The overall goal of this procedure is to deliver a charged substance such as the positively charged neurotransmitter, a subtle choline to a discrete site within the experimental preparation. This is accomplished by preparing micro pipettes from glass capillary tubes using a horizontal micro pipette puller backfilling the micro pipettes with a subtle choline and securing the micro pipette in a micro manipulator. The micro pipette tip is then positioned adjacent to a site of interest.
While a negative retaining current is set to prevent leakage of a subtle choline, a subtle choline is ejected from the micro pipette by applying a positive current at a defined intensity and duration while recording fluorescence and diameter responses in the microcirculation, the signals measure calcium fluorescence underlying endothelium dependent vasodilation of arterials controlling tissue blood flow. I'm Dr.Steve Siegel. This method can be used to provide insight into the cellular signaling events that underlie blood flow control and arterials.
It can also be applied to neurophysiology to study synaptic transmission and the activation of ion channels. I'm Dr.Pune Badger. Generally, individuals new to this technique may struggle because it takes practice to learn how to prepare effective micro pipettes to perform micro-manipulation and to adjust the retain and injection currents to control the delivery of the drug from the micro pipette.
Begin by using a pipette puller to prepare micro iontophoresis, micro pipettes from bo silicate glass capillary tubes with an internal glass filament. A five millimeter taper is preferred to provide stiffness, whereas longer tapers are more flexible. Next, secure a backfill needle attached to a 0.2 micrometer filter onto a syringe containing one molar acetylcholine chloride dissolved in 18.2 mega ohm water.
Now insert the backfill needle into the back end of the micro pipette and advance it to the tip. Inject the acetylcholine into the micro pipette while withdrawing the backfill needle as the micro pipet is filled. To remove any air bubbles, hold the micro pipette with its tip pointing down and gently flick the micro pipette.
Next, secure the micro pipette in a micro pipette holder. Then secure the micro pipette holder in a three axis micro manipulator, like a compact model mounted to a magnetic base. Connect both the positive and negative wires to the micro iontophoresis programmer Using wires with an alligator clip at the end, a silver wire is secured to the edge of the preparation to serve as the reference electrode.
Using the alligator clip, connect the negative wire coming from the micro iontophoresis programmer to the reference electrode. Then connect the positive wire from the micro iontophoresis programmer to the external pen of the micro pipette holder. With the second delegator clip, the circuit is completed when the tip of the micro pipette is advanced into the physiological saline solution.
Over the preparation, this microscope is outfitted with a custom platform to increase the surface area of the XY stage. The platform is fabricated from ferromagnetic stainless steel and it provides sufficient space to position the micro manipulator around the experimental preparation as desired. Position the experimental preparation on the platform.
In this example, muscle tissue has been prepared using the cream master muscle preparation protocol from an associated video. Now position the micro manipulator relative to the preparation so that the micro pipette tip is located above the site of interest. Now, to avoid dilating, the arterials apply a retaining current using the micro pheresis programmer.
The retaining current varies with the size of the micropipet tip and agonist concentration, but is highly reproducible for defined parameters. Next, using the micro manipulator controls while viewing through the microscope carefully position the tip of the micropipet next to an arteriolar wall. With practice, this can be accomplished without breaking the tip or damaging the tissue.
To eject a subtle choline trigger the micro iontophoresis programmer with a TTL pulse of at least five volts. Using commercial software, an electronic trigger can be sent to the computer that simultaneously sends the TTL pulse and starts the recording of digital images from the camera. The duration of the stimulus pulse is controlled by the software.
The micro phoresis programmer controls the ejection current. When it receives the TTL pulse, the retaining current is withdrawn and the ejection current is delivered. This mouse arterial has endothelial cells expressing calcium sensitive GFP, which fluoresces proportionally to rises in intracellular calcium above a threshold level, a subtle choline triggered a fluorescence response as the duration of the acetylcholine stimulus increased.
Endothelial cell calcium fluorescence in the arterial also increased. The intensity of fluorescence increased at the site of stimulation and fluorescence spread over progressively greater distances along the arterial. When performing this procedure, it is important to remember that the effectiveness of micro phoresis is determined by the internal diameter of the tip, the concentration of agonist within the micro pipette, and the intensity and the duration of the ejection current.
After watching this video, you should have a good understanding of how to use micro pheresis to deliver a charged molecule from a micro pipette at a designated site. In the experimental preparation.
