Abstract.SignificanceResealing time based loading efficiency of optoporation is the key parameter for drug or gene delivery. This work describes a comparatively simple optical approach to directly measure the cell membrane resealing time of the gold nanoparticle mediated photoporation.AimTo establish a membrane potential detection optical system, which can provide a direct measurement of resealing time of the optoporated cells.ApproachVoltage sensitive dye has been used to label the gold nanoparticle covered cell before laser activation and the resealing time was estimated from the voltage change due to the fluorescence light intensity change before and after laser activation. The approach has been validated by the simulated data based on diffusion model and Monte Carlo simulation and the experimental data obtained from a flow cytometry analysis.ResultsThe measured resealing time after perforation varied from 28.6 to 163.8 s on Hela cells when the irradiation fluence was increased, with a correlation coefficient (R2) of 0.9938. This result is in agreement with the resealing time (1-2 min) of photothermal porated Hela cells measured by electrical impedance method. The intracellular delivery efficiency of extracellular macromolecular under the same irradiation fluence depends mainly on diffusion velocity rather than pore size.ConclusionThe method described here can be used to directly measure resealing time of optoporated cells for accurately estimating the loading efficiency on discovering the mechanism of optoporation.