MFD-PIE and PIE-FI: Ways to Extract More Information with TCSPC

Abstract

Pulsed interleaved excitation (PIE) is the method of fast alternation of pulsed lasers for quasi-simultaneous observation of fluorophores with different spectral properties. PIE was originally introduced to enable artifact-free fluorescence cross-correlation measurements, while first experiments with alternating laser excitation (ALEX) used the dual excitation of donor and acceptor for single-pair Förster resonance energy transfer (spFRET). In this article, we will review the benefit of PIE for spFRET experiments with multiparameter fluorescence detection (MFD). The direct probing of the acceptor fluorophore in PIE increases the robustness of the quantitative MFD analysis and extends it to even more parameters.