October 13, 2006
A recent toast to James Watson highlights a tolerance for bigotry many want excised from the scientific community.
An expert on the technique shares his secrets.
October 1, 2006|
Since its introduction by Bert Sakmann and Erwin Neher in 1978, patch-clamping techniques have become widely accessible with commercially available equipment. And while the science of patch clamping might require a re-education in basic electrical principles, the real art lies in forming the seal between the micropipette and the cell membrane. We asked Areles Molleman, a researcher and lecturer in cellular and membrane physiology at the University of Hertfordshire, UK, and the author of Patch Clamping: An Introductory Guide to Patch Clamp Electrophysiology, for his tips on forming that perfect seal. His advice follows.
When you place the micropipette in its holder, you can easily make electrical contact between you and the ?hot? electrode of the amplifier. If you are not connected to electrical ground then you?ll act as a giant antenna causing very big noise. This can harm some amplifiers, especially if you gathered static. Use your free hand to touch a grounded surface, or wire yourself up to electrical ground before placing the micropipette. The latter will also enhance noise reduction during recording as you are now part of the Faraday cage.
Apply four to six inches of water pressure to the pipette solution before crossing the bath surface and approaching the cell. You can do that through the extra channel that patch pipette holders have, by mouth, or better, by a simple u-tube. The pressure will keep the tip clean from any debris in the bath. Of course your pipette solution must be very clean as well. Use a micropore filter when filling if in doubt.
Every rig will be a little different in the feel of the manipulators and the optics. It is perfectly normal in the beginning to break several pipettes while locating the pipette near a cell, and take forever to do it. Don?t be discouraged but analyze what went wrong each time. You?ll get faster, but be prepared to start over again when moving to another rig.
Patching with the superfusion on can waste chemicals, possibly expensive drugs, and it increases the chance of pipette contamination. However, if your cells are not well attached to the substrate and you make a tight seal without superfusion, turning it on can easily reposition the cell and break the seal. Another reason to apply superfusion constantly is your microclimate control. Unless you are certain that the temperature and pH will not swing wildly, keep the superfusion on.
A large-bore pipette is good for whole-cell recording for several reasons. It is easy to break through the membrane using suction pulses, there is less chance of resealing during the recording, and pipette solution is quickly exchanged with the cytoplasm. So, in whole-cell recording the size of the pipette is dictated by the success rate in getting seals; within that constraint use the largest pipettes possible. These will be typically around 2MΩ in resistance. Smaller-bore pipettes are needed for single-channel recording.
Good contacts are essential to a good seal. Pipettes come in at an angle toward the cell, usually about 45 to 60 degrees. In placing the pipette, try to choose a part of the cell that is as perpendicular to the pipette axis as possible. This is not difficult with round cells but in the case of flat cells it sometimes helps to use the hill created by the nucleus. Approach spindle-shaped cells 90 degrees to their axes.
When is the time to switch off the over-pressure and apply suction to get a seal? During the approach an oscillatory test pulse of a few millivolts will indicate the resistance that the pipette ?sees? (the larger the current response the lower the resistance). Getting near a cell will increase the resistance, lowering the current response. In many cases, a drop of about 10% indicates the moment to switch to suction, but this varies from cell type to cell type and depends on the overpressure applied. That said, once established for a cell type and a rig it is reliably constant, so take notes. The switch from pressure to suction (usually applied by mouth) should be smooth and quick. If you leave the pipette without pressure or suction for a while, the membrane could wobble and contaminate your pipette tip, making good seals impossible.
If the resistance you are monitoring is decreasing significantly in the process of getting a seal, give up. Your tip will be contaminated and, as said above and unlike in intracellular recording, you have little or no hope of forming a good seal with this pipette. Recognizing failure and starting afresh with a new pipette at the right moment is the single most time-saving measure you can take as a starting patch clamper.
Do you have a micromanipulator with impossibly low drift specifications but you still see the micropipette traveling through the microscope field? Before you complain to the manufacturer, ensure that your manipulator is mechanically isolated to reduce vibration. Drift can be caused by loading the manipulator too heavily, or by torsion forces coming from the cables and tubes connected to the headstage. Prevent this by taping these down as well as possible.
After a seal is established, apply the intended holding potential. This is usually what you think will be a reasonable resting membrane potential for the cell. This will depolarize the patch of membrane under the pipette, but once you break through, the cell won?t receive a big, destabilizing current jolt from the amplifier. Breaking through can be done by a suction pulse or a current pulse. Suction is better for bigger pipettes (<3MΩ), while pulses are better for small pipettes (>6MΩ). Anything in between is trial and error, but once you have a method that works for your cells in your rig, stick to it. Sometimes a combination can do the trick. To do that, use a small amount of continuous suction while applying current pulses of increasing size. If you see a lot of noise appearing, go back to suction pulses.
October 13, 2006
January 7, 2010