Fig. 1: Automated multiwell plate smFRET measurements.

Illustration of the automated single-molecule detection setup implementing a multiwell plate functionality for smFRET experiments. A motorized scanning stage holds the sealed multiwell plate and proceeds from well to well. A liquid dispenser replaces the immersion medium of the water objective and the autofocus maintains the objective focus at a fixed position in solution. Single-molecule fluorescence recordings are performed using a multiparameter confocal fluorescence microscope equipped with pulsed lasers, a high-numerical water objective, and spectrally- and polarization-sensitive single-photon detectors (for simplicity only one polarization is illustrated; see also Supplementary Information). An integrated open-source software suite (available on Github) with graphical user interfaces performs data acquisition, processing, analysis, and visualization. Data can be visualized for each well or ranges of wells (A01, A02, …, H12).