title: A pressure-polishing set-up to fabricate patch pipettes that seal on virtually any membrane, yielding low access resistance and efficient intracellular perfusion.
abstract: When performing whole-cell configuration recordings, it is important to minimize series resistance to reduce the time constant of charging the cell membrane capacitance and to reduce error in membrane potential control. To this end, an existing method was improved by widening the patch pipette shank through the calibrated combination of heat and air pressure. The heat was produced by passing current through a filament that was shaped appropriately to ensure a homogeneous heating of the pipette shank. Pressurized air was applied to the lumen of a pipette, pulled from a borosilicate glass microcap, via the pressure port of a modified commercial holder. The pipette reshaping was viewed on an LCD monitor connected to a contrast-intensified CCD camera and coupled to a modified bright-field stereomicroscope. By appropriately regulating the timing of air pressure and the application of heating, the pipette shank and, independently, the tip opening diameter were widened as desired. The methods illustrated here to fabricate and use the patch pipettes, using just one glass type, allowed the sealing of a wide variety of cell types isolated from different amphibian, reptilian, fish, and mammalian tissues as well as a variety of artificial membranes made with many different lipid mixtures. The access resistance yielded by pressure-polished pipettes was approximately one-fourth the size of the one attained with conventional pipettes; besides improving the electrical recordings, this minimized intracellular ion accumulation or depletion as well. Enlarged shank geometry allowed for fast intracellular perfusion as shown by fluorescence imaging, also via pulled quartz or plastic tubes, which could be inserted very close to the pipette tip.
authors.affiliation: Dipartimento di Biologia ed Evoluzione, Sezione di Fisiologia e Biofisica, Università di Ferrara, Via L. Borsari 46, 44100, Ferrara, Italy.
authors.forename: Mascia
authors.initials: M
authors.lastname: Benedusi
authors.forename: Marco
authors.initials: M
authors.lastname: Aquila
authors.forename: Alberto
authors.initials: A
authors.lastname: Milani
authors.forename: Giorgio
authors.initials: G
authors.lastname: Rispoli
mesh_terms: D000388:Air; D000818:Animals; D002138:Calibration; D002460:Cell Line; D002462:Cell Membrane; D017097:Electric Impedance; D005898:Glass / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D042541:Intracellular Space; D008567:Membranes, Artificial; D018408:Patch-Clamp Techniques / Q000295:instrumentation*; D010477:Perfusion / Q000295:instrumentation*; D011312:Pressure
chemical_list: D008567:Membranes, Artificial