Time-dependent fluorescence definition

In the previous chapter we presented an overview of protein fluorescence. We described the spectral properties of the aromatic amino acids and how these properties depend on protein structure. We now extend this discussion to include time-resolved measurements of intrinsic protein flu( scence. Prior to 1983, most measurements of time-resolved fluorescence were performed using TCSPC. The instruments employed for these measurements typically used a flashlamp etcitation source and a standard dynode-chain-type PMT. Such instruments provided instrument response functions with a half-width near 2 ns, which is comparable to thedecay time of most proteins. The limited repetition rate of the flashlamps, near 20 kHz. resulted in data of modest statistical accuracy, unless the acquisition times were excessively long. Given the complexity of protein intensity and anisotropy decays, and the inherent difficulty of resolving multiexponential processes. ii was difficult to obUun definitive information on the decay kinetics of proteins. 

Another aspect of optical pumping is related to the coherent excitation of two or more molecular levels. This means that the optical excitation produces definite phase relations between the wave functions of these levels. This leads to interference effects, which influence the spatial distribution and the time dependence of the laser-induced fluorescence. This subject of coherent spectroscopy is covered in Chap. 7. 

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. 

Triplet—triplet energy transfer from benzophenone to phenanthrene in polymethylmethacrylate at 77 and 298 K was studied by steady-state phosphorescence depolarisation techniques [182], They were unable to see any clear evidence for the orientational dependence of the transfer probability [eqn. (92)]. This may be due to the relative magnitude of the phosphorescence lifetime of benzophenone ( 5 ms) and the much shorter rotational relaxation time of benzophenone implied by the observation by Rice and Kenney-Wallace [250] that coumarin-2 and pyrene have rotational times of < 1 ns, and rhodamine 6G of 5.7 ns in polymethyl methacrylate at room temperature. Indeed, the latter system of rhodamine 6G in polymethyl methacrylate could provide an interesting donor (to rose bengal or some such acceptor) where the rotational time is comparable with the fluorescence time and hence to the dipole—dipole energy transfer time. In this case, the definition of R0 in eqn. (77) is incorrect, since k cannot now be averaged over all orientations. 

---