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18 May 2009Principles and applications of fluorescence lifetime correlation spectroscopy
Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photon
counting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new technique
with 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 mixture
enables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties and
concentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed and
information on interaction of the species can be obtained. This technique is particularly helpful for distinguishing
different states of the same fluorophore in different microenvironments. The first application of that concept represents
the simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesicle
diffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dye
is 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 labeled
oligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNA
molecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.
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Lenka Beranová, Jana Humpolícková, Martin Hof, "Principles and applications of fluorescence lifetime correlation spectroscopy," Proc. SPIE 7356, Optical Sensors 2009, 735612 (18 May 2009); https://doi.org/10.1117/12.822857