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 electrop
hysiology 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.
