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Outside-out patch

Outside-out patch clamp formation technique. In order: top-left, top-right, bottom-left, bottom-right

The name "outside-out" seems to have been coined in a 1981 paper by Hamill, et al on Improved Patch-Clamp Techniques. It emphasizes both this technique's similarity to the inside-out technique and the fact that it places the external surface of the cell membrane and its channels on the outside of the vessel formed by the glass electrode (labeled "pipette" in the image at right) and the patch of membrane.

The formation of an outside-out patch begins with a whole-cell patch. After the seal is formed, the electrode can be slowly withdrawn from the cell, allowing a bulb of membrane to bleb out from the cell. When the electrode is pulled far enough away, this bleb will detach from the cell and reform as a convex membrane on the end of the electrode (like a ball open at the electrode tip), with the original outside of the membrane facing outward from the electrode. As the graphic at the right shows, this means that the fluid inside the pipette will be simulating intracellular fluid, while a researcher is free to move the pipette and the bleb with its channels to another bath of solution. While multiple channels can exist in a bleb of membrane, single channel recordings are also possible in this conformation if the bleb of detached membrane is small and only contains one channel. The picture at right illustrates the second of these cases.

Outside-out patching gives the experimenter the opportunity to examine the properties of an ion channel when it is isolated from the cell and exposed to different solutions on the extracellular surface of the membrane. The experimenter can perfuse the same patch with a variety of solutions in a relatively short amount of time, and if the channel is activated from the extracellular face, a dose-response curve can then be obtained. This ability to measure current through the exact same piece of membrane in different solutions is the distinct advantage of the outside-out patch relative to the cell-attached method. On the other hand, it is more difficult to accomplish. The longer formation process involves more steps that could fail and results in a lower frequency of usable patches.

Loose patch

Loose patch clamp technique

Loose patch clamp is different from the other techniques discussed here in that it employs a loose seal (low electrical resistance) rather than the tight gigaseal used in the conventional technique. This kind of technique was used as early as the year 1961, as described in a paper by Strickholm on the impedance of a muscle cell's surface, but received little attention until being brought up again and given a name by Almers, Stanfield, and Stühmer in 1982--after patch clamps had been identified as a major tool of electrophysiology.

To achieve a loose patch clamp on a cell membrane, the pipette is pushed toward the cell slowly, until the electrical resistance of the seal between the muscle cell and the pipette increases to a few times greater resistance than that of the electrode. The closer the pipette gets to the membrane, the greater the resistance of the seal becomes, but if too strong a seal is formed, it could become difficult to remove the pipette without damaging the cell. For the loose-patch technique, the pipette does not get close enough to the membrane to push hard against it and form a gigaseal or a permanent connection, nor to pierce the cell membrane. Notice in the image at right that the membrane is intact, and that the lack of a tight seal creates a gap through which ions can pass, though not as easily as moving down the pipette.

A significant advantage of the loose seal is that the pipette that is used can be repeatedly removed from the membrane after recording, and the membrane will remain intact. This allows repeated measurements in a variety of locations on the same cell without destroying the integrity of the membrane. This flexibility has been especially useful to researchers for studying muscle cells under real physiological conditions, obtaining recordings quickly, and doing so without resorting to drastic measures to stop the muscle fibers from contracting A major disadvantage is that the resistance between the pipette and the membrane is greatly reduced, allowing current to leak through the seal. This leakage can be corrected for, however, which offers the opportunity to compare and contrast recordings made from different areas on the cell of interest. Given this, it has been estimated that the loose patch technique can resolve currents smaller than 1 mA/cm².