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J R Lakowicz

Publications and source records attributed to J R Lakowicz.

At least 19 recordsLinked to original sources
PubMed2001

New color chemosensors for monosaccharides based on azo dyes.

Sugar detection is important for many applications. New developments in sugar signaling would provide new technologies to monitor glucose and other sugars. Azo dye 1 presents a new way to build molecular color sensors for monosaccharides. The boronic acid group is used as chelator group for monosaccharides and linked directly in resonance with the aromatic dye. Dye 1 shows a color change, from orange to purple, in the presence of sugar at neutral pH. [structure: see text]

Azo Compounds↗
PubMed2001

Radiative decay engineering: biophysical and biomedical applications.

Fluorescence spectroscopy is a widely used research tool in biochemistry and molecular biology. Fluorescence has also become the dominant method enabling the revolution in medical diagnostics, DNA sequencing, and genomics. To date all the fluorescence observables, including spectral shifts, anisotropies, quantum yields, and lifetimes, have all been utilized in basic and applied uses of fluorescence. In this forward-looking article we describe a new opportunity in fluorescence, radiative decay engineering (RDE). By RDE we mean modifying the emission of fluorophores or chromophores by increasing or decreasing their radiative decay rates. In most fluorescence experiments the radiative rates are not changed because these rates depend on the extinction coefficient of the fluorophore. This intrinsic rate is not changed by quenching and is only weakly dependent on environmental effects. Spectral changes are usually caused by changes in the nonradiative rates resulting from quenching or resonance energy transfer. These processes affect the emission by providing additional routes for decay of the excited states without emission. In contrast to the relatively constant radiative rates in free solution, it is known that the radiative rates can be modified by placing the fluorophores at suitable distances from metallic surfaces and particles. This Review summarizes results from the physics literature which demonstrate the effects of metallic surfaces, colloids, or islands on increasing or decreasing emissive rates, increasing the quantum yields of low quantum yield chromophores, decreasing the lifetimes, and directing the typically isotropic emission in specific directions. These effects are not due to reflection of the emitted photons, but rather as the result of the fluorophore dipole interacting with free electrons in the metal. These interactions change the intensity and temporal and spatial distribution of the radiation. We describe the unusual effects expected from increases in the radiative rates with reference to intrinsic and extrinsic biochemical fluorophores. For instance, the decreased lifetime can result in an effective increase in photostability. Proximity to nearby metallic surfaces can also increase the local field and modify the rate of excitation. We predict that the appropriate localization of fluorophores near particles can result in usefully high emission from "nonfluorescent" molecules and million-fold increases in the number of photons observable from each fluorophore. We also describe how RDE can be applied to medical testing and biotechnology. As one example we predict that nearby metal surfaces can be used to increase the low intrinsic quantum yields of nucleic acids and make unlabeled DNA detectable using its intrinsic metal-enhanced fluorescence.

Energy Transfer↗
PubMed2001

Intrinsic fluorescence from DNA can be enhanced by metallic particles.

High sensitivity detection of DNA is essential for genomics. The intrinsic fluorescence from DNA is very weak and almost all methods for detecting DNA rely on the use of extrinsic fluorescent probes. We show that the intrinsic emission from DNA can be enhanced many-fold by spatial proximity to silver island films. Silver islands are subwavelength size patches of metallic silver on an inert substrate. Time-resolved measurements show a decreased lifetime for the intrinsic DNA emission near the silver islands. These results of increased intensity and decreased lifetime indicate a metal-induced increase in the radiative rate decay of the DNA bases. The possibility of increased radiative decay rates for DNA bases and other fluorophores suggest a wide variety of DNA measurements and other biomedical assays based on metal-induced increases in the fluorescence quantum yield of weakly fluorescent substances.

Animals↗
PubMed2001

Long-wavelength long-lifetime luminophores.

We describe a new approach to making luminophores that display long emission wavelengths, long decay times, and high quantum yields. These luminophores are covalently linked pairs with a long-lifetime resonance-energy-transfer donor and a long-wavelength acceptor. The donor was a ruthenium (Ru) metal-ligand complex. The acceptor was the Texas Red. The donor and acceptor were covalently linked by polyproline spacers. The long-lifetime donor results in a long-lived component in the acceptor decay, which is due to RET. Importantly, the quantum yield of the luminophores approaches that of the higher quantum yield acceptor, rather than the lower quantum yield typical of metal-ligand complexes. The emission maxima and decay time of such tandem luminophores can be readily adjusted by selection of the donor, acceptor, and distance between them. Luminophores with these useful spectral properties can also be donor-acceptor pairs brought into close proximity by some biochemical association reaction. Luminophores with long-wavelength emission and long lifetimes can have numerous applications in biophysics, clinical diagnostics, DNA analysis, and drug discovery.

Algorithms↗
PubMed2001

Fluorescence lifetime characterization of novel low-pH probes.

The structures and functions of the cellular acidic compartments are strongly dependent on the pH gradients across vesicular membranes. Measurement and imaging of the vesicular pH require fluorophores with appropriate pK(a) values. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulness to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases that selectively accumulate in acidic vesicles after being protonated. They have higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffer pH increased the quenching effects of their basic side chains and substantially reduced their steady-state fluorescence and lifetimes. The apparent pK(a) values determined from their lifetime responses were shifted to near neutral values because of the dominant intensity contribution from their protonated species. One unique property of LysoSensor DND-189 is its nonmonotonic lifetime responses of the maxima occurring between pH 4 and 5. LysoSensor DND-192 did not show significant lifetime changes over a wide pH range. LysoSensor DND-160, which was the only excitation and emission ratiometric probe, showed significant pH-dependent lifetime changes as well as its spectral shifts. Its apparent pK(a) values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Because of the pH-dependent absorption spectra, the apparent pK(a) values could be manipulated between 3 and 5 by changing the excitation and/or emission wavelengths. These results indicate that LysoSensor DND-160 is a promising probe for lifetime imaging to determine lysosomal pH.

Buffers↗
PubMed2001

Evaluation of two synthetic glucose probes for fluorescence-lifetime-based sensing.

We evaluated two anthracene derivatives with covalently attached boronic acid groups for fluorescence-lifetime-based sensing of glucose. These anthracene derivatives also contained alkyl amino groups, which quenched the anthracene emission by photo-induced electron transfer. Both anthracene derivatives displayed increased intensities and lifetime in the presence of glucose, as seen from the frequency-domain measurements. A fluorescence lifetime change from 9.8 to 12.4 and 5.7 to 11.8 ns is observed, after the addition of glucose, for the anthracene substituted with one and two boronic acid groups, respectively. This results in a change in the phase angle up to 15 degrees and 30 degrees and in the modulation up to 12 and 25% at 30 MHz for these compounds, respectively. Titration curves in the presence of BSA and micelles are also presented to show the potential interferences from biomolecules. Dissociation constants were evaluated for both compounds, and association with glucose was found to be reversible. Importantly, the apparent glucose binding constants are about 5- to 10-fold smaller with phase, modulation, or mean lifetime than with intensities measurements, shifting the glucose-sensitive range to physiological values. Combining these results and the use of a simple UV-LED as excitation source, the results show an interesting potential of these two compounds in the development of lifetime base devices using synthetic probes for glucose.

Anthracenes↗
PubMed2001

Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor.

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.

Adrenergic beta-Agonists↗
PubMed2001

Optical determination of glutamine using a genetically engineered protein.

We have developed a reagentless optical biosensor for glutamine based on the Escherichia coli glutamine binding protein (GlnBP). Site-directed mutagenesis was performed to engineer single cysteine mutants which were covalently modified with environmentally sensitive sulfhydryl-reactive probes. The fluorescence emission of acrylodan and 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS) attached to GlnBP mutant S179C was shown to decrease 65 and 35%, respectively, upon titration with increasing amounts of glutamine (0 to 6.4 microM; K(Dapp) 160 nM). No significant changes in the fluorescence intensity were observed for the structurally similar amino acids glutamate, asparagine, and arginine. Time-resolved intensity decays showed a 2.4-fold decrease in mean lifetime for GlnBP S179C-acrylodan upon the addition of glutamine, indicating the possibility of a lifetime-based assay. Anisotropy decay measurements for GlnBPS179C-acrylodan showed a 13-ns rotational correlation time in the ligand-free state, whereas multiple correlation times were assigned in the glutamine-bound conformation. The decrease in fluorescence intensity of S179C-acrylodan was adapted to polarization sensing of glutamine. The engineered GlnBP is a first step toward the development of a nonenzymatic biosensor capable of determining glutamine concentrations in cell cultures.

Biosensing Techniques↗
PubMed2001

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C.

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant differences between full-length TnI and TnI(1-159) in their effect on Trp-26. Our results provide the first indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.

Acrylamide↗
PubMed2001

Texture analysis of fluorescence lifetime images of nuclear DNA with effect of fluorescence resonance energy transfer.

BACKGROUND: Fluorescence lifetime imaging microscopy (FLIM) is becoming an important tool in cellular imaging. In FLIM, the image contrast is concentration insensitive, whereas it is sensitive to the local environment and interactions of fluorophores such as fluorescence resonance energy transfer (RET). METHODS: Fluorescence microscopy, lifetime imaging, and texture analysis were used to study the spatial distribution of fluorophores bound to nuclear DNA. 3T3-Swiss albino mice fibroblast nuclei were labeled with Hoechst 33258 (Ho), an AT-specific dye, and 7-aminoactinomycin D (7-AAD), a GC-specific dye. Ho is a RET donor to the 7-AAD acceptor. RESULTS: Texture analysis of 50 alcohol-fixed nuclei quantitatively showed changes of spatial distribution of apparent donor lifetimes. RET increased the spatial heterogeneity in the phase and modulation lifetime images. In most of the doubly stained cells (about 80%), the phase and modulation lifetime distributions were spatially homogeneous. In about 20% of the cells, we noticed that lower phase and modulation lifetimes caused by RET were correlated with regions of high Ho intensity in the nuclei. CONCLUSIONS: The spatial lifetime heterogeneity of Ho in presence of 7-AAD seems to be caused by RET between closely spaced strands in the three dimensionally condensed regions of DNA.

3T3 Cells↗
PubMed2001

On the possibility of long-wavelength long-lifetime high-quantum-yield luminophores.

We describe an approach to creating a new class of luminophores which display both long wavelength emissions exceeding 600 nm and long lifetimes. These luminophores are based on resonance energy transfer (RET) from a long lifetime donor to a short lifetime but long wavelength acceptor. We demonstrated the possibility of obtaining these desirable spectral properties using donors and acceptors noncovalently bound to DNA. The donor was a ruthenium (Ru) metal-ligand complex in which one of the diimine ligands intercalated into double-helix DNA. The acceptors were either nile blue, TOTO-3, or TO-PRO-3. Upon binding of the acceptor to donor-labeled DNA, we found that the acceptor quantum yield was remarkably enhanced so that the wavelength-integrated intensities of the donor and acceptor bound to DNA were many-fold greater than the intensity of the donor and acceptor alone when separately bound to DNA. The origin of this effect is efficient energy transfer from the donor. Under these conditions the effective overall quantum yield approaches that of the acceptor. Importantly, the increased quantum yield can be obtained while maintaining usefully long apparent acceptor lifetimes of 30 to 80 ns. The effect of an increased quantum yield from a low quantum yield donor may find use in assays to detect macromolecular binding interactions. These results suggest the synthesis of covalently linked donor-acceptor pairs with the desirable spectral properties of long wavelength emission, high quantum yield, and moderately long lifetimes for gated detection.

Acridine Orange↗
PubMed2001

Enzyme fluorescence as a sensing tool: new perspectives in biotechnology.

The technology for fluorescence protein-sensing is advancing rapidly owing to the continued introduction of new concepts, new fluorophores, and proteins engineered for sensing-specific analytes. Concerns about the reversibility and selectivity of engineered proteins are being addressed by developing biosensors that are based on the utilisation of coenzyme-depleted enzymes. Such biomolecules do not consume the substrate and can exhibit conformational changes upon the binding of the analyte, which can be easily detected as fluorescence change. In addition, concerns about the stability of biosensors can be overcome by using thermostable enzymes isolated from thermophilic microorganisms. Finally, the development of new techniques such as polarization-based sensing, anisotropy-based sensing and lifetime-based sensing, all of which can be accomplished with light-emitting diodes as the light source, is prompting the design of a new class of specific and stable biosensors, as has occurred with blood glucose measurement. These biosensors represent a valid alternative to the conventional clinical chemistry diagnostics.

Biosensing Techniques↗
PubMed2001

Frequency-domain fluorescence microscopy with the LED as a light source.

We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz--250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10--15 microm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)(2)phe-C(12),with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.

3T3 Cells↗
PubMed2001

On the effect of sodium dodecyl sulfate on the structure of beta-galactosidase from Escherichia coli. A fluorescence study.

An understanding of the structure-function relationship of proteins under different chemical-physical conditions is of fundamental importance for an understanding of their structure and function in cells. In this paper we report the effects of sodium dodecyl sulfate and temperature on the structure of beta-galactosidase from Escherichia coli, as monitored by fluorescence spectroscopy. The structure of the protein was studied in the temperature range of 10-60 degrees C in the absence and presence of sodium dodecyl sulfate by frequency-domain measurement of the intrinsic fluorescence intensity and anisotropy decays. The time-resolved fluorescence data in the absence of SDS indicated that at 10 degrees C the tryptophanyl emission decays were well described by a three exponential decays model, and that the temperature increase resulted in shortening of the long-lived component with little change in the short- and middle-lived components. The addition of SDS to the protein solution also affected the long-lived component. The effects of the detergent and temperature on the enzyme structure were also investigated by means of quenching experiments and anisotropy decays. The obtained results showed that the presence of SDS confers more flexibility to the protein structure, and suggest a strict relation between enzyme activity and protein flexibility.

Acrylamide↗
PubMed2001

Fluorescence properties of albumin blue 633 and 670 in plasma and whole blood.

We have determined the fluorescence characteristics of two long wavelength dyes, albumin blue 633 (AB633) and 670 (AB670), in plasma and blood to evaluate the possibility of making direct fluorescence sensing measurements in blood. Using binding and lifetime measurements we were also able to show that these dyes bind selectively to human serum albumin (HSA) in plasma and blood. By measuring changes in the mean lifetime of AB670 with changes in the HSA concentration, we showed that lifetime-based sensing can be used to monitor HSA concentrations using these albumin blue dyes. Anisotropy measurements for AB633 and AB670 in plasma and blood revealed high anisotropy values for these dyes in these media. Exploiting these high anisotropies, we were also able to determine HSA concentrations in plasma and blood mimics using changes in AB670 anisotropy with HSA concentration. These results show that, apart from being able to make fluorescence measurements directly in plasma and blood, it is possible to sense directly for specific plasma/blood components using fluorescent probes that bind preferentially to them.

Anisotropy↗
PubMed2001

Texture analysis of fluorescence lifetime images of AT- and GC-rich regions in nuclei.

We used intensity and fluorescence lifetime microscopy (FLIM) of 3T3 nuclei to investigate the existence of AT-rich and GC-rich regions of the nuclear DNA. Hoechst 33258 (Ho) and 7-aminoactinomycin D (7-AAD) were used as fluorescence probes specific for AT and GC base pairs, respectively. YOYO-1 (Yo) was used as a dye that displays distinct fluorescence lifetimes when bound to AT or GC base pairs. We combined fluorescence imaging of Ho and 7-AAD with time-resolved measurements of Yo and took advantage of an additional information content of the time-resolved fluorescence. Because a single nucleus could not be stained and measured with all three dyes, we used texture analysis to compare the spatial distribution of AT-rich and GC-rich DNA in 100 nuclei in different phases of the cell cycle. The fluorescence intensity-based analysis of Ho- or 7-AAD-stained images indicates increased number and larger size of the DNA condensation centers in the G2/M-phases compared to G0/1-phases. The lifetime-based study of Yo-stained images suggests spatial separation of the AT- or GC-rich DNA regions in the G2/M-phase. Texture analysis of fluorescence intensity and lifetime images was used to quantitatively study the spatial change of condensation and separation of AT- and GC-rich DNA during the cell cycle.

3T3 Cells↗
PubMed2000

Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer.

BACKGROUND: DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin. METHODS: We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD). RESULTS: The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells. CONCLUSIONS: Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.

3T3 Cells↗