We developed an electric-field exposure microchannel system with 230-nm thin-layer gold electrodes and interfaced it with a single living cell imaging station and a 10-ns electric-pulse (10 nsEP) generator. This design allows us to image intracellular molecules and structures, membrane transport, and viability of single leukemic cells (HL60) while the cells are exposed to 10 nsEPs of 0-179 kV/cm, permitting the study of subcellular responses within a nanosecond regime. The electrodes confine a thin-layer section of the cells exposed to 10 nsEPs, offering unprecedented high spatial resolution (230 nm in the z-direction of imaging plane and electric field) for imaging intracellular molecules of single cells affected by 10 nsEPs. We found that nucleic acids, membrane transport rates, and viability of single cells depend on the number and electric-field-strength (E) of 10 nsEPs, showing the cumulative effect of 10 nsEPs on intracellular molecules and structures and suggesting the possibility of tuning them one-pulse-at-a-time. Using a lower E (51 kV/cm) of 10 nsEPs, we could manipulate nucleic acids of single living cells without disrupting their cellular membrane and viability. As E increases to 80, 124, and 179 kV/cm, membrane integrity and viability of cells exhibit higher dependence on the number of 10 nsEPs in a nonlinear fashion, showing that a critical E and pulse number are needed to surmount cellular transport barriers and membrane integrity.