A Tale of Two Grounds

It was the best of rigs, it was the worst of rigs,

It was the age of 0.07 pA RMS, it was the age of 0.5 pA RMS, ±5 pA p-p at 60 Hz,

It was the epoch of separating signal ground from power ground, it was the epoch of connecting all power grounds to the signal ground,

It was one really annoying run on sentence written by some old white dude, it was a kick ass blog post written by a middle aged white d00d.

There's one thing I've learned here as postdoc that I've actually never seen discussed in other places. When I first heard it, I did a "Watch you talkin' about" head turn, thinking it was crazy.

That thing is, to get really good noise on your patch clamp rig (good meaning low here), you can make 2 grounds. One ground connects everything that is physically close to the headstage. This gets connected to the signal ground plug. The second ground connects everything else. That gets connected to power ground.

Figure 1: Nat's newly refurbished set up (which is tight if I do say so myself). Axopatch 200B and Sutter MP-285. Green As indicate things attached to signal ground. Magenta Bs are connected to power ground.

So what goes on the signal ground?

• Microscope
• chamber
• condenser
• bent piece of metal that can be put right in front of headstage/chamber to further shield (not shown)

That all gets connected to the gold pin at the back of the headstage (or equivalently at the signal ground input on the back of the amplifier). Note that you'll need to break the power ground connection (with a 3 to 2 adaptor). Doing this will get you most of the way there.

What goes on power ground?

• Faraday cage
• air table surface
• manipulator

This is connected to power ground (either attach it to an exposed copper pipe or to power ground through the case of the manipulator). Doing this will get you to the super low RMS values specified in teh Axon manual.

Now I no longer fear denoiseing the setup.

The only tricky things I've run into are that most of the BNC inputs on the Digidata are connected to power ground. So usually I have to break the ground connection to connect the gain value output from the amplifier to the digitizer input (I've never had the amplifier signal output to digitizer analog in cause this).

I just turned on my amplifier, and with a pipette holder on, the PATCH mode RMS noise is 0.099, and WHOLE-CELL mode is 0.48. SHU-WEET!

What is a junction potential?

It's clear to me that a number of visitors to this humble blog arrive each day via a Googly search for the term "junction potential." I can only imagine that some must be fellow electrophysiologists, perhaps in their formative larval stages, searching for more information about this important topic. So, as a service to these folks, I thought a post or two about junction potentials, would be in order. First, what is a liquid junction potential? Then, How do you measure and correct for them?

So, what is a liquid junction potential? Sure, maybe you could look in some of the Electrophysiology Bibles. Or maybe you could even hit up an electrochemistry textbook. But it's 2009, and you've got two things on your side: Google, and me. So forget that, and allow me to regale you with the story of the liquid junction potential:

Long ago, in a galaxy far far away, there was a Gedanken experiment...

Figure 1: Set up of the Gedanken. No, it ain't to scale, though aspartate is bigger than potassium. Not shown is the impermeable wall separating the two solutions. Hey, it's my Gedanken thank you very much.

And in this Gedanken experiment there was a pipette filled with your typical pseudo-intracellular solution: You know the drill, high potassium (light blue), low calcium, and an anion species that's usually not chloride. This anion could be something like methanesulfonate, gluconate, or my own personal favorite, aspartate. The main thing to note is that all of these are bigger than chloride, and bigger than potassium. Thus, they have a lower mobility, meaning they don't diffuse as quickly as the accompanying cation.

Now, what happens when we stick this pipette into a bath solution that has your typical extracellular saline, made to mimic extracellular fluid (i.e., mostly sodium chloride)? Well, the chemical gradients favor the pipette constituents diffusing into the bath, and the bath constituents diffusing into the pipette. But remember, the aspartate is big, so it doesn't diffuse as quickly as any of the other ionic species. That slower diffusion of the anion leaves a net negative charge in the pipette. This charge separation across the junction between two solutions is THE LIQUID JUNCTION POTENTIAL!!!11!!!1!


Figure 2*: The Gedaken imposed barrier is removed, and ions are diffusing down their electrochemical gradients. The bigger, slower aspartate can't keep up relative to the smaller, faster potassium, sodium and chlorides. They get left behind in the pipette, generating an excess of negative charge.

Note that a liquid junction potential would also occur if the bath solution has cations and anions with significantly different mobilities. It just turns out that sodium and chloride have pretty similar mobilities, so that their contribution to the liquid junction potential is much smaller. But if you have N-methyl-d-glucamine (NMDG) as the main cation in your pipette solution, you'll have an excess of positive charge in the pipette solution, and a corresponding slightly positive junction potential.

Next up, how to measure the liquid junction potential.

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*-Note that these figures were created using Inkscape, a very cool and usable opensource vector
graphics drawing program (a la Illustrator). Check it out, download it, play around with it!

A pictorial presentation of pipette pulling

In the interests of both responding to Dr. A's request for pipette pulling related pics, and appeasing the apparently still irked electrophysiology gods, I present to you a brief montage of the glorious task of patch pipette fabrication!

First, what the heck are we even doing? Well, we're gonna pull a glass needle, fill it with salt solution, stick it on a plastic holder with a wire inside, maneuver it to a cell, apply a little suction, and let the magic of "seal formation" occur. Next, assuming we're doing whole cell voltage clamp, we break the seal membrane with more suction, gaining control of the voltage across the cells' remaining membrane, while recording the current (also filling the cell with our pipette solution). Sheesh, when you distill it down to two sentences, it pretty much trivializes what I spent years learning and do everyday.

The opening of the pipette tip will be ~1 µm, while the cell is on the order of ~10 µm. Obviously, if the pipette tip is too big, then we'll just suck up the entire cell. Not good. But, as the pipette tip gets smaller, the resistance between the pipette interior and the cell interior gets larger. Also not good. In fact, that causes a whole host of problems that are left as an exercise for the reader to derive (ok, just kidding. There's a series of posts reserved for this, with current working title: "Dr. RseriesLove, or, How I learned to stop worrying and love the fact that my currents are all wrong)

I start by cutting the capillary glass, by scoring it with a diamond pencil and breaking it off to the correct length (so it'll fit in my particular set up, given the headstage position, etc.).

Then I smooth the ends of the capillary glass with a bunsen burner flame, because jagged end (even how it comes from the factory) will scrape off the silver chloride on the wire that transmits the current from the ions in solution to the electrons in the amplifier circuitry (as well as tearing up the O-ring in the headstage holder).


Then we move onto the puller itself. There are many different kinds of pullers, but having been in a number of electrophysiology labs, I would say most people use pullers made by Sutter Instruments. The basic puller operation is to melt the glass capillary while pulling on either end, drawing the ends apart. Now to get a nice wide tip patch pipette, we use computer controlled application of the heat, allowing you to stop the heating a certain time after the capillary begins to pull apart. Over repeated heating/cooling cycles, you can make the perfect pipette.
Here's the puller, a P-97, and if you unscrewed the 5 screws on the font panel, you could peer in and see the brushless super quiet 92 mm fan we installed (way in the back of course, a real pain in arse to reach). The smoked plexiglass cover opens to reveal:


The business end of the puller. The circle thumbscrews clamp down on the ends of the capillary and maintain tension. The capillary feeds through the box filament, which gets hot when the puller is activated (sorry for the flash glare here). When the glass separates, we're left with a pair of pipettes, which we fire polish by bringing them close to a red hot wire (observing under the microscope). Finally, we're ready to patch!




The pipette is filled with intracellular solution, stuck in the polycarbonate holder (which has the silver wire in it), and stuck into the headstage of the amplifier. The suction tube allows you to provide postive pressure while you're approaching the cell, or negative pressure to form the seal and to breakthrough. The cells are sitting in an extracellular-like solution in the chamber, and the pipette approaches under micromanipulator control (here, a piezoelectric based Sutter MP-285), all the while watching through the microscope. In fact this pipette in this picture is making a GOhm seal on a little HEK cell. Of course, when I applied suction to break through, this cell was terribly leaky (again, Electrophysiologicus, patron of patchers, I'm like so over that hubris- could we maybe move on now?).

If you look closely, the tip of the pipette is wrapped with a thin strip of Parafilm. This helps reduce the capacitance of the pipette, but isn't nearly as time consuming or messy as using Sylgard. A requirement for setting the series resistance compensation. All of which are good topics for future posts!

Hope this was at least mildly useful to some people out there, and marginally enjoyable to others. If anything's not clear, just fire up the comments and lemme know.