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https://doi.org/10.1117/12.200...
Article . 2013 . Peer-reviewed
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8-spot smFRET analysis using two 8-pixel SPAD arrays

Authors: INGARGIOLA, ANTONINO; PANZERI, FRANCESCO; N. Sarkhosh; GULINATTI, ANGELO; RECH, IVAN; GHIONI, MASSIMO ANTONIO; S. Weiss; +1 Authors

8-spot smFRET analysis using two 8-pixel SPAD arrays

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.

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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
18
Average
Average
Top 10%