Effects of Nanosecond Pulsed Electric Fields on theIntracellular Function of HeLa Cells As Revealed by NADH AutofluorescenceMicroscopy

摘要

The fluorescence lifetime of the endogenous fluorophore of reduced nicotinamide adenine dinucleotide (NADH) in HeLa cells is affected by the application of nanosecond pulsed electric fields (nsPEFs). In this study, we found that after nsPEF application, the fluorescence lifetime became longer and then decreased in a stepwise manner upon further application, irrespective of the pulse width in the range of 10–50 ns. This application time dependence of the NADH fluorescence lifetime is very similar to the time-lapse dependence of the NADH fluorescence lifetime following the addition of an apoptosis inducer, staurosporine. These results, as well as the membrane swelling and blebbing after the application of nsPEFs, indicate that apoptosis is also induced by the application of nsPEFs in HeLa cells. In contrast to the lifetime, the fluorescence intensity remarkably depended on the pulse width of the applied nsPEF. When the pulse width was as large as 50 ns, the intensity monotonically increased and was distributed over the entire cell as the application duration became longer. Asthe pulse width of the applied electric field became smaller, themagnitude of the field-induced increase in NADH fluorescence intensitydecreased; the intensity was reduced by the electric field when thepulse width was as small as 10 ns. These results suggest that themechanism of electric-field-induced apoptosis depends on the pulsewidth of the applied nsPEF.