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Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density

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Although Fluorescence Fluctuation Spectroscopy (FFS) are well-known techniques to assess number densities or concentrations of molecules, clusters or particles in biological environments (cells, solutions, surfaces), they fail to provide quantitative results when the species of interest are not evenly labeled. We proposed to combine this kind of methods with photobleaching and theoretically demonstrated that whatever the initial distribution of fluorescent molecules labeling the entities to count, the brightness (as measured by FFS) always decay linearly, thus providing two outputs, the single fluorescent label brightness and a parameter depending on the mean and variance of the distribution of fluorescent labels. The pbFFS method has been experimentally demonstrated using a streptavidin base layer of biomimetic samples to estimate the surface density of streptavidin and its propensity to bind a top layer of biotin molecules. The base layer density estimated with pbFFS agrees well with spectroscopic ellipsometry measurements.

We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides
information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic
systems. The surface density measured by pbFFS is consistent with the results of spectroscopic ellipsometry, a standard surface technique. However, pbFFS has two main advantages: it enables in situ characterization (no dedicated substrates are required) and can be applied to low masses of adsorbed molecules, which we demonstrate here by quantifying the density of biotin-Atto molecules that bind to the streptavidin layer. In addition to molecules immobilized on a surface, we also applied pbFFS to molecules diffusing in solution, to confirm the distribution of fluorescent labels found on a surface. Hence, pbFFS provides a set of tools for investigating the molecules labeled with a variable
number of fluorophores, with the aim of quantifying either the number of molecules or the distribution of fluorescent labels, the latter case being especially relevant for oligomerization studies.


View online : Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density” Julius Sefkow-Werner, Elisa Migliorini, Catherine Picart, Dwiria Wahyuni, Irène Wang, and Antoine Delon Anal. Chem.