Single-dye calibration for accurate instrument response function in time-correlated single photon counting over the...

Accurate determination of the instrument response function (IRF) is essential for time-resolved fluorescence measurements in modern time-correlated single photon counting (TCSPC) systems, which achieve time resolutions below 20 ps. We propose malachite green as a versatile standard dye for IRF measurement. Malachite green exhibits S1 and S2 emission bands that span the entire visible and near-infrared spectrum, with lifetimes for both states less than 1 ps, while providing sufficient photon yield for measuring the IRF of a TCSPC apparatus. Additionally, its absorption bands cover most of the ultraviolet and visible wavelengths. Using malachite green as a single-dye standard, we demonstrate accurate IRF determination across all visible and near-infrared detection wavelengths. Combined with a home-built fluorometer employing a Cassegrain reflector and a fast hybrid detector, IRFs of ∼18 ps are obtained over the entire detection range, which are limited by the transit-time spread of the hybrid detector.