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Proceedings Paper

Parallel multispot smFRET analysis using an 8-pixel SPAD array
Author(s): A. Ingargiola; R. A. Colyer; D. Kim; F. Panzeri; R. Lin; A. Gulinatti; I. Rech; M. Ghioni; S. Weiss; X. Michalet
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Paper Abstract

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

Paper Details

Date Published: 13 February 2012
PDF: 8 pages
Proc. SPIE 8228, Single Molecule Spectroscopy and Superresolution Imaging V, 82280B (13 February 2012); doi: 10.1117/12.909470
Show Author Affiliations
A. Ingargiola, Politecnico di Milano (Italy)
Univ. of California, Los Angeles (United States)
R. A. Colyer, Univ. of California, Los Angeles (United States)
D. Kim, Nesher Technologies, Inc. (United States)
F. Panzeri, Politecnico di Milano (Italy)
R. Lin, Univ. of California, Los Angeles (United States)
A. Gulinatti, Politecnico di Milano (Italy)
I. Rech, Politecnico di Milano (Italy)
M. Ghioni, Politecnico di Milano (Italy)
S. Weiss, Univ. of California, Los Angeles (United States)
X. Michalet, Univ. of California, Los Angeles (United States)

Published in SPIE Proceedings Vol. 8228:
Single Molecule Spectroscopy and Superresolution Imaging V
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling; Ingo Gregor, Editor(s)

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