All experiments were conducted with a prism-type total internal reflection fluorescence (TIRF) microscope (Nikon) equipped with a 488-nm laser (Coherent Sapphire, 200 mW), a 561-nm laser (Coherent Sapphire, 200 mW), and two Andor iXon EMCCD cameras (28 (link),29 (link)). Flowcells and ssDNA curtains were prepared as previously described (28 (link),29 (link)). In brief, lipid bilayers were prepared with 91.5% DOPC, 0.5% biotinylated-PE and 8% mPEG 2000-DOPE. The ssDNA substrate was generated using rolling circle replication with a biotinylated primer, a circular M13 ssDNA template, and Phi29 DNA polymerase, as described (28 (link),29 (link)). The biotinylated ssDNA was injected into the sample chamber and attached to the bilayer through a biotin–streptavidin linkage. The flow cell was then attached to a microfluidic system and sample delivery was controlled using a syringe pump (Kd Scientific) (28 (link),29 (link)). For all two-color images, we used a custom-built shuttering system to avoid signal bleed-through during image acquisition. With this system, images from the green (GFP) and the red (mCherry) channels are recorded independently, these recordings are offset by 100 ms such that when one camera records the red channel image, the green laser is shuttered off and vice versa (28 (link),29 (link)).
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