Principles and Applications of Fluorescence Lifetime CorrelationSpectroscopy

摘要

Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photoncounting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new techniquewith several experimental benefits. In FLCS experiments, pulsed excitation is used and data are stored in a special time-tagged time-resolved mode. Mathematical treatment of TCSPC decay patterns of distinct fluorophores and their mixtureenables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties andconcentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed andinformation on interaction of the species can be obtained. This technique is particularly helpful for distinguishingdifferent states of the same fluorophore in different microenvironments. The first application of that concept representsthe simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesiclediffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dyeis considerably quenched in the layer near the light-absorbing surface. This concept was also used in other applications:a) investigation of a conformational change of a labeled protein, b) detection of small amounts of labeledoligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNAmolecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.