Figure 3.

(A) Pseudo coloured wide-field image of HJ–origami immobilized on a PEG surface with 200 mM MgCl₂ added to the buffer, excited with 532-nm laser light (scale bar = 10 µm). Green spots indicate donor (Cy3) only HJs, orange spots reveal FRET to the acceptor Cy5. (B) Fluorescence time transient of the circled HJ in (A) (upper trace). The Cy3 and Cy5 intensities show anti-correlated behaviour until the acceptor bleaches (at 9 s). The FRET efficiency changes rapidly between 0.2 (iso I) and 0.7 (iso II) (lower trace). Based on a two-state Hidden Markov Model (HMM) these changes are recognized and counted. Only FRET efficiencies between 0.15 and 0.8 are taken into account to rule out dye bleaching or dark states. (C) Transition rates in dependence of the magnesium concentration. All curves show decreasing rate values at higher Magnesium concentrations.