Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Hi, I am Alex Derber from the laboratory of Dr.Albert Bos. I'm the Department of Electrical Engineering at North Carolina State University. Today we are going to demonstrate a novel surgical procedure for implanting electrodes in the manica sex During early metamorphic stages.

In this procedure, which is called Early Metamorphic insertion technology, the implanted electrodes are integrated in the tissue of the insect during the pupil metamorphic stage. A major benefit of this procedure is that it results in mechanically stable and electrically reliable coupling with the neuromuscular tissue and allows for the studying of the flight neurophysiology of the insect. In the adult phase, IC Mensa undergoes a metamorphic lifecycle that lasts about 40 days.

Mensa begins its lifecycle in an egg stage. After seven to 10 days, the larva will have hatched and reached the third star larval stage. There are a total of five star larva stages, and in each of these stages, the larvae undergoes energy harvesting and growth.

Five to six days after the fifth install larval stage, the insect will enter the early pupil stage. During the pupil stage, there's extensive program degeneration and subsequent regeneration of the insects tissues. In preparation for emergence as an adult moth, the insect spends 17 to 19 days.

In the early pupil stage, before entering the late pupil stage, during the late pupil stage, we surgically insert an electrically conductive electrode in the dorsal thorax of the pupil. The tissue develops around the implanted electrodes and secures their attachment to the insect's body. Over the course of a few days, the electrodes inserted into the thorax of the pupil, emerges as a part of the insect's body in the final adult stage as a moth, thus taking advantage of the rebuilding of the insect's entire tissue system.

During metamorphic development, the electrodes are implanted into the indirect flight muscles in the thorax. These muscle groups indirectly responsible for wing movement are the dorsal ventral and the dorsal longitudinal muscles. Here we will be demonstrating an electrode insertion into the dorsal ventral muscles, indirectly responsible for wing upstroke.

With this insertion, we will wirelessly record electromyogram from the dorsal ventral muscles during ya movement. As the insect is suspended by an electromagnetically levitated platform within an LED arena, that stimulates movement. The procedure for early metamorphosis insertion technology begins with the construction of a printed circuit board that will be used as an interface between the inserted electrodes and the recording instrumentation.

The wire electrodes are soldered on the printed circuit board, which is connected in a flat, flexible cable connector. To begin measure and cut a 0.5 by five centimeter squared piece of copper clad laminate using a fine tip, permanent marker, and scissors using the same fine tip, permanent marker. Draw three 0.1 by five centimeter squared pads as etching mask patterns.

Use a straight edge device such as a ruler to make the markings along the entire length of the copper laminate cutout. Make sure that the markings are bold and do not overlap with each other. If the mark lines overlap with each other, the electrodes will be shorted out.

When making the connection to the flat, flexible cable connector, making the boards useless, take the copper laminate cutout over to a ventilated area, preferably with the fume hood for etching of the markings onto the copper laminate. Be careful when handling the printed circuit board etching as this is toxic. If ingested or swallowed and can irritate the skin upon contact, place the Teflon tape on one end of the copper laminate cutout covering about one centimeter along the length.

Next, attach the copper laminate cutout to the inside of a graduated beaker using scotch tape such that half of the copper laminate strip is submerged in the etching. Place the beaker on a rotating platform for 20 minutes. Remove the etched copper laminate from the beaker and place it in a beaker filled with water for 10 minutes.

For rinsing of the residue, apply iso propyl alcohol to a piece of delicate wipe tissue and wipe the markings To expose the non etched copper pads. Remove all traces of the permanent marker. Cut the printed circuit board in smaller squares approximately one centimeter long.

Now that the printed circuit boards have been etched, the electrodes need to be prepared for attachment. It is suggested that these steps be performed with the visual aid of a microscope. Cut two pieces of coated, a kneeled stainless steel wire with a diameter of 0.11 inch coated and 0.008 inches bare using a sharp blade, two lengths of three centimeters each.

These pieces of stainless steel wire are the active electrodes that will be inserted into the thorax of the insect. Using a razor blade, remove four to five millimeters of the plastic coating from each end of each wire. Next, cut.

One 0.7 centimeter piece of insulated stainless steel wire to create a tip extension for the ground electrode. Gently remove the coating with the razor blade as performed in the previous step or melt it with the heat of a soldering iron. Next, cut one piece of lititz wire to a length of 4.5 centimeters.

Inductor wire can also be used for this solder. The previously prepared 0.7 centimeter piece of stainless steel tip to the lititz ground connection wire. An exposed stainless steel tip should be at the end of the ground connection.

Tape the etched circuit board to the soldering workspace using a Teflon tape. Place the tape over the circuit board such that one to two millimeters of the pads allow for soldering of the electrodes to the board. The masked solder free ends of the pads will be inserted into the FCC connector.

Align the three electrode wires with Teflon tape such that one end of each can be soldered to the corresponding pads on the electrode board. The two active electrodes should be soldered on the pads near the edges of the board. The lited ground electrodes should be soldered on the center pad.

Apply stainless steel flux across the electrode pads for easier soldering. Be careful to Avoid shorting the pad connections by applying too much solder. Immerse the electrodes In acetone in isopropyl alcohol for 10 minutes each.

To clean the solder residues. The solutions can be poured into separate dishes or beakers To improve cleaning performance, you can place the electrodes in an ultrasonic bath during immersion And the cleaning solutions. The insects will be Most active during the transitions between day and night.

Therefore, an artificial day night cycle should be established within an insect chamber using automatic outlet timers. These should be set to simulate a seven hour dark and a 17 hour light cycle. Examine the manduka sex eye pu eye daily to determine appropriate insertion time.

The pu eye are ready for insertion approximately one day after the wings. Exhibit dark spots insertions are most successful when performed seven to four days before we closing here. The darker pu on the bottom is ready for surgical insertion of the electrodes to anesthetize the pui.

Place them in the refrigerator at four degrees Celsius for around six hours. Next, prepare the insertion of workspace. The workspace should include isopropyl alcohol, sharp tweezers blades in a 30 gauge hypodermic needle.

As an option cyanoacrylate adhesive may be used to enhance the electrode fixation, sterilize the needle tweezers, and the electrodes by dipping them into or wiping with iso propyl alcohol. Remove the pupil from the refrigerator after six hours and transfer it to the workspace. Determine the location on the dorsal thorax that corresponds to the muscle group of interest.

Here we are interested in the dorsal ventral muscles responsible for upstroke wing movement. Using a sharp blade or a hypodermic needle, gently scratch a one by one centimeter square rectangle through the exo cuticle layer. Using the tweezers slowly peel off these pieces.

Use the tweezers to remove wing Hair from the exposed region of the thorax. A vacuum can be used optionally to assist the cleaning process. It is important to remove as much of the hair as possible to avoid interfering with the inserted electrodes.

Slowly insert the needle about five millimeters into the meso thax where the wings attach to the thorax to create two insertion points targeting the muscle group. Remember where the holes are made because they can be difficult to find if lost. Stabilize the pupa on the table and use tweezers to guide the two recording electrodes into the two insertion points as an option to enhance the mechanical durability.

Clean all of the hair around the electrodes and generously apply ciano accolate adhesive around each insertion point on the thorax with the wire applicator. Prepare cage for ELO with a rough and textured material covering the walls and ceiling that the insect might climb on. Upon emergence, perforated cardboard boxes or packing paper may be used.

Prepare a rigid fixation. Stick with around six centimeters length and two millimeters diameter. Carefully slide this stick through the hole underneath the protruding pros.

Plastic stirs. A cotton swab or metal wires can be used for this step. Fix both sides of the stick on the cage surface such that the pupa cannot roll around.

Position the pupa inside of the cage such that the meso thax is facing up. Extensive movement may cause damage to the electrodes, loss of heli or render the insertion useless. The ground electrode should be inserted into the abdomen or distal parts of the thorax.

To avoid signal coupling, disin insertion can be done either during the later stages of the pupil development or after the insect emerges. The ground electrode insertion site needs to be prepared in this pupil stage for either the pupil stage insertion or the adult stage insertion. Here in an adult stage ground electrode insertion is demonstrated.

Place them moth in the refrigerator at four degrees Celsius for six hours. For cold treatment immobilization, prepare the insertion workspace including isopropyl alcohol, sharp tweezers, a 30 gauge hypodermic needle cyan a creol adhesive, a piece of wire for application of glue, an optional thermal cauterizer in an optional dental wax stick. As before, dip the hypodermic needle into the iso propyl alcohol before contact with the moth located in an insertion point approximately one to two centimeters away from the recording electrodes along the posterior abdomen slowly insert the needle to puncture the abdomen and provide an insertion side.

The needle should not penetrate more than four millimeters. If hemo lymph emerges at the insertion side, you have inserted the needle too far and the procedure is less likely to be successful. Using tweezers, carefully insert the ground electrode into the insertion site and apply pressure until it is three to Four millimeters deep.

Hold the electrode in place and use a wire to apply glue around the insertion site As an optional step. To further enhance the mechanical strength of the insertion, use the thermal cauterizer and collect a small two to three millimeter beat of wax at the tip. Place the tip close to the insertion site and apply heat such that the wax surrounds the electrode and holds it firmly in place.

Upon cooling, allow the insect one day to recover from the insertion before performing experiments. An adapter board with a flat flexible cable connectors required to connect the electrode board on the insect to the triangle Biosystems international head stage unit. The adapter board uses an etched copper laminate board that needs to be prepared by following the steps presented earlier for preparing the edge to electrode board at the solder station, solder a flat flexible cable connector to one end of the prepared board.

A 10 pin flat flexible cable connector can be soldered to the board with the two pairs of pins on the edges of the connector and the central pin pair in contact with the three copper pads. Next solder three 30 American wire gauge hookup wires to three pads. On the other end of the board slaughter three mini connectors to the three pads on the adapter board for oscilloscope readings as described.

In the next stage, have two of the connectors positioned perpendicular to the edges of the board. The central connector should be angled slightly up away from the plane of the board slaughter the other end of these three wires to the head stage connector. Provided by Triangle Biosystems International.

The head stage circuit board should then be secured on top of the levitation frame in order to assist the reliability of the electrodes and observe the signal to noise ratio. Tethered oscilloscope recordings can be obtained. Before deploying the wireless recording system, connect the oscilloscope to a multichannel AC neuro recording amplifier.

Set the amplifier parameters to a high pass cutoff frequency of one hertz, a low pass cutoff frequency of 20 kilohertz and a gain of 100. Connect each of the female mini wire connectors on the adapter board to the amplifier input channels. Remove the insect with the implanted electrode board from the cage.

When it is in an active state, a piece of tissue can be placed under the moth for its rest on before measurements are taken using tweezers, slide the electrode board into the flat flexible cable connection receptor on the adapter board. Remove the tissue beneath the insect and observe the generation of electromyogram spikes. As the insect flaps its wings capture and save the data on the oscilloscope.

This figure shows the muscle potential signal acquired with the oscilloscope from one of the muscles before and during wing flapping. The signal has been processed with a hundred times amplification and a high pass filter of one hertz and a low pass filter of 20 kilohertz and the quiescent period. No muscle potentials are observed.

The muscle potentials during wing flapping occur at approximately 15 to 20 hertz. Once the oscilloscope measurements have been collected and verified, the wireless EMG signals should be collected. An electromagnetic levitation platform can be built for wireless recording of EMG signals during tethered Manus sex to flight.

The levitation platform consists of a frame designed to balance a tethering mechanism. The free floating levitation allows the frame and therefore the insect to ya. During testing without constraint of tethering wires, the frame can be rapid prototyped using a fused deposition modeling machine.

This magnet loaded frame is levitated by a series of electromagnets in the base platform. This levitating platform is located inside of the LED arena, which was constructed using 60 panels composed of an array of five by seven individual LEDs. The arena is controlled by APIC 18 F 45 20 microcontroller, allowing simulation of both clockwise and counterclockwise rotation, as well as control of the rotational speed.

Set up the TBSI wireless recording system. By connecting the TBSI head stage to the adapter board connector on the levitation platform. Remove the insect from the cage when it is active, preferably during its dawn time.

Using tweezers. Carefully insert the electrode board into the flat flexible cable receptor on the levitating frame, such that the insect is suspended firmly within the setup. Place the magnetic wand near the magnetic switch on the head stage to activate wireless data transmission.

A blue light will come on indicating that the head stage is active and ready for recording. Turn the lights off in the room for complete darkness and open the TBSI neuro aware software on a computer. You can use a red lamp to add additional lighting to the room.

Select the appropriate configuration file. The configuration files used in this procedure is the default W five setting. Then click the start deck button in the upper left hand corner of the initial interface.

To begin viewing signals, select the all channels window tab for observation of EMG signals on the TBSI recording system. To ensure a reliable wireless connection and electrode operation, turn on all the LED arena components, the regulated DC power supply and the microcontroller. Slowly balance the levitation platform within the arena.

Select the record to binary tab on the left panel of the nor aware interface. Designate the recording time and file save destination. Choose the appropriate output settings to save your data.

Click the start button to initiate a recording session within the TBSI recording software. This will save your file as a. next file, which can be imported into commonly used data processing programs.

Observe as the insect flies in the direction that corresponds with the movement of the LEDs. Reverse the direction of the LEDs and confirm that the insect reverses direction Perform this as many times as desired. The arrow shown indicates The rotational direction of the LED pattern.

The arrow becomes yellow. When the insect completes a counterclockwise turn, the arrow becomes blue when the insect completes a clockwise turn. The film playback speech shown is 50%faster than normal.

This Figure shows the muscle potential signal acquired from the wireless instrumentation for one muscle before and during wing flapping in the quiescent period. No muscle potentials are observed. The muscle potentials during wing flapping occur at approximately 15 to 20 hertz.

Here we have just shown a one channel recording. However, a multi-channel recording taken with this setup can be used for observing coordination between multiple muscle groups during flight maneuvers. We've just shown you how to perform an early Metamorphic stage electrode insertion into a man sex to pupa.

It is important to time the insertion in the late pupil stage such that the insertion is not too early or late in the pupil stage. Otherwise, you might run into trouble obtaining a reliable electrode setup. We've also shown you how to assemble electrical components necessary for performing wireless recording of EMG data from indirect flight muscles while the insect is levitated in the LED arena.

Thanks for watching and good luck with your experiments.