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 method, called pbFFS for photobleaching Fluctuation Fluorescence Spectroscopy, which aims at characterizing molecules or particles labelled with a unknown distribution or fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of labels and a more reliable estima-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 labels found on surfaces. Hence, pbFFS provides a set of tools to investigate molecules attached to a variable number of fluorophores, with the aim to quantify either the number of molecules or the distribution of fluorophores, 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. 2021. ⟨hal-03403806v1⟩