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Proceedings Paper

8-spot smFRET analysis using two 8-pixel SPAD arrays
Author(s): Antonino Ingargiola; Francesco Panzeri; Niusha Sarkhosh; Angelo Gulinatti; Ivan Rech; Massimo Ghioni; Shimon Weiss; Xavier Michalet
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Paper Abstract

Single-molecule Förster resonance energy transfer (smFRET) techniques are now widely used to address outstanding problems in biology and biophysics. In order to study freely diffusing molecules, current approaches consist in exciting a low concentration (<100 pM) sample with a single confocal spot using one or more lasers and detecting the induced single-molecule fluorescence in one or more spectrally- and/or polarization-distinct channels using single-pixel Single-Photon Avalanche Diodes (SPADs). A large enough number of single-molecule bursts must be accumulated in order to compute FRET efficiencies with sufficient statistics. As a result, the minimum timescale of observable phenomena is set by the minimum acquisition time needed for accurate measurements, typically a few minutes or more, limiting this approach mostly to equilibrium studies. Increasing smFRET analysis throughput would allow studying dynamics with shorter timescales. We recently demonstrated a new multi-spot excitation approach, employing a novel multi-pixel SPAD array, using a simplified dual-view setup in which a single 8-pixel SPAD array was used to collect FRET data from 4 independent spots. In this work we extend our results to 8 spots and use two 8-SPAD arrays to collect donor and acceptor photons and demonstrate the capabilities of this system by studying a series of doubly labeled dsDNA samples with different donor-acceptor distances ranging from low to high FRET efficiencies. Our results show that it is possible to enhance the throughput of smFRET measurements in solution by almost one order of magnitude, opening the way for studies of single-molecule dynamics with fast timescale once larger SPAD arrays become available.

Paper Details

Date Published: 22 February 2013
PDF: 11 pages
Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 85900E (22 February 2013); doi: 10.1117/12.2003704
Show Author Affiliations
Antonino Ingargiola, Univ. of California, Los Angeles (United States)
Francesco Panzeri, Politecnico di Milano (Italy)
Niusha Sarkhosh, Univ. of California, Los Angeles (United States)
Angelo Gulinatti, Politecnico di Milano (Italy)
Ivan Rech, Politecnico di Milano (Italy)
Massimo Ghioni, Politecnico di Milano (Italy)
Shimon Weiss, Univ. of California, Los Angeles (United States)
Xavier Michalet, Univ. of California, Los Angeles (United States)


Published in SPIE Proceedings Vol. 8590:
Single Molecule Spectroscopy and Superresolution Imaging VI
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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