Simple and Efficient Detection Scheme of Two-Color Fluorescence Correlation Spectroscopy for Protein Dynamics Investigation from Nanoseconds to Milliseconds.
ACS Phys Chem Au
; 4(1): 85-93, 2024 Jan 24.
Article
en En
| MEDLINE
| ID: mdl-38283787
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 (â¼10-2), 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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
ACS Phys Chem Au
Año:
2024
Tipo del documento:
Article
País de afiliación:
Japón