol/phosphatidylcholine membranes with three fluorescent membrane probes

In this work, binary mixtures of phospholipid/ergosterol (erg) were studied using three fluorescent membrane probes. The phospholipid was either saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) or monounsaturated (1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphocholine, POPC) phosphatidylcholine, to evaluate the fluorescence properties of the probes in gel, liquid ordered (l(o)) and liquid disordered (l(d)) phases. The probes have been used previously to study cholesterol-enriched domains, but their photophysical properties in erg-enriched membranes have not been characterized. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) presents modest blue-shifts upon erg addition, and the changes in the fluorescence lifetime are mainly due to differences in the efficiency of its fluorescence dynamic self-quenching. However, the steady-state fluorescence anisotropy of NBD-DPPE presents well-defined values in each lipid phase. N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) presents a close to random distribution in erg-rich membranes. There are no appreciable spectral shifts and the steady-state fluorescence anisotropy presents complex behavior, as a result of different photophysical processes. The probe is mostly useful to label l(d) domains in yeast membranes. 4-(2-(6-(Dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium (di-4-ANEPPS) is an electrochromic dye with excitation spectra largely insensitive to the presence of erg, but presenting a strong blue-shift of its emission with increasing concentrations of this sterol. Its partition coefficient is favorable to l(o) domains in POPC/erg mixtures. Although the fluorescence properties of di-4-ANEPPS are less sensitive to erg than to chol, in both cases the fluorescence lifetime responds monotonically to sterol mole fraction, becoming significantly longer in the presence of sterol as compared to pure POPC or DPPC bilayers. The probe displays a unique sensitivity to sterol-lipid interaction due to the influence of hydration and H-bonding patterns at the membrane/water interface on its fluorescence properties. This makes di-4-ANEPPS (and possibly similar probes) potentially useful in the study of erg-enriched domains in more complex lipid mixtures and in the membranes of living yeast cells.     

An allosteric mechanism controls antigen presentation by the H-2K(b) complex.

The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta2m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides. Interaction of peptides with the heavy-chain binding site, in turn, increased markedly the affinity of the heavy chain for beta2m. Binding of peptide variants of the ovalbumin sequence (257-264) to the heavy chain/beta2m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147-155 to the H-2Kd heavy chain/beta2m heterodimer [(3.0 +/- 1.0) x 10(-6) M-1 s-1 at 37 degrees C]. The slow phase was a result of both the ternary complex assembly from the "free" heavy chain, beta2m, and peptide as well as an intramolecular conformational transition within the heavy chain/beta2m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or beta2m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta2m dissociation from the H-2Kb ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the Kb heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their Kd/nucleoprotein peptide counterparts.     

Pressure-induced phase transitions of lipid bilayers observed by fluorescent probes Prodan and Laurdan

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-dodecanoyl-2-(dimethylamino)naphthalene (Laurdan) in bilayer membranes of 1,2-distearoylphosphatidylcholine (DSPC) were observed as a function of pressure at constant temperature. The emission spectra of Prodan and Laurdan varied with the pressure-induced states of bilayer membranes. The maximum emission wavelength (lambda(max)) of Prodan characteristic of the liquid crystalline (L(alpha)), lamellar gel (L(beta)') and pressure-induced interdigitated gel (L(beta)I) phases of the DSPC bilayer was 480, 440 and 500 nm, respectively. On the other hand, the lambda(max) of Laurdan characteristic of the L(alpha) and L(beta)' phases was 480 and 440 nm in a similar manner as Prodan probe. However, no change in the lambda(max) was observed in spite of the occurrence of the interdigitation of bilayer. Since the lambda(max) reflects the solvent property around the probe molecules, we could speculate about the location of fluorescent probe in the bilayer membranes. In the L(alpha) phase the same chromophore group of Prodan and Laurdan probes distributes around phosphate group of lipid (i.e., polar region). The transformation of bilayer into the L(beta)' phase causes the Prodan and Laurdan molecules to move into the glycerol backbone (i.e., less polar) region. In the ripple gel (P(beta)') phase, the emission spectrum of Prodan shows a broad peak at about 480 nm and a shoulder around 440 nm, which means that the Prodan molecules are widespread over the wide range from the glycerol backbone to the hydrophilic part of bilayer. The P(beta)'/L(beta)I phase transition causes the Prodan molecule to squeeze out from the glycerol backbone region and to move the hydrophilic region near the bilayer surface. Contrarily, the Laurdan molecule was not squeezed out from the glycerol backbone region because the long acyl chain of Laurdan serves as an anchor in the hydrophobic core of bilayer. The ratio of fluorescence intensity of Prodan at 480 nm to that at 440 nm, F(480)/F(440), is available to observation of bilayer phase transitions. The plot of F(480)/F(440) versus pressure seems to be useful for the recognition of bilayer phase transition, especially the bilayer interdigitation.     

Synthesis and spectral characterization of a long-lifetime osmium (II) metal–ligand complex: a conjugatable red dye for applications in biophysics

There is a need for luminescent probes, which display both long excitation and emission wavelengths and long decay times. We synthesized and characterized an osmium metal–ligand complex which displays a mean decay time of over 100 ns when bound to proteins. [Os(1,10-phenanthroline)₂(5-amino-1,10-phenanthroline)](PF₆)₂ can be excited at wavelengths up to 650 nm, and displays an emission maximum near 700 nm. The probe displays a modest but useful maximum fundamental anisotropy near 0.1 for 488-nm excitation, and thus convenient when using an argon ion laser. [Os(phen)₂(aphen)](PF₆)₂ is readily activated to the isothiocyanate for coupling to proteins. When covalently linked to bovine serum albumin the intensity decay is moderately heterogeneous with a mean decay time of 145 ns. The anisotropy decay of the labeled protein displays a correlation time near 40 ns. This relatively long lifetime luminophores can be useful as a biophysical probe or in clinical applications such as fluorescence polarization immunoassays.     

Single-molecule spectroscopy selectively probes donor and acceptor chromophores in the phycobiliprotein allophycocyanin

We report on single-molecule fluorescence measurements performed on the phycobiliprotein allophycocyanin (APC). Our data support the presence of a unidirectional F?rster-type energy transfer process involving spectrally different chromophores, alpha84 (donor) and beta84 (acceptor), as well as of energy hopping amongst beta84 chromophores. Single-molecule fluorescence spectra recorded from individual immobilized APC proteins indicate the presence of a red-emitting chromophore with emission peaking at 660 nm, which we connect with beta84, and a species with the emission peak blue shifted at 630 nm, which we attribute to alpha84. Polarization data from single APC trimers point to the presence of three consecutive red emitters, suggesting energy hopping amongst beta84 chromophores. Based on the single-molecule fluorescence spectra and assuming that emission at the ensemble level in solution comes mainly from the acceptor chromophore, we were able to resolve the individual absorption and emission spectra of the alpha84 and beta84 chromophores in APC.     

Actin-gelsolin interactions. Evidence for two actin-binding sites

We have used a fluorescence enhancement of actin labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-actin) to study the interactions between rabbit skeletal muscle G-actin and either purified platelet gelsolin or a 130-kDa binary complex of platelet actin and gelsolin that is stable in EGTA and can be purified from human platelets. We have delineated four binding reactions. The exchange of Mg2+ for Ca2+ on the divalent cation-binding site of NBD-actin gives a small fluorescence increase. Binding of monomeric NBD-actin to the binary complex results in a 2.5-fold increase in the emission at 530 nm in the presence of Ca2+ and a 2-fold increase in the presence of EGTA. Titration experiments show that, under nonpolymerizing conditions, one additional actin is bound to the 130-kDa species to form a ternary complex. This binding is Ca2+-sensitive. Purified gelsolin does not appear to bind to NBD-actin in the presence of EGTA, as determined by fluorescence enhancement, gel filtration, or sedimentation measurements, but the addition of Ca2+ promotes rapid binding with a 1.6-1.7-fold enhancement of the emission intensity. A comparison of the relative fluorescence yields/NBD-actin molecule for a binary complex of gelsolin and one NBD-actin, a ternary complex of gelsolin and two NBD-actin molecules, and a ternary complex with an unlabeled actin in the EGTA-stable site and an NBD-actin in the second site indicates that the first NBD-actin, in the EGTA-stable site, does not give a fluorescence increase on binding but the second one does. Finally, we have demonstrated that one molecule of 45Ca2+ is "trapped" when the binary complex is formed and cannot be removed by EGTA. A summary model for these reactions is presented that indicates the interaction between actin and gelsolin is not a freely reversible Ca2+-controlled reaction.     

Real-time multiplex PCR assays

The ability to multiplex PCR by probe color and melting temperature (T(m)) greatly expands the power of real-time analysis. Simple hybridization probes with only a single fluorescent dye can be used for quantification and allele typing. Different probes are labeled with dyes that have unique emission spectra. Spectral data are collected with discrete optics or dispersed onto an array for detection. Spectral overlap between dyes is corrected by using pure dye spectra to deconvolute the experimental data by matrix algebra. Since fluorescence is temperature dependent and depends on the dye, spectral overlap and color compensation constants are also temperature dependent. Single-labeled probes are easier to synthesize and purify than more complex probes with two or more dyes. In addition, the fluorescence of single-labeled probes is reversible and depends only on hybridization of the probe to the target, allowing study of the melting characteristics of the probe. Although melting curves can be obtained during PCR, data are usually acquired at near-equilibrium rates of 0.05-0.2 degrees C/s after PCR is complete. Using rapid-cycle PCR, amplification requires about 20 min followed by a 10-min melting curve, greatly reducing result turnaround time. In addition to dye color, melting temperature can be used for a second dimension of multiplexing. Multiplexing by color and T(m) creates a "virtual" two-dimensional multiplexing array without the need for an immobilized matrix of probes. Instead of physical separation along the X and Y axes, amplification products are identified by different fluorescence spectra and melting characteristics.     

亚油酸体系脂质过氧化引起的DNA损伤研究

用含两个双键的不饱和脂肪酸-亚油酸作为模型化合物,分析其过氧化程度,同时检测了由于脂质过氧化而引起的DNA损伤,结果表明:在脂质过氧化过程中,DNA与亚油酸过氧化产物反应生成一种荧光物质、其最大激发波长315nm最大发射波长410nm并随着氧化时间增加而增加,与此同时,双链DNA百分含量明显下降,DNA-溴乙锭复合物荧光显著降低,反映了DNA二级结构受到破坏.上述结果揭示了脂质过氧化产物在自由基引起DNA的损伤中可能起重要作用     

Anthracene based base-discriminating fluorescent oligonucleotide probes for SNPs typing: synthesis and photophysical properties

2- and 9-Anthracenecarboxamide labeled 2'-deoxyuridines were synthesized and their photophysical properties were examined. These oligonucleonucleotide probes are capable of detecting adenine base on a target DNA sequence. It was also found that 2-anthracene based oligonucleotide probe is more efficient than the corresponding 9-anthracene based oligonucleotide in the application for DNA chip based SNP detection, due to its longer emission wavelength and high fluorescence intensity.     

Comparison of chlorophyll fluorescence emission characteristics of wheat leaf tissue and isolated thylakoids as a function of excitation wavelength

Abstract. Chlorophyll fluorescence emission spectra and the kinetics of 685 mm fluorescence emission from wheat leaf tissue and thylakoids isolated from such tissue were examined as a function of excitation wavelength. A considerable enhancement of fluorescence emission above 700 nm relative to that at 685 nm was observed from leaf tissue when it was excited with 550 nm rather than 450 nm radiation. Such excitation wavelength dependent changes in the emission spectrum occurred over an excitation spectral range of 440–660 nm and appeared to be directly related to the total quantity of radiation absorbed at a given excitation wavelength. Experiments with isolated thylakoid preparations demonstrated that changes in the fluorescence emission spectrum of the leaf were attributable to the optical properties of the leaf and were not due to the intrinsic characteristies of the thylakoid photochemical apparatus. This was not the case for the observed excitation wavelength dependent changes in the 685 nm fluorescence induction curve obtained from leaf tissue infiltrated with DCMU. Excitation wavelength dependent changes in the ratio of the variable to maximal fluorescence emission and the shape of the variable fluorescence induction were observed for leaf tissue. Isolated thylakoid studies showed that such changes in the leaf fluorescence kinetics were representative of the way in which the photochemical apparatus in vivo was processing the absorbed radiation at the different excitation wavelengths. The results are considered in the context of the use of fluorescence emission characteristics of leaves as non-destructive probes of the photochemical apparatus in vivo.     

光系统Ⅱ核心复合物的稳态荧光光谱

在83K和160K两个温度下,通过激发波长对荧光发射谱的影响研究了光系统Ⅱ中核心复合物的荧光光谱特性。用不同波长的光激发,核心复合物的发射谱的最大发射峰值不变,用480、489、495和507nm的光分别激发核心复合物,其光谱最大峰值处的荧光强度随不同激发波长下β-胡萝卜素分子的吸收强度的增大而降低,在长波长区域光谱的变化依赖于首先被激发的色素分子。所以,激发波长的不同影响着核心复合物中能量传递的途径。通过高斯解析,分析出核心复合物中至少存在有7组叶绿素a组分,它们是Ch1 a660,Ch1 a670,Ch1 a680,Ch1 a682,Ch1 a684,Ch1 a687和Ch1 a690。     

Use of probes with fluorescence indicator distributed throughout the pharmacophore to examine the peptide agonist-binding environment of the family B G protein-coupled secretin receptor

Fluorescence techniques can provide insight into the environment of fluorescence indicators situated at distinct sites within a ligand as it is bound to its receptor. Here, we have developed a series of analogues of the 27-amino acid hormone, secretin, that incorporate a fluorescent Alexa Fluor 488 into the amino terminus, the carboxyl terminus, and positions 13 and 22. Each probe bound with high affinity and was biologically active, stimulating full cAMP responses in receptor-bearing Chinese hamster ovary-SecR cells. Treatment with 10 mum guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp) shifted the agonist-bound receptor into a G protein-uncoupled low affinity state. Fluorescence spectra for the probes in solution and bound to the receptor demonstrated maximal emission at 521 nm after excitation at 481 nm. Collisional quenching of fluorescence with potassium iodide revealed that Alexa at the amino terminus of secretin was more accessible than at the other three positions within the probes. Of note, quenching constants for each probe were higher when bound in the active state than in the G protein-uncoupled, low affinity state of the receptor, with the most marked changes occurring for the two midregion probes. Anisotropy values and fluorescence lifetimes confirmed this, with higher anisotropy and longer lifetimes observed for position 13 and 22 probes bound to the receptor in its uncoupled state than in its active state. These observations suggest that the amino terminus of secretin as docked to the receptor is most exposed to the hydrophilic aqueous milieu, and that the major changes in conformation and exposure to the medium occur in the midregion of secretin. Photoaffinity labeling studies have demonstrated approximation of each of these ligand residues with distinct receptor residues. Combining the fluorescence data with photoaffinity labeling data provides insights into the conformation and dynamics of a natural peptide ligand docked to a Family B G protein-coupled receptor.     

Synthesis and characteristics of fluorescent BODIPY-labeled gangliosides

A series of new fluorescent-labeled gangliosides bearing the residues of acids labeled by 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) in the polar or/and apolar moiety were synthesized. These are ganglioside GM1 labeled with the residue of 4,4-difluoro-4-bora-3a,4a-diaza-5,7-dimethyl-s-indacenyl-3-propanoic BODIPY-FL-propanoic) and -indacenyl-5-pentanoic (BODIPY-FL-pentanoic) acid in the oligosaccharide moiety of the molecule, and ganglioside GD1a labeled with two residues of BODIPY-FL-pentanoic acid in the oligosaccharide moiety and also with the residue of BODIPY-FL-pentanoic acid and the residue of 4,4-difluoro-4-bora-3a,4a-diaza-5-octyl-s-indacenyl-5-pentanoic acid in the ceramide part of the molecule. Some spectral characteristics and the behavior in the model membrane systems of the synthesized probes were studied. In their emission spectra, the BODIPY-labeled gangliosides included into phosphatidylcholine liposomes at high concentrations (> 1 mol %) exhibit a long-wavelength maximum (at approximately 630 nm) in addition to the usual maximum (at 510-515 nm).     

Fluorescence emission spectroscopy of 1,4-dihydroxyphthalonitrile. A method for determining intracellular pH in cultured cells.

We have developed new methodology for measuring intracellular pH (pHi) in cultured cell monolayers and epithelia by analyzing the emission spectra of the trapped fluorescent pH probe, 1,4-dihydroxyphthalonitrile (1,4-DHPN). This compound is unique since both its acid and base forms possess different fluorescence emission characteristics that can be used to quantitate pHi. The fluorescence difference spectrum between an acid and alkaline solution of 1,4-DHPN has a maximum at 455 nm and a minimum at 512 nm. By determining the ratio of the intensity at these two wavelengths as a function of pH, a calibration curve was constructed. Since the two intensities are determined simultaneously, the measurement is independent of dye concentration, bleaching, and intensity fluctuation of the excitation source. Furthermore, analysis of the emission spectra permitted the detection of light scattering, binding effects, and chemical modification of the probe. A microspectrofluorometer was constructed to analyze low light level emission spectra from intracellular 1,4-DHPN. The instrument consists of a modified Leitz inverted microscope (E. Leitz, Inc., Rockleigh, NJ) with a Ploem illuminator adapted for broadband excitation and objective focusing capability. The emission spectra were collected by focusing the fluorescence from the cell onto the entrance slit of an imaging monochromator, which was scanned by a SIT camera interfaced with a computer. This permitted the acquisition of fluorescence emission spectra extending from 391-588 nm in approximately 33 ms. pHi measured in the cultured toad kidney epithelial cell line, A6, was 7.49 +/- 0.04 (n = 12) with an external pH of 7.6. A6 cells were found to regulate pHi in response to both acute acid and alkali loads and maintained pHi relatively constant over a wide range of external pH values. The technique described in this report overcomes several of the difficulties encountered with other fluorescent pH probes where excitation spectroscopy is required to monitor pH.