title: Single-molecule FRET experiments with a red-enhanced custom technology SPAD.
abstract: Single-molecule fluorescence spectroscopy of freely diffusing molecules in solution is a powerful tool used to investigate the properties of individual molecules. Single-Photon Avalanche Diodes (SPADs) are the detectors of choice for these applications. Recently a new type of SPAD detector was introduced, dubbed red-enhanced SPAD (RE-SPAD), with good sensitivity throughout the visible spectrum and with excellent timing performance. We report a characterization of this new detector for single-molecule fluorescence resonant energy transfer (smFRET) studies on freely diffusing molecules in a confocal geometry and alternating laser excitation (ALEX) scheme. We use a series of doubly-labeled DNA molecules with donor-to-acceptor distances covering the whole range of useful FRET values. Both intensity-based (μs-ALEX) and lifetime-based (ns-ALEX) measurements are presented and compared to identical measurements performed with standard thick SPADs. Our results demonstrate the great potential of this new detector for smFRET measurements and beyond.
authors.affiliation: Dipartimento di Elettronica e Informazione, Politecnico di Milano, 20133 Milan, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Panzeri
authors.affiliation: Department of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA.
authors.forename: Antonino
authors.initials: A
authors.lastname: Ingargiola
authors.affiliation: Department of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA.
authors.forename: Ron R
authors.initials: RR
authors.lastname: Lin
authors.affiliation: Department of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA.
authors.forename: Niusha
authors.initials: N
authors.lastname: Sarkhosh
authors.affiliation: Dipartimento di Elettronica e Informazione, Politecnico di Milano, 20133 Milan, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Gulinatti
authors.affiliation: Dipartimento di Elettronica e Informazione, Politecnico di Milano, 20133 Milan, Italy.
authors.forename: Ivan
authors.initials: I
authors.lastname: Rech
authors.affiliation: Dipartimento di Elettronica e Informazione, Politecnico di Milano, 20133 Milan, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Ghioni
authors.affiliation: Dipartimento di Elettronica e Informazione, Politecnico di Milano, 20133 Milan, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Cova
authors.affiliation: Department of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA.
authors.forename: Shimon
authors.initials: S
authors.lastname: Weiss
authors.affiliation: Department of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA.
authors.forename: Xavier
authors.initials: X
authors.lastname: Michalet
keywords: ALEX; FRET; SPAD; TCSPC; confocal; diffusion; lifetime; single-molecule