Abstract
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 characteristics consistent with 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.
Highlights
1.1 BackgroundFreely-diffusing single-molecule FRET studies have yielded a wealth of new scientific results since their proof-of-principle demonstration almost two decades ago [1, 2]
These results demonstrate that burst analysis following the proper burst search can provide information on point-spread functions (PSFs) size and peak intensity consistent with information obtained by fluorescence correlation spectroscopy (FCS) analysis
We have presented a detailed description of an 8-spot confocal setup and illustrated its use for single-molecule FRET (smFRET) studies with the following examples
Summary
Freely-diffusing single-molecule FRET (smFRET) studies have yielded a wealth of new scientific results since their proof-of-principle demonstration almost two decades ago [1, 2]. In order to ensure the separate detection of fast diffusing individual molecules, low concentrations, small excitation volumes and high excitation intensities are necessary. This is generally obtained with a so-called confocal geometry, where a laser is focused into a single diffraction-limited volume of the sample and a single-pixel detector collects light only from this volume. More complex approach for increased throughput involves extending the single-spot confocal geometry to a multispot confocal geometry. This requires parallel photon-counting capabilities, which have only become available recently with the development of single-photon avalanche diode (SPAD) arrays
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