Dual-color Total Internal Reflection Fluorescence Fluctuation Spectroscopy

We developed a dual-color total internal reflection fluorescence (TIRF) system for single molecule imaging and fluorescence fluctuation spectroscopy (FFS). For a performance analysis, we measured a synthetic binding assay with dual-color global fluorescence correlation spectroscopy (2CG-FCS) and dual-color fluorescence intensity distribution analysis (2D-FIDA). We show that dual-color TIR-FFS is a suitable method for measuring coincidence assays at the surface. In particular, our setup has a very high fluorescence collection efficiency resulting in a two- to three-fold increased molecular brightness. Using a customized workbench setup, we improved the setup alignment, the sample handling and the system versatility.