Intrinsic fluorescence experimental observations

Figure 2.17 shows the excited intrinsic fluorescence obtained in our laboratory by excitation at 3917 A of a sublimation-grown crystal a few micrometers thick,71 at 5 K. Results of other authors with sublimation-grown samples81"84,86 confirm the general lineshape of the fluorescence, but, owing to the various experimental conditions, variations are observed, which can be summarized as follows ... 

In about 2000, my laboratory started to study the interactions of fluorophores with metallic nanoparticles, both solution-based and surface-immobilized. Our findings agreed with other workers whom had observed increases in fluorescence emission coupled with a decrease in the fluorophores radiative lifetime. Subsequently, we applied classical far-field fluorescence descriptions to these experimental observations, which ultimately suggested a modification in the fluorophores s intrinsic radiative decay rate, a rate thought to be mostly unchanged and only weakly dependent on external environmental factors. This simple description, coupled with what seemed like a limitless amount of applications led to a paper published by our laboratory in 2001 entitled Metal-Enhanced Fluorescence , or MEF, a term now widely used today almost a decade later. 

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. 

In other media like micelles, cyclodextrin, binary solvent mixtures, and proteins (47-55), lifetime distributions are routinely used to model the decay kinetics. In all of these cases the distribution is a result of the (intrinsic or extrinsic) fluorescent probe distributing simultaneously in an ensemble of different local environments. For example, in the case of the cyclodextrin work from our laboratory (53-55), the observed lifetime distribution is a result of an ensemble of 1 1 inclusion complexes forming and coexisting. These complexes are such that the fluorescent probe is located simultaneously in an array of environments (polarities, etc.) in, near, and within the cyclodextrin cavity, which manifest themselves in a distribution of excited-state lifetimes (53-55). In the present study our experimental results argue for a unimodal lifetime distribution for PRODAN in pure CF3H. The question then becomes, how can a lifetime distribution be manifest in a pure solvent ... 

Among the best well-known examples of photostability after UV radiation, the ultrafast nonradiative decay observed in DNA/RNA nucleobases, has attracted most of the attention both from experimental and theoretical viewpoints [30], Since the quenched DNA fluorescence in nucleobase monomers at the room temperature was first reported [31] new advances have improved our knowledge on the dynamics of photoexcited DNA. Femtosecond pump-probe experiments in molecular beams have detected multi-exponential decay channels in the femtosecond (fs) and picosecond (ps) timescales for the isolated nucleobases [30, 32-34], The lack of strong solvent effects and similar ultrafast decays obtained for nucleosides and nucleotides suggest that ultrashort lifetimes of nucleobases are intrinsic molecular properties, intimately... 

The usual experimental technique is to observe the fluorescence of a probe molecule incorporated into the system under investigation and make the assumption that the motion of the probe reflects that of its environment. Probe molecules may be divided into two cathodes intrinsic probes (such as fluorescent amino acid residues naturally occurring in a protein under investigation) and extrinsic probes either bound chemically to the macromolecule or simply dispersed in the stem. The criteria that need to be applied when selecting suitable candidates for probe molecules have been discussed by Stryer Ideally the probe should... 

Braga, and Lumb (131,274), although criticized for its simplicity (125,283), accounts well for experimental decay of the singlet excimer. The observed quantum yield of monomer fluorescence is reduced in the presence of excimer from the intrinsic yield according to ... 

So far we have seen that fluorescence polarization, its value, is a result of the fact that the two dipoles are not parallel. This value of polarization is called the intrinsic polarization, P. However, there are other factors that affect polarization and therefore intrinsic pol2irization is rarely the value that is observed experimentally. 

The ability of SPR to probe both kinetic and thermodynamic processes, as well as to provide micro-structural information, make it a very important component of the experimental methodology available to probe molecular interactions occurring at surfaces. Furthermore, it allows some of the limitations of other techniques to be overcome. For example, other methods often require one of the partners to be labelled in some way in order to allow it to be detected. Fluorescent probes, radioactive labels, and attachment of independently detectable molecules (e.g. enzymes) have all been used for this purpose. These suffer from the drawback that they may interfere with the binding of the labelled partner to the unlabelled one, or cause unwanted structural perturbations. SPR observations can be based solely on the dielectric properties of molecules, or their intrinsic light absorption characteristics, and thus require no specific labelling. 

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