Fluorescence lifetime measurement

The important conclusion is that we can measure the rate of energy transfer without ever measuring the energy transfer directly, provided we could measure the total rate constant of decay from the excited state in both cases. In the case of fluorescence, this total rate is the reciprocal of the measured fluorescence lifetime the... 

Two different approaches for measuring fluorescence lifetimes are commonly employed to study FRET-related phenomenon the frequency-domain FLIM (see Chapter 2) and the time-domain... 

Prior to describing the possible applications of laser-diode fluorometry, it is important to understand the two methods now used to measure fluorescence lifetimes these being the time-domain (Tl)/4 5 24 and frequency-domain (FD) or phase-modulation methods.(25) In TD fluorometry, the sample is excited by a pulse of light followed by measurement of the time-dependent intensity. In FD fluorometry, the sample is excited with amplitude-modulated light. The lifetime can be found from the phase angle delay and demodulation of the emission relative to the modulated incident light. We do not wish to fuel the debate of TD versus FD methods, but it is clear that phase and modulation measurements can be performed with simple and low cost instrumentation, and can provide excellent accuracy with short data acquisition times. 

There are many molecular interactions which influence the fluorescence decay times. The measured fluorescence lifetime r is usually shorter than the radiative lifetime tr because of presence of other decay rates which can be dependent on intramolecular processes and intermolecular interactions (Figure 10.3). The measured fluorescence lifetime (r) is given by the inverse of the total rate of dynamic processes that cause deactivation from the excited (mostly singlet Si) state... 

There are two widely used methods for measuring fluorescence lifetimes, the time-domain and frequency-domain or phase-modulation methods. The basic principles of time-domain fluorometry are described in Chapter 1, Vol.l of this series(34) and those of frequency-domain in Chapter 5, Vol. 1 of this series.<35) Good accounts of time-resolved measurements using these methods are also given elsewhere/36,37) It is common to represent intensity decays of varying complexity in terms of the multiexponential model... 

The principal requirements for photomultipliers in both pulse and phase-modulation methods of measuring fluorescence lifetimes are as follows. 

In addition to fluorescence intensity and polarization, fluorescence spectroscopy also includes measurement of the lifetime of the excited state. Recent improvements in the design of fluorescence instrumentation for measuring fluorescence lifetime have permitted additional applications of fluorescence techniques to immunoassays. Fluorescence lifetime measurement can be performed by either phase-resolved or time-resolved fluorescence spectroscopy. 

Capelle et al. (186) have measured fluorescence lifetimes and quenching cross sections of vibrational levels from v = 1 to 31 of the B3n(0 +) state of Br2 molecules. The lifetimes vary from 1.3 (v = 27) to 0.14 psec (i> = 17). Lifetimes must be much shorter than the radiative life since in this absorption region (5130 to 6260 A) photodissociation is predominant (571). 

The extent to which the IC process is blocked by vibrational deficiency can be ascertained by the quantum yield for fluorescence, <1>F. Such information can be extracted from the measured fluorescence lifetimes, X[, of the vibronic levels of the Sj state, providing the oscillator strength of the S i < S0 transition is known. The radiative lifetime, based on the transition strength, rr, can calculated from the oscillator strength through the expression, xr 1.5//V2. With a measured oscillator strength of / 1.2 x 10 4 and an average transition frequency of... 

Three techniques are actually available for measuring the fluorescence lifetime Strobe, Time Correlated Single Photon Counting (TCSPC), and multifrequency and crosscorrelation spectroscopy. Strobe and TCSPC are based on measurement in the time domain, while multifrequency and cross-correlation spectroscopy measure fluorescence lifetimes in the frequency domain. The time domain allows direct observation of fluorescence decay, while the frequency domain is a more indirect approach in which the information regarding the fluorescence decay is implicit. 

Bennett, R.G. (1960). Instrument to measure fluorescence lifetimes in the millimicrosecond region. Review of Scientific Instruments, 31, 1275-1279. 

If students have access to a fluorescence lifetime instrument, it would be useful to see how one can measure fluorescence lifetime. In this case, it will be useful if students can perform the experiments described by following fluorescence lifetime quenching with KI and compare their results with intensity quenching experiments. 

Let us consider kt as the rate constant of the excited state depopulation via the energy-transfer mechanism the measured fluorescence lifetime is thus equal to... 

Time correlated single photon counting is a well-established technique that has been used to measure fluorescence lifetimes since the mid-1960 s. These early experiments, which used a variety of flashlamps and gaseous gap-discharge arcs as the excitation source, were reviewed by Ware [47, 48] in 1971. The traditional light sources have been replaced by laser sources in recent experiments, thus markedly extending the range of applications of this technique. Particularly well suited excitation sources for this method are the mode-locked lasers and synchronously pumped dye lasers which are capable of operation at MHz repetition rates. 

Fig. 11. Block diagram showing the system and method for measuring fluorescence lifetimes with a boxcar integrator.

With a possible resolution of less than 1 ps [65, 66], streak cameras offer the highest temporal resolution of any device currently available. The study of picosecond phenomena is a rapidly expanding field and the state-of-the-art in picosecond techniques was recently reviewed [lc]. Here we are concerned only with the general principles of streak cameras and their application to measuring fluorescence lifetimes. 

In the vast majority of cases reported where a streak camera has been used to measure fluorescence lifetimes, the measurements have been made from a single laser shot. Since a high fluorescence efficiency is necessary for single shot experiments, most of these studies have been concerned with measuring the lifetimes and quenching of organic dye molecules in solution. For example, Yu etal. [67] have made a study of the fluorescence lifetime of malachite green as a function of solvent viscosity and the lifetime and relative yield of erythrosin as a function of water concentration in a water—acetone mixture. The fluorescence lifetimes of these dyes are... 

J/cm laser fluence using 1064 nm excitation. The observation that the measured fluorescence lifetime using 1064 nm excitation was independent of laser fluence therefore provides evidence that thermal effects are unimportant in this work. 

Because the dynamics of phospholipid membranes have been well characterized using AF probes [8-13], fluorescence results obtained with hydrated human SC were compared to aqueous suspensions of unilamellar distearoyl-phosphatidylcholine (DSPC) vesicles. DSPC was also used because its phase transition temperature (55°C) is close to that of SC lipids (65 C). The microenvironment inside DSPC and SC membranes was studied by measuring fluorescence lifetimes, and shifts in emission maxima were compared to excitation maxima (Stokes shifts), along with quenching of a series of AF probes by iodide. Stokes shifts (Av) [6] were calculated as ... 

Figure 11.2 Left Schematic illustrating use of double stranded DNA to mediate distance between fluorophore and nanoparticle surface. Middle Symbols show the average values of experimentally measured fluorescence lifetime. The last measurement was done in the absence of gold nanoparticles as a calibration. The dashed and dashed-dotted curves display the calculated fluorescence lifetime for the molecular dipole oriented radially or tangentially with respect to the gold nanoparticle. Right Fluorescence signal corresponding to the measurements presented the middle panel. Reprinted with permission from reference [22]. (2007) American Chemical Society.

Therefore, the efficiency can also be given in terms of the measured fluorescence lifetimes in the presence and absence of... 

Time-correlated single-photon counting (TCSP) has proven to be a much-used method for measuring fluorescence lifetimes. It is highly sensitive in that it requires only one photon to be incident on the detector per excitation cycle, and statistical analysis of the experimental data gives lifetimes with well-defined error limits. Commercial systems are available which allow lifetimes from 50 ps to many tens of nanoseconds to be measured with relative ease and high precision. 

In a very new report, Fujino et al. challenge the two-isomerization-mechanism concept on the basis of their time-resolved and time-integrated femtosecond fluorescence measurements of B-azobenzene following excitation of the (7t,7t ) State. They use the extremely weak fluorescence (cf. Figure 1.8) as an indicator for the population of the emitting state. From the ratios of their measured fluorescence lifetimes (S2 0.11 ps Sp 0.5 ps) and the radiative lifetimes deduced from the (absorption-spectra-based) oscillator strengths, they determine the fluorescence quantum yields 2.5310 for the emission and 7.5410" for the Si—>So emission. By comparison... 

Pyrene excimer formation still continues to be of interest and importance as a model compound for various types of study. Recent re-examinations of the kinetics have been referred to in the previous section. A non a priori analysis of experimentally determined fluorescence decay surfaces has been applied to the examination of intermolecular pyrene excimer formation O. The Kramers equation has been successfully applied to the formation of intermolecular excimer states of 1,3-di(l-pyrenyl) propane . Measured fluorescence lifetimes fit the predictions of the Kramer equation very well. The concentration dependence of transient effects in monomer-excimer kinetics of pyrene and methyl 4-(l-pyrenebutyrate) in toluene and cyclohexane have also been studied . Pyrene excimer formation in polypeptides carrying 2-pyrenyl groups in a-helices has been observed by means of circular polarized fluorescence" . Another probe study of pyrene excimer has been employed in the investigation of multicomponent recombination of germinate pairs and the effect on the form of Stern-Volmer plots ". 

If the spectral properties of the molecules are too similar for spectral identification, they may have different fluorescence lifetimes such that they could be identified in the time domain. By using pulsed laser excitation and time-correlated single-photon counting, which is the standard method for measuring fluorescence lifetimes [47], the researchers at Los Alamos have... 

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