Figure 2. Experimental setup.

(A) A freestanding Pd membrane containing the nanopores was mounted on a confocal microscope using a polydimethylsiloxane (PDMS) flow cell with a reservoir of ≈ 3L on the dark (cis) side and a flow channel on the detection (trans) side that faced the objective lens. A constant flow in the channel avoided the accumulation of analytes on the detection side. The lasers were focused onto the nanopore by a high numerical aperture (NA) objective lens and the fluorescence signal was detected on single-photon avalanche photodiodes. From the recorded photon arrival times, fluorescence bursts were detected using a change point detection algorithm. (B) A total of eight pores were milled into the Pd membrane, each surrounded by partially milled markers that facilitate the localization of the nanopores in a bright-field image (top). An additional marker was added such that the individual pores in the array could be identified. A scanning electron microscope image of a single pore with markers is shown below. The size and shape of each zero-mode waveguide (ZMW) pore used in this study was determined using transmission electron microscopy (bottom right). (C) Simulated electric field intensity distributions in the xz plane near a freestanding ZMW for pore diameters of 30 nm (top) and 150 nm (bottom). A Gaussian laser beam was focused on the pore at a wavelength of 488 nm, polarized in the x-direction. See Appendix 2—figure 1 for different pore sizes and excitation at 640 nm. (D) Electric field intensity ${\displaystyle |E^2|}$ (top) and total detected signal ${\displaystyle S(z)}$ (bottom) as a function of the z-position along the center of the pore for an excitation wavelength of 488 nm and an emission wavelength of 525 nm, corresponding to the blue detection channel, for pores of 50 nm, 100 nm, and 150 nm diameter. The palladium membrane is indicated by the gray shaded area. See Appendix 2—figure 4 for the corresponding plots for the red channel.