Effects of the Surface Morphology of Pipette Tip on Giga-seal Formation

Reported is a study of applying nanofabrication technology to improve the surface roughness of micro glass pipettes to achieve giga ohm seal resistance in patch clamping processes. The surface roughness of pipette tips was first measured by 3D reconstruction of pipette tips using stereo imaging technique based on high resolution SEM images. Both the SEM images and the reconstructed images show that micro glass pipettes have rough and uneven tips which could be one of the causes of leakage in patch clamping. Then focused ion beam system was used to cut across the very end of the tip, producing a smooth and flat new tip. The average surface roughness Ra of a milled pipette tip was within a few nanometres. Patch clamping experiments were carried out using the polished pipettes on human umbilical vein endothelial cells (HUVEC), which were well known for their extremely flat shape making them very difficult to patch. The results show that above 3 Giga ohm seals were achieved in 60% of the experiments, as opposed to 1.5-2.0 Gfl in average with the conventional pipettes. The highest seal resistance achieved with a focused ion beam polished pipette was 9 Gfl, well above the 3 Gfl resistance, the usually best result achieved with a conventional pipette. The leakage current in single channel recording afterwards was found 0.3 pA, significantly smaller than 2-3 pA usually achieved using conventional pipettes. The research results demonstrate that the surface roughness of a pipette has a significant effect on the giga-seal formation of a patch clamping process.