Benchmark of a New Red-Enhanced Custom Technology Spad Detectorfor Single-Molecule FRET Experiments

Single-molecule spectroscopy has relied on detectors that offer a variety of features enabling single molecule sensitivity. Single-Photon Avalanche Diodes (SPADs) are prominent among detectors used to sample fluorescentlabeled species and identify their physical characteristics, diffusion properties and elucidate molecular interactions. Existing brands of commercial SPADs suffer from either low quantum efficiency in one part of the spectrum, poor timing resolution or lack of temporal stability and/or heterogeneous instrument response functions (IRFs). Finally, some SPADs can fail after exposure to strong intensity signals. In this study, we characterize a new customtechnology SPAD detector addressing these drawbacks and compare its properties to those of a popular commercially available SPAD detector used in single-fluorescence microscopy experiments. We performed two-color single- molecule FRET (smFRET) experiments on doubly labeled DNA using Alternating Laser Excitation (ALEX). We compare results obtained with both types of detectors in identical conditions, using both ratiometric FRET and lifetime FRET analysis. The new detector offers excellent sensitivity in the red part of the spectrum. Lifetime FRET measurements reveal excellent timing resolution, IRF stability, enabling the study of short timescale dynamics. Our results validate this new red-enhanced SPAD detector for both intensity-based and time-resolved single-molecule FRET experiments among many other capabilities.