After deciding on an experimental paradigm, you will need to cleave your fibers to reach your brain region(s) of interest. It is vital that the fiber is cleaved properly, as fiber photometry employs very low levels of light. Proper fiber cleaving ensures the maximum light power can be emitted.
In order to cleave your fibers, you will need a scribe. We recommend getting them from Specialized Products. The ruby and carbide versions both work well. We also recommend using a ferrule jig when cleaving fibers. We 3D print the ferrule jigs in-house to help standardize the fiber cleaving process; if you have ordered a system from us, you likely received one with your system. They allow you to cut fibers in 0.5mm increments from 2mm to 10mm. The jigs eliminate the need for rulers and leave you with a perfectly cleaved fiber, ready for implantation.
Fibers purchased from Neurophotometrics are sold at 10 mm in length, so they will need to be cleaved to size dictated by your experimental needs. Consult a brain atlas to choose the proper coordinates for your implants. For fiber photometry experiments, it is very important that the fiber is implanted at the viral injection site. This differs from optogenetics, where the fiber is usually implanted ~200um above the injection site. We are working with considerably lower levels of light in fiber photometry than in optogenetics, so it is important that the fiber is directly implanted in the brain area you are wanting to record from. It is important to note that many other factors beyond target brain regions go into choosing a fiber length as well. These include surgical method, type of animal subject, and where your target brain region is located relative to the varying thickness of the skull.
It is imperative that fiber optics meant for brain implantation are flat and polished at the tip. Jagged, irregular, or slanted tips will diminish the power efficiency and limit the cell populations that you can reach. Using scissors, or chopping all the way through the fiber, will result in a fractured core. A clean and symmetrical cut allows for fluorescent traces in the nW range to be picked up by your fiber photometry system.
To cleave a fiber with a ferrule jig:
1. Place the fiber in the well of the ferrule jig that corresponds to your desired fiber length (the fiber goes into the side of the ferrule with the measurements).
2. Holding the bottom of the ferrule in place with one finger, lightly score ONE side of the fiber using the scribe. The pressure applied should be light enough that the fiber does not snap off, but firm enough to push it up against the side of the well.
3. Flick the excess fiber off gently with your finger.
4. Check the efficiency of the fiber using a power meter to ensure it is about 80%. This can be accomplished by taking a power measurement out of the patch cord with the fiber attached and dividing it by the power coming out of just the patch cord (sans fiber). If it is slightly below this threshold (3-4%), you may lightly polish the fiber by rubbing it gently on polishing paper placed on a rubber pad.
Mistakes happen! Most likely, you will accidentally chop through a few fibers with the scribe. All is not lost — the fiber might be salvageable. As long as the efficiency is still above 80%, you should be okay. If it is close to this threshold but slightly below, lightly polish the tip of the fiber using the guidelines above. If it is at 70% and below, or if you’ve polished the fiber but still have an efficiency below 80%, it is best to start over with a fresh fiber. A fiber with a low efficiency will effectively lower your SNR.
While the instructions above include the use of a ferrule jig, you can still cleave a fiber without one. Using the same scribe, tape the ferrule to a ruler and carefully score one side of the fiber and flick off the excess. All rules still apply — make sure to use gentle pressure and try not to chop all the way through the fiber. At this time, we only have ferrule jigs for 1.25mm ferrules, so this is the method that you will have to use if you have 2.5mm ferrules.
If you are concerned about the ferrule getting scratched while the animal is in the home cage, there are a few possible solutions. One is to opt for a different home cage lid that is raised. This makes it less likely that the animal will knock the ferrule on the metal grating, creating scratches. You can also use a dust cap, but if you have a home cage with a low lid, the cap can get caught and yank the entire implant out. Also, some animals learn to rip off the dust cap, which can be an issue of its own. If you have the raised cage lid and no dust cap, you can simply clean the ferrule with ethanol and a kim wipe and proceed with your experiment.
We understand that lab life is busy, and it might be easier if your fibers arrive pre-cleaved and ready-to-use. To save you some time, we offer a fiber cleaving service for $3 extra per fiber. Please note, however, that this will increase the time needed to process your order. Generally speaking, we recommend buying fibers at 10mm so they are fully customizable for your experimental needs.
This post was written by Savanna Howard.