Biomedical subjects

I Gryczynski

Publications and source records attributed to I Gryczynski.

At least 55 records · Page 3Linked to original sources

PubMed1996

Fluorescence detection of the anticancer drug topotecan in plasma and whole blood by two-photon excitation.

The anticancer drug topotecan was detected in human plasma and whole blood using two-photon excitation at 730 or 820 nm. These wavelengths are longer than the main absorption bands of hemoglobin. Two-photon excitation of topotecan was demonstrated by a quadratic dependence of the emission intensity on the incident power, compared to a linear dependence for one-photon excitation at 410 nm. The observed emission centered at 525 nm was shown to be topotecan from the similarity of the emission spectrum and decay times observed for one-photon and two-photon excitation. Topotecan was detected at concentrations as low as 0.05 and 1 microM in plasma and whole blood, respectively. Since skin blood and tissues are translucent at long wavelengths, these results suggest the possibility of homogeneous or noninvasive clinical sensing with two-photon excitation.

Antineoplastic Agents↗

PubMed1996

Two-photon induced fluorescence of linear alkanes; a possible intrinsic lipid probe.

We measured the fluorescence emission spectra and intensity decays of the linear alkane tetradecane when excited at 300 nm by two-photon excitation. The unquenched lifetime of tetradecane in neat solution is near 4.4 ns. The emission of tetradecane centered at 210 nm is collisionally quenched by oxygen, n-propanol and water. These results suggest that aliphatic groups in non-polar environments can display good fluorescence, and that the aliphatic side chains of detergents and lipids may serve as an intrinsic fluorescent probe of micelles and bilayers.

1-Propanol↗

PubMed1996

Fluorescence spectral properties of troponin C mutant F22W with one-, two-, and three-photon excitation.

We report the first measurements of protein fluorescence with three-photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three-photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1Lb state of tryptophan from 830 to 900 nm.

Animals↗

PubMed1996

Three-photon induced fluorescence of the calcium probe Indo-1.

We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.

Biophysical Phenomena↗

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

Calcium-induced troponin flexibility revealed by distance distribution measurements between engineered sites.

The contraction of vertebrate striated muscle is regulated by Ca2+ binding to troponin C (TnC). This causes conformational changes which alter the interaction of TnC with the inhibitory protein TnI and the tropomyosin-binding protein TnT. We have used the frequency domain method of fluorescence resonance energy transfer to measure TnT-TnC and TnT-TnI distances and distance distributions, in the presence of Ca2+, Mg2+, or EGTA, in TnC.TnI.TnT complexes. We reconstituted functional, ternary troponin complexes using the following recombinant subunits whose sequences were based on those of rabbit skeletal muscle: wild-type TnC; TnT25, a mutant C-terminal 25-kDa fragment of TnT containing a single Trp212 which was used as the sole donor for fluorescence energy transfer measurements; Trp-less TnI mutants which contained either no Cys or a single Cys at position 9, 96, or 117. Energy acceptor groups were introduced into TnC or TnI by labeling with dansyl aziridine or N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our results indicate that the troponin complex is relatively rigid in relaxed muscle, but becomes much more flexible when Ca2+ binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, releasing the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distribution measurements between troponin subunits.

Adenosine Triphosphatases↗

PubMed1995

Fluorescence anisotropy of tyrosine using one-and two-photon excitation.

We examined the emission spectra and steady-state anisotropy of tyrosyl fluorescence with two-photon excitation from 565 to 578 nm. The emission spectra of phenol and N-acetyl-L-tyrosinamide (NATyrA) were all the same for one-photon excitation (OPE) and two-photon excitation (TPE), and the tyrosine emission from ribonuclease A showed 10-nm shift to longer wavelengths with TPE. Surprisingly, the anisotropy of tyrosine, NATyrA and Leu5-enkephalin in frozen solution were near zero for TPE as compared to near 0.3 for OPE. Low values of the anisotropy near 0.05 were also found for phenol and ribonuclease A. A low anisotropy appears to be a basic characteristic of tyrosine or tyrosyl residues with two-photon excitation.

Biophysical Phenomena↗

PubMed1995

Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation.

We examined the emission spectra of tyrosine- and tryptophan-containing proteins using one-photon (270-310 nm) and two-photon (565-610 nm) excitation. Emission spectra for two-photon excitation of native and denatured human serum albumin and of three purine nucleoside phosphorylases indicated an absence of the tyrosine emission normally seen for one-photon excitation below 290 nm. We examined the one-photon and two-photon excitation spectra of tyrosine-tryptophan mixtures to determine the origin of selective excitation of the tryptophan residues. These results confirmed a short-wavelength shift of the tyrosine two-photon excitation spectrum relative to that of tryptophan, as recently reported by Rehms and Callis (1993) Chem. Phys. Lett. 208, 276-282.

Animals↗

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

Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distances was found for free melittin, which is consistent with that expected for the random coil state, characterized by a Gaussian width (full width at half maxima) of 28.2 A. In contrast, narrow distance distributions were found for melittin complexed with CaM, 8.2 A, or with POPC vesicles, 4.9 A. A somewhat wider distribution was found for the melittin complex with TnC, 12.8 A, suggesting the presence of heterogeneity in the mode of binding between melittin and TnC. For all the complexes the mean Trp-19 to dansyl distance was near 20 A. This value is somewhat smaller than expected for the free alpha-helical state of melittin, suggesting that binding with CaM or TnC results in a modest decrease in the length of the melittin molecule.

Calmodulin↗

PubMed1994

Analysis of anisotropy decays in terms of correlation time distributions, measured by frequency-domain fluorometry.

We describe the theory and practical aspects of analyzing fluorescence anisotropy decays in terms of correlation times distributions. In our model the rotational motions of the fluorophores were described using Gaussian or Lorentzian distributions of the correlation times. The theory is presented both for time and frequency-domain measurements, although the simulations and measurements are focused on the frequency-domain measurements of the anisotropy decays. Analysis of simulated data is presented to illustrate the nature of the data and the resolution which can be expected with presently available frequency-domain measurements. Additionally, we describe experimental data for samples where one can reasonably expect a single exponential and/or discrete multi-exponential correlation time distributions, and for samples where the anisotropy decay might be expected to display a distribution of correlation times. These samples include small single tryptophan peptides in propylene glycol, the single tryptophan residue in S. Nuclease, and the single tryptophan residue in the native and partially unfolded states of ribonuclease T1.

Amino Acid Sequence↗

PubMed1994

Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high repetition rate lasers, a phenomenon we call "light quenching." We now describe the theory of light quenching and some of its effects on the steady-state and time-resolved intensity and anisotropy decays of fluorophores. Light quenching can decrease or increase the steady-state or time-zero anisotropy. Remarkably, the light quenching can break the usual z axis symmetry of the excited-state population, and the emission polarization can range from -1 to +1 under selected conditions. The measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The effects of light quenching are different for a single pulse, which results in both excitation and quenching, as compared with a time-delayed quenching pulse. Time-delayed light quenching pulses can result in step-like changes in the time-dependent intensity or anisotropy and are predicted to cause oscillations in the frequency-domain intensity and anisotropy decays. The increasing availability of pulsed laser sources offers the opportunity for a new class of two-pulse or multiple-pulse experiments where the sample is prepared by an excitation pulse, the excited state population is modified by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.

Biophysical Phenomena↗

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↗

PubMed1994

Distance-dependent fluorescence quenching of tryptophan by acrylamide.

We used GHz frequency-domain fluorometry to investigate the time-dependent intensity decays of N-acetyl-L-trytophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol at 20 degrees C. The intensity decays of NATA became increasingly heterogeneous in the presence of acrylamide. The NATA intensity decays were not consistent with the Collins-Kimball radiation boundary condition (RBC) model for quenching. The steady-state Stern-Volmer plots show significant upward curvature. At low temperature in vitrified propylene glycol (-60%), where translational diffusion cannot occur during the lifetime of the excited state, quenching of NATA by acrylamide was observed. The Smoluchowski and RBC quenching models do not predict any quenching in the absence of translational diffusion. Hence, these frequency-domain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide. The rate for quenching of NATA by acrylamide appears to depend exponentially on the fluorophore-quencher separation distance. Comparison of the time-resolved and steady-state data provides a sensitive method to determine the distance dependence of the fluorophore-quencher interaction. The distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, which is often observed for quenching by acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of quenching data of proteins by acrylamide.

Acrylamide↗

PubMed1993

Characterization of p-bis(O-methylstyryl)benzene as a lifetime and anisotropy decay standard for two-photon induced fluorescence.

We describe the fluorescence spectral properties of p-bis(O-methylstyryl)benzene (bis-MSB) as a standard for time-resolved measurements of two-photon induced fluorescence. Bis-MSB displays the same single exponential intensity decay in several solvents for one- and two-photon excitation. The anisotropy decay displays the same single correlation time of one- and two-photon excitation. The amplitudes of the anisotropy decay are distinct for one- and two-photon excitation. At some excitation wavelengths the anisotropy amplitude appears to be solely the result of one- and two-photon photoselection, but at shorter wavelengths the anisotropy amplitudes are not related by single constant factor. The absorption range of bis-MSB makes it a suitable standard for one- and two-photon excitation of intrinsic protein fluorescence and extrinsic fluorophores.

Fluorescence↗

PubMed1993

Intensity and anisotropy decays of [Leu5] enkephalin tyrosyl fluorescence by 10 GHz frequency-domain fluorometry.

The technique of 10 GHz frequency-domain fluorometry was used to resolve the complex picosecond intensity and anisotropy decays of the tyrosyl emission of [Leu5] enkephalin. Enhanced resolution of anisotropy decay was obtained by using acrylamide quenching of the tyrosyl fluorescence and global analysis of the frequency-domain anisotropy data obtained with different amounts of acrylamide. The data indicates a 44 ps correlation time for local tyrosine motions, and a 219 ps correlation time for overall rotational diffusion of the pentapeptide. Our data are consistent with an initial loss of fluorescence anisotropy from r0 = 0.4 to a value of r0 = 0.326 occurring during the first two picoseconds after excitation.

Acrylamides↗

PubMed1993

Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease.

The dipole relaxational dynamics in the environment of a single tryptophan residue Trp-140 in staphylococcal nuclease was studied by time-resolved (multi-frequency phase-modulation) spectroscopy and selective red-edge excitation. The long-wavelength position of the fluorescence spectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mobile protein groups which relax on the subnanosecond time scale. For these temperatures (0-20 degrees C) the steady-state emission spectra did not show the excitation-wavelength dependent shifts (red-edge effects) for excitation wavelengths from 295 to 308 nm; however, the anisotropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rather than the motion of the tryptophan itself. The motions of the tryptophan surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the difference in position of fluorescence spectra at excitation wavelengths 295 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at -50 degrees C, where the steady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation data at the half-transition point (-40 degrees C) demonstrates the existence of the nanosecond dipolar relaxations. At -40 degrees C the time-dependent spectral shift is close to monoexponential with the relaxation time of 1.4 ns.

Binding Sites↗