Measuring protein surface density on glass substrate using fluorescence fluctuation spectroscopy

Abstract

Surface functionalization implies to know the surface density of proteins, which, for cellular biology, impacts on cells behavior (motility, spreading, and fate). Contradictory, the quantification of this parameter is lacking. We studied the surface density of deposited proteins on glass using the Image Correlation Spectroscopy (ICS) technique as part of the Fluorescence Fluctuation Microscopy (FFM) methods. As the confocal microscope scans the surface, the proteins deposited on it contribute to the image through the confocal point spread function (PSF). The width of the image autocorrelation is equal to the width of the PSF; its amplitude provides the average number of proteins within the PSF, proportional to the surface density. Nevertheless, the assessment of the surface density can be underestimated if the surface coverage is not uniform or overestimated if a background contributes to the fluorescence signal. Hence, we combine the ICS and photobleaching (pICS) to detect these artifacts and estimate the actual surface density. The pICS method makes it possible to have a better control of the estimation of the surface density. We noticed that the surface density of proteins increased with their initial concentration in solutions. Overall, FFM is a quantitative technique to infer the surface density of protein.