Single-Molecule Fluorescence Imaging Setup

Fluorescence images were acquired using a modified Olympus IX81. A nanometre precision XYZ piezo stage (Physik Instrumente GmbH, Germany) on top of a mechanical stage with 1 × 1 cm range (JPK Instruments, Germany) was used for sample positioning. The sample was illuminated through a 60× magnification objective lens (60×, 1.42 NA, Olympus, Austria) and an additional 1.6× magnification tube lens with a 642 nm diode laser (Omicron, Germany). A dichroic filter (ZT405/488/561/640rpc, Chroma, Germany), an emission filter (446/523/600/677 nm BrightLine quad-band band-pass filter, Semrock, USA), and an additional emission filter (HQ 700/75 M, NC209774, Chroma, Germany) were used for imaging of the probes of the immobilized fluorophore–biomolecule conjugates (ATTO647N-cNP77 for Cy-LAPAP and ATTO655-SA for Cou-LAPAP and Rho-LAPAP). The fluorescence signal was detected using an Andor iXonEM+ 897 (back illuminated) EMCCD camera (16 μm pixel size) (Andor Technology, United Kingdom).^{93 (link)} 1500 images were recorded with 20 ms illumination time and 4.15 kW cm⁻² excitation intensity at λ_exc = 640 nm.