Measurement of surface concentration of fluorophores using fluorescence fluctuation spectroscopy

TL;DR

This paper explores using fluorescence fluctuation spectroscopy to measure the surface concentration of fluorophores on substrates with micrometer resolution by analyzing fluorescence intensity fluctuations during substrate translation.

Contribution

It introduces a novel application of fluorescence fluctuation spectroscopy for absolute surface concentration measurement of fluorophores on solid substrates.

Findings

Validated method on fluorescent microspheres

Applied technique to DNA biochips

Achieved micrometer-scale resolution

Abstract

Fluorescence fluctuation spectroscopy is applied to study molecules, passing through a small observation volume, usually subjected to diffusive or convective motion in liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate 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 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.