This video demonstrates a procedure to create and use vibrating probes used to measure bioelectric current from biological samples. First probes are manufactured by taking blank stainless steel probes and electroplating their tips with gold. And then platinum probe settings such as frequency and phase angle are then determined and the probe is calibrated.
Measurements can then be taken from the sample of interest data is obtained that reveals the character of bioelectric currents generated by and flowing around the biological sample. The main advantage of this technique over existing methods is that it is non-invasive, highly sensitive, and has a high signal to noise ratio. This method can answer key questions in the medical field, such as how wound electric fields stimulate cells to promote wound healing.
To begin the process of manufacturing vibrating probes to measure bioelectric, current blank probes are purchased from World Precision Instruments. First, cut the probe 25 to 30 millimeters behind the tip and use a number 11 scalpel to scrape away about five millimeters of paraline insulation at the cut end to ensure a good connection. Now using electrically conductive silver loaded epoxy mount the probe in a gold R 30 connector.
Store the probe overnight at room temperature to allow the epoxy to harden the next day. Plate the probe tip with gold and then platinum. To do so, first, rinse the probe tip in acetone and connect it to the negative output of a nano amp power source.
Now place the probe under a dissecting microscope and place it in a gold plating solution. A reference wire connected to the positive output and placed in solution completes the circuit. Apply a current of five nano amps for five minutes, then increase the current to 20 nano amps until the tip is about half the desired final size.
Now, rinse the probe tip in distilled water and then place it in izing solution. Apply a current of 250 nano amps for five minutes, then increase the current to 500 nano amps until the tip is about 80%of the desired final size. Increase the current to one micro amp and apply current in one second bursts until the final tip diameter of 30 to 35 micrometers is obtained.
Finally, rinse the tip and distilled water probes can be stored at room temperature indefinitely. Now let's see the probe system once manufactured, attach the probe to a piso electric bender. Mounted on a three-dimensional micro positioner probe.
Vibration is controlled by the vibrating probe power supply, which also allows for adjustment of vibration, amplitude, and frequency. The probe power supply sends a reference signal to the lock-in amplifier, which also displays the vibration frequency and phase angle. The amplifier is connected to a computer via an analog digital interface.
Data is recorded using Strat Clyde Electrophysiology software's whole cell program settings for the lock-in amplifier and electrophysiology software can be found in the written portion of this protocol. Now let's see how to set up the new probe. New probes have to be tested and their unique frequency and phase angle determined to do so.
Place the new probe and the reference and earth wires in the calibration chamber containing physiological saline. Turn on the power supply and turn up the frequency until the maximum vibration is observed. This is the resonant frequency of the probe.
Using the probe at this resonant frequency can cause instability and noise in the recording. So the probe is de-tuned by subtracting 10 hertz to give the probes working frequency. Adjust the vibration amplitude so that the probe vibration distance is the same as the tip diameter.
This way, when the probe is vibrated, a double image of the probe tip is seen. To determine the phase angle, place the probe in saline in the calibration chamber and apply a current of 1.5 microamps per centimeter squared. Repeatedly adjust the phase angle on the lock-in amplifier until there is no response.
Adding or subtracting 90 degrees gives the maximum response, and this angle is the probes working phase angle. During an experiment, it's important that these settings of frequency, phase, angle, and amplitude are not changed as this will up to the probe's response. Conveniently, south to north flowing current will produce an upward deflection.
While current flowing north to south should show a downward deflection. The response of the probe to a standardized current of exactly 1.5 microamps per centimeter squared applied to the probe in a calibration chamber is used to calculate the current in the sample prior to sample measurement, calibrate the probe in an appropriate solution, then calibrate the probe began at the end of the experiment in used solution to compensate for osmolality change due to evaporation. When analyzing data measurements from the first half of the experiment can be calculated using the starting calibration values and measurements from the second half calculated, using the end calibration.
When measuring samples, a chamber designed to hold and to mobilize the sample is used. For example, Petri dishes with wire loops to hold eyes are used for cornea measurements. To measure a sample, place the dish containing physiological saline under the dissecting microscope and place the area of interest on the sample into the field of view.
Then place the probe in solution oriented parallel to the sample surface and in focus, so it is on the same level as the point on the sample to be measured. Move the probe approximately one centimeter away from the sample and turn on the vibration while recording on the computer software to establish a stable baseline. Then move the probe into measuring position about 50 micrometers from the surface.
When the new peak value is stable, move the probe back to the reference position and the trace will return to baseline. This can be repeated at regular time points to produce time-lapse data or the sample can be moved slightly and measurements taken at different positions. TE spatial current mapping data now will show some representative results.
This current profile of a cornea wound shows maximum current flows at the wound edge. The time-lapse data shows the time course of current change at the skin wound. The red point shows current before wounding after wounding.
There is a transient inward current which then reverses to become an outward current measurements from rat brain show outward currents at the olfactory bulb and inward currents at the lateral ventricle. Shown here is spatial mapping of current at a cornea wound Following this procedure. Other methods such as selective micro electrodes can be used to answer additional questions such as which ES are responsible for carrying the electrical current after its development.
This technique paved the way for researchers in the field of medical research to study wound healing, embryo development, and spinal cord injury.
