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Surface properties of glass micropipettes and their effect on biological studies.

Malboubi M, Gu Y, Jiang K - Nanoscale Res Lett (2011)

Bottom Line: It is found that surface roughness parameters are strongly related on the tip size.The results of the experiments show that polished pipettes make significantly better seals.The results of this work are of important reference value for achieving pipettes with desired surface properties and can be used to explain biological phenomenon such as giga-seal formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Mechanical Engineering, The University of Birmingham, Edgbaston, Birmingham, B15 2TT UK. mlb@contacts.bham.ac.uk.

ABSTRACT
In this paper, an investigation on surface properties of glass micropipettes and their effect on biological applications is reported. Pipettes were pulled under different pulling conditions and the effect of each pulling parameter was analyzed. SEM stereoscopic technique was used to reveal the surface roughness properties of pipette tip and pipette inner wall in 3D. More than 20 pipettes were reconstructed. Pipette heads were split open using focused ion beam (FIB) milling for access to the inner walls. It is found that surface roughness parameters are strongly related on the tip size. Bigger pipettes have higher average surface roughness and lower developed interfacial area ratio. Furthermore, the autocorrelation of roughness model of the inner surface shows that the inner surface does not have any tendency of orientation and is not affected by pulling direction. To investigate the effect of surface roughness properties on biological applications, patch-clamping tests were carried out by conventional and FIB-polished pipettes. The results of the experiments show that polished pipettes make significantly better seals. The results of this work are of important reference value for achieving pipettes with desired surface properties and can be used to explain biological phenomenon such as giga-seal formation.

No MeSH data available.


Related in: MedlinePlus

Glass micropipette before (a) and after (b) FIB milling.
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Figure 12: Glass micropipette before (a) and after (b) FIB milling.

Mentions: The fact that both the pipette tips and pipette inner walls are rough may help better understanding of the mechanism of applications in which pipettes are in contact with vulnerable biological samples. Patch-clamp technique is taken as an example of pipette applications. Patch-clamping suffers from current leakage between cell membrane and glass surface. Cell membrane is sucked from 5 to 100 μm into a pipette in patch clamping. Optical and electron microscope images of patches show that membrane and pipette are in close contact, but they do not show the surface topography involved in seal formation [20,21]. Surface roughness of glass micropipettes is reported in the literature to play an important role in giga-seal formation [11-13,22,23]. Normally, pipettes are fire polished before experiments to make rough tips smoother. In this study, FIB milling is used for polishing pipette. FIB polishing was found to be a more controllable process. Fire polishing requires a very good timing and positioning. Pipette end can be easily overheated which results in a closed tip or irregular tip shape. Fire polishing melts the glass and makes pipette tips smoother but it also has a blunting effect on tips which change the pipette shape and sharpness. FIB polishing allows working on the very end of pipettes (the last one micron from the tip) without changing other properties of pipettes such as shape and sharpness. To observe the importance of surface roughness of pipette on giga-seal formation, patch-clamp experiments were carried out using conventional and FIB-polished pipettes. FIB milling of pipettes leaves an ultimately smooth surface free from peaks and valleys or sharp spikes. Figure 12 shows images of a pipette before and after FIB milling. Because of the conic shape of pipette only the very end of pipette was cut during milling process in order not to change the pipette opening significantly. Ten recordings were obtained for each type of pipettes. Seal resistances are shown in Figure 13. FIB-polished pipettes formed significantly better seals which made it possible to measure single ion channel currents with considerably lower noise (see Figures 14 and 15). Higher seal resistance for polished pipettes could be explained by their better sealing potential. Contact area between pipette tip and cell membrane is higher for polished pipettes and since there are no peaks or spikes, membrane can get closer to the tip. As a result, it is more difficult for ions to escape form glass-membrane distance and higher seal are achievable.


Surface properties of glass micropipettes and their effect on biological studies.

Malboubi M, Gu Y, Jiang K - Nanoscale Res Lett (2011)

Glass micropipette before (a) and after (b) FIB milling.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3211496&req=5

Figure 12: Glass micropipette before (a) and after (b) FIB milling.
Mentions: The fact that both the pipette tips and pipette inner walls are rough may help better understanding of the mechanism of applications in which pipettes are in contact with vulnerable biological samples. Patch-clamp technique is taken as an example of pipette applications. Patch-clamping suffers from current leakage between cell membrane and glass surface. Cell membrane is sucked from 5 to 100 μm into a pipette in patch clamping. Optical and electron microscope images of patches show that membrane and pipette are in close contact, but they do not show the surface topography involved in seal formation [20,21]. Surface roughness of glass micropipettes is reported in the literature to play an important role in giga-seal formation [11-13,22,23]. Normally, pipettes are fire polished before experiments to make rough tips smoother. In this study, FIB milling is used for polishing pipette. FIB polishing was found to be a more controllable process. Fire polishing requires a very good timing and positioning. Pipette end can be easily overheated which results in a closed tip or irregular tip shape. Fire polishing melts the glass and makes pipette tips smoother but it also has a blunting effect on tips which change the pipette shape and sharpness. FIB polishing allows working on the very end of pipettes (the last one micron from the tip) without changing other properties of pipettes such as shape and sharpness. To observe the importance of surface roughness of pipette on giga-seal formation, patch-clamp experiments were carried out using conventional and FIB-polished pipettes. FIB milling of pipettes leaves an ultimately smooth surface free from peaks and valleys or sharp spikes. Figure 12 shows images of a pipette before and after FIB milling. Because of the conic shape of pipette only the very end of pipette was cut during milling process in order not to change the pipette opening significantly. Ten recordings were obtained for each type of pipettes. Seal resistances are shown in Figure 13. FIB-polished pipettes formed significantly better seals which made it possible to measure single ion channel currents with considerably lower noise (see Figures 14 and 15). Higher seal resistance for polished pipettes could be explained by their better sealing potential. Contact area between pipette tip and cell membrane is higher for polished pipettes and since there are no peaks or spikes, membrane can get closer to the tip. As a result, it is more difficult for ions to escape form glass-membrane distance and higher seal are achievable.

Bottom Line: It is found that surface roughness parameters are strongly related on the tip size.The results of the experiments show that polished pipettes make significantly better seals.The results of this work are of important reference value for achieving pipettes with desired surface properties and can be used to explain biological phenomenon such as giga-seal formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Mechanical Engineering, The University of Birmingham, Edgbaston, Birmingham, B15 2TT UK. mlb@contacts.bham.ac.uk.

ABSTRACT
In this paper, an investigation on surface properties of glass micropipettes and their effect on biological applications is reported. Pipettes were pulled under different pulling conditions and the effect of each pulling parameter was analyzed. SEM stereoscopic technique was used to reveal the surface roughness properties of pipette tip and pipette inner wall in 3D. More than 20 pipettes were reconstructed. Pipette heads were split open using focused ion beam (FIB) milling for access to the inner walls. It is found that surface roughness parameters are strongly related on the tip size. Bigger pipettes have higher average surface roughness and lower developed interfacial area ratio. Furthermore, the autocorrelation of roughness model of the inner surface shows that the inner surface does not have any tendency of orientation and is not affected by pulling direction. To investigate the effect of surface roughness properties on biological applications, patch-clamping tests were carried out by conventional and FIB-polished pipettes. The results of the experiments show that polished pipettes make significantly better seals. The results of this work are of important reference value for achieving pipettes with desired surface properties and can be used to explain biological phenomenon such as giga-seal formation.

No MeSH data available.


Related in: MedlinePlus