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Cover. Analysis of the multimerisation state of human telomerase RNA (hTR) by Single molecule Fluorescence Coincidence Spectroscopy (SmFCS). SmFCS is based on a two-colour excitation/two-colour detection setup and relies upon the coupling of two laser beams (red and blue) to excite two different fluorophores simultaneously [Anal. Chem. (2003) 75, 1664–1670]. For these experiments, hTR was 5-end labelled with either Alexa Fluor 488 or Alexa Fluor 647 fluorophore dyes. The presence of hTR multimers was investigated by detecting coincident bursts of fluorescence photons in the red and blue detection channels as individual molecules (hTR monomer, dimer or higher order multimer) diffuse in and out of the detection volume where the two laser beams overlap. As depicted in the Figure, when an hTR dimer (containing both fluorophores) for instance diffuses through the detection volume, the two colour fluorophores are independently excited by the red and blue laser beams. Upon relaxation, bursts of fluorescence are produced by both fluorophores, detected simultaneously in both the red and blue channels and finally cross-correlated giving rise to a fluorescence coincidence event. Only entities containing both fluorophores can generate coincidence events. hTR monomers will produce only red or blue photons. The histograms of ratio R (Ired/Iblue) is then used to analyse the distribution of coincidence events and to determine in turn the multimerisation state of hTR. For further details see the paper by Ren et al. in this issue [Nucleic Acids Res. (2003) 31, 6509--6515].
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