Measuring, in solution, multiple-fluorophore labeling by combining fluorescence correlation spectroscopy and photobleaching.
J. Phys. Chem. B. 2010-03-04; 114(8): 2988-2996
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1. J Phys Chem B. 2010 Mar 4;114(8):2988-96. doi: 10.1021/jp910082h.
Measuring, in solution, multiple-fluorophore labeling by combining fluorescence
correlation spectroscopy and photobleaching.
Delon A(1), Wang I, Lambert E, Mache S, Mache R, Derouard J, Motto-Ros V, Galland
(1)Laboratoire de Spectrométrie Physique UMR 5588, Université de Grenoble I/CNRS,
BP 87, 38402 Saint Martin d’Hères, France.
Determining the number of fluorescent entities that are coupled to a given
molecule (DNA, protein, etc.) is a key point of numerous biological studies,
especially those based on a single molecule approach. Reliable methods are
important, in this context, not only to characterize the labeling process but
also to quantify interactions, for instance within molecular complexes. We
combined fluorescence correlation spectroscopy (FCS) and photobleaching
experiments to measure the effective number of molecules and the molecular
brightness as a function of the total fluorescence count rate on solutions of
cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here,
photobleaching is used as a control parameter to vary the experimental outputs
(brightness and number of molecules). Assuming a Poissonian distribution of the
number of fluorescent labels per cDNA, the FCS-photobleaching data could be
easily fit to yield the mean number of fluorescent labels per cDNA strand
(approximately = 2). This number could not be determined solely on the basis of
the cDNA brightness, because of both the statistical distribution of the number
of fluorescent labels and their unknown brightness when incorporated in cDNA. The
statistical distribution of the number of fluorophores labeling cDNA was
confirmed by analyzing the photon count distribution (with the cumulant method),
which showed clearly that the brightness of cDNA strands varies from one molecule
to the other. We also performed complementary continuous photobleaching
experiments and found that the photobleaching decay rate of Alexa Fluor 647 in
the excited state decreases by about 30% when incorporated into cDNA, while its
nonradiative decay rate is increased such that the brightness of individual Alexa
labels is decreased by 25% compared to free Alexa dyes.
PMID: 20143802 [Indexed for MEDLINE]