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Proceedings Paper

Determination of protein concentration on substrates using fluorescence fluctuation microscopy
Author(s): Richard De Mets; Irène Wang; Joseph Gallagher; Olivier Destaing; Martial Balland; Antoine Delon
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Paper Abstract

Micro-fabrication and surface functionalization imply to know the equilibrium surface concentration of various kinds of molecules. Paradoxically, this crucial parameter is often poorly controlled and even less quantified. We have used a technique belonging to the family of fluorescence fluctuation microscopy, namely Image Correlation Spectroscopy (ICS), to measure the absolute surface concentration of fibrinogen molecules adsorbed on glass substrates. As these molecules are immobile, the width of the autocorrelation of the confocal image obtained by scanning the sample only reflects that of the confocal Point Spread Function. Conversely, the amplitude of the autocorrelation is directly related to the average number of proteins simultaneously illuminated by the laser beam and therefore to their surface concentration. We have studied the surface concentration of fibrinogen proteins versus the initial concentration of these molecules, solubilized in the solution which has been deposited on the surface. The estimation of this relation can be biased for several reasons: the concentration of fibrinogen molecules in solution is difficult to control; the measurement of the surface concentration of adsorbed molecules can be strongly underestimated if the surface coverage or the molecular brightness is not uniform. We suggest methods to detect these artifacts and estimate the actual surface concentration, together with control parameters. Globally, fluorescence fluctuation microscopy is a powerful set of techniques when one wants to quantify the surface concentration of molecules at the micrometer scale.

Paper Details

Date Published: 4 March 2014
PDF: 11 pages
Proc. SPIE 8950, Single Molecule Spectroscopy and Superresolution Imaging VII, 895007 (4 March 2014); doi: 10.1117/12.2040355
Show Author Affiliations
Richard De Mets, LIPhy, CNRS, Univ. of Grenoble 1 (France)
Irène Wang, LIPhy, CNRS, Univ. of Grenoble 1 (France)
Joseph Gallagher, LIPhy, CNRS, Univ. of Grenoble 1 (France)
Olivier Destaing, INSERM, Institut Albert Bonniot, Univ. of Grenoble 1 (France)
Martial Balland, LIPhy, CNRS, Univ. of Grenoble 1 (France)
Antoine Delon, LIPhy, CNRS, Univ. of Grenoble 1 (France)

Published in SPIE Proceedings Vol. 8950:
Single Molecule Spectroscopy and Superresolution Imaging VII
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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