The system allows for researchers to easily construct an inexpensive and versatile illumination system for optogenetic experiments. Advantages of this technique include the ability to program the LED illumination timing with available wavelengths that span the visible near IR spectrum, and that the LEDs can be used in a variety of contexts To build an electrical circuit, clip the printed circuit board onto the helping hands and insert the jumper wires, connecting them to form the A1 and A3 pinholes to the ground, the D2 jumper wire into the D6 pinhole, and the power supply plus number seven jumper wires into the A7 pinhole. Bend the terminals 45 degrees and add flux.
After soldering, trim the backs of the wires. Insert one end of the soldered female wire to wire connector into the A5 hole. Then connect one end of the resistor to the A5 hole.
Connect the other end of the connector to the ground. Then connect the other end of the resistor to the ground and solder the connection. For the Arduino connector, crimp the ends of the wire to wire connectors and push the crimped ends through the rectangular connector.
To set up the LEDs, tin a wire by heating from below while adding solder from the top. Place a generous amount of solder over the large soldering tip and use the tip to heat the LED base at the contact containing flux. After a few seconds, individually drag the solder across each contact and fix the black wire onto the cathode using a hair clip.
Place a generous amount of solder onto the large soldering tip, and press the tip onto the wire until the solder on the LED base melts. Hold down the wire and remove the soldering iron while holding the wire in place. Place a small amount of solder paste onto the pads for the LED connections and use forceps to place the LED over the pads.
Hold the pre-tinned red wire on the anode and use a hair clip to clip the wire. Add solder to the large tip as demonstrated and press the tip onto the connection until the solder on the LED base melts and the solder paste melts and becomes silver. For making the LED wires, place a 4.76 millimeter shrink tube over the wire cut to length and a 3.18 millimeter shrink tube over the wire to wire connector.
Strip and add flux to the wire ends. Use the helping hands to clip the wire to wire connector and twist the connector end with one wire end. Solder the materials together and attach the other wire to the setup in the same manner and solder.
Then clip the LED wires to the helping hands with tape under the LED base. Next, mix epoxy according to the manufacturer's instructions and spread the epoxy over the top of the soldered LED. Using the spade drill bit, drill a 1.5 centimeter hole through the top of the box onto which the LED will be placed and use a high speed rotary tool to make a notch on one side of the hole to make room for the LED wire.
Tape a 25 to 30 millimeter piece of privacy film onto the black box covering the hole that the LED will illuminate through and tape the LED outside of the black box on top of the hole. For a four LED control system, drill four 0.83 centimeter holes 3.81 centimeters apart into the lid of the box and use a high speed rotary tool to cut a 1.19 by 1.90 centimeter rectangular hole into the top left corner. Using a spade drill bit, drill a 1.5 centimeter hole in the back of the box and insert a grommet into the hole.
For the computer controlled LEDs, sandpaper the area where the microcontroller will be glued in the box as well as the bottom side of the microcontroller holder. Snap the microcontroller onto the holder then epoxy the holder and the box together before securing the holder in the black box. Then place double-sided tape on the clip and secure the clip inside the box.
In samples transfected to express phytochrome B but not phycocyanin bilin, leakiness increases as the amount of reporter DNA increases. In addition, when the entire phytochrome B gene switch including the phycocyanin bilin chromofour producing plasmid are illuminated with far red light, luciferase expression also increases with the increasing amounts of reporter construct within the transfection mix. Similarly, when the light sensitive cells are illuminated with red light, luciferase expression also increases with increased reporter amount.
When comparing induction levels of the red light treated cells to the far red light treated cells, a small decrease in the fold activation is found with increasing amounts of reporter. It is essential to carefully follow each step when building the electrical circuit and to double check the pinhole numbers line by line before soldering each component. This protocol can be used for the optical stimulation of many types of samples or optogenetic tools using a variety of wavelengths and allows the programming of illumination schedules.
