Parallel multispot smFRET analysis using an 8-pixel SPAD array

Abstract

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

Keywords: FRET; SPAD array; high throughput; photon counting; single-molecule.

Figure 1

Schematic representation of the multispot smFRET setup. A linear pattern of 4 excitation spots is generated at an intermediate focal plane using the LCOS. The excitation pattern is projected into the sample via the objective lens (Obj.) through a coverslip (CS). The resulting sample fluorescence is fed into the dual-view input and split into two separate images for the donor and acceptor spectral components. The SPADA acquires the 8 emission spot signals (4 for donor and 4 for acceptor) and provides 8 TTL outputs to the downstream acquisition board.

Figure 2

Burst sizes of a dsDNA sample labeled with donor and acceptor dyes 5 base pairs apart. Scatter plots report the acceptor burst sizes versus the corresponding donor burst sizes for the 4 measurement spots. For each spot, the burst population is split in donor-only bursts (green dots) and in the “high FRET” population (red dots). The data is corrected for background and leakage of donor emission in the acceptor channel.

Figure 3

FRET histograms for the dsDNA samples with distances between dyes of 3, 5, 7 and 9 base pairs. Within each sample quadrant, each histogram refers to a different excitation spot. The red arrows indicate the position (E) of the peak corresponding to the FRET population. For comparison, on each plot, the FRET efficiency of the other samples is reported as vertical dashed lines.