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Fluorescence fluctuation spectroscopy is applied to study molecules, passingthrough a small observation volume, usually subjected to diffusive orconvective motion in liquid phase. We suggest that such a technique could beused to measure the areal absolute concentration of fluorophores deposited on asubstrate or imbedded in a thin film, with a resolution of a few micrometers.The principle is to translate the solid substrate in front of a confocalfluorescence microscope objective and to record the subsequent fluctuations ofthe fluorescence intensity. The validity of this concept is investigated onmodel substrates (fluorescent microspheres) and DNA biochips.

Measurement of surface concentration of fluorophores using fluorescence fluctuation spectroscopy