Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy

¹  Institute of Physical Chemistry, RWTH Aachen University - 52056 Aachen, Germany, EU
²  Institute of Physical and Theoretical Chemistry, Eberhard Karls University - 72076 Tübingen, Germany, EU
³  Institute for Neurobiology and Biophysics 2, Forschungszentrum Jülich - 52425 Jülich, Germany, EU
⁴  PicoQuant GmbH - 12961 Berlin, Germany, EU

Corresponding author: joerg.enderlein@uni-tuebingen.de

Received: 14 April 2008
Accepted: 25 June 2008

Abstract

Dual-focus fluorescence correlation spectroscopy is a method for precisely measuring the diffusion coefficient of fluorescing molecules close to the infinite dilution limit in a reference-free and absolute manner. We apply the method to determine the diffusion coefficients of three fluorescent dyes across the visible spectrum. These values can be used as absolute reference standards for fluorescence correlation spectroscopy. In particular, it is found that the diffusion coefficient of the widely used reference dye Rhodamine 6G is by 37% larger than the value used in most publications on fluorescence correlation spectroscopy over the last three decades.