Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements

Dual-color cross-correlation spectroscopy allows the detection and quantification of labeled biomolecules at ultra-low concentrations, whereby the sensitivity of the assay correlates with the measurement time. We now describe a parallel multifocal dual-color spectroscopic configuration employing multiple avalanche photodiodes and hardware correlators. Cross-correlation curves are obtained from several dual-color excitation foci simultaneously. Multifocal dual-color excitation is achieved by splitting each of two laser beams (488 and 633 nm) into four sub- beams with the help of two 2×2 fan-out diffractive optical elements (DOEs), and subsequent superposition of the two sets of four foci. The fluorescence emission from double- labeled biomolecules is detected by two 2×2 fiber arrays.