"During the last few years, single-molecule fluorescence (SMF) methodologies have revolutionized the accessible information on biomolecular complexes, molecular dynamics, and biological mechanisms. Novel and important structural and dynamic insights can be obtained by studying molecules one by one, particularly into biological processes. In this field, the development of new methodologies and tools, on one hand, and of new improved fluorophores, on the other, are two major lines of research. In the current proposal, a cutting edge SMF methodology will be implemented: SMF Pulsed Interleaved Excitation (PIE) with two-colour coincidence detection, a technique that allows alternating excitation of fluorophores of different colours acting as donor and acceptor in fluorescence resonance energy transfer (FRET) processes. SMF-PIE has several advantages over the conventional SMF schemes to study biological systems, such as accurate determination of FRET efficiencies and inter-fluorophore distances, effective removal of the spectral, or distinction between molecules that have a low FRET efficiency from those that lack acceptor tag. The instrument performance will be calibrated and validated using model samples. Subsequently, SMF-PIE will be applied in three multidisciplinary biophysical approaches: 1) testing the feasibility and performance of newly synthesised fluorescein derivatives for SMF studies; 2) the resonance energy transfer between the new dyes acting as FRET donors and a DNA intercalator fluorophore as acceptor will provide insights into the mechanisms of intercalation of these carcinogenic molecules; 3) SMF-PIE will be employed to study the early pre-fibrillar aggregates from an amyloidogenic protein, the SH3 domain of α-spectrin, providing insights into the amyloid fibril formation mechanisms, a major biophysical and biomedical challenge nowadays."