Quantitative FCS: Determination of the Confocal Volume by FCS and Bead Scanning with the MicroTime 200

Abstract

Fluorescence Correlation Spectroscopy (FCS) is used to determine concentrations and diffusion constants in the picoto nanomolar concentration region with broad applications in Biology and Chemistry. However, the method is correlated to a broad range of measurement parameters and other factors as background contributions which make quantitative results very often difficult to obtain. Quantitative results rely on the size of the confocal volume which has to be determined experimentally. The confocal volume is difficult to measure in situ and is sensitive to saturation and bleaching of the dye molecules, optical aberrations and variations of the index of refraction as observed in biological specimen.