Fig. 2: ABEL-trap-based detection of FRETfluors in a complex sample.
a, Schematic of ABEL trap detection: FRETfluors (blue DNA; coloured stars) are detected in a microfluidic cell atop an inverted microscope. Here 532 nm pulsed laser excitation is scanned across the field of view using x and y acousto-optic deflectors (AODs). A FRETfluor in the trapping region fluoresces when it is co-localized at the scanned laser position, enabling closed-loop feedback control over its position via electrodes that apply x and y voltages to electrokinetically move the particle back to the trap centre. Spectroscopic data are simultaneously acquired (time-correlated single-photon counting (TCSPC); 4× APDs for polarization and red/green channels). FPGA, field-programmable gate array; NA, numerical aperture. b, Raw ABEL trap data showing signals from seven different FRETfluors over 30 s (10 ms binning). Background-subtracted brightness in the red and green channels increases during trapping events (top). FRET efficiency calculated from the red and green brightness traces (middle). The grey dotted lines indicate the expected FRET values for each class of FRETfluor. Fluorescence lifetime decays for the green and red channels during the first trapping event (brown background above; split into green and red backgrounds) versus donor lifetime when the acceptor is blinking or photobleached (grey background) (bottom). The IRF is shown by the grey dotted line.
