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1.
Ignacy Gryczynski Henryk Szmacinski Joseph R. Lakowicz 《Photochemistry and photobiology》1995,62(4):804-808
Abstract— We show that the calcium fiuorophore Indo-1 can be excited by simultaneous absorption of three-photons at 885 nra, 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 17μ 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 Tksapphire lasers, and may provide higher spatial resolution than available using two-photon excitation. 相似文献
2.
Joseph R. Lakowicz Zakir Murtaza Wayne E. Jones Jr. Kihan Kim Henryk Szmacinski 《Journal of fluorescence》1996,6(4):245-249
We report the first observation of polarized emission from a rhenium-phenanthroline complex, Re(CO)3(phen)Cl. Highly luminescent rhenium complexes are known, with quantum yields near 0.5 and lifetimes in excess of 10 s. The detection of polarized emission suggests the use of rhenium complexes as probes of the hydrodynamics of large macromolecular complexes and for use in fluorescence polarization immunoassays with gated detection. 相似文献
3.
RESOLUTION OF TWO EMISSION SPECTRA FOR TRYPTOPHAN USING FREQUENCY-DOMAIN PHASE-MODULATION SPECTRA 总被引:1,自引:0,他引:1
Joseph R. Lakowicz Ranjith Jayaweera Henryk Szmacinski Wieslaw Wiczk 《Photochemistry and photobiology》1989,50(4):541-546
We describe a novel application of frequency-domain fluorometry which allows resolution of the decay times and emission spectra of samples which display multi-exponential decay kinetics. This method does not require any previous knowledge about the decay times or any assumptions about the shape of the emission spectra. We record the wavelength-dependent phase angles and modulations (phase angle and modulation spectra) using a number of light modulation frequencies. The data is analyzed by non-linear least-squares to recover the emission spectra and their associated decay times. Phase and modulation spectra (PM Spec) were used to recover the emission spectra associated with the two decay times of tryptophan at pH = 7 (0.54 and 3.44 ns). The emission spectra of these components are centered at 340 and 355 nm, respectively, with the amplitude of the 0.54 ns component contributing 6% to the total emission. These results are in agreement with previous time-resolved studies by Szabo and Rayner [J. Am. Chem. Soc. 102, 554-563 (1980)]. Control experiments were performed on mixtures of N-acetyl-L-tryptophanamide (NATA) and PPD, which demonstrate our ability to recover the spectra and decay times from two component mixtures. NATA itself displayed a single decay time and only one emission spectrum. 相似文献
4.
We have explored the opportunities for enhanced ratiometric pH sensing using the well-known carboxy seminaphthofluorescein (SNAFL-2) and silver island films (SiFs). Our results show that the metallic surfaces can provide up to a 40-fold increase in probe fluorescence intensity as compared to nonmetallic surfaces with the same probe coverage. However, while the S/N is significantly better for pH sensing, the emission wavelength ratiometric values are similar to that obtained in solution, due to the fact that the emission of both the acidic and basic forms of the probe are enhanced to similar extents. To the best of our knowledge this is the first report of enhanced ratiometric fluorescence sensing on metallic surfaces. 相似文献
5.
We describe imaging of calcium concentrations using the long-wavelength Ca2+ indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry. 相似文献
6.
We measured the wavelength-dependent intensity decays of 4,9-dihydro-4,6-dimethyl-9-oxo-1H-imidazo-1,2a-purine (Yt-base) in propanol to determine the time-resolved emission spectra and rates of spectral relaxation. We found that resolution of the spectral relaxation times was dramatically improved by global analysis of the frequency-domain data with increasing amounts of the collisional quencher CCl4. Collisional quenching preferentially decreases the longer-lived relaxed component of the emission, thereby increasing the fractional contribution of the incompletely relaxed portion of the emission. The data could not be explained by a single spectral relaxation time, and at least two relaxation times are needed to describe the time-dependent emission center of gravity of Yt-base.Dedicated to Professor Stefan Paszyc on the occasion of his 70th birthday. 相似文献
7.
Joseph R. Lakowicz Ewald Terpetschnig Zakir Murtaza Henryk Szmacinski 《Journal of fluorescence》1997,7(1):17-25
Metal-ligand complexes containing ruthenium, osmium, or rhenium display a high photostability, with polarized emission and
decay times from 100 ns to 100 Μs. Such probes have considerable potential in biophysics, clinical chemistry, and fluorescence
microscopy. In this review we sumrecent developments from this laboratory on the spectral properties of conjugatable metalligand
complexes. We also suggest how improved probes can be developed based on the selection of organic ligands. 相似文献
8.
Joseph R. Lakowicz Henryk Szmacinski Ignacy Gryczynski 《Photochemistry and photobiology》1988,47(1):31-41
Abstract— We used frequency-domain fluorescence spectroscopy to measure rotational diffusion and time-resolved emission spectra of indole in methanol on the picosecond timescale. The indole emission was quenched by acrylamide to allow measurements to the instrumental limit of 2 GHz and to eliminate emission from the longer-lived indole molecules, which can no longer provide information on the picosecond (ps) processes. The resolution was adequate to measure rotational correlation times as short as 8 ps at 80†C, and spectral relaxation times as short as 16 ps at 5†C. 相似文献
9.
SITE-TO-SITE DIFFUSION IN PROTEINS AS OBSERVED BY ENERGY TRANSFER AND FREQUENCY-DOMAIN FLUOROMETRY *
Joseph R. Lakowicz Ignacy Gryczynski Jzef Kuba Wieslaw Wiczk Henryk Szmacinski Michael L. Johnson 《Photochemistry and photobiology》1994,59(1):16-29
Abstract 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 ?7 to ?5 cm2/s. This possibility was verified by studies of a methylenechain 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 α-helical state but over a limited range of distances. 相似文献
10.