Troubleshooting patch clamping

Troubleshooting patch clamping

Fabrication of patch-recording electrodes

Electrodes with a small tip diameter are needed to obtain patches that contain only a few or preferably one ion channel. These electrodes will therefore naturally have a high resistance. The range of resistance for a normal single-channel electrode may vary from approximately 7 to 20 MΩ and should normally be pulled from standard-walled glass. A two-stage pull process is normally necessary to obtain patch electrodes with tip diameters of no more than 1 µm. The optimal settings to be used will vary even between electrode pullers of the same model, and these settings have to be found empirically. The first-stage pull settings should allow the glass capillary to thin over a drop of approximately 5 mm. These initial settings are usually not so critical. The more precise second-stage pull settings should create two near-identical electrodes with resistances of around 6–8 MΩ.

For the lowest noise, highest resolution recordings, coat the patch electrodes with a thin layer of Sylgard. The Sylgard coat should be applied from above the shank of the electrode to near the tip opening. Most Sylgard has a tendency to run (into the tip), so keep the tip slightly up above the horizontal. Sylgard is normally applied up to 0.3–0.5 mm from the tip. The Sylgard should then be cured, for example, by drawing it through a heated coil of nichrome wire or by using a heat gun (the curing only takes a few seconds). The Sylgard coat increases the thickness of the wall near the tip of the electrode and its hydrophobic quality prevents solution from creeping up the electrode. Both these features decrease capacitance coupling to the bath, which helps to reduce the background noise level.

Sylgard (Dow Corning no.184) Mix as required by adding ten parts of elastomer to one part of curing agent. Aliquots of mixed Sylgard can be stored for a few months at -20°C or even longer at -80°C.

Heat-polish the tip of the electrode under a dissecting microscope (magnification x400) by bringing it close to a V-shaped piece of platinum/iridium (90/10%) wire, through which variable current is passed to obtain the desired tip opening/resistance (8–15 MΩ).

NOTE: Electrodes can be stored in an air-tight sealed container. Electrodes left in open air and even for prolonged periods (greater than 1 week) in a closed container will attract particulates, and this can interfere with successful patch clamp recording. Unused patch electrodes can be used another day, but it is wise to lightly repolish them (see Step 6) to remove any impurities or grease that may have got accumulated while in storage.

Ready to Patch

Fill only the top 5–8 mm from the tip of the recording electrode with internal solution, and mount the pipette firmly on the headstage holder. Ensure that the AgCl-coated silver wire is submerged in the internal solution. Reduce the length of the Ag/AgCl wire in the electrode holder (only 5 mm longer than the holder can reduce the noise level to 60–70 fA)

NOTE All solutions must be filtered. The internal solution is particularly important and should be filtered with a 0.22 µm pore diameter filter.

To avoid debris from collecting at the tip, apply a small but constant positive pressure (about 5 mm Hg) to the pipette solution when entering the bath solution right up until the moment when the pipette nearly touches the cell and causes a slight indentation of the membrane.

Problem Possible reason Solution
Patches are generally too noisy Noise arising from dielectric coupling Ensure the Sylgard coat on electrodes is sufficiently thick
Keep solution depth in the bath low so that the electrode tip is only minimally submerged
Fill the tip of the electrode with internal solution just enough to cover the tip (2–4 mm) of the silver wire
Poor membrane seals owing to less than optimal electrodes Electrode tips must be cleaned by polishing to remove any debris (or Sylgard) that will compromise the electrical seal with the cell membrane
Keep the surface of the external solution as clean as possible,so that particles are not picked up by the electrode tip when entering the solution
Background noise Internal electrical noise(including 50 Hz noise) Reduce the length of the Ag/AgCl wire in the electrode holder only 5 mm longer than the holder can reduce the noise level to 60–70 fA)
Earth each piece of equipment (amplifier, headstage, oscilloscope, etc.) and associated metal that could act as aerials (e.g., metal stands, microscope, manipulators, etc.). This is achieved by connecting to an earth block attached to the inside of the Faraday cage
Earth the terminal block to the ground socket on the amplifier
Solution lines to and from the bath should be grounded via the insertion of short lengths of metal tubing
Background noise in the electrophysiology rig Inadequate shielding of external electrical signals Check the integrity of your Faraday cage for holes or lack of earthing, etc.
Add an additional earthed screen (usually a piece of copper mesh) in front of the recording chamber
Experimenter acts as an aerial Touch the earthed Faraday cage or attach an earth strap to the wrist