Time correlated single photon counting system

UV-visible spectra were recorded using a Cary 300 spectrophotometer (Agilent). Extinction coefficients were determined using protein solutions with absorbance values < 0.2 (at l_max^Abs). Fluorescence emission and excitation spectra were measured using a Jobin-Yvon Fluoromax-4 spectrofluorometer (Horiba) with a 2 nm excitation/emission slit-width. Quantum yields were determined by the comparative gradient method using fluorescein (in 0.1 M NaOH) as a standard (Φ_F = 0.91) and excitation wavelength (l_exc) at 470 nm. For fluorescence lifetime measurements, the spectrofluorometer was coupled to a time-correlated single photon counting system (Horiba Jobin-Yvon) equipped with a 455 nm NanoLED operating at 1 MHz. Each measurement was terminated when a maximum peak preset of 20,000 photon counts was reached for the fluorescence monitored at 505 nm. The instrument response function was determined using a light scattering solution. Analysis of fluorescence decay profiles was performed using Horiba DAS6 software.