Study of the Tip Surface Morphology of Glass Micropipettes and Its Effects on Giga-Seal Formation

摘要

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 area roughness Sa 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 GΩ seals were achieved in 60% of the experiments, as opposed to 1.5-2.0 GΩ in average with the conventional pipettes. The highest seal resistance achieved with a focused ion beam polished pipette was 9GΩ, well above the 3GΩ resistance, the usually best result achieved with a conventional pipette. 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.