Combining fluorescence fluctuations and photobleaching to quantify surface density

Julius Sefkow-Werner (1) , Elisa Migliorini (1) , Catherine Picart (1) , Dwiria Wahyuni (2) , Irène Wang (3) , Antoine Delon (4)

1 UMR BioSanté - BioSanté
2 Department of Physics
3 LIPhy - Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères]
4 LIPhy - Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères]

We establish a self-calibrated method, called \emph{pbFFS} for \textit{photobleaching Fluctuation Fluorescence Spectroscopy}, which aims at characterizing molecules or particles labelled with an unknown distribution or fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a more 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 that 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 \textit{in situ} characterization (no dedicated substrates are required) and is applicable to low masses of adsorbed molecules, as demonstrated here by quantifying the density of biotin-Atto molecules that bind to the streptavidin layer. Besides molecules immobilized on surfaces, we also applied pbFFS to molecules diffusing in solution, to confirm the distribution of fluorescent labels found on surfaces. Hence, pbFFS provides a set of tools to investigate molecules labelled with a variable number of fluorophores, with the aim to quantify either the number of molecules or the distribution of fluorescent labels, the latter case being especially relevant for oligomerization studies.

Domaines: Biophysique [physics.bio-ph]; Optique [physics.optics]; Biotechnologies

Julius Sefkow-Werner, Elisa Migliorini, Catherine Picart, Dwiria Wahyuni, Irène Wang, et al.. Combining fluorescence fluctuations and photobleaching to quantify surface density. 2022. ⟨hal-03403806v3⟩