Abstract
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.
Highlights
Examining the three-dimensional structure of biomolecules is vital for understanding important biological functions
We benchmarked a linear 32-single photon avalanche diode (SPAD) array equipped with time-correlated single photon counting (TCSPC) readout electronics developed by POLIMI [35], using the same pulsed laser as before, but a simpler excitation optical train based on a cylindrical lens conjugated to the back focal plane of the microscope objective lens, in order to obtain a line illumination pattern, instead of an LCOS-SLM [17] (Fig. 1B)
Comparing the performance of the 48-spot Single-molecule Forster Resonance Energy Transfer (smFRET)-periodic acceptor excitation (PAX) microscope to a standard single-spot μsALEX microscope, we found no difference in the quality of the data but a throughput increase approximately proportional to the number of SPADs, as expected
Summary
Received Date: Revised Date: Accepted Date: 10 April 2019 24 June 2019 22 July 2019. Please cite this article as: M. Received Date: Revised Date: Accepted Date: 10 April 2019 24 June 2019 22 July 2019 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Michaleta aDepartment of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA bDepartment of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics &.
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