Plan apo tirf

DHBs were imaged with a 100× objective (Plan Apo TIRF; Nikon Instruments) excited with a 488 nm Argon ion laser (Coherent OBIS, 4mW). Fluorescence was detected by an electron-multiplying CCD camera (Ixon L897). The images and videos were analyzed with both NIS Elements D Analysis and ImageJ software⁴⁰ , and the particle tracker plugin developed by the Computational Biophysics Lab at ETH Zurich^{41 (link)}. The presence of target nanopores (red circles in Supplementary Fig. 15) were confirmed by fluorescence blinking experiments and the fluorescence intensity of the brightest frame were measured for each nanopore. For the three types of nanopores, 95% confidence ovals were produced for the relationship between fluorescence intensity and pixel size. Minimum time scale is 20 ms for CCD camera because the maximum frame number of the CCD camera is 50 in ROI (region of interest, 7.1 × 7.1 μm²).

All motility experiments were performed on an inverted fluorescence microscope (Nikon ECLIPSE TE300) equipped with a 532 nm laser (Coherent Sapphire) for rhodamine excitation (~3 mW on the sample) and a 488 nm Laser Physics argon laser for QDs-655 nm excitation (~3 mW on the sample). Images were acquired in total internal reflection configuration, through Nikon Plan Apo TIRF, 1.45 oil immersion objective. 91 nm pixel size images were obtained by projecting the fluorescence signal onto a iXon 3 EMCCD camera, after an additional 3× magnification through an achromatic doublet telescope. At the beginning of each record, one F-actin image was acquired and used afterwards to overlay the trajectory of the moving QD with its correspondent actin filament (Fig. 1b). Depending on the ATP concentration, different integration times (50–100 ms) were used at constant EM gain = 300.