2-GHz frequency-domain fluorometer

Lakowicz, Joseph R.; Laczko, Gabor; Gryczyński, Ignacy

doi:10.1063/1.1139215

Cited by 222 publications

(124 citation statements)

References 29 publications

Supporting

Mentioning

120

Contrasting

Order By: Relevance

“…We note that the recovered rotational correlation time for indole, 30 ps, is very close to the range of values reported in the literature for different solvent and temperature conditions [13]. We did not succeed in recovering reasonable rotational parameters attributable to the mixture components solely by analysis of the mixture data assuming two rotational species; this data set in fact fit best to a single rotational rate which represented a value weighted more toward the p-terphenyl (background) value (data not shown).…”

Section: Dynamic Polarization Measurementssupporting

confidence: 63%

A method for on-line background subtraction in frequency domain fluorometry

et al. 1991

View full text Add to dashboard Cite

Time-resolved fluorescence determinations on biochemical samples are often complicated by contributions from background. In the time-correlated single-photon counting method, background subtraction is a routine procedure. A limitation of frequency domain fluorometry, however, arises from the difficulty of performing this operation. This limitation has become increasingly significant as frequency domain methods are being applied to evermore complex biological systems using the higher-frequency capabilities of modern instrumentation. We have devised a method for such a correction in the frequency domain, regardless of the complexity of the background decay, based on measurement of the background phasor and subsequent subtraction from the sample phasor. This method is applicable to both lifetime and dynamic polarization measurements, and it can be readily implemented on commercially available frequency domain fluorometers. Decay curves may be accurately recovered from samples containing background contributions ranging from less than 5% to greater than 90% of ihe total signal intensity.KEY WORDS: Frequency domain; background; lifetime; dynamic polarization. INTRODUC~ONPhase fluorometry has become a widely utilized technique for performing time-resolved fluorescence measurements. As this technique becomes increasingly applied to complex samples at low concentrations, notably biological samples, unavoidable background fluorescence has come to significantly limit its overall sensitivity. Fluorescent contaminants in reagents, parasitic light, and scattered light (Rayleigh and Raman) are among the most common sources of background. 153background is attributable to a single exponential decay, then a multicomponent analysis, in which one component can be clearly assigned to the background, is adequate to recover the sample's time-resolved parameters. Usually background contributions are more complex, though, rendering this approach inappropriate.A method to correct frequency domain data for background signals, whether they originate from contaminant fluorescence or scattered light, has been reported by Lakowicz et al. [1]. In their method, the fraction of the total steady-state signal attributable to the background must be determined in a separate experiment under identical instrument conditions. This measurement is necessitated by the fact that, in their method, the signal acquisition is based on phase and modulation data alone, so that the AC contributions of the background must be calculated a posteriori from the independently determined DC values. Moreover, in their approach a rigorous error propagation analysis cannot be achieved during data acquisition but must be performed sepa-1053-0509/91/0900-0153506.50/0 9 1991 Plenum Publishing Corporation 154 Reinhart, Marzola, Jameson, and Gratton rately. Consequently the method is not a true on-line procedure.Another approach to blank correction was recently proposed by Swift and Mitchell [2] specifically for the case of parallel data acquisition. In parallel phase fluoro...

show abstract

Section: Dynamic Polarization Measurementssupporting

confidence: 63%

A method for on-line background subtraction in frequency domain fluorometry

et al. 1991

View full text Add to dashboard Cite

show abstract

“…3 Bottom. Typical uncertainties in the phase and modulation values are 0.20 and 0.005, respectively (25,26). CCD FT refers to the pixel-by-pixel analysis (49).…”

Section: Experimental Methodsmentioning

confidence: 99%

Fluorescence lifetime imaging of free and protein-bound NADH.

Lakowicz

Szmacinski

Nowaczyk

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

692

582

View full text Add to dashboard Cite

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 challengi 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, lifetimeimages 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 ginmodulation 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 an imang procedure that allows speyific 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.The phenomenon of fluorescence is widely utilized for research in the biosciences. These applications have been focused on two divergent types of measurements, timeresolved fluorescence and fluorescence microscopy. In the former, one takes advantage of the high information content ofthe time-dependent fluorescence decays to uncover details about the structure and dynamics of macromolecules (1-3). Such measurements are performed almost exclusively by using picosecond laser sources coupled with high-speed photodetectors. In contrast, fluorescence microscopy, in combination with dyes, stains, fluorophores, or fluorophorelabeled antibodies, is most often used to determine the localization of species of interest, usually proteins or other macromolecules (4-6). The acquisition of two-dimensional (2D) fluorescence images is typically accomplished with low-speed accumulating detectors. Consequently, the high information content of time-resolved fluorescence is not usually available for studies of microscopic biological samples. This is particularly disadvantageous when one considers the sensitivity of fluorescence decay times to chemical and environmental factors of interest, such as local pH, cation concentration, oxygen, and polarity, to name a few.In the present report we combine measurements of fluorescence lifetimes with 2D imaging to create images in which the lifetimes at each pixel are used to create contrast in the images. While there have been reports...

show abstract

“…8, August 1989 phase and demodulation information from the sample emission is preserved in the beat frequency. 1 The key elements of our systems are the acousto-optic modulators. The modulators are custom made by IntraAction Corp. (Belwood, IL).…”

Section: Apparatusmentioning

confidence: 99%

“…The original modulator used in a cross-correlation frequency domain fluorometer was the Debye-Sears tank, which is a standing-wave acousto-optic modulator that uses a liquid medium for the acoustic-wave propagation. 1 More recently, standing-wave acousto-optic modulators have been used for frequency domain measure· ments to amplitude modulate the output of a mode-locked laser and to obtain high (40-320MHz) and very low (de to 10 MHz) modulation frequencies. 11 In principle, using acousto-optic modulators, it should be possible to use uncollimated cw sources, such as high-pressure xenon arc lamps, which can provide good intensity over a wide spectral range.…”

Section: Introductionmentioning

confidence: 99%

Wide-band acousto-optic light modulator for frequency domain fluorometry and phosphorimetry

Piston

Marriott

Radivoyevich

et al. 1989

Review of Scientific Instruments

View full text Add to dashboard Cite

Multifrequency-phase and modulation tluorornetry allows for accurate analysis of fluorescence decay in the frequency domain. Essential to these frequency domain methods is a high-frequency modulation of the light source. Techniques for generating wide-band modulation oflight are currently limited to the use of Pockel's cells and intrinsically modulated sources such as modelocked lasers and synchrotron radiation. We present a method that employs two acousto-optic modulators in series for use with cw light sources. This modulator system gives two orders of magnitude more intensity output than the Pockel's cell modulator and requires Jess than onetenth of the rf driving power. ln addition, the Pockel's cell system is limited to modulation frequencies less than 250 MHz, whereas the particular implementation discussed here gives a quasicontinuous distribution of modulation frequencies from de to 320 MHz. To obtain this range offrequencies, acoustic standing waves are set up simultaneously in each i:nodulator, and the desired modulation frequency is achieved by choosing the proper combination of the two standing-wave frequencies. Light modulation is obtained at twice each of the individual standingwave frequencies and at the sum and difference of twice the two acoustic frequencies. Data are presented to illustrate the use of this system for the measurement of phosphorescence as well as fluorescence decay.

show abstract

2-GHz frequency-domain fluorometer

Cited by 222 publications

References 29 publications

A method for on-line background subtraction in frequency domain fluorometry

A method for on-line background subtraction in frequency domain fluorometry

Fluorescence lifetime imaging of free and protein-bound NADH.

Wide-band acousto-optic light modulator for frequency domain fluorometry and phosphorimetry

Contact Info

Product

Resources

About