Obtaining Gigaohm Seals

There are multiple factors which should be attended to in order to successfully obtain gigaseals. They include: state and type of preparation; mechanical vibration; state of the solutions; condition of the recording pipette; pressure/suction applied to the recording pipette; control of the micromanipulator; angle of approach; and pipette placement onto the cell surface. Failure to attend to any of these factors can easily lead to hours of frustration. As my old mentor, JL used to say "In patch clamping, everything works against you . . . it's the little things that kill you." So in being mindful of his advice, let me describe how I approach each of these important factors.

State and Type of Preparation

The time between media changes has been thought to affect patching success. The standard recommendation is to change the culture media the night before you do your experiments. Apparently, the membrane properties of some cell types can change as they take up nutrients from the media (sorry I don't have the reference). For mammalian neurons, unless you use a heated stage the cells will start to appear granular after about an hour outside the incubator. This is a good indicator that you should get some fresh cells, otherwise you may start to have problems getting seals or finding channels. Besides, how can you trust results from deteriorating cells? Regarding the type of preparation. I have found that I have had to vary different aspects of my patching technique to accomodate different cell types. More on that later.

Mechanical Vibration

Vibration is your enemy. Get rid of it or at least minimize it. First make sure your air table is pressurized correctly. Too much or too little air pressure will prevent vibration damping. All your equipment should be firmly attached to the surface of the air table. Tape down all those loose wires and tubes so that NOTHING can move during your experiment and especially during the critical moments of seal formation. In addition, put your rig somewhere away from air flow and foot traffic.

State of Solutions

Sterile filter all your solutions. Dust, bacteria and other contaminants will definitely prevent seal formation. I use 0.22 µm disposable filter disks attached to 60 mL syringes. This arrangement works well so I freshly filter the solutions as I dispense them. Also, check the pH of your solutions occasionally because bacteria can sometimes start to grow in them.

Recording Pipette

Fire polish the recording pipette tip so that the glass curves inward. See how much firepolishing your pipettes take before they seal over. I have obtained best results with heavily firepolished pipettes. But balance the firepolishing with consideration of the resistance, otherwise you may start out with a pipette resistance several time higher than what you want.

Pressure/Suction

Keep positive pressure on the recording pipette as you take it through the air/bath interface. This is where it can pick up the most contaminants - from dust and debris floating on the bath surface. Once you are in the bath don't bring the recording pipette out again. Its not necessary to use a tremendous amount of positive pressure. For example you can blow hard into the connecting tube and clamp it off. After you are in the bath you may be able to release the pressure entirely before you attempt to form a seal. I like to keep the tube in my mouth as I bring the recording electrode to the cell surface since I have the most control this way. You can either keep the pressure on as you touch the cell surface or you can try releasing the pressure first. I have had the best success keeping pressure on all the way to the cell. Once the pipette touches the cell, the situation is very fluid. Perhaps it will seal immediately. Or perhaps nothing will happen and you will want to push the pipette harder against the cell. Some cells such as muscle cells or frog oocytes can take lots of abuse. Neurons are generally very delicate and you can't push too hard. I have had best success watching the cell and looking for the "right" amount of membrane displacement in order to get seal formation. For some reason this works better for me than watching my oscilloscope and looking for a sudden increase in the resistance. Either way, once you are on the cell, release the pressure and first give the membrane a chance to seal by itself. If nothing happens after about 30 s then try applying gentle and continuous suction. As an example, I have obtained seals just by holding the tube close to my mouth as I inhale! Other times you may have to apply forceful suction to obtain seals. But as a rule, don't use more suction than necessary or you will obtain short-lived seals or break into the cell.

Micromanipulator Control

If you can afford one, get a motor controlled micromanipulator or hydraulic system. I use a Huxley-type manipulator, which can be problematic. The best manipulator I have ever used was an Eppendorf 5171. I got GOhm seals virtually every time!

Angle of Approach

This may not be such a critical factor, as long as you try to make the recording pipette as perpendicular as possible to the cell surface. Usually an angle of at least 45 degrees is adequate. You may find that the angle of approach affects how much pressure you must apply to the cell surface to form a seal.

Movement of Recording Pipette

For manual manipulators, try to make the movement as smooth and vibration-free as possible. The actual moment of contact between pipette and cell should be a single smooth movement withoug need for further adjustment after the initial placement. You have to judge how much pressure to apply to the cell at the same time as you place the electrode on the cell. Otherwise if you make adjustments after the initial contact you will disturb the process of seal formation and reduce your chances of obtaining a GigaOhm seal.