Wednesday, March 25, 2009

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.

8 comments:

Dr. Jekyll and Mrs. Hyde said...

Aside from you forgot to mention how despite the fact that everyone in the lab does virtually the same thing, yet somehow we all like different pipette programs.... :)

I'm way too lazy to flame polish the back ends--I just rechloride every week and change the o-ring every month or whenever I'm having problems. What do you prefer about doing the flame polishing instead?

Nat Blair said...

Rechloriding every month and replacing the O-ring every 6 months?

That was the way I was first taught in Rich Lewis's lab, and how we did it in Bruce's lab. At this point I doubt I could force myself to stop doing it that way. But I'm stuck in my ways, not doubt about that!

Neuropharma said...

I LOVE THIS POST. Thanks Nat :)
Looking forward for more :)

I have several comments (if you don't mind):

NB, "when you distill it down to two sentences, it pretty much trivializes what I spent years learning and do everyday"

Exactly! A PhD student from another lab came to ours to do some electrophysiology experiments he thought would be cool to include in his thesis research. He was shocked to discover that it would take him such a long time to learn the technique (he's starting from level 0) and said that it seemed so easy when reading it from some published paper! PI said, "no one can understand an electrophysiologist but another electrophysiologist!" You read it and it sounds piece of cake, but no-one would know what the experimenter went through to get the results but someone who really tried doing it before. The student ended up changing his mind; he doesn't want to do cool electrophysiology anymore!

DJMH, "somehow we all like different pipette programs"

True! The funny thing is that we have a postdoc who asks me to always return the puller to the program she uses after every time I pull mine! One day we were nearly going to end up fighting because I forgot to do so and she pulled the "wrong" electrodes! Even funnier is that she never gives me those "wrong" electrodes, she throws them away! Hope no such problems happen in your labs too because of that.

Another thing: we never flame-polish electrodes in our lab either. We replace the silver wire whenever we feel the need to, yes, nearly every week. We don't have to waste time waiting for the wire to rechloride. We have a container filled with Clorox and a bunch of silver wires. Whenever we want to change the wire we're using, we take one from the container and put the "un-cholrided" one instead to get rechlorided until we need it again.

Nat Blair said...

Glad you liked it Neuropharma! I've got a lot of posts lined up, waiting to finalize. The pictures are taken and the currents are recorded. I just have to beat them into posting shape.

True! The funny thing is that we have a postdoc who asks me to always return the puller to the program she uses after every time I pull mine!

Totally lame. I'd not do it just to spite her. Or just hit the "reset" button after I was finished. What, she's so goddamn lazy she can't hit a total of three buttons?

We have a container filled with Clorox and a bunch of silver wires.

*shudder* I'll admit that I'm an anal retentive freak when it comes to patching, but I hate using bleach. First, I always ended up with little bleach spots all over my clothes. Secondly, there's just something that gives me the hee-bee-jee-bees about using bleach on the wire. It's irrational I know.

Instead, I've always used a battery powered chlorider. Perhaps the subject of another post.

Comrade PhysioProf said...

SUCK!!!! SUCK HARDER!!!!!!!

Dr. Jekyll and Mrs. Hyde said...

In fact, my very first patch experience happened to be an in vivo patch, with my PI hovering over me saying, "Blow....blow... Ok now give a little suck. Yeah, a little more."

But I got the cell.

Neuropharma, that postdoc needs an attitude readjustment. Tell her to write a grant and buy her own damn puller.

I'm ok with the KCl/battery combo but it seems to wear off faster than bleaching....that might just be my imagination though.

E-phys at a PUI? really? said...

You totally rock

Nat Blair said...

Thanks E-phys. Flattery will get you everywhere as they say.

I still have lots of ephys methods and techniques posts lined up. Final paper stuff, lots of experiments, and some sick kids have force that into hiatus.