Determination of the confocal volume for quantitative fluorescence correlation spectroscopy

Abstract

Single molecule detection methods, in particular those based on fluorescent labels offer the possibility to gain not only qualitative but also quantitative insight into the function of complex biological systems. Fluorescence Correlation Spectroscopy (FCS) is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness in the pico- to nano-Molar concentration range, with broad applications in Biology and Chemistry. Although FCS in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts FCS to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by FCS-curve fitting, a FCS dilution series and confocal bead scanning. The results are compared in the view of quantitative FCS measurement and analysis.