Well, today puts things again in the realm of micropipette prep, and again things are not coming out precisely as expected. I’ve found that even though I thought previously that backfilling was not taking place in the pipettes that were finely tipped, apparently it is. The pipettes do not seal off on the end, even with a decent amount of elongation, it’s simply that filling them is difficult. The backfill is not visible after letting the electrodes sit for a period of time (10-30 mins), but becomes quite apparent after attempting to fill and being treated with a nice bubble surrounded by fluid towards the tip, and towards the main portion of the pipette. When I have this figured out, I’m going to write a little guide, and perhaps post it on the Wiki I’m thinking of starting, so that at least the other google empowered folks who are attempting to do the same thing won’t have to go through the same steps I did over a number of days to get things working.
That said, I’m more and more convinced that this is the sole problem being encountered. Low impedence electrodes with fluid continuity do not have the awful noise issues experienced before, and perhaps as soon as this technique is down, recording will be no problem.
What I thought initially would be hard, proved to be exceptionally easy then exceptionally impossible. I’d heard from others long before I’d gotten into the business of pulling my own electrodes that this would be a road fraught with dismay and frustration, or at least fraught with cursing and many discarded electrodes that did not work out. When I started in on this, I got our lab’s shared laser puller up and running. This part wasn’t hard, and merely required reading the directions. Then after less than half a day of pulling electrodes, I was getting ones that were around 30-40 Megohm. What, I found soon after, however, was that that was 30-40 Megohm with a tiny bit of air still holding out near the tip, and that filling the very tip of the electrode by capillary action is necessary to get a proper electrode. As an aside on this matter, if you have crazy noise issues that creep into your signal and you’re sure it isn’t clogged: you may have a bubble in there somewhere. It seems however, that getting this capillary action going becomes exponentially harder and slower as the tip diameter closes down. Currently, this is showing itself in the sense that I can only get 4-10 Megohm micropipettes that actually sip from the KCL I’m placing them in.
I’m writing this, and posting it, both for my own reference, and for the fact that I couldn’t really easily find anything on methodology for pulling electrodes. Perhaps I should start a wiki for such things? It would not have to be limited in scope only to that particular topic, but rather be a site covering lab technique and the various ins and outs. Personally, this rather appeals to me for the reason that a lot of the manual lab work requires what seems to be almost a black art, and either someone in your lab can show you this art, to some extent, or you just have to spend enough time banging into things that you figure it out on your own. I will have to think about this…
So, I’ve recently been training up on doing electrophysiology in the lab, and I thought I’d blog about the experiences a bit (mainly technical details) as I go along since I’ve encountered a number of issues along the way, and some I’ve resolved so far, whereas others still need some work to get going.
This is basically both for personal reference, as well as for anyone who happens to be interested in these details. One of my standard solutions when looking for information about things that don’t seem to be working correctly is through googling, and I’ve really not been able to find any good information on all this stuff.
