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

TL;DR

This study combines Fluorescence Correlation Spectroscopy and photobleaching to accurately quantify the number of fluorescent labels on molecules in solution, improving single-molecule analysis methods.

Contribution

It introduces a combined FCS and photobleaching approach to determine the number of fluorophores per molecule, accounting for statistical distribution and variable brightness.

Findings

FCS-photobleaching data fit yields mean fluorophore number (@ 2)

Photon count distribution confirms variability in brightness

Method effectively characterizes labeling on cDNA molecules

Abstract

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 (@ 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.