Characterization of surface properties of glass micropipettes using SEM stereoscopic technique M. Malboubi a,⇑ , Y. Gu b , K. Jiang a a School of Mechanical Engineering, University of Birmingham, Birmingham B15 2TT, UK b School of Medicine, University of Birmingham, Birmingham B15 2TT, UK article info Article history: Available online 15 February 2011 Keywords: Glass micropipette Surface properties SEM stereoscopic technique Giga-seal formation Patch clamping abstract SEM stereoscopic technique is used in this research to determine the three dimensional surface struc- tures of pipettes. Surface properties of a pipette, both at the tip and at the inner wall, play an important role in experiments involving direct contact between a pipette and a biological sample. Tip surface prop- erties of pipettes of different sizes were measured and it was found that the smaller pipette has smaller surface roughness. The inner surface properties of pipette are also measured by cutting half of the pipette using focused ion beam milling. The results show that average surface roughness of pipette tip is 42.2 and 8.3 nm for pipettes with tip diameters 8.8 and 1.1 lm, respectively. Pipette inner wall has the average surface roughness of 39.0 nm and 28.4 nm for pipettes with tip diameters 13 and 9 lm. The study takes patch clamping as an example. The result of this work can be used to explain one of the major sources of leakage in giga-seal formation in patch clamping. Ions can escape through liquid between the valleys and the cell membrane. The results suggest that as long as the membrane and pipette surface are close enough, the length of the contact is in the second order of importance. This is in good agreement with the practical knowledge in patch clamping that the smaller pipette makes a better seal. The fact that both the tip and the inner wall are rough may promote future FEA analysis and experiments which involve membrane and glass interactions. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction Glass micropipettes have been used for decades to study mi- cron-sized biological samples. A micropipette can have different functions in different applications; it could be a microchannel for delivering liquids, genes or sperm to targets [1–3], or a microelec- trode in voltage, current or patch clamp studies [4,5]. In some applications such as patch clamping, cell aspiration or fertilization, a micropipette makes physical contact with samples. In these applications, the surface properties of micropipettes are of great importance to getting high quality results. A rough surface is not favorable because it increases the chance of tip contamination and damage to the delicate biological samples [6]. Although the effect of pipette roughness in various applications is emphasized in the literature, there is no numerical report of surface properties of pipettes. The round shape of the pipette and its fragility make it difficult to measure the surface properties by tactile measurement devices, such as AFM. The authors have reported the measurement of surface properties of a patch clamp micropipette tip by SEM ste- reoscopic technique earlier [7]. In this paper the surface properties of pipettes with different sizes have been measured with the same method. The surface properties of the inside wall of pipette is also measured by cutting half of the pipette using FIB milling. The influ- ence of a rough pipette surface on patch clamping is analyzed in the paper. The findings in this study could also be used in other techniques and future pipette-cell interaction models. 1.1. Patch clamp technique Patch clamping is an electrophysiology technique which allows the study of cellular ion channel activities. It was first introduced by Neher and Sakmann in 1976 and currently is considered to be the gold standard for measurement of ion channel activities [5]. In this technique, a glass micropipette is used to isolate a patch of cell membrane from an external solution. Once the pipette tip has made contact with the cell surface, suction is applied to pipette 0167-9317/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2011.02.029 Abbreviations: AFM, atomic force microscopy; DEM, digital elevation model; FEA, finite element analysis; FIB, focused ion beam; S 10z , ten point height of selected area; SEM, scanning electron microscopy; S a , average height of selected area; S dq , root mean square gradient; S dr , developed interfacial area ratio; S sk , skewness of selected area; S p , maximum peak height of selected area; S q , root- mean-square height of selected area; S ku , kurtosis of selected area; S v , maximum valley depth of selected area; S z , maximum height of selected area. ⇑ Corresponding author. E-mail addresses: mlb@contacts.bham.ac.uk (M. Malboubi), K.Jiang@bham.ac.uk (K. Jiang). Microelectronic Engineering 88 (2011) 2666–2670 Contents lists available at ScienceDirect Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee