Measurement of surface concentration of fluorophores by fluorescence fluctuation spectroscopy

Fluorescence fluctuation spectroscopy is applied to study molecules passing through a small observation volume, usually subjected to diffusive or convective motion in a liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate or embedded in a thin film, with a resolution of a few micrometers. The principle is to translate the solid substrate in front of a confocal fluorescence microscope objective and to record the subsequent fluctuations of the fluorescence intensity. The validity of this concept is investigated on model substrates (fluorescent microspheres) and DNA biochips. (c) 2006 Optical Society of America.