The overall goal of this protocol is to create a large volume tissue illuminator that is well suited to optogenetic manipulations in non human primates. This method can help answer key questions in the field of neuroscience, such as how do populations of neurons in the different brain regions contribute to behavior, and how can temporally specific neuronal firing patterns in one region impact another region? The main advantage of this technique is that it uses light, and thus allows optogenetic manipulation of large behaviorally relevant brain volumes with minimal tissue damage.
The implications of this technique extend toward translational medicine because of the similarities between the human and non-human primate brains. Generally, individuals new to this method will struggle because it takes some practice to apply the proper amount of pressure when pulling these fibers. To begin this procedure, use a pair of sharp shears to cut a section of 250 micrometer diameter plastic optical fiber that is at least 10 centimeters longer than the desired total illuminator length.
Next, remove 15 to 20 centimeter of polyethylene jacket from one end of the plastic optical fiber using a 22-gauge wire stripper. Then, secure the distal-most three to five centimeters of the stripped end of the fiber in a table vice clamp. Hold the jacketed end of the fiber taut in one hand, with a constant steady pull.
The fiber should be parallel to the floor, pulled away from the vice. Maintain this constant tension on the fiber throughout heating and cooling, so that the fiber remains straight and taut as it thins. Using the lowest setting of a dual temperature heat gun, heat the stripped section of the fiber until it is thinned to a diameter of 60 to 100 micrometers, or about the diameter of a human hair.
Maintain the constant tension on the fiber while allowing it to cool. Failure to maintain tension or overheating the fiber may cause the fiber to curl and or break. To help maintain constant tension on the fiber as you heat it, use your non-dominant hand so you don't have to let go when you're using the heat gun.
Also, you can gently wrap the fiber around your palm to get an even better grip. Once the fiber has fully cooled, and the desired diameter has been achieved, pinch the fiber three centimeters from the narrowest point on both sides. With a quick sharp pull along the axis of the fiber, separate the sides to create a tapered tip.
Then, examine the tapered tip in low power under a dissection microscope. If the tip is forked or curled, discard the fiber. Even with a lot of practice, about a third of fibers will have forked or curled tips.
We just discard those fibers rather than trying to repair them, since it's so quick and inexpensive to pull another one. Prepare to etch the tapered tip by placing a piece of lab tape near the end of the tip, leaving the desired tip length exposed. Here the last five millimeters are exposed.
The tape protects the fiber from etching above the desired length of light emission. After that, fold a small square of five micrometer silicon carbide lapping sheet over between the thumb and forefinger. Place the tip of the fiber between the two sides of the lapping sheet, and gently etch the fiber using small circular motions.
Frequently rotate the fiber so that all sides are etched evenly. Next, remove about five millimeters of the polyethylene jacket from the opposite end of the illuminator using a 22-gauge wire stripper. Then, cut the opposite end flat with a hot knife to smooth the surface.
Affix a connector on the end of the illuminator opposite to the etched tip. This allows the illuminator to connect to an optical cable or laser. Afterward, polish the opposite end flat with successively finer lapping sheets.
Subsequently, insert the flat opposite end into a 260 micrometer inner diameter stainless steel ferrule until it is flush with the end of the ferrule. Visually confirm that the opposite end is smoothly and evenly polished with a fiber microscope. Tape the ferrule vertically on the edge of the table with the smaller opening of the ferrule pointing down.
Then, fill a one milliliter syringe with plastic epoxy and attach a blunt 18-gauge needle to the syringe. Following this, use the needle to apply epoxy down into the ferrule. Fill the ferrule completely with epoxy.
Insert fiber into the ferrule so that it is flush with the bottom of the ferrule. If needed, wipe any excess epoxy from the polished surface of the fiber with a wet lint-free wipe prior to drying. In this step, prepare a tower microdrive prior to each testing session.
Then affix a 25-gauge guide tube with a beveled tip to the lower clamp on the drive. Secure the large volume illuminator in the upper clamp on the same drive. This clamp is attached to the drive motor.
Afterward, thread the large volume illuminator through the guide tube fully, and confirm that the tip of the illuminator extends past the tip of the guide tube. Alternately, lab tape may be applied to the fiber so that the tape, rather than the fiber itself, is clamped, to protect the fiber. Next, soak the guide tube and illuminator in an antiseptic solution for sterilization.
Place a custom sterilized grid with one millimeter hole spacing inside the clean chamber, and secure it at a prespecified orientation with the screw on the side of the chamber. Retract the sterilized illuminator from the guide tube by pulling the illuminator up using sterile blunt forceps. The tip of the illuminator should be five to 10 millimeters above the tip of the guide tube.
Affix the microdrive to the holder and place the guide tube into the target grid hole. Then, lower the microdrive stage until the guide tube is just through the level of the dura. At the same time, lower the illuminator manually with sterile forceps.
If there is any resistance, retract the illuminator five to 10 millimeters, wait 10 minutes, and try again. If there is still resistance on the second attempt, retract the illuminator tip into the guide tube, lower the guide tube 0.25 millimeters, and try again. Connect the ferrule on the other end of the illuminator to the larger optic setup or laser.
This figure shows that the error rates increased significantly with illumination. And the raster plots here show the inhibition of neurons spanning the 2.5 millimeter thickness of cortex. And the local field potential showing the light artifact spanning 4.5 millimeters.
Once mastered, a large volume illuminator can be constructed using this technique in 10 to 15 minutes, plus epoxy drying time. Calibration adds another half hour or so, so to maximize efficiency, we usually make several illuminators at once. While attending this procedure, it's important to remember that many of the illuminators won't meet quality control standards.
That's okay. If they fail tip inspection, we discard them. But if they fail calibration, we usually repolish them and repeat the calibration.
After its development, this technique helped pave the way for researchers in the field of neuroscience to use optogenetics to suppress neuronal firing and modulate behavior in non-human primates. After watching this video, you should have a good understanding of how to construct a large volume tissue illuminator.
