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.

Single-molecule FRET experiments with a red-enhanced custom technology SPAD

PANZERI, FRANCESCO;INGARGIOLA, ANTONINO;GULINATTI, ANGELO;RECH, IVAN;GHIONI, MASSIMO ANTONIO;COVA, SERGIO;
2013

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.
Single Molecule Spectroscopy and Superresolution Imaging VI
9780819493590
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11311/823948
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