The fluorescence emission of the liposome is recorded simultaneously with resistivepulse sensing (Figure ). The fluorescence signals reported in this work were obtained by defining a 5.4 × 5.4 m (18 × 18 pixel) region of interest (ROI) located at the end of the nanopipette to quantify the fluorescence intensity of a multipassing liposome (Figure and Figure ). The fluorescence intensity is recorded in analog-to-digital units (ADU) and converted to the number of photons by calibrating the camera detector gain from a series of gradient images. Details regarding the conversion are shown in Figure . We observed that the liposomes do not emit fluorescence when located directly outside of the nanopipette orifice resulting in liposomes displaying 'on/off' fluorescence behavior during multipassing, as discussed in a later section. A supplementary video has been provided showing the multipassing of a fluorescent liposome at 10 fps (200× slower than real time) (Video S1). corresponding to a liposome entering and leaving the nanopipette at -1.0 V and +1.0 V, respectively. The asymmetric shape of the resistive pulse arises from the conical shape of the nanopipette and indicates the direction that the liposome is traveling when translocating through the orifice. A resistive pulse corresponding to a liposome entering the nanopipette exhibits a sharp decrease in current at the leading edge of the pulse, indicating when the liposome first passes through the nanopipette orifice. The current gradually returns to the opennanopipette baseline current as the liposome travels up into the conically-shaped nanopipette and no longer blocks the orifice (Figure ). The opposite shape is observed for a liposome exiting the nanopipette, where the leading edge of the resistive pulse is gradual as the liposome travels down the nanopipette, followed by a sharp increase in current back to the baseline as the liposome exits the nanopipette orifice (Figure ). The baseline currents of the i-t traces were fit using asymmetric least squares shown by the dashed orange line in Figures and (see Supporting Information). The charging current following potential switching results in nonlinear baseline currents. The asymmetric least squares fit to the baseline current is used to calculate the maximum blockage current of a liposome translocation (Figure ). The variations seen in the intensity of the fluorescence signal during liposome multipassing (Figure ) is hypothesized to result from three primary factors. First, the fiber illuminator emits light in a single optical mode, which can produce interference fringes within the waveguide-like nanopipette. As the liposome travels ~25 m into the nanopipette (depending on the internal delay time), it traverses these interference fringes in the excitation light which produces variability in the liposome's emission intensity. In addition, vertical movement of the liposome through the objective lens depth of field, ~1 m, and possible lensing effects from the curved nanopipette, contribute to the fluctuations in the observed fluorescence intensity (Figure ). Lastly, the overall fluorescence emission intensity decreases as the fluorescent labels associated with the liposome slowly photobleach within the excitation light (vide infra).