High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Maya Segal, Antonino Ingargiola, Eitan Lerner, Sang Yoon Chung, Jonathan A. White, Aaron Streets, S. Weiss, X. Michalet

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used o increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.

Original languageEnglish
Pages (from-to)21-45
Number of pages25
StatePublished - 1 Oct 2019
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2019 Elsevier Inc.


This work was supported in part by NIH grants R01 GM095904, R01 GM069709, R01GM130942, by NSF awards MCB 1244175, MCB 1818147, EAGER 1842951, and by a seed grant from the UCLA Jonsson Comprehensive Cancer Center. S. Weiss discloses intellectual property used in the research reported here.

FundersFunder number
National Science Foundation1842951, MCB 1818147, MCB 1244175
National Institutes of HealthR01 GM069709, R01GM130942
National Institute of General Medical SciencesR01GM095904
Jonsson Comprehensive Cancer Center


    • Freely-diffusing
    • High-throughput
    • SPAD array
    • Single-molecule FRET


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