8-spot smFRET analysis using two 8-pixel SPAD arrays

Abstract

Single-molecule Förster resonance energy transfer (smFRET) techniques are now widely used to address outstanding problems in biology and biophysics. In order to study freely diffusing molecules, current approaches consist in exciting a low concentration (<100 pM) sample with a single confocal spot using one or more lasers and detecting the induced single-molecule fluorescence in one or more spectrally- and/or polarization-distinct channels using single-pixel Single-Photon Avalanche Diodes (SPADs). A large enough number of single-molecule bursts must be accumulated in order to compute FRET efficiencies with sufficient statistics. As a result, the minimum timescale of observable phenomena is set by the minimum acquisition time needed for accurate measurements, typically a few minutes or more, limiting this approach mostly to equilibrium studies. Increasing smFRET analysis throughput would allow studying dynamics with shorter timescales. We recently demonstrated a new multi-spot excitation approach, employing a novel multi-pixel SPAD array, using a simplified dual-view setup in which a single 8-pixel SPAD array was used to collect FRET data from 4 independent spots. In this work we extend our results to 8 spots and use two 8-SPAD arrays to collect donor and acceptor photons and demonstrate the capabilities of this system by studying a series of doubly labeled dsDNA samples with different donor-acceptor distances ranging from low to high FRET efficiencies. Our results show that it is possible to enhance the throughput of smFRET measurements in solution by almost one order of magnitude, opening the way for studies of single-molecule dynamics with fast timescale once larger SPAD arrays become available.

Keywords: FRET; SPAD; SPAD arrays; high-throughput; multi-spot; photon-counting; single molecule; smFRET.

Figure1

Schematic representation of the multispot setup. See Section 2 for a detailed description of the system.

Figure2

FRET histograms for the 7 bp sample. Each plot corresponds to one channel of the multispot system. A gaussian fit (grey line) of the FRET population and the fitted peak position (red dashed lines) are shown with the estimated FRET efficiency for each channel (text box).

Figure 3

FRET histograms for the 27 bp sample. Each plot corresponds to one channel of the multispot system. A gaussian fit (grey line) of the FRET population and the fitted peak position (red dashed lines) are shown with the estimated FRET efficiency for each channel (text box). Histograms for the donor-only sample are shown in light gray for comparison.

Figure 4

FRET peak position as a function of the base-pair separation between donor and acceptor dyes for the 5 dsDNA samples. Each “+” symbol represents the estimated FRET population peak position for each of the 8 channels of the multispot setup. The maximum difference between channels is reported as “Δ” for each sample. The red line reports the FRET population peak position as measured with the reference single-spot μs-ALEX setup. The theoretical dependence of FRET versus distance (grey lines) assuming a constant Förster radius R₀ and a donor-acceptor distance computed according to a cylindrical dsDNA model. Two values for R₀ (6.5 nm and 5 nm) were used. See text for details (section 3.2).

Figure 5

FRET histograms computed from 10 seconds of data acquired by and pooled from 8-channels, reported for all 5 dsDNA samples, illustrating the high-throughput performance of the multispot setup.

Figure 6

(right) FRET peak position estimation for the 5 samples as a function of measurement time (black lines). The light-blue regions represent ±3σ confidence limits of the peak position. (left) Gaussian fit and peak position (red dashed lines) of the FRET peaks after 60 seconds of measurements.