Lossless photon recording of two-color fluorescence correlation spectroscopy for protein dynamics investigations from nanoseconds to milliseconds

Nanosecond-resolved fluorescence correlation spectroscopy (ns-FCS) based on two-color fluorescence detection is a powerful strategy for the investigation of fast dynamics of biological macromolecules labeled with donor and acceptor fluorophores. The standard methods of ns-FCS utilize two single-photon avalanche diodes (SPADs) for the detection of single-color signals (four SPADs for two-color signals) to eliminate the afterpulse artifacts of SPAD at the expense of the efficiency of counting fluorescence photons. In this study, we demonstrated that hybrid photodetectors (HPDs) enable the lossless recording of all the fluorescence photons in ns-FCS based on the minimal system using only two HPDs for the detection of two-color signals. Unexpectedly, HPD was found to show afterpulses at the yield (<10-4) much lower than that of SPADs (~10-2), which could still hamper the correlation measurements. We demonstrated that the simple subtraction procedure could cancel the afterpulse artifacts clearly. The developed system showed the better signal to noise ratio compared to the conventional method for the correlation measurements in the time domain longer than a few nanoseconds. The fast chain dynamics of the B domain of protein A in the unfolded state was observed by the new method.