On the Resolution Capabilities and Limits of Fluorescence Lifetime Correlation Spectroscopy (FLCS) Measurements

Quantitative tests were performed in order to explore the practical limits of FLCS. We demonstrate that: a) FLCS yields precise and correct concentration values from as low as picomolar to micromolar concentrations; b) it is possible to separate four signal components in a single detector setup; c) diffusion times differing only 25% from each other can be resolved by separating a two component mixture based on the different fluorescence lifetimes of both components; d) most of the inherent technical limitations of conventional FCS are easily overcome by FLCS employing a single detector channel confocal detection scheme.