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Determining the number of fluorescent entities that are coupled to a givenmolecule (DNA, protein, etc.) is a key point of numerous biological studies,especially those based on a single molecule approach. Reliable methods areimportant, in this context, not only to characterize the labeling process, butalso to quantify interactions, for instance within molecular complexes. Wecombined Fluorescence Correlation Spectroscopy (FCS) and photobleachingexperiments to measure the effective number of molecules and the molecularbrightness as a function of the total fluorescence count rate on solutions ofcDNA (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 thenumber of fluorescent labels per cDNA, the FCS-photobleaching data could beeasily fit to yield the mean number of fluorescent labels per cDNA strand (@2). This number could not be determined solely on the basis of the cDNAbrightness, because of both the statistical distribution of the number offluorescent labels and their unknown brightness when incorporated in cDNA. Thestatistical distribution of the number of fluorophores labeling cDNA wasconfirmed by analyzing the photon count distribution (with the cumulantmethod), which showed clearly that the brightness of cDNA strands varies fromone molecule to the other.

Measuring, in solution, multiple-fluorophore labeling by combining Fluorescence Correlation Spectroscopy and photobleaching