Simple and Efficient Detection Scheme of Two-Color Fluorescence Correlation Spectroscopy for Protein Dynamics Investigation from Nanoseconds to Milliseconds

Yutaka Sano; Yuji Itoh; Supawich Kamonprasertsuk; Leo Suzuki; Atsuhito Fukasawa; Hiroyuki Oikawa; Satoshi Takahashi

doi:10.1021/acsphyschemau.3c00040

Simple and Efficient Detection Scheme of Two-Color Fluorescence Correlation Spectroscopy for Protein Dynamics Investigation from Nanoseconds to Milliseconds

ACS Phys Chem Au. 2023 Nov 10;4(1):85-93. doi: 10.1021/acsphyschemau.3c00040. eCollection 2024 Jan 24.

Authors

Yutaka Sano^{1

2}, Yuji Itoh^{1

2

3}, Supawich Kamonprasertsuk^{1

2}, Leo Suzuki^{1

3}, Atsuhito Fukasawa⁴, Hiroyuki Oikawa^{1

2

3

5}, Satoshi Takahashi^{1

2

3}

Affiliations

¹ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan.
² Department of Chemistry, Graduate School of Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
³ Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8577, Japan.
⁴ Electron Tube Division, Hamamatsu Photonics K. K., Iwata, Shizuoka 438-0193, Japan.
⁵ MOLCURE, Kawasaki, Kanagawa 212-0032, Japan.

Abstract

Nanosecond resolved fluorescence correlation spectroscopy (ns-FCS) based on two-color fluorescence detection is a powerful strategy for investigating the fast dynamics of biological macromolecules labeled with donor and acceptor fluorophores. The standard methods of ns-FCS use 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 utilizing photon data in the calculation of correlograms. Herein, we demonstrated that hybrid photodetectors (HPDs) enable the recording of fluorescence photons in ns-FCS based on the minimal system using two HPDs for the detection of two-color signals. However, HPD exhibited afterpulses at a yield with respect to the rate of photodetection (<10^-4) much lower than that of SPADs (∼10^-2), which could still hamper correlation measurements. We demonstrated that the simple subtraction procedure could eliminate afterpulse artifacts. While the quantum efficiency of photodetection for HPDs is lower than that for high-performance SPADs, the developed system can be practically used for two-color ns-FCS in a time domain longer than a few nanoseconds. The fast chain dynamics of the B domain of protein A in the unfolded state was observed using the new method.