RT Journal Article SR Electronic T1 Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules JF bioRxiv FD Cold Spring Harbor Laboratory SP 085027 DO 10.1101/085027 A1 A. Ingargiola A1 E. Lerner A1 S. Chung A1 F. Panzeri A1 A. Gulinatti A1 I. Rech A1 M. Ghioni A1 S. Weiss A1 X. Michalet YR 2016 UL http://biorxiv.org/content/early/2016/11/04/085027.abstract AB We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristic identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription.Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.