Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltagesensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is ~1 nm. The ion-channel-like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable "conventional" pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.

The exclusion of polar dyes by healthy cells is widely employed as a simple and reliable test for cell membrane integrity. However, commonly used dyes (propidium, Yo-Pro-1, trypan blue) cannot detect membrane defects which are smaller than the dye molecule itself, such as nanopores that form by exposure to ultrashort electric pulses (USEPs). Instead, here we demonstrate that opening of nanopores can be efficiently detected and studied by fluorescent measurement of Tl+ uptake. Various mammalian cells (CHO, GH3, NG108), loaded with a Tl+-sensitive fluorophore FluxOR™ and subjected to USEPs in a Tl+-containing bath buffer, displayed an immediate (within<100 ms), dose-dependent surge of fluorescence. In all tested cell lines, the threshold for membrane permeabilization to Tl+ by 600-ns USEP was at 1–2 kV/cm, and the rate of Tl+ uptake increased linearly with increasing the electric field. The lack of concurrent entry of larger dye molecules suggested that the size of nanopores is less than 1–1.5 nm. Tested ion channel inhibitors as well as removal of the extracellular Ca2+ did not block the USEP effect. Addition of a Tl+-containing buffer within less than 10 min after USEP also caused a fluorescence surge, which confirms the minutes-long lifetime of nanopores. Overall, the technique of fluorescent detection of Tl+ uptake proved highly effective, noninvasive and sensitive for visualization and analysis of membrane defects which are too small for conventional dye uptake detection methods.

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In recent years, research into biological and medical effects of millimeter waves (MMW) has expanded greatly. This paper analyzes general trends in the area and briefly reviews the most significant publications, proceeding from cell-free systems, dosimetry, and spectroscopy issues through cultured cells and isolated organs to animals and humans. The studies reviewed demonstrate effects of lowintensity MMW (10 mW/cm 2 and less) on cell growth and proliferation, activity of enzymes, state of cell genetic apparatus, function of excitable membranes, peripheral receptors, and other biological systems. In animals and humans, local MMW exposure stimulated tissue repair and regeneration, alleviated stress reactions, and facilitated recovery in a wide range of diseases (MMW therapy). Many reported MMW effects could not be readily explained by temperature changes during irradiation. The paper outlines some problems and uncertainties in the MMW research area, identifies tasks for future studies, and discusses possible implications for development of exposure safety criteria and guidelines.

Intense nanosecond-duration electric pulses (nsEP) open stable nanopores in cell plasma membrane, followed by cell volume changesdue to water uptake or expulsion, as regulated by the osmolality balance of pore-impermeable solutes inside and outside the cell. The size of pores opened by 50, 60-ns EP (10 Hz, ~13 kV/cm) and 5, 600-ns EP (1 Hz, ~6 kV/cm) in GH3 cells was estimated by isoosmotic replacement of bath NaCl with (polyethylene glycols and sugars. Such replacement reduced cell swelling and/or turned it into a transient or sustained shrinking, depending on the availability of pores permeable to the test solute. Unexpectedly, solute substitutions showed that for the same integral area of pores opened by 60- and 600-ns treatments (as indicated by cell volume changes), the pore sizes were similar. However, the 600-ns exposure triggered significantly higher cell uptake of propidium. We concluded that 600-ns EP opened a greater number of larger (propidium-permeable pores), but the fraction of the larger pores in the entire pore population was insufficient to contribute to cell volume changes. For both the 60- and 600-ns exposures, cell volume changes were determined by pores smaller than 0.9 nm in diameter; however, the diameter increased with increasing the nsEP intensity.