Combining fluorescence fluctuations and photobleaching to quantify surface density

TL;DR

The paper introduces pbFFS, a novel method combining fluorescence fluctuations and photobleaching to accurately quantify surface molecule density and label distribution in situ, applicable to both surface-immobilized and diffusing molecules.

Contribution

The paper presents a complete theoretical framework and experimental validation of pbFFS, enabling in situ, reliable measurement of molecule surface density and fluorophore distribution, even at low molecule counts.

Findings

pbFFS provides consistent surface density measurements with standard techniques.

It enables in situ analysis without dedicated substrates.

It accurately quantifies fluorophore distribution on molecules.

Abstract

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 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 more standard surface techniques, such as ellipsometry. Compared to those techniques, pbFFS has two main advantages: it enables 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 labels found on surfaces. Hence, pbFFS provides a set of tools to investigate molecules attached to a variable number of fluorophores, with the aim toquantify either the number of molecules or the distribution of fluorophores, the latter case being especially relevant for oligomerization studies.