48-spot single-molecule FRET setup with periodic acceptor excitation

Abstract

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of bindingunbinding 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 is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time.Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. 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 such as DNA transcription by bacterial RNA polymerase.

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

“…C) Two patterns of 4x12 spots were generated in the sample by two high power (1 W) lasers (532 nm and 635 nm) and their associated LCOS-SLMs. The 5.4 µm distance between neighboring spots, matched, after magnification, the 500 µm distance between SPADs in the corresponding two 4x12 SPAD arrays (SPAD diameter: 50 µm) [30].…”

“…This requires careful magnification adjustments, and cumbersome alignment steps, including adjusting a rotational degree of freedom. For these reasons, we chose a more flexible (if more expensive) solution using programmable liquid crystal on silicon spatial light modulators (LCOS-SLMs) [27,28,29,30]. These devices can be used in direct space [31] or reciprocal space [32], as used in holography.…”

“…After the development of larger SPAD arrays by our POLIMI collaborators, we upgraded our multispot smFRET setup with two 12x4 SPAD arrays [36,30] (Fig. 1C).…”

“…The phase images are generated with a custom LabVIEW program that computes the phase pattern using user inputs and supports automated pattern scanning as described in Ref. [30]. The LCOS_LabVIEW repository is available on GitHub (link).…”

“…The overlap of the two excitations with respect to the optical axis is quantified by fitting the peak position and the Gaussian waist of each spot ( Figure 3C). Details of the analysis are provided in the pattern_profiling alignment notebook (link) [30].…”