Extended Data Fig. 9: Increasing scaling of PRE-smFRET to 9 independent samples within one FOV.

(a) Bright field image of arrays printed with contactless array printer. Each microdroplet group contains 9 printed spots, each containing an anchor oligonucleotide with an orthogonal binding domain. (b) Schematic of one microdroplet group as imaged in one FOV using a microscope with a 60x objective lens, projected onto Teledyne Kinetix cameras. (c) Single- molecule fluorescence image of 9 FRET-labelled DNA duplexes, each with a distinct single-stranded tether domain and associated FRET signal, obtained with our pTIRF imaging platform. Image false colored by FRET efficiency. (d) FRET efficiency histogram of pTIRF images as in (c), colored by location as shown in (b). Solid lines show individual Gaussian fits to histograms extracted from each printed spot. (e) FRET efficiency, from the center of Gaussian distributions in (d), against dye distance, estimated from molecular dynamics simulations. Red solid line shows fit to Extended Data Equation 1 resulting in measured Förster distance, R₀, of 59.8 ± 1 Å, in agreement with the reported R₀ of 60.7 Å for LD555-LD655 on DNA.