Troubleshooting pipette drift

[NB: This post refers to mechanical drift, not drift in the recorded electrode potential.]


Every electrophsyiologist, at some point or another, will encounter pipette drift. Pipette drift is exactly what it sounds like: a continuous movement of the pipette tip relative to the bath over time. Depending on the cause, pipette drift can range from the obvious to the barely noticeable. Generally, though, pipette drift is worth isolating and correcting regardless of how trivial it may seem. Indeed, for a patch clamper conducting an experiment on an adherent cell over a time period greater than a few minutes, even very slow drift can cause problems.


Detecting pipette drift
For patch clampers working on adherent cells, a common symptom of pipette drift could reasonably be termed, "sudden death syndrome" - i.e. when a cell with an otherwise perfectly good seal, small and steady leak and good capacitance transients, suddenly just packs up on you. Inspection via the microscope objective will often be sufficient to identify pipette drift as the cause, as the pipette tip will clearly have pulled away from the cell, or broken on the bottom of the plate.

If you're not sure that the pipette is drifting, load a patch pipette and bring the tip down close to the bottom of the bath. Find any random small speck on the glass bottom to serve as a reference point, and bring the pipette tip as close as possible to it. At one minute intervals, inspect the position of the pipette relative to the speck. Lateral (x,y) drift will be obvious, as the tip will slowly move away from the speck. Vertical (z) drift can be obvious too, if the pipette cracks on the bottom of the plate. If the pipette is drifting up and away from the reference point, the twiddling the focus nob will reveal a lengthening in the distance between the focal planes of the speck and pipette tip.

Isolating pipette drift

• Obstruction - Obviously, the first thing to do if the pipette is behaving strangely is to check that it isn't resting against the side of the bath, or pushed up against the microscope condenser (in an inverted scope) or objective (in an upright). Call it the Homer Simpson test, if you will.
  
• Pipette holder* -
  
• Check rubber O-ring - Remove the pipette holder from the stage and open it up. Check the integrity of the O-ring that secures the pipette inside the holder. These have a limited lifetime, and when worn by repeated use will no longer hold the pipette firmly. Replace if damaged. [NB: a good way to increase the longevity of the O-ring is to fire polish the ends of your pipette glass before pulling pipettes from it.]
• Connection to headstage - Check that the connector between the pipette holder and headstage is snug. If it's loose, the pipette holder will sometimes rotate, which can alter the position of the tip relative to the bath.
  
• Headstage and micromanipulator cables - Ensure that neither the headstage nor the micromanipulator are under tension from their respective electrical leads and/or tubes. Tension in the tube used for changing the internal pressure to the pipette holder can sometimes twist the holder in the headstage socket. These are common sources of drift on newly assembled rigs, and can be rectified by fastening cables/tubes securely, whilst leaving sufficient slack between the fastening point (e.g. to a magnetic mounting post or platform) and the equipment being served by the cables or tubes.
  
• Micromanipulator mechanism - If all else fails, check the micromanipulator itself. Most operator's manuals contain instructions on checking for micromanipulator drift. One way is to remove the headstage and secure a long, rigid piece of material to the micromanipulator arm. For example, take a glass Pasteur pipette and heat-pull the tip to a fine point that can be adequately inspected in the bath through the microscope objective. Again, check for relative drift. There are numerous reasons for why a micromanipulator might be drifting, many of which will dependent on the type used. If a liquid hydraulic manipulator is being used, obviously check for bubbles in the lines and follow the operator's manual for instructions on how to remove them. Otherwise, the best policy is to have the micromanipulator inspected and repaired by a trained service engineer.
• Stage movement - On rare occasions, it's the stage that is drifting and not the pipette. Check for tension in any tubes or cables connecting external appliances to the stage (e.g. a solution switcher, or bath heater). Alternatively, dust and gunk can build up in the stage runners (especially after an overflow incident) causing resistance. Either way, you should be able to identify these problems by moving the stage around to the extremes of its movable axes: if the action feels spongy, and the stage visibly "relaxes" to a different position after release, then look for sources of resistance.

* [Edit: Apparently a custom pipette holder is available from G23 Instruments that contacts the pipette at two points with two separate O-rings for enhanced stability ].