A Quantitative and Reliable Calibration Standard for Dual-Color Fluorescence Cross-Correlation Spectroscopy

Dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS) allows binding analysis of biomolecules. Combining cross- and autocorrelation amplitudes yields binding degrees and concentrations of bound and unbound species. However, non-ideal detection volume overlap reduces the cross-correlation, causing overestimation of the K_d . The overlap quality factor that relates measured and true cross-correlation amplitudes has been difficult to determine, because neither a perfect 1 : 1 labeled sample nor perfectly overlapping volumes are readily accomplished. Here, we describe how a stochastically labeled sample can be used for quantitative calibration. Lipid vesicles doped with green and red fluorescent dyes yield highly reproducible relative cross-correlations and allow determination of the setup-dependent overlap quality factor. This reliable, affordable and quick-to-prepare calibration standard expedites any quantitative co-localization or binding analysis by dcFCCS.