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FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
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Single-molecule FRET experiments with a red-enhanced custom technology SPAD.

Francesco Panzeri1, Antonino Ingargiola2, Ron R Lin2

  • 1Dipartimento di Elettronica e Informazione, Politecnico di Milano, 20133 Milan, Italy.

Proceedings of Spie--The International Society for Optical Engineering
|December 28, 2013
PubMed
Summary

Red-enhanced Single-Photon Avalanche Diodes (RE-SPADs) show great potential for single-molecule fluorescence resonant energy transfer (smFRET) studies. These new detectors offer excellent sensitivity and timing for analyzing freely diffusing molecules.

Keywords:
ALEXFRETSPADTCSPCconfocaldiffusionlifetimesingle-molecule

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Molecular analysis

Background:

  • Single-molecule fluorescence spectroscopy is vital for studying individual molecular properties.
  • Single-Photon Avalanche Diodes (SPADs) are standard detectors for these applications.
  • Red-enhanced SPADs (RE-SPADs) offer improved sensitivity and timing performance.

Purpose of the Study:

  • Characterize the performance of RE-SPADs for single-molecule fluorescence resonant energy transfer (smFRET).
  • Evaluate RE-SPADs in a confocal geometry using alternating laser excitation (ALEX).
  • Assess suitability for analyzing freely diffusing molecules.

Main Methods:

  • Utilized doubly-labeled DNA molecules with varying donor-to-acceptor distances.
  • Performed intensity-based (μs-ALEX) and lifetime-based (ns-ALEX) measurements.
  • Compared RE-SPAD performance against standard thick SPADs.

Main Results:

  • RE-SPADs demonstrate high sensitivity across the visible spectrum.
  • Excellent timing resolution was observed with the new detectors.
  • Characterization confirmed RE-SPADs' suitability for smFRET studies.

Conclusions:

  • RE-SPADs are highly promising for smFRET applications.
  • The new detectors offer significant advantages over standard SPADs.
  • This technology has broad potential for advanced molecular studies.