Polarized fluorescence decay

Fisz, J. Jn 1996, Polarized fluorescence decay surface for a mixiure of non-interacting species in solution, Chetn. Phys. Lett. 259 579-587. 

Fisz, J. J.. 1996, Polarized fluorescence decay surface for many-ground- and many-excited-state cies in solution, Chem. Phys. Utl. 262 507-518. 

Thus, the polarized fluorescence decay components contain information about the motional dynamics of the molecule. 

The attachment of pyrene or another fluorescent marker to a phospholipid or its addition to an insoluble monolayer facilitates their study via fluorescence spectroscopy [163]. Pyrene is often chosen due to its high quantum yield and spectroscopic sensitivity to the polarity of the local environment. In addition, one of several amphiphilic quenching molecules allows measurement of the pyrene lateral diffusion in the mono-layer via the change in the fluorescence decay due to the bimolecular quenching reaction [164,165]. 

The first photoelectric fhiorimeter was described by Jette and West in 1928. The instrument, which used two photoemissive cells, was employed for studying the quantitative effects of electrolytes upon the fluorescence of a series of substances, including quinine sulfate [5], In 1935, Cohen provides a review of the first photoelectric fluorimeters developed until then and describes his own apparatus using a very simple scheme. With the latter he obtained a typical analytical calibration curve, thus confirming the findings of Desha [33], The sensitivity of these photoelectric instruments was limited, and as a result utilization of the photomultiplier tube, invented by Zworykin and Rajchman in 1939 [34], was an important step forward in the development of suitable and more sensitive fluorometers. The pulse fhiorimeter, which can be used for direct measurements of fluorescence decay times and polarization, was developed around 1950, and was initiated by the commercialization of an adequate photomultiplier [35]. 

The case of several populations of fluorophores having their own fluorescence decay i (t) and time constants characterizing r (t) deserves particular attention. In Section 5.3, it was concluded that an apparent or a technical emission anisotropy r(t) can be obtained by considering that the measured polarized components, I(t) and I (t), are the sums of the individual components (i.e. of each population) and by using Eq. (6.43). Hence... 

Evolution of fluorescence spectra during the lifetime of the excited state can provide interesting information. Such an evolution occurs when a fluorescent compound is excited and then evolves towards a new configuration whose fluorescent decay is different. A typical example is the solvent relaxation around an excited-state compound whose dipole moment is higher in the excited state than in the ground state (see Chapter 7) the relaxation results in a gradual red-shift of the fluorescence spectrum, and information on the polarity of the microenvironment around a fluorophore is thus obtained (e.g. in biological macromolecules). 

It should be recalled that, in polar rigid media, excitation on the red-edge of the absorption spectrum causes a red-shift of the fluorescence spectrum with respect to that observed on excitation in the bulk of the absorption spectrum (see the explanation of the red-edge effect in Section 3.5.1). Such a red-shift is still observable if the solvent relaxation competes with the fluorescence decay, but it disappears in fluid solutions because of dynamic equilibrium among the various solvation sites. 

Homotransfer does not cause additional de-excitation of the donor molecules, i.e. does not result in fluorescence quenching. In fact, the probability of de-excitation of a donor molecule does not depend on the fact that this molecule was initially excited by absorption of a photon or by transfer of excitation from another donor molecule. Therefore, the fluorescence decay of a population of donor molecules is not perturbed by possible excitation transport among donors. Because the transition dipole moments of the molecules are not parallel (except in very rare cases), the polarization of the emitted fluorescence is affected by homotransfer and information on the kinetics of excitation transport is provided by the decay of emission anisotropy. 

When the excitation light is polarized and/or if the emitted fluorescence is detected through a polarizer, rotational motion of a fluorophore causes fluctuations in fluorescence intensity. We will consider only the case where the fluorescence decay, the rotational motion and the translational diffusion are well separated in time. In other words, the relevant parameters are such that tc rp, where is the lifetime of the singlet excited state, zc is the rotational correlation time (defined as l/6Dr where Dr is the rotational diffusion coefficient see Chapter 5, Section 5.6.1), and td is the diffusion time defined above. Then, the normalized autocorrelation function can be written as (Rigler et al., 1993)... 

Intramolecular charge transfer in p-anthracene-(CH2)3-p-Ar,Af-dimethylaniline (61) has been observed174 in non-polar solvents. Measurements of fluorescence-decay (by the picosecond laser method) allow some conclusions about charge-transfer dynamics in solution internal rotation is required to reach a favourable geometry for the formation of intramolecular charge-transfer between the donor (aniline) and the acceptor (anthracene). 

The polarization properties of light in combination with fluorescence can be used as a powerful tool for determining motional properties of membranes. This is possible due to the fact that the time scale of interest for membrane lipids falls within the time frame of the fluorescence decay phenomena (0-100+ ns). This, coupled with high sensitivity, low perturbing properties of fluorescent probes, and the large number of available probes, makes the fluorescence approach the method of choice for membrane motional studies. 

Fluorescence anisotropy decay measurements, which are based on the excitation of probes with polarized light and subsequent polarized fluorescence emission, can... 

The effect of exciplex dissociation (process MC) on the over-all kinetics of molecular fluorescence decay has been examined by Ware and Richter34 for the system perylene-dimethylaniline in solvents with dielectric constants (e) varying from 2.3 to 37. In low dielectric media (e = 2.3-4) the perylene fluorescence response may be fitted to a two-component exponential curve and exciplex emission is also observed, whereas in more polar solvents (e > 12) exciplex fluorescence is absent (at ambient temperatures) and the molecular fluorescence decays exponentially. These observations are consistent with both an increase in exciplex stability toward molecular dissociation with solvent polarity (Eq. 13) and the increased probability of dissociation into solvated ions... 

Fluorescence techniques have been used with great success in the study of PEO-fe-PSt micelles [64]. In this study, the effect of polymer concentration on the fluorescence of pyrene present in water at saturation was studied. Three features of the absorption and emission spectra change when micellization occurs. First, the low-energy band of the (S2-So) transition is shifted from 332.5 to 338 nm. Second, the lifetime of the pyrene fluorescence decay increases from 200 to ca. 350 ns, accompanied by a corresponding increase in the fluorescence quantum yield. Third, the vibrational fine structure changes, as the transfer of pyrene from a polar environment to a nonpolar one suppresses the permissibility of the symmetry-forbidden (0,0) band. 

The time-resolved emission spectra were reconstructed from the fluorescence decay kinetics at a series of emission wavelengths, and the steady-state emission spectrum as described in the Theory section (37). Figure 4 shows a typical set of time-resolved emission spectra for PRODAN in a binary supercritical fluid composed of CO2 and 1.57 mol% CH3OH (T = 45 °C P = 81.4 bar). Clearly, the emission spectrum red shifts following excitation indicating that the local solvent environment is becoming more polar during the excited-state lifetime. We attribute this red shift to the reorientation of cosolvent molecules about excited-state PRODAN. 

Compounds 268a and 269a showed large Stokes shifts in polar solvents. Such large Stokes shifts have been observed in many TICT (twisted intramolecular charge transfer) molecules. Fluorescence decay time measurement indicated that there were two kinds of excited states, fast and slower decaying components, and the latter was the emission from the more polar state. 

Fig. 11. Environment-dependent single-emitter excited-state lifetimes, (a) Calculation of the decay rates of a single emitter at variable distance to the interface for parallel and perpendicular orientation of its dipole moment, (b) Annular illumination SCOM images for two orthogonal polarizations revealing the orientation of single emitter dipoles embedded in thin film of PMMA. (c) Examples of two fluorescence decays for circled spots A and B in (b). For more details see [50].

If, from another point of view, fluorescent decay leads to a considerable population of the ground state level c, (see, e.g., Fig. 1.2), then molecules on this level may exhibit angular momenta polarization, being particularly noticeable for thermally unpopulated, high lying levels. 

Examples, as described by Eqs. (4.41), (4.42) and (4.43) show what kind of information one may obtain directly by registering oscillations in the fluorescence decay. These are the lifetime r = T-1 of the state, the factors affecting it, the precession frequency uij/ and, consequently, the value of the Lande factor gj>, as well as its sign (by the initial phase of oscillations), and, finally, the degree of polarization V. A favorable condition for registration should be the validity of T = 2tt/(Qu>ji) [Pg.135]

---