Biomedical subjects

H Szmacinski

Publications and source records attributed to H Szmacinski.

At least 37 records · Page 2Linked to original sources

PubMed1995

Metal-ligand complexes as a new class of long-lived fluorophores for protein hydrodynamics.

We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes that can be used to measure rotational motions of proteins. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. In this report, we show that the asymmetric complex Ru(bpy)2(dcbpy) (PF6)2 displays a high anisotropy value when excited in the long wavelength absorption band. For covalent linkage to proteins, we synthesized the N-hydroxy succinimide ester of this metal-ligand complex. To illustrate the usefulness of these probes, we describe the intensity and anisotropy decays of [Ru(bpy)2(dcbpy)] when covalently linked to human serum albumin, concanavalin A (ConA), human immunoglobulin G (IgG), and Ferritin, and measured in solutions of increased viscosity. These data demonstrate that the probes can be used to measure rotational motions on the 10 ns to 1.5 microseconds timescale, which so far has been inaccessible using luminescence methods. The present probe [Ru(bpy)2(dcbpy)] can be regarded as the first of a class of metal-ligand complexes, each with different chemical reactivity and spectral properties, for studies of macromolecular dynamics.

Biophysical Phenomena↗

PubMed1995

On the possibility of calcium imaging using Indo-1 with three-photon excitation.

We show that the calcium fluorophore Indo-1 can be excited by simultaneous absorption of three-photons at 885 nm, a wavelength readily available from Ti:sapphire lasers. Three-photon excitation was demonstrated by the emission intensity of Indo-1 which depended on the cube of the laser power, and by a higher anisotropy than was observed for two-photon excitation. Excitation of Indo-1 becomes a two-photon process when the wavelength is decreased to 820 nm. Three-photon excitation was accomplished at a low 17 microM concentration of Indo-1. Examination of the spatial profile of the excited Indo-1 showed a smaller volume for three- versus two-photon excitation. These results suggest that three-photon excitation may be useful in fluorescence microscopy using the long wavelength output of Ti:sapphire lasers, and may provide higher spatial resolution than available using two-photon excitation.

Calcium↗

PubMed1994

Functional significance of the conformational dynamics of the N-terminal segment of secreted phospholipase A2 at the interface.

The kinetic and fluorescence properties of several pig pancreatic phospholipase A2 (PLA2) with substitutions and deletions in the N-terminal region and of tyrosines 52 and 73 are characterized. The substitutions Ala-1-D-Ala or -Gly, Trp-3-Phe, Gln-4-Nle, Arg-6-Glu, Tyr-52-Phe, and Tyr-73-Phe had at the most only a modest effect on the interfacial catalytic activity on the anionic interface to which they bind with high affinity. The observed rate of hydrolysis in the scooting mode by deletion mutants lacking one or more successive residues from the N-terminal region was lower by 50-95%. Detailed kinetic analysis of the deletion mutant lacking Ala-1 (des-1-AMPA) showed that the 50% decrease in the rate is due to a 5-fold increase in the interfacial Michaelis-Menten parameter, KM*, without a significant change in kcat. These results and direct measurements show that the primary effect of Ala-1 deletion is to lower the affinity for the active site directed ligands. Although the affinity of these mutants for anionic interface remains the same as for the wild type, the affinity for zwitterionic neutral diluents is considerably lower. Significant differences in the fluorescence quantum yields and the heterogeneity in the frequency-domain fluorescence intensity decays of these enzymes suggest that both in solution and at the interface the N-terminal region is an ensemble of conformations rather than a discrete state. Additional results suggest that the interfacial microenvironment of Trp-3 in des-1-AMPA is more polar and Trp-3 is more accessible to quenching by acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Synthesis of squaraine-N-hydroxysuccinimide esters and their biological application as long-wavelength fluorescent labels.

We describe the synthesis and the fluorescence spectral characterization of two conjugatable long-wavelength fluorescence probes. These probes consist of a squaraine moiety, which is a cyanine-type chromophore with a central squarate bridge, and a reactive N-hydroxysuccinimide group for coupling with amino functions. One form is water soluble due to the presence of a sulfobutyl group; the other is water insoluble. The water-insoluble form was reacted with taurine to achieve water solubility and this squaraine-taurine conjugate displayed a very high affinity for bovine serum albumin. The squaraines exhibit short lifetimes and low quantum yields in water, with a significant increase in lifetime and quantum yield when bound to proteins. Their absorption maxima around 635 nm in water and 640 nm when bound to proteins allow excitation with the newly commercially available diode laser sources at 635, 645, and 650 nm. The spectral properties and photostabilities of the water-soluble squaraine probes are compared with those of the commercially available CY5-NHS ester.

Cyclobutanes↗

PubMed1994

Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2.

We describe the first fluorescence lifetime images of cells. To demonstrate this new capability we measured intracellular images of Ca2+ in COS cells based on the Ca(2+)-dependent fluorescence lifetime of Quin-2. Apparent fluorescence lifetimes were measured by the phase-modulation method using a gain-modulated image intensifier and a slow-scan CCD camera. We describe methods to correct the images for photobleaching during acquisition of the data, and to correct for the position-dependent response of the image intensifier. The phase angle Quin-2 images were found to yield lower than expected Ca2+ concentrations, which appears to be the result of the formation of fluorescent photoproducts by Quin-2. Fluorescence lifetime imaging (FLIM) does not require wavelength-radiometric probes and appears to provide new opportunities for chemical imaging of cells.

Animals↗

PubMed1994

Fluorescence lifetime imaging microscopy: homodyne technique using high-speed gated image intensifier.

In the previous sections we demonstrated imaging of intracellular Ca2+ using our approach to FLIM. What other analytes can be imaged using FLIM? We have now characterized the lifetime of a good number of ion indicators. Based on these studies we know that Cl- can be imaged using FLIM with probes such as SPQ or MQAE, pH can be imaged using resorufin and probes of the SNAFL and SNARF (Molecular Probes) series, and Mg2+ can be imaged using Magnesium Green, Mag-quin-2, or Mag-quin-1 (Molecular Probes). At present, the probe for K+, as PBFI, are just adequate as a lifetime probe, but it seems likely that newer probes for Na+ (Sodium Green) and K+ will be practical for effective imaging. Of course, imaging of oxygen is possible using a wide variety of fluorophores. It should be noted that a wide variety of substances and/or phenomena are known to alter decay times, acting as quenchers. These include the phenomena of resonance energy transfer, collisional quenching, temperature effects, and viscosity effects. Also, the FLIM method is not limited to microscopic objects but can be possibly used in remote imaging of any object. Hence, FLIM will allow the imaging of the chemical and physical properties of objects based on the effects of the local environment on the decay kinetics of fluorophores. The instrumentation for FLIM is presently complex and requires a moderately complex laser source, a gain-modulated image intensifier, and a slow-scan CCD camera. However, one can readily imagine the instrumentation becoming rather compact, and even all solid-state, owing to advances in laser and CCD technologies and, more importantly, advances in probe chemistry. To be specific, the dye laser shown in Fig. 1 may be replaced by a simpler UV laser, such as the 354 nm HeCd laser which has become available (Fig. 11). Intensity modulation of a continuous wave sources can be accomplished with acoustooptic modulators. The scientific slow-scan CCD cameras are presently rather expensive, but they are used in the present instrumentation because of their linearity and high dynamic range. However, the increasing use of CCD detectors suggest that even the scientific-grade CCD cameras will soon become less costly. Additionally, the frame rates of these detectors continue to increase in response to the need for faster imaging. Furthermore, the performance of the video CCD cameras is increasing, as seen by the introduction of 10-bit video analog-to-digital (A/D) converters.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoquinolines↗

PubMed1994

Site-to-site diffusion in proteins as observed by energy transfer and frequency-domain fluorometry.

We report measurements of the site-to-site diffusion coefficients in proteins and model compounds, which were measured using time-dependent energy transfer and frequency-domain fluorometry. The possibility of measuring these diffusion coefficients were shown from simulations, which demonstrate that donor (D)-to-acceptor (A) diffusion alters the donor frequency response, and that this effect is observable in the presence of a distribution of donor-to-acceptor distances. For decay times typical of tryptophan fluorescence, the simulations indicate that D-A diffusion coefficients can be measured ranging from 10(-7) to 10(-5) cm2/s. This possibility was verified by studies of a methylene-chain linked D-A pair in solutions of varying viscosity. The D-A diffusion was also measured for two labeled peptides and two proteins, melittin and troponin I. In most cases we used global analysis of data sets obtained with varying amounts of collisional quenchers to vary the donor decay time. Unfolding of troponin I results in more rapid D-A diffusion, whereas for melittin more rapid diffusion was observed in the alpha-helical state but over a limited range of distances.

Diffusion↗

PubMed1993

Optical measurements of pH using fluorescence lifetimes and phase-modulation fluorometry.

We measured the pH-dependent fluorescence decay times of the seminaphthofluoresceins (SNA-FL), seminaphthorhodafluors (SNARF), and BCE-CF using phase-modulation fluorometry. The phase and modulation values were found to be strongly pH-dependent in the physiological pH range, over the easily accessible range of light modulation frequencies from 10 to 300 MHz, making these probes useful as lifetime-based pH sensors. The phase and modulation values are dependent on excitation and emission wavelength as well as pH. This dependence allows the range of pH sensitivity to be chosen by selection of the wavelength(s) and enables increased precision of the pH measurements by use of phase and/or modulation measurements at several wavelengths. These probes can be excited using a a green He-Ne laser at 543 nm, which allows their use in low cost instrumentation. Phase and modulation measurements are especially suitable for sensing applications because they are insensitive to the changes in signal intensity that result from photobleaching, probe washout, and/or light losses.

Fluoresceins↗

PubMed1993

Homogeneous model immunoassay of thyroxine by phase-modulation fluorescence spectroscopy.

We describe a homogeneous competitive model immunoassay for determination of thyroxine by multifrequency phase-modulation fluorescence. Using a nonradiative energy transfer transduction mechanism, B-phycoerythrin conjugated to thyroxine is the energy donor and a carboxymethylindocyanine dye conjugated to anti-thyroxine antibody is the energy acceptor. Energy transfer from B-phycoerythrin to the acceptor results in a decreased lifetime and/or phase angle. The fluorescence lifetime change reflects the extent of energy transfer. In the competitive immunoassay format, the donor-thyroxine conjugate and an analytical sample of thyroxine compete for acceptor-antibody binding sites, resulting in a phase angle change which is dependent on the amount of thyroxine in the sample. Dose-response curves of phase angle versus thyroxine concentration are comparable to steady-state intensity curves. Since phase-modulation lifetime measurements are largely independent of total signal intensity, sources of optical interference are minimized. The potential for whole blood measurements exists since the energy transfer lifetime method can be extended to longer wavelengths.

Antibodies↗

PubMed1993

Calcium-dependent fluorescence lifetimes of Indo-1 for one- and two-photon excitation of fluorescence.

We characterized the fluorescence intensity decays of Indo-1, which is commonly used as an emission wavelength-ratiometric calcium probe. The apparent lifetime of the long-wavelength side of the emission of Indo-1 is dependent on Ca2+. This long-wavelength emission displays the characteristics of an excited-state reaction, that is, a negative preexponential component in the multiexponential analysis. The emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 x 10(-49) cm4 s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. Hence, Indo-1 can be used for calcium imaging based on one- or two-photon excitation, using either emission wavelength ratios or lifetime imaging methods.

Calcium↗

PubMed1992

Frequency domain imaging of absorbers obscured by scattering.

Multiple pixel, frequency domain measurements of phase shift, theta, and modulation, m, in a phantom containing an absorber obscured by a relatively non-absorbing scattering solution are presented in combination with a theory of photon migration imaging. Results employing a single point source show that two dimensional theta measurements made in the presence (theta presence) and in the absence (theta absence) of an absorber can be used to create delta theta images. delta theta (theta absence-theta presence) images can be used to detect as well as locate the three dimensional position of the absorber. Images of mpresence measured in the presence of the absorber normalized by mabsence also provided detection and two dimensional location of its position. Images of % mpresence/mabsence at higher modulation frequencies provided greater resolution as predicted by photon migration theory. Neither theta nor m images alone could be used to detect or locate the presence of the absorber.

Light↗

PubMed1992

Fluorescence lifetime imaging.

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.

Algorithms↗

PubMed1992

Fluorescence lifetime imaging of free and protein-bound NADH.

We introduce a methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image and not on the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. This represents a challenging case for lifetime imaging because the NADH decay times are just 0.4 and 1.0 ns in the free and bound states, respectively. In the present apparatus, lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier and recorded with a charge-coupled device (CCD) camera. The intensifier gain is modulated at the light-modulation frequency or a harmonic thereof. A series of stationary phase-sensitive images each obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle or modulation of the emission at each pixel, which is in essence the lifetime image. We also describe am imaging procedure that allows specific decay times to be suppressed, allowing in this case suppression of the emission from either free or bound NADH. Since the fluorescence lifetimes of probes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules, this method allows imaging of the chemical or property of interest in macroscopic and microscopic samples. The concept of FLIM appears to have numerous potential applications in the biosciences.

In Vitro Techniques↗

PubMed1992

Fluorescence lifetime imaging of calcium using Quin-2.

We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.

Aminoquinolines↗

PubMed1991

Resolution of end-to-end diffusion coefficients and distance distributions of flexible molecules using fluorescent donor-acceptor and donor-quencher pairs.

We used time-dependent fluorescence energy transfer, time-dependent collisional quenching, and global analysis of the data resulting from these through-space and contact interactions to recover the end-to-end distance distributions and diffusion coefficients of flexible fluorescent molecules. The fluorescence decays of covalently linked tryptamine-acceptor and tryptamine-quencher pairs were measured by the frequency-domain method. These data were fit using numerical solutions of the differential equation, which predicts the time- and distance-dependent population of the excited state donors in the presence of energy transfer or collisional quenching, followed by transformation to the frequency domain for nonlinear least-squares comparison with the experimental data. We found that the energy transfer data for the donor-acceptor pair alone were adequate to recover the starting distribution and the end-to-end diffusion coefficient; however, the resolution is dramatically improved by the use of both the through-space and contact interactions.

Mathematics↗

PubMed1991

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching.

We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T1, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40 degrees C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T1, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.

Acrylamide↗

PubMed1991

Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry.

We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.

Mathematics↗

PubMed1990

Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra.

We describe a new fluorescence method that allows the resolution of both the decay times and emission spectra of mixtures of fluorophores. This method is completely general and does not require any assumptions or knowledge of the decay times or emission spectra of the individual fluorophores. We use the phase angle spectra and modulation spectra of the mixture, measured over a range of suitable light modulation frequencies and emission wavelengths. These data are analyzed by nonlinear least-squares analysis to recover the emission spectra and the associated decay times. The principle of the method and the nature of the data are illustrated by using two-component mixtures with increasing spectral overlap. We then demonstrate the recovery of minor components, of structure emission spectra, and of a three-component mixture with completed overlapping emission spectra. And finally, we describe the resolution of a two-component mixture with decay times of 0.8 and 1.4 ns using modulation frequencies up to 774 MHz.

Fluorescent Dyes↗