Figure 1.

Design and demonstration of combined extension and FRET measurements with FluoRBT. (a) A simplified schematic of the experimental setup is shown. Evanescent excitation with 845- and 532-nm light is achieved using a set of small mirrors positioned under the objective. Emitted light is spectrally separated via a dichroic beamsplitter; fluorophore emission is further isolated with a set of optical filters and an IR block. A pair of magnets above the sample is used to apply forces and torques on DNA. See Fig. S1. (b–e) Simultaneous measurements of FRET and extension (Δz) were collected during opening and closing of a hairpin under tension. (b) A DNA tether was stretched using a magnetic bead, a separate rotor bead was used as an evanescent scattering probe of Δz, and a FRET dye pair (green star, Quasar 570; orange star, Quasar 670) reported locally on the state of an incorporated hairpin, consisting of 15 basepair stem region and eight nucleotide loop. (c) Simultaneously acquired Δz (top, 2000 Hz raw and 20 Hz averaged), donor and acceptor intensities (middle, 20 Hz), and calculated FRET efficiency (bottom) are shown for a single DNA hairpin fluctuating between closed and open states under constant force. Arrowheads show start of donor excitation (∼2 s), acceptor bleaching (∼48 s), and donor bleaching (∼57 s). (d) Correspondence between Δz (vertical axis) and FRET efficiency (color scale) is shown for four separate hairpin tethers. (e) Data from 13 hairpin tethers were pooled together to generate a 2D histogram of 6180 instantaneous measurements, presented as a contour plot. Contours are equally spaced; color bar indicates the relative number of observations for each area. The mean Δz extension and FRET efficiency of both states for each molecule are shown with colored symbols. The mean Δz position of the closed state and the mean FRET efficiency of the open state were set to zero in our analysis procedure, as described in Supporting Material.