When performing whole-cell configuration recordings, it is important to minimize series resistance, to reduce time constant of charging the cell membrane capacitance and error in membrane potential control. To this goal, it was improved an existing method widening the patch pipette shank, through the calibrated combination of heat and air pressure. The heat was produced by passing current in a filament shaped appropriately to ensure an homogeneous heating of the pipette shank. The 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 a LCD monitor, connected to a contrast-intensified CCD camera, coupled to a modified bright-field stereomicroscope. By appropriately regulating timing of air pressure and heating application, the pipette shank was widened as desired, independently by the tip opening diameter. The methods illustrated here to fabricate and use the patch pipettes, using just one glass type, allowed to seal on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes made with many different lipid mixtures. The access resistance yielded by pressure polished pipettes resulted ~4-fold smaller than the one attained with conventional pipettes; besides improving the electrical recordings, this minimized intracellular ion accumulation or depletion as well. Enlarged shank geometry allowed fast intracellular perfusion, as shown by fluorescence imaging, also via pulled quartz or plastic tubes, that could be inserted very close to the pipette tip.
A Pressure-Polish Setup to Fabricate Patch Pipettes Yielding Low Access Resistance and Efficient Intracellular Perfusion
BENEDUSI, Mascia;MILANI, Alberto;AQUILA, Marco;RISPOLI, Giorgio
2011
Abstract
When performing whole-cell configuration recordings, it is important to minimize series resistance, to reduce time constant of charging the cell membrane capacitance and error in membrane potential control. To this goal, it was improved an existing method widening the patch pipette shank, through the calibrated combination of heat and air pressure. The heat was produced by passing current in a filament shaped appropriately to ensure an homogeneous heating of the pipette shank. The 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 a LCD monitor, connected to a contrast-intensified CCD camera, coupled to a modified bright-field stereomicroscope. By appropriately regulating timing of air pressure and heating application, the pipette shank was widened as desired, independently by the tip opening diameter. The methods illustrated here to fabricate and use the patch pipettes, using just one glass type, allowed to seal on a wide variety of cell types, isolated from different amphibian, reptilian, fish, and mammalian tissues, and on artificial membranes made with many different lipid mixtures. The access resistance yielded by pressure polished pipettes resulted ~4-fold smaller than the one attained with conventional pipettes; besides improving the electrical recordings, this minimized intracellular ion accumulation or depletion as well. Enlarged shank geometry allowed fast intracellular perfusion, as shown by fluorescence imaging, also via pulled quartz or plastic tubes, that could be inserted very close to the pipette tip.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
