Have you checked to make sure that your rig is perfectly level and that there is plenty of slack in all of the cables that connect to the mainuplator and head stage?

Another idea, that a constant slow z drift can be your objective drifting and not certainly your electrode (check with 2 pipetts in the meantime). Agree with previous suggestions, that vibration may originate from streched cables, perfusion, heat, holder. If only your electrode is vibrating, but the tissue bellow not, than it is not your table.

If sutter had checked and said there was no problem, it seems, your setting has some problem somewhere. Check your air table, check the electrode holder, and the connection between the holder and the small tube. Check your camera, whether it is stable, maybe camera is not stable and electrode is not really drifting.

Anyway, good luck!

Temperature can also be another variable worth considering. Cool air from an AC vent can cause the pipette and other parts of your setup to change dimensions.  Temperature changes in your recording chamber can also cause the electrode and even the chamber itself to expand and contract.  

As mentioned above, the focus can drift too.  This is especially prevalent in older scopes where the focus raises and lowers the entire top portion of the microscope rather than just the objective carrier (like my old Zeiss FS1).

I have also had problems caused by old and new electrode holders.  It seems the holder needs to be "broken in" to become stable but will again lead to drift issues if it is  in a state of disrepair.

Finally, vibrations especially in the frequency range you refer to are actually amplified by  many conventional pneumatic (air) vibration isolation tables.  This is made worse if the vibrations are in the x-y axes as most air tables are designed to dampen high frequency vibrations in the z axis.  We ran into this problem a few years back because of the motion characteristics of the new building we were in.  The solution was to use some fantastic spring-loaded vibration isolation tables from Kinetic Systems with the isolator provided by a small company called Minus-K.  

The last thing I would suspect is the Sutter Manipulators.  We have 20 or so and have never had a problem with drift in any of them. Typically I would not state something so strongly, but we have performed many simultaneous dual recordings from soma and dendrites while doing calcium imaging and uncaging and things are typically rock-solid.

The reason may be a tensile force originating either from a cable coming out of your headstage or even from a suction tubing connected to the pipette holder. Both should be loose in sufficiently long segments and firmly fixed e.g. to a micromanipulator stand (not the micromanipulator itself!) at couple of points. Optimal setings must be find by a trial and error way.

From my experience the major source of pipette position drift is in the pipette holder. The idea to fix the pipette in place using a rubber ring is not really great – at least from the viewpoint of positioning accuracy. If you think about it, it is a bit strange to spend thousands of euros on ultraprecise micro or nanomanipulators and then finally rely on a rubber seal holding the pipette in place. One thing that helped to virtually eliminate the drift was to drill a little hole in the side of the cap screw of the pipette holder and put a little screw there to clamp the pipette firmly against the wall of the holder cavity.

Further to my point about the problem with the drift due to the rubber ring. Quick search on the net brough up a page demonstrating alternative design of the pipette holder clamp: http://www.neobiosystems.com/ezPatch.php

Not much else to add but two other possible sources  for pipette drift are the temperature of the solution, and unfixed stands. If you are doing experiments at "physiological" temperatures, make sure your pipette is in contact with the bath  solution for a, at least, couple of minutes before you attempt patching any cell. The higher temperature of the solution will affect the pipette. The second source I had had, was that the stand was not fixed to the table. We were relying on the weight of a heavy stand that was not fixed (screwed) to the table. Additionally, it turned out that the surface of the table was not perfectly smooth. In that case, the movement was not a slow drift but a sudden jerk.

Finally, I also would recommend you to leave the ROE of the micromanipulator at its slowest speed (position 9 for a Sutter micromanipulator). If I leave it at a fast position (4 or below), pipette drift becomes obvious. However, be advised that, in my opinion, position 9 serves more like a break. It is really hard to move the manipulator at this position, when it moves it does it in a jerky way and you will lose your cell..

The drift in Z could indeed come from the lens. This could be tested as suggested by looking at two pipettes, or at the tissue / bottom of the perf chamber.

Cables and variable temperature can of course affect the stability, but I would think first about the holder. We had a bad stretch with those from Axon. We are now using those from G23 - which are recommended by Axon and turned out to be much much more stable.

(http://g23instruments.com/shop/category_4/Microelectrode-holders---ISO-Range.html?sessid=W0zAMtSEbd2JUZkVFFydV2FD7xspiVMMmBPVCstiCiGEJnhP44egTMhgAX8cCc2Q&shop_param=cid%3D%26).

I also think about the holder, may be you could check if without pressure in your pipette you have still got a drift. If the holder is not good, your pipette can slide with the pressure. An other hypothesis can be your system to fix the postion of the pipette with the good axe, may be it is not completely blocked and slide slowly.

Hi Philip,

I second Philippe's comment. The pipette holder, in my experience, turned out to make an enormous difference. I would recommend video-recording your pipette in the recording conditions and reviewing it at high speed. In my case, I just took a screenshot every 2 seconds and turned it into a video file.

I used a manual micromanipulator and compared it with movement from two axon headstages with different pipette holders and realized the problem was in the axon headstage / holders. https://www.youtube.com/watch?v=JlAYViKUClw&list=UUs3-qUELPY-2AOHa7Q_db5w

I then compared long vs. short plastic pipette holders we had in the lab and realized the shorter ones dramatically outperformed the longer ones https://www.youtube.com/watch?v=_-4f0T2Hifo&list=UUs3-qUELPY-2AOHa7Q_db5w

I imagine the plastic (or the rubber holding the pipette) is changing its shape due to temperature fluctuations, and minimizing the size of the plastic pipette holder produced improved stability in my case.

I tried many things to maximize stability of my patch-clamp setup, including wrapping the faraday cage with trash bags to minimize air flow and temperature fluctuation through the cage. Although it looks ridiculous, there was a lot of improvement, so I continue to use it this way. Some of the techniques I tried improved stability, some did not, but in every case video recording pipette drift was crucial to critically evaluating (A) how much of a problem there was and (B) which changes to my setup improved the situation.

Note finally that I had a LOT of "drift" was actually more of a random "wiggle", such that hour-long pipette location change was minimal, but minutes-long instability was resulting in poor patches for long durations in a way that I never would have noticed if I didn't video record the movement.

https://www.youtube.com/watch?v=_-4f0T2Hifo&list=UUs3-qUELPY-2AOHa7Q_db5w

https://www.youtube.com/watch?v=JlAYViKUClw&list=UUs3-qUELPY-2AOHa7Q_db5w

The other thing to consider are air currents leading to temperature changes. So if the pipette holder isn't the problem the next most likely thing to fix the problem would be preventing any microclimate changes. This may involve a Faraday-cage type solution with an emphasis on preventing currents of air. You can sometimes troubleshoot this problem by just opening a window which (depending on your lab situation) can immediately indicate whether or not this is a likely factor. Also, just throwing a light sheet of material over the whole setup will give you an idea if this is going to help or not.

My 30 years experience in patch clamp tells that all (cables, tubes, O-ring, tables, objectives, temperature, etc.) have effects. We use a screw in pipette holder to fix the pipette and to avoid rubber O-ring problems (Pavel Novak also mentioned this). Most impressive (often not detectable) are air small flows through different parts of set up (specifically, all mechanics to which the pipettes are attached including micromanipulator, stands, arms, ect. which have been made from different metals and having different temperature sensitivity to expand). That is why there can be not predictable moves of the pipette tip also.

Dear Philip - check if the cables of the head-stage (holder) are not stretched when electrode is in working position; make sure that rubber gaskets holding the electrode are not too old; switch of the bath heating if attached and on; no remaining tension should follow after the manipulator is brought to its working position (due to old grease; Scientifica have solved this problem with using holding magnets); as mentioned by others, also the air flow and the temperate fluctuations may cause a drift of the pipette. It is one of those irritating problems when there can be many solutions and need methodical check-up of various options. Good luck. Saak       

Hi all,

I am having some difficulty with fixing the pipette drift in my rig. I am wondering if you could give me some advice on this as it looked like I ran out of ideas to try.

- I am trying to do whole-cell recording from pyramidal neurons in hippocampal dissociated cultures at DIV 14 - 21. 

- Problem: the pipette seemed to drift in x/y dimension while the cells stay stable. The drifting is most dramatic during the first 5 min when pipettes are moved to a new position. The direction of this movement is not very consistent. The drifting distance within 5 min is about 1/5 - 1/10 of a cell diameter. After 5 min, the drifting is on and off every once in a while. After 2 hour, it drifted the whole cell diameter. It persists even when the manipulator is turned off. I can still patch with this but it is quite impossible to keep a long recording (~ 20 min is the longest one I got yesterday).

- Model of manipulator: MP 285

- What I have tried and did not help:

* changing the old holder with a brand-new pipette holder

(Warner: 64-0839 (QSW-T15P))

* Checked all of the cables connecting to the headstage and made sure they are loosen

* Tried tightening the pipettes to the holder at different levels

* Turned off the perfusion

* change the O-ring in the old pipette holder. Look similar to this one:

http://g23instruments.com/shop/article_ISOS15/ISO-S-1.5G-micro-electrode-holder.html?sessid=JCa98KerM1ndZ0txuVvMswIPrGLKkK3A4yJa8Iv4x6LM8Nn7CFAdTOIzLkDXWn6j&shop_param=cid%3D6%26aid%3DISOS15%26

- I also checked the air table and it seemed to still be floating. The air tank is not empty.

- I have a cage covering the air table and curtains surrounding me. I also do not use any heater for the bath. So I guess microclimate change would be very unlikely...

Do you have any thought on this? Thanks so much!

Best,

Huong

Why the baseline keep drifting after offset even before patching the cell with the Cs-gluconate internal solution but not K-gluconate internal?

Sutters can drift based on where in the limit range you are operating.  It may help to change where on the axes you are (near the top limit vs. near the middle, for example).  Just because they are not seeing it doesn't mean it isn't happening in your configuration.  You can also try fiddling with the posts/clamps its sitting on, sometimes you'll discover a loose connection somewhere that causes motion.  The fact that its always the same magnitude and in the same direction sounds like there is a dribble of current to one of the stepper motors that causes motion.  I've had that many times with sutters, never once with the scientifica (except for when I had some tension in the cables going to the manipulator) taping or attaching all the cables to the post the manipulator is sitting on was what finally did the trick in my case.   Good luck.

I recently had a chat with a guy from Luigs & Neumann and I want to add what I learned there:

He said that tension in the fixation screws of the microscope to the air table is sometimes a source of drift, too. Loosen some of the screws and test again will show weather this is the case. Also he confirmed that Teflon holders (like Axon Instruments) are problematic, because they "flow", an intrinsic Teflon property it seems.

Sometimes it can be the tissue instead of the pipette. try putting some weight on top of your tissue, or slowing the flux of the solution which might be moving your sample.