Photoselective excitation

If no significant rotation of the fluorophores occurs within the excited state fluorescence lifetime, then a high degree of anisotropy will be retained within the sample reflected by a value of r close to r0 (ra is a spectroscopic parameter and is the value of r the instant the excited state population is created). If significant rotation of the photoselected excited states occurs, on the other hand, the estimate of r from Equation 2.25 is consequently small (<0.01) because the optical order created at the instant of excitation is rapidly lost. 

Fluorescence polarisation spectroscopy is still very much used to probe the rotational dynamics of single molecules, either on surfaces or in solution [152]. In bioa-nalytical assays the fluorescence emission intensity is measured as a function of rotational speed. When a solution of fluorophores is excited with polarised light, the fluorophores selectively absorb those photons that are parallel to the transition moment of the fluorophore, resulting in photoselective excitation. The fluorophore molecules rotate to varying extents during the fluorophore lifetime. If the fluores-... 

This is the consequence of photoselective excitation of luminophores by polarized light, which selectively excites a population of molecules oriented with respect to the electrical vector of excitation. Emission also occurs with the light polarized along a fixed axis in the luminophore. The angle between these moments determines the maximal polarization of fluorescence. - Luminescence polarization is defined as follows ... 

Thus, when a population of fluorophores is illuminated by a linearly polarized incident light, those whose transition moments are oriented in a direction close to that of the electric vector of the incident beam are preferentially excited. This is called photoselection. Because the distribution of excited fluorophores is anisotropic, the emitted fluorescence is also anisotropic. Any change in direction of the transition moment during the lifetime of the excited state will cause this anisotropy to decrease, i.e. will induce a partial (or total) depolarization of fluorescence. 

If excited molecules can rotate during the excited-state lifetime, the emitted fluorescence is partially (or totally) depolarized (Figure 5.9). The preferred orientation of emitting molecules resulting from photoselection at time zero is indeed gradually affected as a function of time by the rotational Brownian motions. From the extent of fluorescence depolarization, we can obtain information on the molecular motions, which depend on the size and the shape of molecules, and on the fluidity of their microenvironment. 

Of considerable interest is the fact that not only the steady-state anisotropy but also its kinetics depend on the excitation wavelength. In this case another red-edge effect connected with site photoselection may be observed. Dipole-orientational relaxation may occur not only by rotation of the dipoles surrounding the fluorophore but also by rotation of the aromatic group itself. If red-edge excitation results in the photoselection of fluorophores whose energy of interaction with the environment already corresponds to that in the excited state, then the relaxation-associated rotation should not be observed and the rotation that occurs should be completely Brownian in character.(22)... 

Experiments involving anisotropy of phosphorescence or of the absorption of the triplet state rely upon the same principles as the measurement of fluorescence anisotropy. All are based upon the photoselection of molecules by polarized light and the randomization of polarization due to Brownian motion occurring on the time scale of the excited state. Anisotropy is defined as... 

Evidently, correlation functions for different spherical harmonic functions of two different vectors in the same molecule are also orthogonal under equilibrium averaging for an isotropic fluid. Thus, if the excitation process photoselects particular Im components of the (solid) angular distribution of absorption dipoles, then only those same Im components of the (solid) angular distribution of emission dipoles will contribute to observed signal, regardless of the other Im components that may in principle be detected, and vice versa. The result in this case is likewise independent of the index n = N. Equation (4.7) is just the special case of Eq. (4.9) when the two dipoles coincide. 

Fluorescence excitation spectroscopy is thus a powerful technique for obtaining molecular information about systems of cellular size. At present, the technique is restricted to single small objects because of the requirement of angular integration of the emitted fluorescence. As work progresses, similiar information will be obtainable from spectra taken at a particular angle with respect to the exciting beam. This will allow extension of the photoselection concept to suspensions of particles and perhaps to individual cells. 

The polarization study on naphthalene was complemented by Lavalette 39) who determined the polarized excitation spectrum, again using photoselection. The polarization of the strong Tm - Ti band at 4170 A was monitored as a function of the wavelength of polarized excitation into the singlet bands. As expected, a minimum polarization value of —0.18 was obtained at 2900 A near the 0—0 band of the S2 5q Mg) transition. 

Even in situations in which the molecules of interest are randomly oriented (e.g. in solution) the orientational distribution of emitting molecules may not be isotropic, due to the fact that the incident (excitation) beam photoselects molecules based upon the relative orientation of the absorption transition dipoles, p abs> with respect to the incident polarization vector, if [7,9,10]. The probability of absorption is proportional to I jfabs if I thus, for example, molecules oriented such that if... 

Fluorophores with dipoles perpendicular to excitation light will not absorb. Fluorophores with dipoles parallel to excitation light will absorb the most. Thus, polarized excitation will induce photoselection in the fluorophore absorption. The electric vector of excitation light is oriented parallel to or in the same direction as the z-axis. Emitted light will be measured with a polarizer. When emission is parallel to the excitation, the measured intensity is called I. When the emission is perpendicular to the excitation light, the measured intensity is called 11 - Light polarization and anisotropy are obtained according to Equations (11.1) and (11.2) ... 

Figure 11.1 Photoselection of differently oriented molecules with plane-polarized (a) and unpolarized (b) light. Excited molecules are shaded. After Albrecht, A.C. (1961). Journal of Molecular Spectroscopy, 6, 84. Figure published by Dorr, F. (1971), Creation and Detection of the Excited States, pp. 53-122, Dekker, New York.

This challenge has been met by a new technique based on the excitation of the surface I fluorescence, initiated in our laboratory, which allowed us to set up a powerful tool for photoselection of surface and subsurface states. This technique combines the superradiant 2D character of the surface emission, its very good spectral resolution at T < 80 K, and its strong sensitivity to gas coating. 

The pump pulse in time-resolved pump-probe absorption spectroscopy is often linearly polarized, so photoexcitation generally creates an anisotropic distribution of excited molecules. In essence, the polarized light photoselects those molecules whose transition moments are nominally aligned with respect to the pump polarization vector (12,13). If the anisotropy generated by the pump pulse is probed on a time scale that is fast compared to the rotational motion of the probed transition, the measured anisotropy can be used to determine the angle between the pumped and probed transitions. Therefore, time-resolved polarized absorption spectroscopy can be used to acquire information related to molecular structure and structural dynamics. 

Kottis and Lefebvre (322) have suggested that if polarized light is used to excite randomly oriented molecules to the triplet state, observation of the changes in the AMg = +1 ESR spectrum can reveal the correlation of the polarization properties of the excitation with the principal axis system of the triplet zero-field tensor. Such photoselection experiments have been carried out successfully by Lhotse and coworkers (323) and El-Sayed and Siegel (324) on a number of aromatic systems. Piette and collaborators (325) have studied the effect of metal complexation on the zero-field parameters and lifetimes of the phosphorescent triplet of aromatic-metal complexes with similar photoselection technique. The changes in... 

---