Department of Molecular and Cellular Physiology, Stanford University School of Medicine
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Johnson, B. E., Brown, A. L., Goodman, M. B. Pressure-polishing Pipettes for Improved Patch-clamp Recording. J. Vis. Exp. (20), e964, doi:10.3791/964 (2008).
Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (<3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (<5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.
Using a filament puller such as a Sutter P-97 Flaming/Brown Microelectrode Puller, pull a set of approximately 10-20 pipettes.
Pressure polishing pipettes
Notes about the microforge
We use a microforge assembled from off-the-shelf commercially available parts. (You can also buy a pressure-polishing kit from a few companies, e.g., ALA Science, GlassWorx). Regardless of the microforge, it is essential to have optics that are good enough to see the tip opening, which is often only a few µm in diameter. We use a long-working distance 100x lens for this purpose. Also, coat the polishing coil with high-melting temperature glass in order to minimize depositing vaporized metal on the pipettes.
Part 3: Anticipated Results
Moderate pressure polishing can reduce the resistance of a pipette without an appreciable change in opening diameter. If desired, this protocol can be used to make pipettes with sub-micron openings and resistances between 5-10MΩ.
We originally developed this method to make pipettes useful for patch-clamp recording from 2-3 µm neurons in C. elegans 1. It is in routine use in the Goodman laboratory. We pressure-polish pipettes not only for recording from tiny C. elegans neurons, but also for obtaining macropatch and single-channel recordings of ion channels expressed in Xenopus oocytes. For a given tip-opening diameter, the blunt shape produces a lower-resistance pipette. Lower pipette resistance leads to lower series resistance and lower-noise recordings.
The pressure-polishing technique was developed in collaboration with S. R. Lockery.
|Glass Capillaries||Consumable||Sutter Instrument Co.||BF150-85-10 (or similar)||The method works with any type of capillary except those made from quartz. However, we recommend using capillaries with a filament.|
|Inverted Microscope||Microscope||Leica Microsystems||DM IL (or similar)||Any simple inverted microscope will do. However, it is essential to have sufficient magnification to see pipettes during polishing. We use a 100x metallurgical lens with a long working distance for this purpose.|
|Microforge Kit||Tool||ALA Science||CPM-2||Supplies polishing coil, controller and foot pedal.|
|Air pressure source||Tool||ALA Science||PR-60||Accessory for providing high pressure, includes a suitable valve. Alternatively, compressed air from the building can be used together with a filter/regulator (set to 40 PSI) and a 4-way valve (see below)|
|4-way valve||Tool||Cole-Parmer||A-98150-01||Fittings will be needed to couple valve to tubing|
|Pipette Holder with Luer Connector||Tool||World Precision Instruments, Inc.||MPH-6S20||Use a male luer-to-barb fitting to connect pipette holder to high-pressure tubing|
|Tygon® tubing||Tool||Cole-Parmer||06408-62||Use this tubing to connect pipette holder to 4-way valve|
1. Goodman, M. B. & Lockery, S. R. Pressure polishing: a method for re-shaping patch pipettes during fire polishing. J Neurosci Methods 100, 13-15 (2000).
2. Sutter Instruments. P-97 Pipette Cookbook, Rev. D (Sutter, 2008).