ABSTRACT
The ion channels located on the cell fine structures play an important role in the physiological functions of cell membrane. However, it is impossible to achieve precise positioning on the nanometer scale cellular microstructures by conventional patch-clamp technique, due to the 200 nm resolution limit of optical microscope. To solve this problem, we have established a high-resolution patch-clamp technique, which combined commercial scanning ion conductance microscopy (SICM) and patch-clamp recording through a nanopipette probe, based on SICM feedback control. MDCK cells were used as observation object to test the capability of the technique. Firstly, a feedback controlled SICM nanopipette (approximately 150 MOmega) non-contactly scanned over a selected area of living MDCK cells monolayer to obtain high-resolution topographic images of microvilli and tight-junction microstructures on the MDCK cells monolayer. Secondly, the same nanopipette was non-contactly moved and precisely positioned over the microvilli or tight-junction microstructure under SICM feedback control. Finally, the SICM feedback control was switched off, the nanopipette slowly contacted with the cell membrane to get a patch-clamp giga-ohm sealing in the cell-attached patch-clamp configuration, and then performed ion channel recording as a normal patch-clamp electrode. The ion channel recordings showed that ion channels of microvilli microstructure opened at pipette holding potential of -100, -60, -40, 0, +40, +60, +100 mV (n=11). However, the opening of ion channels of tight-junction microstructure was not detected at pipette holding potential of -100, -40, 0, +40, +100 mV (n=9). These results suggest that our high-resolution patch-clamp technique can achieve accurate nanopipette positioning and nanometer scale high-resolution patch-clamp recording, which may provide a powerful tool to study the spatial distribution and functions of ion channel in the nanometer scale microstructures of living biological samples.