22 February 2013 Single-molecule FRET experiments with a red-enhanced custom technology SPAD
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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.
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Francesco Panzeri, Francesco Panzeri, Antonino Ingargiola, Antonino Ingargiola, Ron R. Lin, Ron R. Lin, Niusha Sarkhosh, Niusha Sarkhosh, Angelo Gulinatti, Angelo Gulinatti, Ivan Rech, Ivan Rech, Massimo Ghioni, Massimo Ghioni, Sergio Cova, Sergio Cova, Shimon Weiss, Shimon Weiss, Xavier Michalet, Xavier Michalet, } "Single-molecule FRET experiments with a red-enhanced custom technology SPAD", Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 85900D (22 February 2013); doi: 10.1117/12.2003187; https://doi.org/10.1117/12.2003187
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