We're looking to develop an AFM cantilever to potentially quantitatively study the biting mechanisms of the mosquito. Our protocol offers a new method for creating accurate, controllable, and reliable AFM probes in order to study animal biting or singing processes. Our protocol aims at accurately simulating the animal biting process, causing current methodologies to either be uncontrollable, unreproducible in a reliable manner, or in certain cases completely inaccurate.
The impact of our work is twofold. The first, we create a protocol that enables other researchers to fabricate their own custom bio hybrid AFM cantilevers. Secondly, our work paves the way for creating other protocols that merge testing systems with biological material for creating more realistic testing scenarios.
To begin, place a dead mosquito on the glass slide. Then gently place a scalpel blade over the labium, near the mosquito's head. Make an incision across the entire upper half of the labium with a shallow penetration depth.
Next, with a pair of tweezers, firmly hold the mosquito's head. And with another pair of precision tweezers, lightly pinch the labium between the tapered tip and the incision. Then pull the tweezers holding the labium towards the tapered tip.
Place the mosquito under the microscope to verify if the labrum's tip is present. Now clamp and close the tips of a set of precision tweezers and place the tip of the tweezers right beside the labrum's tip. Use the tweezer tip to apply a gentle force on the labrum perpendicular to the length of the fascicle.
Continue pushing the labrum across the glass slide until separation from the other fascicle members is achieved. Finally, inspect the sample under a microscope to verify the separation. To begin, take the glass slide containing the mosquito labrum.
Position a scalpel blade approximately 200 micrometers from the labrum's tip. Then apply gentle pressure to cut the tip of the labrum all the way through. With precision tweezers, locate and isolate the labrum's tip on the glass slide.
Then using the same tweezers, gently pinch the labrum. Once the specimen is firmly pinched, gradually release the clamping force. Under a microscope, examine the tweezers tips to confirm the presence of the labrum's tip on one of them.
Begin by placing a small amount of epoxy on the edge of a new glass slide. Then position the glass slide under the probe station and focus on it. Mount a tipless AFM cantilever to the clamp assembly.
Secure the base of the cantilever, leaving its end free and suspended in space. Next, attach the manipulator to the probe station. Adjust the z-axis of the manipulator to elevate the cantilever a few millimeters above the glass slide containing the epoxy.
Then using the manipulator, adjust the position of the AFM cantilever along the x and y direction over the epoxy on the glass slide. Carefully align the cantilever's tip directly above the epoxy. After that, slowly lower the tipless cantilever in the z direction toward the epoxy on the glass slide until it touches the epoxy.
Afterward, gently move the cantilever in the x and y direction away from the epoxy, ensuring it is separated from the slide. Raise the cantilever in the z direction using the manipulator. Rotate the manipulator 90 degrees around the cantilever's long axis, and place it sideways on the probe station.
Then place the precision tweezers with the labrum's tip under the probe station camera so that the entire length of it is visible. Afterward, position the manipulator assembly with the clamp and cantilever under the camera so that the entire length of the cantilever is visible. Rotate the manipulator to adjust the cantilever perpendicular to the labrum's tip.
Using the degrees of freedom of the manipulator, slowly move the tip less cantilever in the x-y directions, such that the glue on the cantilever contacts the cut end of the labrum's tip. Then cure the epoxy to solidify the connection between the cantilever and the labrum. Gently move the cantilever away from the tweezers, ensuring the labrum's tip is attached to the cantilever beam.
