ID: 2194

  • Title:
    Visualizing Electropores through Ca2+ Puffs: Contrasting Millisecond and Nanosecond PEFs

    Silkunas, Mantas - Frank Reidy Research Center for Bioelectrics, ODU, Norfolk, USA; Institute for Digestive System Research, LUHS, Lithuania.
    Silkuniene, Giedre - Frank Reidy Research Center for Bioelectrics, ODU, Norfolk, USA; Institute for Digestive System Research, LUHS, Lithuania.
    Gudvangen, Emily - Frank Reidy Research Center for Bioelectrics, ODU, Norfolk, USA.
    Pakhomov, Andrei - Frank Reidy Research Center for Bioelectrics, ODU, Norfolk, USA.

    The mechanism of electroporation, despite its widespread application, remains incompletely understood due to the lack of available tools capable of investigating individual pores. The resolution limit of optical microscopy impedes the observation of individual pores in live cells.

    We have recently introduced a novel technique that combines total internal reflection fluorescence (TIRF) microscopy and whole-cell patch clamp to visualize Ca2+ influx as fluorescence puffs, control the membrane potential, and measure the transmembrane current. HEK293 cells were placed on glass coverslips that had an electrically conductive but optically transparent indium tin oxide (ITO) layer, and loaded with a fluorescent Ca2+ indicator. Brief hyperpolarization pulses to below -220 mV generated transient electroporation lesions, manifested as dynamic bright spots of fluorescence within the cell’s footprint. The depth of field of TIRF imaging is limited to ~100 nm, thus enabling clear detection of discrete Ca2+ puffs instead of a diffuse fluorescence cloud when other microscopy methods are used.

    This method was modified to analyze pore formation by applying electric pulses extracellularly, without patch clamp. HEK293 cells were loaded with a fluorescent indicator Cal520-AM and with EGTA-AM, to minimize the fluorescence background following electroporation. Electric pulses of various duration were applied between the ITO layer and a rod electrode positioned above the target cell. We tested 300-ns pulses at up to 6 kV/cm, and 1-ms pulses at up to 0.6 kV/cm. We also tested different pulse polarities (making the ITO layer either cathode or anode), in a physiological solution with 10 mM Ca2+ or without Ca2+ as a control. Electroporation pulses were synchronized with time-lapse TIRF image acquisition at approximately 200 frames per second.

    Discrete membrane electroporation lesions were detected and monitored as Ca2+ puffs after a millisecond pulse in the presence of extracellular Ca2+. As of the time when this abstract is written, we have not observed any discrete puffs after nanosecond pulse treatments, with either pulse polarity; instead, fluorescence intensity increased uniformly in the entire cell footprint. This result is consistent with expectations that nanosecond pulses create numerous nanometer-size membrane lesions (“supraelectroporation”), which contrasts relatively large localized lesions in case of millisecond-pulse electroporation. Elimination of Ca2+ from the extracellular solution prevented any fluorescence increase after the application of either milli- or nanosecond pulses, indicating that intracellular Ca2+ stores were not involved in puff formation.

    Total Internal Reflection Fluorescence (TIRF) microscopy; Patch clamp, Ca ions influx; Electropermeabilization.


    Topic 1:
    1. Biological responses (molecular, subcellular, cellular and intercellular)

    Topic 2:
    4. Diagnostics, analytics, experimental techniques

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