Lifetime fluorescence

After excitation, molecules remain in the excited state a short time before returning to the ground state. The lifetime of the excited state is equal to the mean time during which molecules stay in the excited state. This time is considered as the fluorescence lifetime. This time goes from the nanosecond (1 O s) to the picosecond (1 O s). 

The mathematical definition of the fluorescence lifetime arises from the fact that non radiative and radiative processes participate in the deexcitation of the molecule. 

The radiative lifetime ir is equal to 1 / k,-. It is the real lifetime of emission of a photon that it should be measured independently of the other processes that deactivate the molecule. However, since these processes occur in parallel to the radiative one, it appears that it is impossible to eliminate them during the measurement of the radiative lifetime. Therefore we are going to measure a time characteristic of all the deexcitation processes. This time is called the fluorescence lifetime and is lower than the radiative lifetime. 

A fluorophore can have one or several fluorescence lifetimes. So in proteins, it is difficult to assign one specific fluorescence lifetime to a specific Trp residue. Cautions should be taken simply because from one protein to another the structure and the dynamics of the environment of tlie Trp residue differ inducing by that a variation in the fluorescence lifetime(s) of the fluorophore. Protein denaturation yields a uniform fluorescence lifetime equal to 2.5 ns, indicating that the structure and thus the dynamics of the protein in the tertiary structure play an important role in the determination of the fluorescence lifetime 

Let s see how can we describe the intensity decay with time. Let us consider No the population of fluorophores having reached the excited state. The velocity of decrease of this population with time t is equal to  

A fluorophore can have one or several fluorescence lifetimes, depending on several factors  

1 A ground-state heterogeneity resulting from the presence of equilibrium between different conformers. Each conformer presents a specific fluorescence lifetime. 

However, the data has to be interpreted carefully. In Fig. 4 we can see that a small change in the orientation of aj and Q, corresponds to a large change in z, (e.g. from position O H)- Hence the error of Q, is amplified in the calculation of the depth z,. 

The lifetime of the first excited state of single pentacene molecules in p-terphenyl was measured by time correlated single photon counting (TCSPC) [10]. The expected lifetime of the 5 -state of pentacene is about 20 ns, and it was necessary to optimize the excitation laser pulse duration carefully. While short pulses are advantageous. 

Transform limited pulses of a duration of 9 ns with a very stable center frequency were generated as follows A tunable single mode dye laser with a bandwidth of 2 MHz was pulsed with the help of an acousto-optical modulator. For the fluorescence lifetime experiments pulses of 9 ns FWHM and a separation of 1 ps were used. The laser pulses were focused onto a pinhole with a diameter of 5 pm and illuminated about 100 pm of the sample crystal. A set of lenses imaged the emerging fluorescence onto a fast photomultiplier. Optical cutoff filters removed scattered laser light from fluorescence. Recording times up to 1200 s were necessary to obtain reliable statistics for the fluorescence decay curves. The laser had to be stabilized onto a fixed frequency during the full measurement time. 

---

Strickler S J and Berg R A 1962 Relationship between absorption intensity and fluorescence lifetime of molecules J. Chem. Phys. 37 814-22... 

The vast majority of single-molecule optical experiments employ one-photon excited spontaneous fluorescence as the spectroscopic observable because of its relative simplicity and inlierently high sensitivity. Many molecules fluoresce with quantum yields near unity, and spontaneous fluorescence lifetimes for chromophores with large oscillator strengths are a few nanoseconds, implying that with a sufficiently intense excitation source a single... 

Chemical reactions can be studied at the single-molecule level by measuring the fluorescence lifetime of an excited state that can undergo reaction in competition with fluorescence. Reactions involving electron transfer (section C3.2) are among the most accessible via such teclmiques, and are particularly attractive candidates for study as a means of testing relationships between charge-transfer optical spectra and electron-transfer rates. If the physical parameters that detennine the reaction probability, such as overlap between the donor and acceptor orbitals. 

Figure C1.5.12.(A) Fluorescence decay of a single molecule of cresyl violet on an indium tin oxide (ITO) surface measured by time-correlated single photon counting. The solid line is tire fitted decay, a single exponential of 480 5 ps convolved witli tire instmment response function of 160 ps fwiim. The decay, which is considerably faster tlian tire natural fluorescence lifetime of cresyl violet, is due to electron transfer from tire excited cresyl violet (D ) to tire conduction band or energetically accessible surface electronic states of ITO. (B) Distribution of lifetimes for 40 different single molecules showing a broad distribution of electron transfer rates. Reprinted witli pennission from Lu andXie [1381. Copyright 1997 American Chemical Society.

Ambrose W P, Goodwin P M, Martin J C and Keller R A 1994 Alterations of single-molecule fluorescence lifetimes in near-field optical microscopy Science 265 364-7... 

Pirotta M, Guttler F, Gygax FI, Renn A, Sepiol J and Wild U P 1993 Single molecule spectroscopy fluorescence lifetime measurements of pentacene in p-terphenyl Chem. Phys. Lett. 208 379-84... 

Dunn R C, Holtom G R, Mets L and Xie X S 1994 Near-field fluorescence imaging and fluorescence lifetime measurement of light harvesting complexes in intact photosynthetic membranes J. Chem. Phys. 98 3094-8... 

Cline-Love L J and Shaver L A 1976 Time correlated single photon technique fluorescence lifetime measurements Anal. Chem. 48 370A-371A... 

Here t. is the intrinsic lifetime of tire excitation residing on molecule (i.e. tire fluorescence lifetime one would observe for tire isolated molecule), is tire pairwise energy transfer rate and F. is tire rate of excitation of tire molecule by the external source (tire photon flux multiplied by tire absorjDtion cross section). The master equation system (C3.4.4) allows one to calculate tire complete dynamics of energy migration between all molecules in an ensemble, but tire computation can become quite complicated if tire number of molecules is large. Moreover, it is commonly tire case that tire ensemble contains molecules of two, tliree or more spectral types, and experimentally it is practically impossible to distinguish tire contributions of individual molecules from each spectral pool. 

A different example of non-adiabatic effects is found in the absorption spectrum of pyrazine [171,172]. In this spectrum, the, Si state is a weak structured band, whereas the S2 state is an intense broad, fairly featureless band. Importantly, the fluorescence lifetime is seen fo dramatically decrease in fhe energy region of the 82 band. There is thus an efficient nonradiative relaxation path from this state, which results in the broad spectrum. Again, this is due to vibronic coupling between the two states [109,173,174]. 

Another example of the role played by a nonradiative relaxation pathway is found in the photochemistry of octatetraene. Here, the fluorescence lifetime is found to decrease dramatically with increasing temperature [175]. This can be assigned to the opening up of an efficient nonradiative pathway back to the ground state [6]. In recent years, nonradiative relaxation pathways have been frequently implicated in organic photochemistry, and a number of articles published on this subject [4-8]. 

The fluorescence lifetime tp can be measured directly and is the lifetime of the state, taking into account all decay processes. It is related to and by... 

Table 7.11 Fluorescence quantum yield

The limits of lifetime detection and resolution in on-the-flight fluorescence lifetime detection in hplc were evaluated for simple, binary systems of polycycHc hydrocarbons (70). Peak homogeneity owing to coelution was clearly indicated for two compounds having fluorescence lifetime ratios as small as 1.2 and the individual peaks could be recovered using predeterrnined lifetimes of the compounds. Limits of lifetime detection were deterrnined to be 6 and 0.3 pmol for benzo[b]fluoranthene and benzo[k]fluoranthene, respectively. 

Fluorescent lifetimes of trivalent rare earth transition metal chemistry. G. E. Peterson, Transition Met. Chem. (N.Y.), 1966, 3, 202-302 (173). 

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. 

The fluorescent lifetime of chlorophyll in vivo was first measured in 1957, independently by Brody and Rabinowitch (62) using pulse methods, and by Dmitrievskyand co-workers (63) using phase modulation methods. Because the measured quantum yield was lower than that predicted from the measured lifetime, it was concluded that much of the chlorophyll molecule was non-fluorescent, suggesting that energy transfer mechanisms were the means of moving absorbed energy to reactive parts of the molecule. 

Fluorescence lifetime measurements can increase the analytical specificity when analyzing mixtures (1-4) and can indicate changes in chemical binding of the fluorophores under various environmental conditions (5). 

Depolarization measurements, coupled with fluorescence lifetimes, are correlated with rates of molecular rotation to obtain estimates of molecular conformation, volume, and shape. 

The quantity riV/RT is equal to six times the rotational period. The rotational relaxation time, p, should he shorter than the fluorescence lifetime, t, for these equations to apply. It is possible to perform calculations for nonspherical molecules such as prolate and oblate ellipsoids of revolution, but in such cases, there are different rotational rates about the different principal axes. 

---