Finite-difference time-domain simulations of the dipole emission in the proximity of the freestanding ZMW. (A) A scheme of the simulation setup. The dipole is placed in the center of the pore in the xy plane at varying z-positions. The detected signal is monitored on the detection (i.e., lower) side. (B-C) From top to bottom: The z-profiles of the excitation probability, the detection efficiency η, the emitter quantum yield Φ, and the total detected signal, along the center of the nanopore are shown for the blue (B, λex =488 nm, λem =525 nm) and red (C, λex =640 nm, λem =670 nm) channels. The total detected signal S(z) is defined as the product of the excitation intensity, detection efficiency, and quantum yield. (D-E) Predicted fluorescence lifetimes τ of BSA-Alexa488 and Kap95-Alexa647. The position of the palladium membrane is indicated as a gray shaded area. The weighted averages of the fluorescence lifetime based on the detected signal S(z) are shown as colored horizontal dashed lines. The gray dashed line indicates the measured fluorescence lifetime in the absence of the ZMW. The predicted lifetimes are 1.98, 1.88, and 1.88 ns for BSA–Alexa488, and 1.22, 1.95, and 1.15 ns for Kap95–Alexa647, for pore diameters of 50, 100, and 150 nm, respectively. The quantum yields and fluorescence lifetimes were estimated based on a literature values of Φ0 = 0.8 and τ0 = 4.0 ns for Alexa488 (Sanabria et al., 2020), and Φ0 = 0.33 and τ0 = 1.17 ns for Alexa647 (Hellenkamp et al., 2018), and measured lifetimes in the absence of the ZMW of 2.3ns for BSA–Alexa488 and 1.37 for Kap95–Alexa647 (compare Appendix 7—Figure 1).