A 16 Channel Spad Array for High-Throughput Tcspc Measurements of Single-Molecule FRET of Freely Diffusing Molecules

Abstract

Single-molecule techniques probe the spectroscopic properties of biomolecules one molecule a time. These approaches allow detecting and quantifying subpopulations which would be masked in ensemble measurements. Single-molecule FRET (smFRET), which indirectly measures the distance between fluorophores, has been extensively used to characterize the conformation of biomolecules or molecular interactions in complex biomolecular assemblies among many applications. FRET efficiency is typically computed ratiometrically from photon counts detected in two spectral channels. However, this approach requires careful correction and calibrations to account for background and differences in quantum yield and detection efficiency between the two channels. Alternatively, time-correlated single-photon counting (TCSPC) measurements can provide the same result from the donor fluorescence lifetime only, which is much less sensitive to these experimental aspects.In both approaches, a fundamental limitation remains the long acquisition time needed to accumulate sufficient statistics from freely-diffusing molecules, severely limiting the throughput of these techniques and preventing the study of rapidly evolving systems. To circumvent these limitations, we have developed a multi-spot setup using sensitive SPAD arrays, achieving high-throughput data acquisition for FCS and smFRET. These systems had so far been limited to counting applications.In this work, we present the first proof-of-principle TCSPC multispot system employing a simple line-excitation, a new dedicated SPAD array with low jitter and integrated multichannel TCSPC electronics[1]. This system allows single-molecule detection on all 16 channels using a total excitation power of 100 mW. We report measurements on short doubly-labeled dsDNA molecules and demonstrate uniform performance across the 16 detection channels. These results are a first step toward extending current single-spot TCSPC spectroscopic techniques (ns-ALEX or PIE) to a multispot geometry for high-throughput time-resolved smFRET measurements.[1] Cuccato, A et al. IEEE Phot. J. 5-5, p6801514, 2013.