Fluorophore environment

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

A mixture of noninteracting fluorophores might be observed by spatially variant FRET in a specimen, which is blurred because of optical resolution issues, will result in different lifetimes being measured for zm and zy- and zm > %f. In many instances, a single frequency measurement will be insufficient to determine the number of fluorophores or the number of fluorophore environments in a sample. 

The measurement of fluorescence lifetimes is an integral part of the anisotropy, energy transfer, and quenching experiment. Also, the fluorescence lifetime provides potentially useful information on the fluorophore environment and therefore provides useful information on membrane properties. An example is the investigation of lateral phase separations. Recently, interest in the fluorescence lifetime itself has increased due to the introduction of the lifetime distribution model as an alternative to the discrete multiexponential approach which has been prevalent in the past. 

Temperature variation induces modification of global and local motions of the fluorophore environment and of the fluorophore itself, modifying its fluorescence emission feature. 

The temperature variation can affect not only the fluorescence intensity of the spectrum but also its emission bandwidth. However, this is dependent on the fluorophore environment and the fluorophore. Figure 10.17 shows the fluorescence emission spectrum of Trp residues of the protein LCA. In the range of temperatures studied, a shift to the red was not observed, and so we are far from denaturing temperatures. In addition, the emission bandwidth (54 nm) does not change with temperature. 

Intensity, position of the emission wavelength, lifetime are some observables that are going to characterize a fluorophore. Modification of the temperature and / or the viscosity of the medium will affect the values of these observables. Each fluorophore has its own fluorescence properties and observables. These properties are intrinsic to the fluorophore and are modified with the environment. We shall see in the next chapter that these modifications do not follow the same rules for all fluorophores. Therefore, it is important to understand the nature of the fluorophore environment before taking conclusions that could be misleading in the interpretation of the studied phenomenon. Also, we are going to see that fluorophore structure can influence its fluorescence lifetimes. This will vary from a fluorophore to another. 

Chattopadhyay A, Mukherjee S (1993) Fluorophore environments in membrane-bound probes— red edge excitation shift study. Biochemistry 32(14) 3804-3811. doi 10.1021/ bi00065a037... 

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