Single-molecule fluorescence resonant energy transfer (smFRET) allows identifying sub-populations within doubly-labeled molecules, based on the distances between the donor (D) and acceptor (A) fluorescent labels. 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 techniques is their low throughput, which renders measurements time-consuming and prevents from studying kinetic phenomena in real-time.
Here we demonstrate a high-throughput smFRET setup which multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode (SPAD) arrays. Using two excitation lasers, one of which is alternated on the 10 us time scale, allows identifying and sorting species with one or two active fluorophores, extending the range of measurable FRET efficiencies and enabling proper fluorescence-aided molecular sorting. The performance of the system is demonstrated with a set of doubly-labeled double-stranded DNA oligonucleotides with different D-A distances. 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.
Antonino Ingargiola, Maya Segal, Angelo Gulinatti, Ivan Rech, Ivan Labanca, Piera Maccagnani, Massimo Ghioni, Shimon Weiss, and Xavier Michalet, "A 48-spot single-molecule FRET setup with periodic acceptor excitation (Conference Presentation)," Proc. SPIE 10500, Single Molecule Spectroscopy and Superresolution Imaging XI, 1050005 (Presented at SPIE BiOS: January 27, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2289127.5751629913001.
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