High-throughput, single-molecule analysis with a multipixel SPAD array

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 allow sampling of a large number of distinct individual molecules. However, to allow separating photon bursts from individual molecules requires low concentrations. This leads to long acquisition times (minutes to hours) in order to obtain sufficient statistics. It also prevents from studying dynamic phenomena happening on time scales larger than the burst duration but smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate while keeping the same low concentrations required for individual molecule burst identification. In this work we present a realization of this idea using multispot excitation and detection. The donor excitation spots are created by a LCOS spatial light modulator while fluorescent emission of donor and acceptor dyes is collected and refocused on a custom 8-pixel SPAD array. smFRET measurements were performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores and compared with results obtained with a conventional single-spot acquisition approach. Identical results obtained with shorter acquisition time demonstrate the potential of this approach for high-throughput smFRET analysis on freely diffusing molecules.