Wednesday, October 27, 2010

How do I measure the liquid junction potential?

And so it emerges from the depths.....

For years innocent readers thought they had escaped...

A horror so horrible it inspires screams...of horror

It is...the...long awaited post on how to measure a junction potential!!

It's clear from the search terms that bring people here, plenty of folks are curious about this topic. The "What is a Liquid Junction Potential" post is one of the most read. So allow me to finally follow up with how to actually measure it. Although there are the standard descriptions about junction potentials in various texts and bibles of the field, a step by step 'how-to' is lacking. Seeing as I bridge these two in real life lab,why not here?

Ok, you have some internal solutions, and you want to report the correct voltages in your paper.

You've got to account for the junction potential between the internal solution and the bath solution present when you first stick in the pipette. Now, you can calculate it if you want; there's a program built into Clampex. But, are you sure that the constants are correct for what's in your solution, especially if it has something weird in it? TEA-methanesulfonate for example. Or NMDG-aspartate - a killer internal for recording sodium current by the way. Are you even sure you made the solution correctly?

It's easy, so measure it!

When I measure junction potentials, I follow the advice of my old granpappy, as discussed in E. Neher, Methods Enzymol. 1992;207:123-31.

Here's what you want to do:
1) Get your stuff together: battery powered chlorider (don't tell me you're using bleach - GAH!!), silver wire, 3 M KCl, 5+ mL of each of the internal solutions, plus a few mL of the typical bath solution, a few patch pipettes.

2) Make a 'flowing KCl' bridge, by taking a patch pipette, breaking off most of the tip, and filling it with 3 M KCl. Chloride a silver wire and use this as the bath ground. The high concentration of KCl, and the similar mobility of K+ and Cl- will help keep the junction potential at this electrode constant, even as the bath solutions changes. I fashioned a little jig to hold the bath pipette (on left below, a piece of plexiglass on a piece of white teflon).

3) Rechloride your silver wire on the headstage, fill a normal resistance patch pipette with one of the solutions. If you're measuring only a single internal/bath pair, then you can put the internal in the pipette. But since setting this up is somewhat annoying, I often measure multiple internals relative to the same bath solution. In that case, I put the bath solution in the pipette.
4) Whatever solution you put in the pipette, add it to the bath.

5) Put your amplifier in current clamp mode (slow versus fast doesn't matter), set the 'meter' to 'Vm', make sure that there's no external command signal coming into the amplifier.

6) lower pipette into solution.

7) Use the "pipette offset" potentiometer until the meter reads zero mV. Wait a minute or so; if the voltage drifts by more than a few tenths of a mV, then you might need to rechloride the wires.

8) Completely exchange the bath solution with the solution to be measured.

9) Read the meter, which shows the junction potential.

10) Replace the bath with the same solution as in the pipette, check that Vm is back close to zero. After that you can measure any other solutions.

11) Now, to get the total transmembrane voltage in your experiments, you have to add your command voltage (or recorded in the case of a current clamp expt) to the measured junction potential. NOTE: If you switched the bath and pipette locations, then you MUST reverse the polarity. So, in the 8.2 mV above, that's actually a junction potential of -8.2, and is added to the command (e.g., a step from -80 to 0 in Clampex becomes -88 to -8 mV). If you're an anal retentive scientist *whistles innocently*, then you already account for the junction potential in your voltage protocols, resulting in nice round numbers.

There you go, and hope that helps. Questions? Let em rip in the comments.

53 comments:

Balazs said...

That`s it, Nath, respect! I am so glad you finally found the time for this and I hope it feels really good to have this done. I came by Google again, second hit on the list, amazing, I am sure it will help many of us! Thanks!!

Nat Blair said...

THanks Balazs, and you're welcome! Once I sat down to do it, it wasn't that hard (it took me some time to find the photos, which I thought I lost after a OS reinstall).

Hopefully this will help people out there. And you're right, my blog has some impressive Google juice to have the 2nd hit on a search for 'junction potential'

Juan said...

Awesome post, it's really informative. I have a question. Why do you use a "flowing KCl bridge"? Would it work if I just use the Ag/AgCl ground pellet currently installed in my rig?

Thanks!

Nat Blair said...

Juan,

Glad you found this helpful! That's why I put it out there.

You definitely don't want to use the Ag-AgCl pellet as the ground.

That's because there's a junction potential between the Ag-AgCl pellet and the bath solution. The magnitude of this potential changes when you switch the concentration of Cl- in the bath (say, from typical external solution of ~150 mM Cl- to 6-10 mM for low Cl- pipette solutions), the junction potential at the pellet will change dramatically.

Using the flowing KCl pipette will allow you to keep the Ag-AgCl to 3 M KCl potential constant, and will keep the junction potential between the flowing KCl -> bath solution largely constant (as the high concentration of KCl will dominate the junction potential when paired with lower concentration solutions - i.e. the ones you are measuring).

So, after you zero the potential with the same bath solution and pipette solution, you've accounted for both of those junction potentials. Now, when you change the bath solution to the solution you're measuring, the resulting voltage is only the result of the junction potential of the pipette/bath solution pair.

Is that helpful?

Thanks again for stopping by and asking a question!

Juan said...

Thanks a lot! That makes sense. So, if I understand correctly, it would be possible to use the Ag-AgCl pellet as ground as long as you leave the bath solution constant while changing only the solution in the pipette. Is this right?

It would also be awesome if you could post how to make a battery powered chlorider. Yes, I've been using Clorox for years!

Juan

Anonymous said...

Hello,

I have a question for the webmaster/admin here at junctionpotential.blogspot.com.

Can I use part of the information from your blog post above if I provide a backlink back to your site?

Thanks,
Daniel

Nat Blair said...

Yeah, it's definitely ok to use a Ag-AgCl pellet for the ground, assuming your bath solution has a constant [Cl-].

The chlorider is a good idea for a post. I think I even made myself a little diagram of the wiring to hook up the switch and the battery.

Nat Blair said...

@Anony Daniel,

If you're serious, then contact me via my profile page.

vy9 said...

Hi,

This is really helpful. I came across it when googling "LJP, current clamp"! I have started doing current clamp recordings using Multiclamp and Clampex and have a pretty basic question on LJP compensation. Any help would be really helpful!

Does the LJP show up as a voltage difference between Vm on the amplifier (MC in my case) and recorded Vm on Clampex? That is, if I inject current such that a cell is at -70 mV as seen on MC, and I have a LJP of 5 mV, will Clampex record -65 mV instead? And then during offline compensation, I change the recorded voltage to -70 mV?

Or will Clampex record -70mV if MC voltage reading is at -70mV, and I have to change the clampex recording to -75 mV during compensation (which will beat the purpose of recording from a cell at a Vm of -70 mV?)

I am pretty confused.. Thanks!

patch clamper said...

Nice post. I'm unsure why you would determine the junction potential and manually correct for it as you described rather than just use the pipette offset knob on the amplifier to make the TRACK current 0 prior to forming a seal.
I believe the amplifier (axopatch 200) uses this offset setting to correct for the junction potential and will display true voltages if this is set properly. Am I incorrect on this?

I tried to record with no amplifier correction (the pipette offset of 0), and my amplifier was overloaded. How would you record with no amplifier correction if you wanted to correct the junction potential manually?

Also, what's wrong with using bleach to chloride a wire?

Nat Blair said...

@vy9 and patch clamper - Thanks for your comments! If you are wondering about these things, definitely other people are too.

The voltage displayed on the amplifier doesn't include the junction potential, and regardless of whether you're in voltage clamp or current clamp, the voltages all have to be offset by whatever the measured (or calculated) junction potential is.

Now it's possible that the software will include this offset for you, after you enter the appropriate value. But I think it's better to do the offset after the recording, just so you don't get confused when Vcmd output by your amp doesn't equal the Vm displayed in Clampex.

patch clamper - When you zero the current in track you're accounting for everything but the junction potential. To prove it to yourself, try measuring the junction potential. Zero the current with the typical pipette and bath solutions you use, then put the pipette solution in the bath and record the voltage.

As for bleach to chloride wires, it's actually ok, but from an aesthetic standpoint, it repels me. Plus when I did it, I would always get little bleach spots on my clothes.

patch clamper said...

I haven't tried to measure the JP to test the TRACK circuit, but I will.

I'm curious because the Axopatch 200B manual states that the PIPETTE OFFSET knob does correct for the JP.

page 13:

"...Adjust the PIPETTE OFFSET control until the TRACK volgate is zeroed. At this point, the tracking circuit does not have to put out a voltage in addition to the pipette offset voltage to achieve zero current.
This specifies the proper setting of the PIPETTE OFFSET control to zero junction potentials and electrode asymmetries."

page 91:

"The PIPETTE OFFSET control is a ten-turn potentiometer used to add up to +/-250 mV to the pipette
command potential in order to compensate for the total offset of the liquid-liquid and liquid-metal
junction potentials in the electrode and bath...".

Nat Blair said...

True, but the other thing to remember, which I sadly didn't say in the post, is that when you get a seal, there's no longer a junction potential (neither of the ions are freely diffusing now). So when you offset it initially with just the pipette in the bath, that was ok, but that offset is now inappropriate.

Anonymous said...

Very good post.

patch clamper said...

I think the light just turned on for me with your last comment.

It's been unclear to me for some time why people measure the JP and account for it, because the Axopatch manual makes it sound like the amplifier accounts for the JP when you properly adjust PIPETTE OFFSET.

If I understand properly (please correct me if I am wrong):

When you first put the pipette in the bath, you DO use the pipette offset knob to achieve a zero current, which does compensate for JP and any other electrode asymmetries under that initial non-patched condition only.

Then, after you make the seal there is no longer a JP and so the voltages will be off by whatever the JP was initially. This is why you need to measure the JP of the solutions yourself and then apply the appropriate correction to your recordings.

Thank you for your information!

Nat Blair said...

Yes, that's it!

This was great, it helped both of us clarify what is going on.

yixin said...

I am a new patch clamper. I'm wondering how about I offset it immediately after I get the seal? Then after I rupture the membrane, can I do voltage or current clamp recording without considering the LJP? And is that offset appropriate,why?

Anonymous said...

I am a new patch clamper. I'm wondering how about I offset it immediately after I get the seal? Then after I rupture the membrane, can I do voltage or current clamp recording without considering the LJP? And is that offset appropriate,why?

Anonymous said...

I am a new patch clamper. I'm wondering how about I offset it immediately after I get the seal? Then after I rupture the membrane, can I do voltage or current clamp recording without considering the LJP? And is that offset appropriate,why?

yixin said...

I am a new patch clamper. I'm wondering how about I offset it immediately after I get the seal? Then after I rupture the membrane, can I do voltage or current clamp recording without considering the LJP? And is that offset appropriate,why?

Anonymous said...

Awesome post. Do you mind if I ask what your source is for this information?

Nat Blair said...

Years of experience and that Neher paper.

Why do you ask?

Anonymous said...

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Jamie said...

Thanks for this, really great couple of posts on LJPs you have. Just one question - why do all the other methods use 3M KCl agar, rather than this method which seems far simpler and easier? Is there any theoretical reason agar would better? I can only assume it's something to do with diffusion of the solutions. Any help would be greatly appreciated!

Nat Blair said...

Jaime -

Thanks, glad you found it helpful!

For the agar bridge, are people using it for measuring the liquid junction potential or during typical recordings?

You might use an agar bridge when you're doing typical recordings, because it will retard diffusion of the KCl out into the bath. Given that you might have 3 orders of magnitude less KCl in your external solution, you don't want all that ground KCl changing the bath [KCl].

But that reduction of diffusion isn't complete, so overtime you'll get a gradient of KCL leaving the bridge, and external solution entering the agar bridge. That gradient makes calculating the junction potential difficult. That's why a flowing KCL bridge (like the broken pipette) is preferred for the liquid junction potential measurement. The overall length of the measurement is short, so loading the bath with KCl from the ground pipette isn't an issue.

If I needed to use an agar bridge during regular recordings (perhaps you want to change external Cl- concentration, which would play havoc with the junction potential between it and the Ag/AgCl ground pellet), I'd just use my regular external solution, ~150 mM NaCl. The increased resistivity of this relative to 3 M KCl isn't going to be an issue with patch clamp recordings, where the vast majority of the series resistance is the access resistance between the pipette and the cell interior.

(That's not the case for something like the squid axon. There you have a silver wire shoved down the axon, and the resistance of the axoplasm between that wire and the membrane is the small, or smaller, than the resistance between the outside of the membrane and your ground electrode).

Brad said...

Thanks for the great post.

I was calculating the LJP by changing out the pipette solution and leaving the bath the same with a AgCl pellet ground in place on the rig as Juan was suggesting. However, it seems like we would have the same problem with different Cl concentrations interacting with the AgCl on the chlorided patch electrode which we changed the solutions out on.

Also, when I first put the electrode into the bath after changing to the internal solution, having zeroed my offset with ACSF, the voltage is initially 8 mV, but quickly starts changing and keeps going. Is this because the electrodes are not chlorided well or is it the two different solutions interacting like a battery?

How do you make/use a battery powered chlorider? I just jumped a 9V battery from a silver wire to my electrode through 3M KCl. I got some deposits once I figured out which way to run it, but they are whitish not dark grey like I get with bleach. Maybe this is the aesthetic difference?

Nat Blair said...

Glad you liked it Brad. Hope it was helpful.

Yes, anytime the Ag/AgCl solution changes its [Cl-] solution, you'll get a big change in the potential at that location. So if you are ever changing bath Cl-, use a bridge. You might not need a 3 M KCl bridge though, as KCl diffusing out into the bath might affect the cell(s), raising the typically low KCl in the bath.

As for rapid shifts in potential after zeroing it, it might relate to the way you chlorided it. If the wire is whitish, then reverse the polarity on it, and it should get covered with a black coating. That's what you want. If it's white, I would guess there's little AgCl on it, and that will certainly make the potential very unstable.

Brad said...

So you think leaving the bath the same (with the pellet) and changing out the pippet solution is a bad idea?

Nat Blair said...

No, as long as the bath solution stays the same (and contains some Cl-, say 5-10 mM), you can use the pellet as the bath electrode. Then the only junction potential present will be between the pipette solution and the bath solution.

The thing is, in order to measure the junction potential between your pipette and bath solution, you must change the bath solution. Thus necessitating the KCl bridge.

Anonymous said...

Hi All,

These comments just make it complete!

Nice theories, but do they work in practice? The LJP calculation tool in Clampex gives me 16 mV but I got only 7 mV when recorded as per Nat`s protocol. I repeated many times over and over, re-did solutions etc. Any idea what should I look at? Anyone out there with the same discrepancies between measured and calculated LJP?

Morven said...

Thanks for your very useful post! I have one question on the polarity of the junction potential:

I calculated my junction potential using pclamp's calculator and it came out to be +14mV.

I then measured the junction potential the way you described with internal solution in my pipette, and zeroed the amp with the same internal solution in the bath. When I change the bath solution to my external solution the amp reads -10mV.

Is one of my solutions really wrong, or does pclamp calculate the junction potential with respect to the bath? I can deal with an error of 4mV but not 24mV!

Andrew Yee said...

Hi Nat,

Thanks for the post! Would you mind correcting or confirming my understanding of this?

I have a similar situation as Morvern. Using a K-methanesulfonate internal solution I measured my junction potential to be -7.5 mV (by zero-ing in the pipette solution and then switching to my external solution). Clampex however estimates my junction potential to be +8 mV. I assume that this is because the program theoretically switches the two solutions around (ie. measuring the junction potential relative to the external solution).

From my understanding of junction potentials, since the methanesulfonate anion is relatively big (and less mobile) than the other major ions involved, my pipette should theoretically be more negative than the extracellular solution because of the residual negative charge caused by the methanesulfonate being left behind in the pipette.

Therefore, in order to have a true holding potential of -60 mV, I would need to set my command potential at -52.5 mV because the pipette (and the cell) are already 7.5 mV more negative upon breaking in, due to the methanesulfonate anions.

Is this correct??

Clampex gives the equation:

Vm = Vp + Vljp

So, in current clamp, the true membrane potential (Vm) is the recorded potential (Vp) plus the junction potential (Vljp). In voltage clamp, I assume that the true membrane potential (Vm) is the command potential (Vp=Vcmd) plus the junction potential (Vljp).

Thus, the command potential (Vcmd) I would need to set to get a true holding potential (Vm) would be:

Vcmd = Vm - Vljp

Therefore, to hold my cell at a true membrane potential of -60 mV, I would need to set my command potential at (-60)-(-7.5) = -52.5 mV.

Cheers,
Andrew

Nat Blair said...

Yes Andrew, you analyzed it exactly correctly. Well done!

Yoni said...

Thank you for a very helpful post!

I have one question though that is bothering me and you kind of referred to that in one of your answers. As far as I understand, once you create a gigaseal the bath solution is out of the equation for calculating LJP. The LJP that you have now (after breaking into the cell) is the one between your internal solution and the cell content. How do you measure this LJP? And how does measuring the LJP between the internal and external solutions help you estimate the cell's correct membrane potential once you're in the cell?

Thanks,

Yoni

Nat Blair said...

Yes Andrew, you've analyzed it completely correctly. If you switch the "sides" of the bath and pipette solution, that is similar to switching the connections on a battery. That will end up changing the polarity of the voltage.

Nat Blair said...

Yoni,

Once you have a gigaseal, the ions in the bath and pipette are no longer able to diffuse across the pipette tip. So, in this case, there's no longer any junction potential.

Anonymous said...

Hi Nat!
First, thank you for another great and helpful post!

I have encountered one problem when measuring ljp of my aCSF, namely, once I measured the ljp and want to check for the reversion of the potential when filling the recording chamber with my internal (KMeSO4-based) I observe strongly increased potential. I do not understand why.

I basically do what you have described in your blog, with slight differences. I use a seperate bath, filled with my internal and connect it via an agarbridge (3M KCl) to my recording chamber. In the start configuration I have the internal in the pipette, the recording chamber and my "ground-bath". Once the pipette makes contact with the internal in the recording chamber I use the pipette offset. BTW when entering my amp is in i=0 mode, to make sure there is no command voltage interfering. In the next step I exchange the internal in the recording chamber for my aCSF solution, "dip" the pipette in and read the potential. In my last step I exchange the aCSF for my internal solution to check for reversion of the potential to 0 mV, but instead the potential increases. Not to forget, I exchange the agarbridge every time I change the solution for a fresh one. As ground I use a Ag/AgCl pellet connected with the bath by an agarbridge.

Do you have any idea where in this procedure my error is?

Thorsten

realillusion said...

Thanks for the post. Very educational. I have a question, so i use KCL intracellular solution (150mM), does that mean the junction potential is very minimal? as both ions can diffuse efficiently. And is it a good way to measure resting membrane potential using such internal solution, by reading Vm right after break through and switch to I=0?

Pam said...

Nat,

This was very instructive, you explained it all very well. One question I had in reading these posts was what your response was to the people who said that they got different results when they measured the junction potential using your method versus using the LJP tool in Clampex. Could you comment?

Pam said...

Nat,

This was very instructive, you explained it all very well. One question I had in reading these posts was what your response was to the people who said that they got different results when they measured the junction potential using your method versus using the LJP tool in Clampex. Could you comment?

Nat Blair said...

Being a dyed-in-the-wool empiricist, I always trust what I can measure over what is calculated. My own measurements are very consistent over time, and when performed on different setups, etc. So, at least it isn't as though the values are jumping around.

To be totally honest, I've never played around with the JP calculator. But, I've often wondered if the parameters such as activity coefficient and mobilities of some ions we use in electrophysiology are taken from solid experimental values.

Are you getting very different values when comparing things? Or is it more in the range of ±5 mV?

Pam said...

I haven't tried yet, will soon! Thanks!

Pam said...

I haven't tried yet, was waiting for your comment! Thanks!

Pam said...

Looks like the response I posted didn't get posted! Thanks for your comment; I haven't tried to do the measurement yet, was waiting for your reply. And I completely agree with you about trusting what I can measure!

Anonymous said...

Hello all,

Very good post, I just wanted to add a couple of pointers for everyone from my experience with LJPs.


First off, here:

http://web.med.unsw.edu.au/phbsoft/Figl%20App%20Note2004.pdf

is another very well resource for measuring LJPs with Axon, both experimentally and with the software.

All of these resources can also be found at the website of Dr. Barry, who actually developed Axon's software calculator:
http://web.med.unsw.edu.au/phbsoft/LJP_Calculator.htm

Now for the technicality of correcting for the LJP with the amplifier, you have to be careful of your pipette resistances to not saturate your signal with your offset command (that is correcting the LJP before the experiment by applying an external command). A little trick I have found to work is to zero everything with the junction null in the bath and once cell attached, go to I=0 and display Vm. Since there is no JP once cell attached the previous zeroing command will give you the junction potential when in gigaOhm and I=0. Zero the Vm and you are corrected. This method works also for low resistance electrodes and on top of everything it gives you a good idea of the quality of the patch, since leaky membranes or seals will give you numbers far away from your LJP.

Try that or otherwise or adjust your holding command with the appropriate LJP offset. Be careful though of your polarity when you do the calculations (the experimental setup will here be super important).

Hope it makes sense and helps

Nat Blair said...

Thanks for the links anonymous.

In the end your procedure is basically the same as what I do. Personally I don't like the idea of zeroing the offset a second time, after getting cell attached. I prefer to have the raw data stored with the files to be exactly what the computer control created. But, I agree it's a slight difference in approach.

lysmata said...

Can someone explain to me how a microelectrode "feel" the potential in a resting potential measurement, when its surrounded by 3M KCl? And when we speak about "current injection" there is a variation of ionic concentration or not? Thanks at all

Anonymous said...

I am not very clear about the measurement of the membrane potential. Especially on the qualitative aspect of the phenomenon. What 's happened at the microelectrode? How a reference electrode inside a pipette, filled, for example, of 3M of KCl can "feel" the potential and can translate it in a current?

And also about Voltage and current clamp...What is meant by current injection? There is an augmentation of ionic concentration? If so, what kind of ion? And this, doesn't affect the cytoplasmic concentration of that ion?

Anonymous said...

Great blog, thatnks. You might be able to clear this up for me, it's been debated in the lab a bit.....

If we have an LJP correction of -9mV (we're certain about the direction of correction) and don't do an a priori correction, then if we command the cell to hold at -80 mV in voltage clamp what potential do we really need to hold at to achieve -80 mV - is it -71 or -89???? Thanks.

Nat Blair said...

Sorry for the delay in comments- when they first arrived, I tried to publish them, but had a Blogger glitch. Then it slipped my mind.

@lysmata - don't forget that what the electrode is feeling is the difference in potential between the bath and the pipette. And when doing the experiment, you've set the potential equal to zero when first putting the pipette in the bath.

Nat Blair said...

@Anonymous2 - if you're certain that the pipette junction potential is -9 mV, then for whole cells experiments (or outside-out patch), you'll want to command the voltage to -71 in order to get -80 mV.

Nat Blair said...

@Anonymous1 -

You can calculate how many ions are moving given the amount of current (current is amperes, which is coulombs per second, which can be converted to charges per second. Given the number of charges (which is really ions), and Avogadro's number, you can convert to moles. It turns out to be a small amount of ions.

In practice, you only have to worry about changes in bulk ionic concentrations in two main cases: 1) during long injections of large currents.
2) for changes in Ca2+ ions, where the starting concentration in the cytoplasm is ~10^-7 M.


As far as the microelectrode feeling the potential, when there is a complete circuit, the electric field propagates through that circuit at the speed of light. Then the voltage drops across each part of the circuit depending on its resistance, relative to the total resistance and the potential (as with any electric circuit).

Anonymous said...

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