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Fluorescence phase-shift method

Jablonski (48-49) developed a theory in 1935 in which he presented the now standard Jablonski diagram" of singlet and triplet state energy levels that is used to explain excitation and emission processes in luminescence. He also related the fluorescence lifetimes of the perpendicular and parallel polarization components of emission to the fluorophore emission lifetime and rate of rotation. In the same year, Szymanowski (50) measured apparent lifetimes for the perpendicular and parallel polarization components of fluorescein in viscous solutions with a phase fluorometer. It was shown later by Spencer and Weber (51) that phase shift methods do not give correct values for polarized lifetimes because the theory does not include the dependence on modulation frequency. [Pg.9]

Fluorescence Lifetimes. Fluorescence lifetimes were determined by the phase shift method, utilizing a previously-described phase fluorimeter. The emission from an argon laser was frequency doubled to provide a 257 nm band for excitation. Fluorescence lifetimes of anisole and polymer 1 in dichloro-methane solution were 2.2 and 1.4 nsec, respectively. Fluorescence lifetimes of polymer films decreased monotonically with increasing DHB concentration from 1.8 (0) to 0.7 nsec (9.2 x 10 3 MDHB). Since fluorescence lifetimes (in contrast to fluorescence intensities) are unaffected by absorption effects of the stabilizer, these results provide direct evidence in support of the intensity measurements for RET from polymer to stabilizer. [Pg.110]

Fig. 6. Experimental arrangement for lifetime measurements by the phase-shift method, using laser excitation. The laser beam is amplitude-modulated by a Pockel cell with analysing Nicol prism and a small part of the beam is reflected by a beam splitter B into a water cell, causing Rayleigh scattering. This Rayleigh-scattered light and the fluorescence light from the absorption cell are both focused onto the multiplier cathode PMl, where the difference in their modulation phases is detected. (From Baumgartner, G., Demtroder, W., Stock, M., ref. 122)). Fig. 6. Experimental arrangement for lifetime measurements by the phase-shift method, using laser excitation. The laser beam is amplitude-modulated by a Pockel cell with analysing Nicol prism and a small part of the beam is reflected by a beam splitter B into a water cell, causing Rayleigh scattering. This Rayleigh-scattered light and the fluorescence light from the absorption cell are both focused onto the multiplier cathode PMl, where the difference in their modulation phases is detected. (From Baumgartner, G., Demtroder, W., Stock, M., ref. 122)).
B) Phase-shift methods. The phase shift method for determining fluorescence lifetimes is based on the principle that if fluorescence is excited by suitably modulated light source, emitted radiation will also be similarly modulated. With reference to a scattering substance, emission from a fluorescent substance will introduce a time lag due to finite time between absorption and emission. This, by definition is the lifetime of the excited state. The time lag will cause a phase-shift relative to the exciting light. Phase fluorimetry requires a modulated light source and a phase sensitive detector. [Pg.309]

Methane was the first polyatomic molecule other than CO2 for which vibrational energy-transfer pathways and rates were investigated. This study of methane involved the utilization of the phase-shift method for determining the lifetimes of vibrationally excited states. The asymmetric stretching vibration, v-, of methane at 3010 cm (see Fig. 1) was excited by a chopped He-Ne laser operating on the 2947.9 cm Ne transition. Fluorescence was detected from both the mode and the bending mode of methane at 1306 cm . Rates were extracted from phase-shift measurements, and Ref. 16 provides an excellent discussion of the background for, and use of the phase-shift method. [Pg.187]

The phenomena discussed above can be studied using various techniques, and not all methods are equally suitable in a particular case. Two principally different kinds of methods for measuring fluorescence lifetimes exist, namely, pulse methods and modulation or phase-shift methods. Phase-shift methods, despite the fact that they have been known for a longer time, have not found widespread use during the last decade. However, important technical advances have been made in phase-shift methods which in fact have inspired many researchers to apply them more frequently. Nevertheless, pulse methods are still the most widely used today, in particular for high time resolution. If carried out properly both types of methods must and will give the same result. Details of the measuring problem will determine which method is more appropriate in a particular case. [Pg.345]

If the laser is tuned to the center frequency coik of an absorbing transition /) k), the detected fluorescence intensity /pi monitored on the transition A ) m) is proportional to the laser intensity /p as long as saturation can be neglected. In the phase-shift method the laser intensity is modulated at the frequency f = jlTt (Fig. 6.87a) according to... [Pg.348]

Phsise-Shift Method. In the phase-shift method atoms or molecules are excited by a sinusoidally modulated light or electron beam, as illustrated in Fig. 9.27. Fluorescence light is recorded with a photomultiplier tube. This light will thus jiilso be modulated, but because of the delay in the excited state a phase-shift is introduced [9.149]. At the same time, the contrast in... [Pg.321]

This method is based on the excited-state lifetime. The light intensity emitted from molecules excited by a short pulse of light decays exponentially with time. This decay pattern is unique for each molecule and can be used for analytical purposes. Alternatively, a phase shift method can be employed to measure the fluorescence lifetime. A sinusoidally varying excitation light source is used and the phase shift between the excitation waveform and the emission waveform can be used to detect the analytical signal. [Pg.96]

Fig.11.12a,b. Lifetime measurement with the phase shift method, (a) Experimen-tal arrangement and modulation of laser light and fluorescence, (b) analogous electrical circuit... [Pg.556]

The phase shift method is not so well suited to the measurement of non-exponential decays (if, for example, the fluorescence from several levels with different lifetimes overlap). Although measurements at different modulation frequencies enable one to fit the measured phase shifts q to a sum of exponential I Cj (exp(-t/T ), the decay curve cannot be viewed directly, and the fit may not be unambiguous. [Pg.558]

The molecules are excited by a light pulse with a trailing edge short compared to the mean lifetime t of the excited level. The subsequent decay of the level population, monitored by the decay of the fluorescence intensity, is either viewed directly on a scope or is monitored with a boxcar integrator or a transient recorder (see Sect.4.5.10). This method does not suffer from the influence of induced emission since the exciting light is already switched off when the fluorescence is observed. It is especially adapted to the use of pulsed or mode-locked lasers as excitation sources. From the decay curve the mean lifetime can be derived directly. Deviations from exponential decays, caused for instance by cascade effects, can be seen immediately. The accuracy is comparable to that of the phase shift method. [Pg.558]

For fluorescent compounds and for times in die range of a tenth of a nanosecond to a hundred microseconds, two very successftd teclmiques have been used. One is die phase-shift teclmique. In this method the fluorescence is excited by light whose intensity is modulated sinusoidally at a frequency / chosen so its period is not too different from die expected lifetime. The fluorescent light is then also modulated at the same frequency but with a time delay. If the fluorescence decays exponentially, its phase is shifted by an angle A([) which is related to the mean life, i, of the excited state. The relationship is... [Pg.1123]

This chapter presents new information about the physical properties of humic acid fractions from the Okefenokee Swamp, Georgia. Specialized techniques of fluorescence depolarization spectroscopy and phase-shift fluorometry allow the nondestructive determination of molar volume and shape in aqueous solutions. The techniques also provide sufficient data to make a reliable estimate of the number of different fluorophores in the molecule their respective excitation and emission spectra, and their phase-resolved emission spectra. These measurements are possible even in instances where two fluorophores have nearly identical emission specta. The general theoretical background of each method is presented first, followed by the specific results of our measurements. Parts of the theoretical treatment of depolarization and phase-shift fluorometry given here are more fully expanded upon in (5,9-ll). Recent work and reviews of these techniques are given by Warner and McGown (72). [Pg.181]

At present, two main streams of techniques exist for the measurement of fluorescence lifetimes, time domain based methods, and frequency domain methods. In the frequency domain, the fluorescence lifetime is derived from the phase shift and demodulation of the fluorescent light with respect to the phase and the modulation depth of a modulated excitation source. Measurements in the time domain are generally performed by recording the fluorescence intensity decay after exciting the specimen with a short excitation pulse. [Pg.109]

Fluorescence lifetime measurements are an important aspect of photophysical research. In the past few months the phase-shift measurement technique has become more widely used. This is largely due to the successful achievement of a multifrequency modulation apparatus. An apparatus made from commercially available components has been described and shown to have an accuracy of 10 ps. The performance was checked using mixtures of acridine and quinine sulphate and least-squares-ht procedures. A series of papers from the Illinois group give very detailed account of the state of the art and show the power of the method. The colour delay error arising from the wavelength error in photodetectors can be determined and fluorescence decay times can be obtained with an accuracy of a few picoseconds. ... [Pg.7]

Over a substantial number of years the phase-shift or frequency-domain method has been employed for the measurement of fluorescence lifetimes. The technique requires the continuous excitation of a fluorescent sample with a source of varying intensity. The fluorescence response would normally be expected to increase and decrease to reflect the changes in excitation intensity. However, in a frequency-domain experiment the excitation beam is modulated at a high frequency, (o = 2nf, to produce a sinusoidally changing intensity given by ... [Pg.663]

Phase shift fluorimetry, the other important method for measuring fluorescent lifetimes, also continues to be developed and improved. The effects of Inaccurate reference lifetimes on the interpretation of frequency domain fluorescence data can be removed or minimized by a least squares analysis method.The direct collection of multi-frequency data for obtaining fluorescence lifetimes can be achieved by the use of digital parallel acquisition in frequency domain fluorimetry. Frequency domain lifetime measurement has been used for on-line fluorescence lifetime detection of eluents in chromatography. An unusual use of frequency domain measurement which has been reported is for the examination of photon migration in living tissue. Photons in the... [Pg.7]

Pulse fluorometry has been favoured over phase techniques since hitherto no general method has been available for determining the proportions and lifetimes of fluorescence components in complex systems. Weberhas presented an exact solution of the problem using the values of the phase shifts and relative modulation of the overall fluorescence of as many light-modulation frequency as there are components. The simplicity and speed of the numerical methods involved... [Pg.43]

Tvvo vidcl used approaches are used for lifetime measurcnienis. ilie lime-domain approach and the frt i/iu niy-domain approach. In tinte-domain measurements. a pulsed source is employed and the time-depcndcnr decay of fluorescence is measured. In the frequency-domain method, a sinusoidallv modulated source is used to excite the sample. The phase shift and demodulation of the fluorescence emission relative lo the excitation waveform provide the lifetime information. ( onimercial instrumentation is available to implement both techniques. ... [Pg.422]

In contrast to pulse methods described above, the phase-shift technique usually employs a continuous light source whose intensity is modulated by various means at some frequency /. The fluorescence response of the system is then also modulated at that frequency, albeit with some phase delay 0 and a reduced modulation depth m, as compared to the exciting light. "" From either of these quantities the fluorescence lifetime can be extracted. For a single-exponential decay the relationship between lifetime t, the modulation frequency /, phase shift 0, and the modulation depth m are given by tan(0) = /t and m = (1 -t-... [Pg.359]

There are two methods that have been used to determine fluorescence lifetimes in DNA sequencing. In the first method, known as the frequency-domain or phase-modulation method, the excitation beam is intensity modulated. The a.c. portion of the resulting emission is phase-shifted relative to the laser modulation this phase-shift contains information about the fluorescence lifetime, or lifetimes if more than one fluor is present [140]. McGown and coworkers [144,145] used this method for four-color sequencing. In that work, 488 nm or 514 nm laser light was electronically modulated with a Pockels cell before being focused onto a capillary column. Detection, made normal to the laser direction, was optically filtered to reduce laser scatter and was focused onto the detector of a... [Pg.492]

Fluorescence decays are generally measured using the time-correlated single photon counting (TCSPC) technique [43, 44], although the phase-shift [45] method has been also used (see Chap. 14). A brief description of TCSPC apparatus with nanosecond and picosecond time resolution is given below in order to illustrate the essential components and requirements for each time resolution. [Pg.554]

If multiple scattering processes occur an erroneously long lifetime will be obtained, as for several of the above-mentioned methods. The opposite effect is obtained if, in addition to the fluorescence light, non-shifted stray light from the modulated light source is recorded. If the modulation is not perfectly sinusoidal, the first Fourier component can be isolated and the phase shift for this component will still yield the lifetime. [Pg.272]

The mean fluorescence lifetime may also be determined by continuous intensity measurements, if the exciting light intensity is modulated at a high frequency. Fluorescence is excited by light modulated sinusoidally at a known frequency (ajln Hz). The emission is a forced response to the excitation, and is therefore modulated at the same frequency, but with a phase shift, due to the time-lag between absorption and emission. The intensities of the two beams are monitored by photomultipliers. The difference in phase (0) between the two intensities is determined electronically. The lifetime r is given by cox = tan<. The modulation frequency must be made comparable to the decay rate, e.g., around 30 MHz for a mean lifetime of 30 ns. Such frequencies can be achieved by using a hydrogen lamp actuated by a suitably modulated current source. Commercial equipment is available. The method has been applied to quinine sulphate, fluorescein, and acridine, for example, with a precision of 1-2%. It is especially useful for very short (sub-nanosecond) lifetimes. [Pg.155]


See other pages where Fluorescence phase-shift method is mentioned: [Pg.271]    [Pg.22]    [Pg.1990]    [Pg.311]    [Pg.349]    [Pg.349]    [Pg.179]    [Pg.18]    [Pg.555]    [Pg.3426]    [Pg.152]    [Pg.668]    [Pg.1990]    [Pg.345]    [Pg.197]    [Pg.96]    [Pg.147]    [Pg.100]    [Pg.323]    [Pg.643]    [Pg.38]   
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