A Pressure-Polish Setup to Fabricate Patch Pipettes Yielding Low Access Resistance and Efficient Intracellular Perfusion

Abstract

When performing whole-cell configuration recordings, it is very important to minimize series resistance. This goal is achieved here by using a method able to enlarge the cone-shape section of the pipette, without increasing the tip opening diameter, by using a calibrated combination of heat and air pressure. The heat was produced by passing current in a glass-coated platinum wire, shaped appropriately to ensure an homogeneous heating of pipette shank, connected to a regulated high-current generator. The pressurized air (∼4 atm) was applied to the pipette lumen through a 0.2 µm filter (to avoid pipette clogging), via the pressure port of a modified commercial holder. The pipette reshaping was viewed on an LCD monitor (and recorded on-line on a computer), connected to a contrast-intensified CCD camera coupled to a bright-field stereomicroscope. The hot halogen lamp of the microscope illuminator was replaced with a variable white LED source, to avoid the loss of fine control of the platinum wire temperature. By pressurizing the pipette lumen during fire-polishing, the pipette shank was widened as desired, without increasing the tip opening diameter: these pressure-polished pipettes, tested on many cell types, yielded routinely access resistances ∼4-fold smaller than the ones attained with conventional pipettes. The pressure-polished pipettes minimized therefore intracellular ion accumulation or depletion, and errors in membrane potential control, in the presence of large membrane currents. Moreover, they allowed to study rapid voltage-activated currents (by reducing the time constant of charging the cell membrane capacitance), the efficient incorporation in the cytosol of large molecules (that was followed with fast fluorescence imaging), and to position pulled quartz capillaries inside the pipette very close to its tip, resulting in fast intracellular perfusion.