16-Ch time-resolved single-molecule spectroscopy using line excitation

Abstract

Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times. We report a time-resolved multispot system employing a 16-channel SPAD array and TCSPC electronics, which overcomes the throughput issue. Excitation is obtained by shaping a 532 nm pulsed laser into a line, matching the linear SPAD array geometry. We show that the line-excitation is a robust and cost-effective approach to implement multispot systems based on linear detector arrays.

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

“…Alternatively, it is possible to use a line or sheet illumination pattern ( Fig. 1B) and rely on out-of-focus light rejection by the geometry of the detector array itself, as we demonstrated with a linear array [17] and others have demonstrated with a 2D array [34] (although the latter demonstration was not a single-molecule experiment, the concept is applicable to smFRET). The drawback of these approaches, beside the increased background signal and inefficient excitation power distribution 3 The 532 nm laser used in this setup was a high power (1 W) ps-pulsed laser (68 MHz).…”

“…The detector modules include integrated active quenching circuits (iAQCs) designed to rapidly reset the SPADs in which an avalanche has been created upon absorption of an incoming photon. The modules are also equipped with timing electronics enabling single-photon counting, and, in some cases, with time-correlated single-photon counting electronics, enabling single-photon timing with ≈ 50 ps timing resolution [17]. It is worth noting that alternative SPAD array designs using standard CMOS fabrication technology have also been developed during the past two decades [18].…”

“…We next benchmarked a linear 32-SPAD array equipped with TCSPC readout electronics developed by POLIMI [35], using the same pulsed laser as before, but a simpler excitation optical train based on a cylindrical lens conjugated to the back focal plane of the microscope objective 4 lens, in order to obtain a line illumination pattern, instead of an LCOS SLM [17] (Fig. 1B).…”

“…A linear SPAD array geometry, combined with a linear illumination pattern such as demonstrated in ref. [17] would significantly speed up data acquisition in this type of fast kinetics experiment, as well as offer the exciting possibility of tracking the evolution of individual molecules along their reaction path. , which allow precise injection of volumes (pL) of sample within a mixing region (ellipse).…”

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

“…Compared to our previous works 16,48 , a second alternating excitation laser was incorporated, and the corresponding alignment hurdles were solved, permitting sorting of single-molecules according to their D-A stoichiometry. In particular, we have shown that the setup allows identifying singly and doubly-labeled species over the full range of FRET efficiencies, opening the door to a much wider range of assays than was previously possible.…”