We have developed an instrument for spectral cross-talk-free dual-color fluorescence cross-correlation spectroscopy (FCCS), which provides a readout modality for the study of enzyme activity in application areas such as high-throughput screening. Two spectrally distinct (approx250 nm) fluorophores, Cy3 and IRD800, were excited simultaneously using two different excitation sources: one poised at 532 nm and the other at 780 nm. The fluorescence information was processed on two different color channels monitored with single-photon avalanche diodes (SPADs) that could transduce events at the single-molecule level. The system provided no color cross-talk (cross-excitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET), significantly improving data quality. To provide evidence of cross-talk-free operation, the system was evaluated using bright microspheres ((lambda)_(abs) velence 532 nm, (lambda)_(em) velence 560 nm) and quantum dots ((lambda)_(abs) velence 532 nm, (lambda)_(em) velence 810 nm). Experimental results indicated that no color leakage from the microspheres or quantum dots into inappropriate color channels was observed. To demonstrate the utility of the system, the enzymatic activity of APE1, which is responsible for nicking the phosphodiester backbone in DNA on the 5' side of an apurinic/apyrimidinic site, was monitored by FCCS using a double-stranded DNA substrate dual labeled with Cy3 and IRD800. Activity of APE1 was also monitored in the presence of an inhibitor (7-nitroindole-2-carboxylic acid) of the enzyme using this cross-talk-free FCCS platform. In all cases, no spectral leakage from single-molecule events into inappropriate color channels was observed.
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