Gabor Laczko

A 10‐GHz frequency‐domain fluorometer

We developed a frequency‐domain fluorometer which operates from 4 to 2000 MHz. The modulated excitation is provided by the harmonic content of a laser pulse train (3.76 MHz, 5 ps) from a synchronously pumped and cavity dumped dye laser. The phase angle and modulation of the emission are measured with a microchannel‐plate photomultiplier (PMT). Cross‐correlation detection is performed outside the PMT. The high‐frequency signals for cross correlation were obtained by multiplication of the output from a 500‐MHz frequency synthesizer. The performance was verified in several ways, including measurement of known time delays and examination of standard fluorophores. The detector displayed no detectable color effect, with the 300–600‐nm difference being less than 5 ps. The precision of the measurements is adequate to detect differences of 20 ps for decay times of 500 ps. A correlation time of 53 ps was found for indole in water at 20 °C. The shortest correlation time we measured was 15 ps for indole in methanol/w...

Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer.

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.

Single Molecule Studies of Multiple-Fluorophore Labeled Antibodies. Effect of Homo-FRET on the Number of Photons Available Before Photobleaching

Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the behavior of individual and complex molecular systems in real time. SMD enables the characterization of complex molecular interactions and reveals basic physical phenomena underlying chemical and biological processes. We present here a systematic study of the quenching efficiency of Förster-type energy-transfer (FRET) for multiple fluorophores immobilized on a single antibody. We simultaneously monitor the fluorescence intensity, fluorescence lifetime, and the number of available photons before photobleaching as a function of the number of identical emitters bound to a single IgG antibody. The detailed studies of FRET between individual fluorophores reveal complex through-space interactions. In general, even for two or three fluorophores immobilized on a single protein, homo-FRET interactions lead to an overall non-linear intensity increase and shortening of fluorescence lifetime. Over-labeling of protein in solution (ensemble) results in the loss of fluorescence signal due to the self-quenching of fluorophores making it useless for assays applications. However, in the single molecule regime, over-labeling may bring significant benefits in regards to the number of available photons and the overall survival time. Our investigation reveals possibilities to significantly increase the observation time for a single macromolecule allowing studies of macromolecular interactions that are not obscured by ensemble averaging. Extending the observation time will be crucial for developing immunoassays based on single-antibody.

Time-resolved fluorescence emission spectra of labeled phospholipid vesicles, as observed using multi-frequency phase-modulation fluorometry

Abstract Multi-frequency phase-modulation fluorometry was used to determine the time-resolved spectral parameters of two different fluorescent probes, 6-palmitoyl-2-[(2-trimethylammonium)ethyl[methylamino]naphthalene chloride (Patman) and 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS) in lipid vesicles. The frequency-domain measurements permitted calculation of time-resolved emission spectra, the time-resolved decays of the emission center of gravity and of the emission spectral width. Nanosecond spectral relaxation was found using both probes, and these rates increased with temperature. The detailed time-domain information available using our method indicated that spectral relaxation of TNS is mainly a continuous process, whereas relaxation of Patman shows the characteristic features of a stepwise relaxation. Our results indicate that complex excited-state processes can be quantified using frequency-domain fluorometry.

New trends in photobiology: Gigahertz frequency-domain fluorometry: Resolution of complex decays, picosecond processes and future developments

Abstract We describe the principles, instrumentation and applications of frequency-domain fluorescence spectroscopy. This method is useful for the resolution of multi-exponential decays and complex anisotropy decays on the picosecond timescale. The present instrumentation allows measurements to 2 GHz, which has been used to measure rotational correlation times as short as 7 ps. In the future it may be possible to extend the frequency range to 10 GHz, which should allow still faster processes to be quantified. It should be emphasized that resolution of fast processes is not obtained at the expense of losing information on the nanosecond timescale. Additionally, the GHz frequency-domain measurements are performed using low excitation intensities, which do not damage the samples.

Picosescond fluorescence lifetime standards for frequency- and time-domain fluorescence.

We characterized a series of dimethylamino-stilbene derivatives as standards for time-domain and frequency-domain lifetime measurements. The substances have reasonable quantum yields, are soluble in solvents available with a high purity, and do not show significant sensitivity to oxygen quenching. All the fluorophores displayed single exponential intensity decays, as characterized by frequency-domain measurements to 10 GHz. The decay times vary from 880 to 57 ps, depending on structure, solvent, and temperature, which is a useful range for modern picosecond time-domain or gigahertz frequency-domain instruments. These fluorophores may be used either to test an instrument or as reference compounds to eliminate color effects. We also characterized two-fluorophore mixtures, with the decay times spaced twofold (150 and 300 ps), with varying proportions. These mixtures are useful for testing the resolution of other time- and frequency-domain instrumentation. The excitation wavelength ranges from 260 to 430 nm, and the emission from 350 to 550 nm. The decay times are independent of the excitation and emission wavelengths.

Transient effects in quenching detected by harmonic-content frequency-domain fluorometry

Abstract Transient effects in the diffusional quenching of indole fluorescence by acrylamide were detected by frequency-domain fluorometry. The samples are excited with a repetitive train of picosecond pulses, and the frequency response is measured using the harmonic content up to 1 GHz. The decay of unquenched indole in aqueous buffer is dominantly a single exponential. In the presence of acrylamide the partially quenched indole fluorescence became distinctly more complex. Analysis of the frequency response of the fluorescence yields the apparent interaction radius (7.1 A) and mutual diffusion coefficient (1.1 × 10 −5 cm 2 s ).

Time-resolved emission spectra of hemoglobin on the picosecond time scale

We used front-face illumination to examine the steady-state and time-resolved emission from the intrinsic tryptophan emission of human hemoglobin (Hb). Experimental conditions were identified which eliminated all contributions of scattered light. The sensitivity obtained using front-face optics was adequate to allow measurement of the wavelength-dependent frequency response of the emission to 2 GHz. The intensity decays displayed pico- and nanosecond components in the emission at all wavelengths from 315 to 380 nm. The contribution of the picosecond component decreased from 72 to 37% over this range of wavelengths. Frequency-domain measurements were used to calculate the time-resolved emission spectra and decay-associated emission spectra. These spectra indicate that the picosecond components of the emission display maxima near 320 nm, whereas the nanosecond components are centered at longer wavelengths near 335 nm. The nanosecond components appear to be due to residual impurities which remain even in highly purified samples of Hb. However, we cannot eliminate the possibility that some of these components are due to Hb itself.

Enhanced resolution of anisotropic rotational diffusion by multi-wavelength frequency-domain fluorometry and global analysis

Abstract Frequency-dependent differential polarized phase and modulation data were used to measure time-dependent anisotropy decays of 9-aminoacridine and Y-base. The data were obtained at various excitation wavelengths, at which the fundamental anisotropies ( r 0 ) varied from −0.17 to 0.373. The different values of r 0 result in different contributions of the multiple correlation times to the anisotropy decay. The multi-wavelength data were analyzed globally, revealing both molecules to be anisotropic rotators. The rotational motions of Y-base are more anisotropic than those of 9-aminoacridine. In propylene glycol at −5 ° C the two correlation times for Y-base were found to be 2.1 and 12.1 ns. For 9-aminoacridine the correlation times were 9.5 and 14.1 ns.

Picosecond resolution of oxytocin tyrosyl fluorescence by 2 GHz frequency-domain fluorometry

The technique of frequency-domain fluorometry has been extended to 2000 MHz using the harmonic content of a picosecond laser source and a microchannel plate photomultiplier tube. This new instrument was used to resolve complex subnanosecond intensity and anisotropy decays of the tyrosyl emission of oxytocin. The intensity decay was found to contain at least three exponential components, 80, 359 and 927 ps. The anisotropy analysis revealed a 29 ps torsional motion of the tyrosine residue as well as a 454 ps overall rotational correlation time. The time resolution of this method should permit the comparison of experimental results with theoretical models for motions of proteins.