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Z Gryczynski

Publications and source records attributed to Z Gryczynski.

At least 37 records · Page 2Linked to original sources
PubMed1999

Polarization sensing of fluorophores in tissues for drug compliance monitoring.

We used a new method, polarization sensing, to monitor the concentration of the fluorophore rhodamine 800 in an intralipid suspension and in chicken tissue. Rhodamine 800 (Rh800) could be excited at 648 nm using a laser pointer. We developed a simple device for measuring the combined emission from a highly polarized reference film and the unpolarized or orthogonally polarized emission of Rh800 from the scattering intralipid or tissue. The concentration of Rh800 in this medium was revealed by large changes in the polarization (P) with values of P ranging from 0.8 to -0.9. It is possible to vary the sensitive Rh800 concentration range by variation of the detected emission wavelengths, orientation of the excitation polarizer, or fluorophore concentration in the reference film. Polarization sensing of fluorophores in tissue requires only steady-state detection, and can be accomplished with simple and/or portable electronics. Such devices may find use in electronic detection of ingested medicines based on transdermal detection of nontoxic long-wavelength fluorophores.

Animals↗
PubMed1999

Polarization sensing with visual detection.

We describe a new approach to fluorescence sensing which relies on visual determination the polarization. The sensing device consists of a fluorescent probe, which changes intensity in responses to the analyte, and an oriented fluorescent film, which is not affected by the analyte. An emission filter is selected to observe the emission from both the film and the sensing fluorophore. Changes in the probe intensity result in changes in the polarization of the combined emission from the sensor and reference. The degree of polarization can be detected visually using a dual polarizer with adjacent sections oriented orthogonally to each other. The emission passing through the dual polarizer is viewed with a second analyzing polarizer. This analyzer is rotated manually to yield equal intensities from both sides of the dual polarizer. This approach was used to measure the concentration of RhB in intralipid and to measure pH using 6-carboxyfluorescein. The analyzer angle is typically accurate to 1 degree, providing pH values accurate to +/- 0.1 pH unit at the midpoint of the titration curve. We also describe a method of visual polarization sensing that does not require an oriented film and that can use the same fluorophore for the sample and reference. These approaches to visual sensing are generic and can be applied to a wide variety of analytes for which fluorescent probes are available. Importantly, the devices are simple, with the only electronic component being the light source.

Algorithms↗
PubMed1999

Anisotropy-based sensing with reference fluorophores.

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.

Anisotropy↗
PubMed1999

Polarization-based oxygen sensor.

A new approach to oxygen sensing based on the luminescence polarization observed from a novel type of sensor is described. The oxygen sensor consists of an oxygen-sensitive silicone film containing tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride [Ru(dpp)3Cl2] and an oxygen-insensitive film of Styryl 7 in poly(vinyl alcohol). Polarizers are used to select orthogonally polarized emission components from Ru(dpp)3Cl2 and Styryl 7. The polarization of the combined emission was found to be highly sensitive to the partial pressure of oxygen. This method of polarization sensing is generic and can be used with any fluorophore which displays an analyte-dependent change in intensity.

Fluorescence Polarization↗
PubMed1999

High Throughput Screening with Multiphoton Excitation.

Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons to excite the lowest singlet state of the fluorophore. MPE is typically accomplished with short but high-intensity laser pulses, which allows simultaneous absorption of two or more photons. The intensity of the multiphoton-induced fluorescence is proportional to the square, cube, or higher power of the instantneous photon flux. Consequently, two-photon or multiphoton excitation only occurs at the focal point of the incident beam. This property of two-photon excitation allows the excited volume to be very small and to be localized in the center of each well in the HTS plate. We show that two-photon-induced fluorescence of fluorescein can be reliably measured in microwell plates. We also show the use of 6-carboxy fluorescein as a pH probe with two-photon excitation, and measure 4'-6-diamidino-2-phenylindole (DAPI) binding and two-photon-induced fluorescence. In further studies we measure the time-dependent intensity decays of DAPI bound to DNA and of calcium-dependent fluorophores. Finally, we demonstrate the possibility of three-photon excitation of several fluorophores, including indole, in the HTS plate. These results suggest that MPE can be used in high-density multiwell plates.

Journal Article↗
PubMed1999

Fluorescence spectral properties of the anticancer drug topotecan by steady-state and frequency domain fluorometry with one-photon and multi-photon excitation.

Topotecan is an antitumor agent with activity against a variety of cancers. We examined the steady-state and time-resolved fluorescence spectral properties of topotecan with one- and two-photon excitation. Topotecan was found to display a high two-photon cross section near 20 GM for wavelengths within the fundamental output of a Ti:sapphire laser, 800-880 nm. In frozen solution the anisotropies of topotecan are near the theoretical maxima for one-photon and two-photon excitation with colinear electronic transitions. The intensity and anisotropy decays of topotecan fluorescence were found to be homogeneous (single exponentials) in phosphate-buffered saline and propylene glycol. The steady-state and time-resolved data indicate that topotecan binds to a double-helical DNA oligomer d(AT)10 resulting in increased anisotropies and multiexponential intensity and anisotropy decays. Subnanosecond components in the anisotropy decay of the DNA-topotecan complex suggest loose binding of the drug to DNA. Loose binding of topotecan to DNA is also revealed by accessibility of topotecan to collisional quenching by iodide.

Antineoplastic Agents↗
PubMed1998

Time resolved emissions in the picosecond range of single tryptophan recombinant myoglobins reveal the presence of long range heme protein interactions.

We have analyzed the time resolved fluorescence emission in the subnanosecond range of recombinant wild-type SW myoglobin and its single TRP mutants W7F and W14F. These recombinants carry a methionine at the N1 terminal end. The emission of Trp-7 in the met form of W14F showed residual lifetime components much shorter than those estimated after excitation energy transfer to the heme. We propose that in this recombinant the N1 methionine is close to Trp-7, thereby producing an extra quenching due to either collisions or electron transfer with its sulfur. When the measurements were repeated on its CO-form, the extra quenching of Trp-7 was much decreased, indicating a heme linked conformational change involving the amino terminal end of the protein. This hypothesis is supported by ligand linked conformational changes in myoglobin, reported by Ansari et al. and by Giardina et al. At neutral pH the lifetimes of W7F were consistent with estimations based on the atomic coordinates of SW myoglobin. Those of the wild-type were exactly the combination of the lifetimes of the two mutants. This suggest that the mutations did not affect the overall structure of the protein. However, in the ferric form, substitution of Trp-14 in W14F resulted in low stability at acid pH, as evident from lifetimes modifications at pH 4.8, while no modifications were produced by titrations of W7F to pH 4.5. This suggests a role of Trp-14 in the structural stability of myoglobin.

Animals↗
PubMed1998

Temperature titration: a new approach to the thermodynamics of oxygen binding to hemoglobin.

A cell was constructed in order to study hemoglobin's reaction with gaseous ligands. The temperature of the hemoglobin sample is systematically altered within a given temperature range (275-310 degrees K), while the percentage of oxygen in the equilibrating gas is kept constant. The equilibration time of the sample at each temperature step depends on sample concentration, ligand affinity, and absolute temperature; in most cases, the equilibration time is on the order of minutes. The construction of the optical compartment allows the experimenter to vary the optical pathlength using specially designed spacers, thus making it possible to study hemoglobin-ligand interactions over a wide range of protein concentrations (0.1-200 mg/ml). Optical glass is used in the construction of the cuvette in order to optimize its optical stability over a long period of time. At equilibrium the absorption spectrum of the sample is collected and decomposed into the relative contributions of oxy-Hb, deoxy-Hb, and ferric-Hb, thus revealing the fraction of oxyhemoglobin as well as any baseline drifts and protein degradation. Temperature steps of 1 degree K are already sufficient to change the absorption spectra in a significant way. This type of setup is also advantageous in that the experimenter can change the sample at any point (temperature) without having to restart the entire experiment. This makes it possible to study the oxygen binding characteristics of unstable hemoglobins. Analyses of the binding curves obtained with this technique immediately yield the overall oxygen binding constants beta i together with the respective standard enthalpies delta H(i).

Hemoglobins↗
PubMed1998

Entropy-driven intermediate steps of oxygenation may regulate the allosteric behavior of hemoglobin.

When the oxygen binding isotherms of human, bovine and fallow deer (Dama-Dama) hemoglobins are measured at different temperatures either by optical or calorimetric techniques, analyses according to the Adair's formalism show that at least one of the intermediate steps of ligation has a positive enthalpy change, i.e., absorbs rather than emitting heat, indicating that it is entropy rather than enthalpy driven (Bucci, E., et al. 1991. Biochemistry. 30:3195-3199; Bucci, E., et al. 1993. Biochemistry. 32:3519-3526; Johnson, C., et al. 1992. Biochemistry. 31:10074-10082; Johnson, C., et al. 1995. Biophys. Chem. 59:107-117). This phenomenon is confirmed in systems in which the beta82 lysines of human hemoglobin are covalently cross-linked by acylation with dicarboxylic acids of increasing length, namely the fumaryl (four-carbon-long), adipoyl (six-carbon-long), and sebacoyl (10-carbon-long) residues. Consistently in all of the systems here reported, the enthalpy excursions are masked by compensatory entropy changes, which keep the free energy of ligand binding constant for the first three steps of oxygenation. Furthermore, the cooperativity index and the overall oxygen affinity seem to be correlated to the positive enthalpy excursions of the intermediate steps of ligation. Fumaryl-Hb (hemoglobin cross-linked with a fumaryl residue, four carbons) with the lowest absorption of heat has the highest affinity and lowest cooperativity index. Adipoyl-Hb (hemoglobin cross-linked with an adipoyl residue, six carbons) has the highest absorption of heat and the highest cooperativity index. It appears that nonuniform heat release by the intermediates of oxygenation is part of the allosteric phenomena in hemoglobin systems. There is not enough information that would allow assigning these phenomena to the interplay of the various conformations described for hemoglobin besides the classic T (Fermi et al. 1984. J. Mol. Biol. 175:159-174) and R (Shanaan. 1983. J. Mol. Biol. 171:31-59), as listed at the end of the Discussion. The possibility cannot be excluded that entropy-driven steps characterize new conformational transitions still to be described.

Adipates↗
PubMed1998

Fluorescence anisotropy controlled by light quenching.

We demonstrated that fluorescence anisotropy can be effectively decreased or increased in the presence of light quenching, depending on relative polarizations of excitation and quenching pulses. For parallel light quenching, anisotropy decreases to 0.103 and z-axis symmetry is preserved. In the presence of perpendicular light quenching, the steady-state anisotropy of a pyridine-2-glycerol solution increases from 0.368 for an unquenched sample to 0.484 for a quenched one. We show that the angular distribution of transition moments loses z-axis symmetry in the presence of perpendicular light quenching. In these cases we used more general definitions of anisotropy. Induced by light quenching, anisotropy can be applied in both steady-state and time-resolved measurements. In particular, the systems with low or no anisotropy can be investigated with the proposed technique.

Fluorescence Polarization↗
PubMed1997

Two-photon excitation by the evanescent wave from total internal reflection.

We report the first observation of two-photon excitation of fluorescence using the evanescent wave from total internal reflectance (TIR). The evanescent wave at 770 nm from a fs Ti:Sapphire laser was used to excite the calcium probe Indo-1 at a quartz-water interface. The emission intensity of Indo-1 depended quadratically on the incident power at 770 nm, when incidence angles were above and below the critical angle (theta c) for TIR. The time-resolved intensity and anisotropy decays with TIR at 770 nm demonstrated the origin of the signals as Indo-1 and eliminated the possibility of scattered light contributing to the signal. The emission from Indo-1 was further demonstrated to be due to two-photon excitation by the increased anisotropy observed both from the steady-state and time-resolved data. Comparison of the intensities for one-photon and two-photon evanescent wave excitation revealed a smaller effective excited volume for two-photon excitation, indicating that the excited fluorophores are located closer to the interface with two-photon excitation. These results suggest that total internal reflectance can be combined with two- or multiphoton excitation for studies of surface absorption, immunoassays, or pattern photobleaching.

Fluorescence↗
PubMed1997

Time-resolved fluorescence of hemoglobin species.

We used time-resolved fluorescence in the pico- to nanosecond time range to monitor the presence of tetramers, dimers and monomers in carbonmonoxyhemoglobin (COHb) solutions and to investigate how their distributions change under different experimental conditions. Comparison of fluorescence lifetime computed from the atomic coordinates of COHb (Vasquez et al., 1996) with those experimentally measured allowed identification of molecular species present in the hemoglobin solution. It was possible to observe modification of the distribution of tetramers, dimers, monomers and species with disordered hemes produced by different experimental conditions. Protein concentration affected the detectable lifetimes, indicating increasing amounts of dimers and monomers at low protein concentrations, while the amount of inverted hemes was not modified. Titration with up to 1 M NaCl modified only the extent of dissociation of hemoglobin into dimers, without affecting heme inversion and monomer formation. Hyperbaric pressure increased the amounts of dimers and monomers. This is the first time that monomeric subunits of hemoglobin have been detected at neutral pH in the normal system.

Carboxyhemoglobin↗
PubMed1997

Effect of central metal substitution on linear dichroism of porphyrins: evidence of out-of-plane transition moments.

Absorption anisotropy and emission anisotropy measurements in poly(vinyl) alcohol (PVA) films of different porphyrin derivatives are reported. Wavelength dependent absorption anisotropy in oriented PVA films, and wavelength dependent excitation spectrum of emission anisotropy of fluorescent porphyrin derivatives in isotropic PVA films indicate the presence of multiple transition moments with different well-defined orientation. Comparison of linear dichroism and orientation behavior in stretched PVA films of deuteroporphyrin III (C2V symmetry) and its iron derivative reveals significant out-of-plane transition moment components. A considerable participation of out-of-plane polarized absorption components is also observed for metal derivatives of non-symmetrical protoporphyrin IX. It appears that central metal substitutions in porphyrin rings do not produce 'circular' degeneration of electronic transition moments. Instead, the presence of metal induces absorption components orthogonal to the porphyrin plane.

Anisotropy↗
PubMed1996

Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin.

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 lysines of the beta-cleft (DecHb). The Adair's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is present at the second step of oxygenation, which has an affinity 27 times lower than at the first step. Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity with an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol Chem. 269, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31-59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation.

Allosteric Regulation↗
PubMed1996

Two-color two-photon excitation of fluorescence.

We report the observation of two-photon excitation of an organic fluorophore with two different wavelengths, a phenomenon we refer to as two-color two-photon (2C2P) excitation. Ultraviolet emission of p-terphenyl at 340 nm was observed when the sample was illuminated with both 375 and 750 nm pulses from a picosecond dye laser. The emission of p-terphenyl was about 100-fold and more than 1000-fold less for illumination at only 375 or 750 nm, respectively. Observation of the 2C2P signal required temporal and spatial overlap of the 375 and 750 nm pulses. The amplitude of the signal depended on the polarization of each beam. 2C2P excitation can have applications in fluorescence microscopy and elsewhere when spatially localized excitation is desirable.

Fluorescence Polarization↗
PubMed1996

On the possibility of evanescent wave excitation distal from a solid-liquid interface using light quenching.

Evanescent wave illumination with total internal reflection is often used to provide excitation near a quartz-water interface. We now show that evanescent illumination at one wavelength and incident angle, coupled with light quenching at a second wavelength and incident angle, can be used for selective excitation of fluorophores located up to 5000 A into the aqueous phase. The displacement of the fluorophore population from the solid-liquid interface depends on the angles of incidence of the excitation and quenching beams and the optical power of the quenching beam. Light quenching with an evanescent wave was demonstrated to be experimentally possible using Pyridine2 and a light-quenching wavelength of 736 nm. The use of combined evanescent wave excitation and evanescent wave quenching could provide selective excitation of fluorophores in the cytoplasmic region of cells and may provide improved response times for optical sensors based on evanescent excitation.

Fluorescence↗
PubMed1995

Heme-protein interactions in horse heart myoglobin at neutral pH and exposed to acid investigated by time-resolved fluorescence in the pico- to nanosecond time range.

We measured the steady state and time-resolved emission intensity decay of horse heart myoglobin at various pH values from neutral to pH 4.42. The steady state intensity was reversibly increased with the decreasing pH, almost doubling at pH 4.5. Frequency domain data for emission decay were analyzed separately for each pH and simultaneously by global analyses. The results indicated the presence of four lifetime components, conserved throughout the pH titrations at 40, 116, 1363, and 4822 ps, respectively. The titration affected only their fractional intensities. Assignments of the lifetimes were based on the Förster theory of radiationless dipole-dipole interaction and the atomic coordinates of the system. We assigned the two shorter lifetimes to Trp-14 and Trp-7, respectively, in the presence of normal hemes. The 1363-ps lifetime was assigned to Trp-7 with inverted hemes (i.e. rotated 180 degrees around the alpha-gamma-meso axis of the porphyrin ring). The 4822-ns lifetime was assigned to reversibly heme-dissociated myoglobin. Lorentzian lifetime distributions were narrow for the lifetimes at 40, 116, and 4822 ps, indicating a homogeneous protein structure. Instead the lifetime at 1363 ns had a broad, pH-independent distribution consistent with small angle wobblings of inverted hemes inside the heme pocket. These analyses revealed the presence of three species originating from heme-protein interactions: the native form of crystalline myoglobin, the conformation with disorder hemes, and the reversibly dissociated heme-free myoglobin. There was increased heme inversion and heme dissociability at lower pH, consistent with the titration of the proximal and distal histidines inside the heme pocket.

Animals↗