This procedure begins by positioning the pipette tip near the polishing filament at 100 times magnification. Next turn on air pressure and heat. The filament.
The pipette tip will expand to form a blunt shaped tip. Turn off the heat to obtain a smaller tip opening. Turn off the pressure and heat again.
Hi, I'm Brandon Johnson from the laboratory of Dr.Miriam Goodman in the Department of Molecular and Cellular Physiology at Stanford University. Today we will show you a procedure for pressure polishing patch pipettes. We use this procedure in our laboratory to study ion channels and membrane patches and NC elgan neurons.
So let's get started. Pull a set of six to 10 patch pipettes. Consult a local expert or our visualized experiment making patch pipettes and sharp electrodes with a programmable polar for help.
To begin the procedure, set a compressed airline pressure to about 40 PS.I then connect the compressed airline to a four-way control valve. Connect a length of plastic tubing to the four-way valve and attach a high pressure pipette holder to the loose end of the plastic tubing. Use a micro forge rig to perform the polishing.
We use the Ccpm two kit from a LA Scientific. This rig has a high pressure pipette holder, a platinum heating filament controlled by a foot pedal and a microscope equipped with a low and high magnification 10 times in 100 times long. Working distance air objectives are ideal because it is critical to be able to observe the pipette opening during the protocol.
Next, verify that the pressure valve is in the off position. Then place a pipette into the pipette holder and tighten the fitting to finger tight for the safety of the lab. Make sure the pipette is firmly held in the pipette holder.
Otherwise, the air pressure will cause the pipette to be ejected from the holder. As an optional step, you can coat the pipette with a hydrophobic insulator such as wax to decrease capacitance and improve noise characteristics. Now place the pipette in holder in the micro forge position the pipette holder onto the microscope stage so that both the tip and the polishing filament are visible at low 10 times power on the micro forge.
Bring the tip approximately 50 microns from the filament, then switch to the high power 100 Objective, both the pipette tip and the polishing filament should be in focus and in the field of view. If not, adjust the height of the filament. Since the pipette position cannot be adjusted, the pipette tip and filament should be on the opposite sides of the field of view.
Avoid positioning the pipette tip too closely to the filament because it will expand as it heats and could bump into your pipette. Once the tip and filament are properly positioned, turn the valve in order to direct pressurized air into the pipette and heat the pipette tip until the taper behind the tip begins to balloon outwards. Stop the heating before the tip balloons too extensively.
Experimentation is required to determine the appropriate distance, heat, and time for the desired shape. As an optional step, you can also polish the tip in the absence of pressure in order to reduce the tip opening diameter. Also, keep in mind that these polishing steps can be repeated to fine.
Tune both the shape and dimensions of the pipette tip. When the polishing is complete. Switch off the pressure point the pipette tip in a safe direction.
Remove the pipette and place it in a clean storage box where it will be protected from dust. For the safety of the lab, make sure to turn off the pressure before removing the pipette. Otherwise, it can be ejected from the pipette holder.
Upon removal, We've just shown you how to pressure polish pipettes for improved patch clamp recordings. When doing this procedure, it's important to remember to coat the polishing coil with high melting temperature glass in order to minimize deposition of vaporized metal on the pipettes. And to use caution while pipettes are exposed to pressurized air.
Make sure the pipette is firmly held in the holder and to turn off the air before removing the pipette from the holder. So that's it. Thanks for watching.
Happy pressure polishing and good luck with your experiments.
