Fluorescence photoselection

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. 

This equation shows that, at time t, each anisotropy term is weighted by a factor that depends on the relative contribution to the total fluorescence intensity at that time. This is surprising at first sight, but simply results from the definition used for the emission anisotropy, which is based on the practical measurement of the overall ly and I components. A noticeable consequence is that the emission anisotropy of a mixture may not decay monotonously, depending of the values of r, and Ti for each species. Thus, r(t) should be viewed as an apparent or a technical anisotropy because it does not reflect the overall orientation relaxation after photoselection, as in the case of a single population of fluorophores. 

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. 

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... 

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. 

Since the presence of polarizers induces photoselection, the global fluorescence intensity recorded in the presence of polarizers is lower than that obtained in their absence. This is a consequence of the decrease in the number of absorbing and emitting fluorophores. 

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. 

Other related systems have been studied. These include crocetin" " and rhodopsin and synthetic pigments." The effect of photoselection upon the dichroic ratio in immobilized systems of bacteriorhodopsin in purple membrane have been examined." Photochemical studies of artificial bac-teriorhodopsins have been reported." A streak camera has been used to measure a fluorescence lifetime of less than 2 ps for bacteriorhodopsin at room... 

Fluorescence polarization cannot attain the +1 theoretical limits for maximum beam polarization owing to the nature of the absorption and emission processes, which usually correspond to electric dipole transitions. Although the excitation with linearly polarized radiation favours certain transition dipole orientations (hence certain fluorophore orientations, and the so-called photoselection process occurs), a fairly broad angular distribution is still obtained, the same happening afterwards with the angular distribution of the radiation of an electric dipole. The result being that, in the absence of fluorophore rotation and other depolarization processes, the polarization obeys the Lev shin-Perrin equation,... 

The principle of an FP measurement is depicted in Fig. 10. Upon illumination with Hnearly polarized light, the fluorophores in solution experience the highest probability of absorbing a photon when they have an orientation parallel to the incoming light vector. If the molecules were stationary or if fluorescence were an instantaneous process, the emission of this photoselected fluorophore ensemble... 

Time-resolved optical experiments rely on a short pulse of polarized light from a laser, synchrotron, or flash lamp to photoselect chromophores which have their transition dipoles oriented in the same direction as the polarization of the exciting light. This non-random orientational distribution of excited state transition dipoles will randomize in time due to motions of the polymer chains to which the chromophores are attached. The precise manner in which the oriented distribution randomizes depends upon the detailed character of the molecular motions taking place and is described by the orientation autocorrelation function. This randomization of the orientational distribution can be observed either through time-resolved polarized fluorescence (as in fluorescence anisotropy decay experiments) or through time-resolved polarized absorption. 

The results of a photoselection experiment on a dilute solution of PMPrS in a rigid glass consisting of a 7 3 volume ratio of 3MP and isoP at 77°K are shown in Figure 5. The polarization of the fluorescence, excited at four different wavelengths throughout the lowest absorption band, is shown... 

Steady-State Anisotropy Following continuous excitation with vertically polarized light, a distribution of fluorophores whose transition vectors for the absorption process are vertically aligned will be photoselected, creating an excited state population, which possesses a degree of anisotropy (r) or optical order, in an otherwise isotropic distribution of fluorophores. Measurement of the intensity of fluorescence, via an emission polarizer in planes parallel (z n) and perpendicular (zx) to the vertical plane allows estimation of r from... 

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. 

The loss of r will result through molecular motion within the excited state fluorescence lifetime (rf) until the photoselected population achieves an isotropic orientation. If the anisotropy decays following a simple, single relaxation mechanism, it will be described by Equation 2.31... 

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 ... 

---