Rxivist logo

48-spot single-molecule FRET setup with periodic acceptor excitation

By Antonino Ingargiola, Maya Segal, A. Gulinatti, I. Rech, Ivan Labanca, Piera Maccagnani, M. Ghioni, Shimon Weiss, Xavier Michalet

Posted 26 Jun 2017
bioRxiv DOI: 10.1101/156182 (published DOI: 10.1063/1.5000742)

Single-molecule FRET (smFRET) allows measuring distances between donor (D) and acceptor (A) fluorophores on the 3-10~nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between D and A dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Download data

  • Downloaded 700 times
  • Download rankings, all-time:
    • Site-wide: 21,241 out of 94,912
    • In biophysics: 756 out of 4,144
  • Year to date:
    • Site-wide: 54,600 out of 94,912
  • Since beginning of last month:
    • Site-wide: 31,977 out of 94,912

Altmetric data

Downloads over time

Distribution of downloads per paper, site-wide


Sign up for the Rxivist weekly newsletter! (Click here for more details.)