In patch clamps, operators are required to make a very-fine-tipped glass micropipette for their electrode. Due to the small bore of the glass tip (around 1-2 um), air bubbles are sometimes trapped permanantly at the tip of the glass after backfilling with the internal solution and finger tapping on the glass.
In other words, is there a technique that could fill the internal solution to the very-fine-tipped glass pipette more effectively than just by finger tapping?
Thank you very much in advance!
Are you using filamented glass?
http://www.sutter.com/PDFs/technote_FilamentGlass.pdf
If your glass is not filamented, you may have to front-fill your pipette using suction until there's enough solution in the taper to allow bubble-free back-filling.
I agree with Christoph. Front-filling helps and so does warming to the solution to 37, if that wont compromise the integrity. Also, you can vigorously 'pluck' the sides of the pipette to dislodge large bubbles .
Electrodes pulled from filament glass are so easy to fill, just use it !
Our group currently uses the B150-86-10 fire polished but non-filamented glass capillaries. We planned to use the filamented one before, but it seems that for our application the filamented one we currently have does not suitable for our puller or its protocol. Our puller has a pretty low RAMP value.
Any clues please?
Dear Dr. Kwan,
I faced this problem many years ago, when I worked with a Canadian colleague, Dr. T. P. Hicks, who used to solve this problem by first 'boiling' the pipette into distilled water. The pipette was fixed in a plexiglass and immersed in distilled water and then boiled.
With this process, the pipette became completely filled, without air bubbles. Then the excess of water was withdrawn with a syringe (from the top opening of the pipette), and thereafter the pipette was back-filled with the adequate recording solution , and left undisturbed for several hour to equilibrating the ion concentrations inside.
Of course, when possible, using glass capillaries with filament would be the easiest solution...
Brilliant! I tried something in the "high-temperature" direction previously but using a lighter to "fire polish" the pipette tip instead, I thought heat can change the volume of the air bubble while creating a convection to drive the air bubble away.
However, maybe due to inappropriately high temperature induced by the lighter's fire (even in a flash/short time), the tip of the micropipette actually sealed up or changed shape.
Anyway, this boiling technique sounds the best solution to us at this moment because our group has purchased quite a lot of the non-filamented glass capillaries.
I agree with Kevin and Christoph. Someone should make a YouTube video of effective "plucking" to rid the electrode of visible bubbles. Even with these bubbles gone, you may encounter smaller bubbles not visible to the naked eye. These will be evident when you first touch down to the bath with the electrode: the resistance is too high. These smaller bubbles can be expelled with positive pressure. Find them on the screen (in the optical axis) and then expel them, staying well above the preparation. Provided that you are exchanging bath volumes at a reasonable rate, there shouldn't be a problem with accumulation of internal solutes in the bath.
The lab where my rig is located is run by a former post-doc of Erwine Neher, who swears by an approach taught to him using sharp-toothed forceps. Pinch the pipette (at the middle of its body) with the forceps and drag the pipette through the teeth to create vibrations through the glass. repeat 5-10 times. This usually dislodges most bubbles. Typically, I can tell there are no bubbles in the pipette when I apply positive pressure to the pipette (once its inserted into the chamber) and do not observe changes resistance.
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