Measuring, in solution, multiple-fluorophore labeling by combining fluorescence correlation spectroscopy and photobleaching.
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.
Delon
Antoine
A
Laboratoire de Spectrométrie Physique UMR 5588, Université de Grenoble I/CNRS, BP 87, 38402 Saint Martin d'Hères, France. adelon@ujf-grenoble.fr
Wang
Irène
I
Lambert
Emeline
E
Mache
Silva
S
Mache
Régis
R
Derouard
Jacques
J
Motto-Ros
Vincent
V
Galland
Rémi
R
D018076:DNA, Complementary / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D038761:Photobleaching; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012996:Solutions