Switched out the filament on our puller and now the patch electrodes appear different and my new recordings have different decay slopes then before. Its the only thing that has changed.
Agreed. I haven't systematically sampled the effect of the pipette taper on current kinetics, but access resistance has a major effect on the shape of the synaptic currents.
The electrode geometry will affect the electrode capacitance and therefore the shape of the capacitance transients in response to a change in voltage. What measurements are you making? How are the transients different?
Hello,
Well Chris may be right. You should check the access or series resistance of your electrodes. What are their resistances measured in the bath recording solution in both conditions.
What do you mean by switched out ? Replacing the old filament by a new one? You may have therefore increased the efficacy of the heating current and got sharper electrodes.
Yes it can. You want to keep your access res as low as possible and, even more important, constant between recordings and within the same recording.
You are talking only about decay slopes. Are rise times also changed? If a too low access resistance is causing the slowing of decays you should also have slower rise times. What is the access resistance with your new pipettes and in what range are the kinetics of the responses? Access resistance does not influence kinetics in a linear fashion.
Yes, in two general ways: the decay slope contributed by the electrode depends on the capacitance of the electrode and the resistive access to that capacitor (I.e. A capacitor will charge more slowly if the resistance to get to the capacitor is higher) . So the geometry of the electrode can affect both it's resistance and capacitance, and thus the decay time constant.
But another related factor is that the specific capacitance is nor generally uniform over the length of the electrode. The electrode capacitance is mostly formed by the glass insulating wall with saline on either side. The thickness of the glass wall of the electrode tends to increase with distance from the tip. So the time constant of the specific capacitance is decreasing as you get farther from the tip. This becomes a factor because the decay time that is observed is due to the sum of all these different time underlying constants. From a practical standpoint it means that capacity compensation circuits on electrophysiology amplifiers are never going to be completely effective, because they can compensate at one, or at best a few time constants. The compensation can't deal with the continuous nature of the actual capacitance. From the point of view of the question, the this aspect of the geometry of the pipette absolutely affects decay time. If one could fashion an electrode where the glass wall thickness increases more quickly with distance from the tip (or coat the electrode with an insulator), then this stray capacitance would be minimized, and decay time improved.
The geometry will have an impact on the capacitance of th pipette and the serie resistance. Moderne amplifier are able to compensate these parameters and to avoid the impact of the pipette on the measured currents. You can have problems if you do not make accurate compensation or if you have a large serie resistance
Two factors need to be considered in pipette design: (1) current amplitude and (2) current kinetics.
If you're dealing with very small currents (e.g., single-channel voltage clamping), the pipette's resistance is rarely an issue because you can readily clamp to the desired potential without having to worry about saturating your pipette's ability to mediate the corresponding current.
From your description of altered rise & decay times, I assume that you're not looking at single-channel currents, but at macroscopic current (e.g., whole-cell). Here, series resistance (a combination of factors, including the pipette resistance, the opening of the access hole you create when you go into the whole-cell configuration, etc.) issues come into play. Depending on the voltage you dial in (e.g., a large voltage step) the system may not be able to clamp fast enough. You seeing altered slopes is the result of the clamp not having reached steady state, yet. Essentially, your amplifier is still attempting to get to the dialed-in voltage while you're recording... obviously not a good thing. The way around this is to supercharge the membrane, which is possible with most amplifiers these days (consult your amplifier manual on Series Resistance Compensation (Rser comp). Of course, there are limitations to what you can achieve with Rser comp, so the design of your pipette is very important, especially if you're dealing with fast & large currents (e.g., voltage-clamping Nav channels). Here, a short, stubby design is preferred to allow for sub-millisecond control of the potential.
You may want to consult Sutter Instruments Pipette Cookbook:
http://www.sutter.com/PDFs/pipette_cookbook.pdf
Also good (but possibly overkill) is Brown/Flamings book:
Brown & Flaming "Advanced Micropipette Techniques for Cell Physiology" IBRO Handbook Series; Methods in Neurosciences (available used from book sellers for a couple dollars, ISBN 978-0471909521)
I think there is also a chapter on pipette design in Neher & Sakman's "Single channel recording" (ISBN 978-1441912305). Don't have my copy handy; I think one of my students has it! :-)
UPDATE (20Mar2014): Book recovered! Yes, Chapter 2 has a wonderful discussion on pipette geometry.
And, of course, always a good thing to consult - the reference for all those who enter the world of patch-clamping: Axon Instruments "The Axon Guide" (available as PDF online from different sites; Molecular Devices, sadly does not sell it, I believe).
Another thought: you should make it a habit to note down your pipette resistance (open, in-bath) for every single experiment you do. That way, you can catch day-to-day variations in your pipette resistance and adjust your pulling accordingly. Humidity, etc. can significantly affect your pull. Changing the filament, of course, will drastically change how your pipettes emerge from the pull. Since you can visually make out a difference in your pipettes between before and after your filament exchange, you will have to spend a couple hours figuring out the new numbers for your puller that produce the same pipettes as before.
UPDATE 20Mar2014:
I interpreted the rise and decay in your question as referring to an ionic current (e.g., Nav or Cav), hence me thoughts on current amplitude and kinetics. It now appears to me though that you were talking about the pipette's capacitive transient. If so, I agree with what has been said already. Minimize your pipette capacitance using wax, Sylgard, or parafilm (neat! never tried this before). This is especially important if you use large OD pipettes (e.g., Kimax 51 #34500-99, OD around 1.8 mm).
Everybody has provided a good answer. In my opinion, the best patch clamp pipettes are those with rather short tappers (which decrease the likelihood of "clogging" or "sealing over") but more importantly, low tip resistance, which I prefer between 3 and no more than 10 MegaOhms when in the bath. We routinely measure this value before approaching the cells.I have the luck of using switching clamp amplifiers, so that I can get away with using higher resistances when necessary, but still, for me 7 Meg seems like a magic number. Another way to reduce capacitance is lowering as much as possible the level of solution in the recording chamber. But as many have already pointed out, it is the tip resistance what really matters. After that, a perfect Gigaseal, a clean cell access and a rather high access (whole cell) resistance) will help to give you good Vm control and nice recordings.
I agree with the comments made by the other answerees. The puller is very likely making your electrodes have different tip shape and electrical series resistance.
Your answers are all there.
Note that decay time is usually more sensitive than rise time to clamp conditions.
Practical solutions, in addition to series resistance compensation:
Ideally, you want to patch the cell with the largest diameter possible to get a series resistance as low as possible. However, when you use 1-2 MOhms pipettes, patching becomes a real challenge. But then, you get nice kinetics.
Note, however, that you can a dramatic decrease in clamp quality (hence current kinetics), 50 microns away. There is a nice paper from Z. Nusser about that.
If you use big pipettes, I strongly suggest polishing the tip (there are cheap solutions to do that). This will dramatically improve the quality and stability of the recordings.
It is also important to put wax on the pipette, as far as possible (i.e. as close as possible to the tip). This decreases a lot capacitance problems. This is what people use when doing single channel recordings.
Of course, this means painful and time-consuming processes, but this is the price to pay to get good recordings.
That parafilm idea is excellent! Pippettes tips can also get polished by applying between 50 and 70% of the melting heat to the tips (pushing back the tip inside the filament) for a few seconds; as this tends to close the aperture, it is wise to use it on wide open tips (~2-4Megs), and adjust the timing according to results. One thing that has not been mentioned and I cannot repeat enough is that internal solutions, tubing and pipettes MUST be absolutely clean and free of grease to get the best seals. It is painful to keep re-learning that!
I used bees wax in the past (and Sylgard of course), but I will definitely try the thin stripes of parafilim. Thank you for the tip.
From my experience the most crucial parameter is a series resistance (a combination of factors, including the pipette resistance, the opening of the access hole you create when you go into the whole-cell configuration, etc.). When it is large your time parameter evaluations are wrong as well as holding (in the case of huge currents). Try to improve this factor. If you menage to have Rs less than 15 MOhms your pipette geometry is good. Nevertheless don't forget to compensate pipette capacitance before cell touching. Good luck
Nathaniel Blair, I have never heard of parafilm for that! How do you apply it to the pipette? Sounds great!
I agree that keeping series resistance as low as possible is critical. Use electronic series resistance compensation if you can (but be careful for oscillations here that may lead to cell recording loss). We have found that going to higher temperatures reduces series resistance (I think the glass expands during an increase in temperature). So you could try recordings at higher temperatures, but this will change a lot of things in your cells. Currents will tend to get faster at higher temperatures. When you are in cell attached mode, before you break-in, you should apply the Cfast compensation of the patch amplifier. This will get rid of pipette capacitance. You can reduce pipette capacitance with dental wax coating of the pipette. Its pretty fast. We can get the pipette capacitance down to 4-6 pF and this can be compensated well by the amplifier.
Nathaniel and I hail from the same spot, and we all use the parafilm trick routinely. Just wanted to note that we experimented with a variety of dental wax types about a year back to see if we could beat parafilm, and found that the wax not worth the trouble for our uses.
It doesn't take long to get competent at wrapping with parafilm. Also it's a bit of a part trick when another physiologist is watching so there's that. One tip learned from painful experience: Don't wrap too tight. The parafilm will creep up on the tip during a recording and push the cell off your pipette.
Thank you all for the responses. After i changed the filament on the puller, i did a ramp test to adjust the patch electrode tip close to what it was before, ~3-4 megaohms. However, it is variable now between 2.8-15 megaohms, i used the ones ~3-10 megaohms. The new tips also appear to have less taper, and thats why i ask if it may be an issue for whole cell decay slopes. I'm finding that my decay slopes are greater with these generally smaller tips with less taper, could electrode Rs be the issue? i would assume that greater Rs would lead to slower decay slopes.
Thank you Dr. Lossin for the resources and the tip, i will start keeping track of my pipette resistances.
I think impedance of pipette is more important that geometry for changing rise and decay of currents. My suggestion is measuring new pipette impedance and compare that with previous ones and correcting your pulling program. Also looking of geometry of tip of your pipettes under microscope would be beneficial.
Besides improving the protocol, you may also pay attention to the humidity, according to Sutter manual. It is also said the two wheels pulling pipettes apart may be ruined and lead to unstable pipette.
Try to use thick walled pipette glass to pull your pipettes. You can also try to lower the level of your external bath solution so that the pipette is only exposed to fluid at its tip. You can also blow into the pipette while you pull it to get a wider taper. See the papers of Miriam Goodman (Stanford Univ.). She has a cool technique to get pipettes with a wide taper (low Rs) for recordings from small neurons of C. Elegans.
As Chun-feng told, humidity is very important and you maybe need to regenerate or change Drierite granules if they color have changed to pink.
