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

Francesco Panzeri, Antonino Ingargiola, Ron R. Lin, Niusha Sarkhosh, Angelo Gulinatti, Ivan Rech, Massimo Ghioni, Sergio Cova, Shimon Weiss, Xavier Michalet

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review

9 Scopus citations

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.

Original languageEnglish
Title of host publicationSingle Molecule Spectroscopy and Superresolution Imaging VI
DOIs
StatePublished - 2013
Externally publishedYes
EventSingle Molecule Spectroscopy and Superresolution Imaging VI - San Francisco, CA, United States
Duration: 2 Feb 20133 Feb 2013

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume8590
ISSN (Print)1605-7422

Conference

ConferenceSingle Molecule Spectroscopy and Superresolution Imaging VI
Country/TerritoryUnited States
CitySan Francisco, CA
Period2/02/133/02/13

Keywords

  • ALEX
  • FRET
  • SPAD
  • TCSPC
  • confocal
  • diffusion
  • lifetime
  • single-molecule

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