This protocol describes how to generate carbon fiber electrodes. First, we'll fill the glass capillaries with carbon fibers that have had their sizing removed. Next, we'll pull the fiber capillary assembly into two electrodes and cut the fiber.
Then we'll arrange the electrodes to dip into epoxy. To seal the fiber into the electrode, we'll store the fiber electrodes on a rack in a hot oven. These electrodes are subsequently used to detect catecholamine release from vesicles in single cells with carbon fiber and barometry.
Successful recordings show low noise and well distinguished upward spikes on the recording trace. Hi, I'm Carrie Pike from Dr.Amy Harkins laboratory in the Department of Pharmacological and Physiological Science at St.Louis University School of Medicine. I'm going to show you a procedure for generating low noise carbon fiber electrodes to be used in pyrometry for measuring catecholamine release from single cells.
Let's begin To prepare the carbon fibers for making electrodes. First, cut a bundle of carbon fibers to a length that is about 1.25 times the length of the capillary pipette in the fume hood. Remove the sizing by boiling about 100 milliliters of acetone in a container and adding the fibers to the hot boiling acetone for 30 minutes or longer.
After boiling, remove the fibers from the heated acetone and transfer them to a clean beaker containing 50 to a hundred milliliters of fresh acetone. Transfer the fibers once again to aluminum foil, which can be transferred out of the hood. Dry the fibers by placing the bundle on an open 150 millimeter Petri dish and letting the carbon fibers air dry overnight.
Now that the carbon fibers are prepared, proceed to preparing the glass capillary tubes to fire polish the tubes. Use micro hematocrit glass capillary tubes, which are 7.5 centimeters long with a 1.5 millimeter outer diameter and a 1.3 millimeter inner diameter. Hold the tube in the center and expose one end to the flame.
Rotate the tube about three times while it is in the flame. Invert the tube and place the opposite end into the flame and rotate again three times. Both sides of the capillary tube are now fire polished.
Place the fire polish tubes in a glass dish and transfer them to the oven until they are ready to be filled with carbon fibers. Before starting to thread a carbon fiber into a capillary tube, be sure to wear a protective face mask. Then use plastic tubing connected to a vacuum with a filter to suck the fiber into the glass tube.
Next, cut the fiber using a scalpel. Be sure to leave a quarter of an inch of fiber out of both ends of the tube. The threaded carbon fiber is now ready for pulling to pull the pipette fiber electrode.
Place the capillary tube into the electrode polar and tighten the knobs to hold the capillary tube in place. Next, set the polar at the optimal settings and manipulate the switches to execute two poles. Once the pipette is pulled in half, separate the two halves by cutting the bare fiber at the center.
Using scissors. Remove each electrode half and pull on the protruding fiber to break the glass seal. Trim the fiber with scissors so that only one eighth inch of fiber is left outside of the tube.
Repeat these steps until about 16. Fiber electrodes are pulled before proceeding to seal the electrodes to make the epoxy glass seal.First. Heat a pre-made aliquot of epoxy on a hot plate that is set to 55 degrees Celsius for six minutes In a fume hood while the epoxy is heating, wrap about 16 of the pulled electrodes into a bundle.
Using tape after the epoxy is heated for six minutes, add 0.7 grams of hardener to the hot epoxy. Mix this by swirling the vial. Dip the bundle of electrodes into the hot epoxy plus hardener solution.
Dip the bundle twice each time for about five seconds, so that a sufficient amount of epoxy will enter the glass tips through capillary action. Undo the bundle and place the electrodes onto a cardboard holder with slots. Place this tray into an oven set to 100 degrees Fahrenheit.
After at least 48 hours, examine each electrode under a dissecting microscope. Discard the electrodes that did not fill with epoxy. Have a smash tip.
Have more than one fiber or can't be used for other reasons. After inspecting each electrode, place them back on the cardboard holder and back into the oven. Electrodes are usually good for about one month after fabrication and require cutting of the carbon fiber before using for pyrometry on a cell.
To cut the carbon fiber, place the carbon fiber electrode carefully onto a glass slide with Teflon tape. Visualize the tip under a dissecting microscope. Next, use a clean scalpel blade to cut perpendicularly across the carbon fiber to leave a flat clean surface for placement of the carbon fiber against a cell.
Immediately prior to recordings. Backfill the capillary with three molar potassium chloride using a filling syringe, and make sure to dry the end opposite the fiber. To remove excess salt.
Place the electrode onto the silver, silver chloride pellet of the electrode holder. Attached electrically to the pyrometry head stage. Tighten the electrode holder to keep the electrode stable.
Position the electrode adjacent to the cell and proceed to recording RO metric events. This protocol describes how to generate and cut tric electrodes. During tric recordings, a carbon fiber electrode is placed against the surface of a secretory cell.
Exocytotic activity is observed as tric spikes, which indicates the electrochemical current caused by the transfer of electrons after oxidation of the catecholamines. With practice making and cutting EmPro metric electrodes, noise levels can be greatly improved over many of the commercially available electrodes. I've just shown you how to fabricate and parametric carbon fiber electrodes when doing this procedure.
It's important to pay close attention to the details of the protocol for consistent and low noise electrodes. So that's it. Thanks for watching and good luck with your experiments.
