Fiber Photometry experiments are relatively simple to perform and allow for long-term detection of dynamic neural signals in animals. They are increasingly used in neuroscience research, however, sometimes researchers can encounter some issues with signal collection. Today, I’ll outline potential reasons for bad signals and provide important experimental considerations to help ensure your success!

01. Virus

1. Monitor Your Freeze-Thaw Cycles: Check if the virus has undergone repeated freeze-thaw cycles, as this can lower active virus titration and impact transfection efficiency. Therefore, in fiber photometry experiments, the virus should be used immediately after thawing to minimize damage.

2. Affinity to Target Neurons: The same virus can show varying degree of transfection efficiency with different cell types. Always test new virus can cell type combinations.

Virus Injection

1. Stereotaxic Surgery: The accuracy of your stereotaxic surgery can also affect fluorescence signals. You should always practice with dyes first to tune in your surgery skills before moving on to injecting your virus. Another point to note is that you should always make sure that your stereotaxic instrument is well tuned and that your animal is secured correctly onto the instrument. Check to make sure that horizontal height difference of your leveled skull surface is less than 0.03mm。

2. Virus Injection: Using a finer diameter glass electrode can reduce injection damage. Especially for shallow brain regions, using a pulled glass electrode can minimizing virus leakage. Always leave the needle in place for 10 minutes after injection.

3. Check your Virus Injection With Immuno-histology: Examine your brain sections for fluorescence expression. Sample focusing, transfection rate and fluorescence quenching can all affect the ability to visualize fluorescence expression on your sections.

Considerations:

If the virus injection was unsuccessful, it could be due to misalignment of the skull during surgery, leading to incorrect needle placement (this issue is common for small target brain areas), the virus flowing into the ventricle post-injection, or failing to leave the needle in place, causing the virus to leak out of the injection site.

03. Fiber Implant

1.Perform optic fiber implant in the same surgery session with your viral injection. This can minimize damage to your animal.

2. Choosing Appropriate Holders for procedures requiring different injection depth and location.

3. Check your stereotaxic equipment and your holder make sure all attachments are secure and everything is tight.

The expression area of the virus injection and the implantation location of the optic fiber should be close together. According to literature, a distance greater than 100µm will significantly affect signal strength.

Matching your Fiber Specs

1. Sensitivity to Ambient Light: Fiber photometry experiments is sensitive to ambient light. It is recommended to conduct experiments in a stable and dark environment. Black ceramic pins and black ceramic sleeves are preferred to avoid interference from ambient light on fluorescence signals.

2. Larger Numerical Aperture Using fibers with a larger numerical aperture aids in the transmission of fluorescence signals. Keeping the same core diameter from your fiber implant, patch cable, patch cord to your recorder is crucial for signal transduction.

3. Use High-Intensity Lasers To Bleach Your Fiber Ahead of Time This reduces the auto fluorescence of fiber and other materials, minimizing noise interference.

05. Fiber Photometry Settings

1. Check Your Light Path: This includes the ports connecting the fiber to the machine and the ports connecting the fiber to the mouse (clean them with alcohol!).

2. Increase Exposure Time: Increasing exposure time on your CMOS sensor helps in capturing fluorescence signals.

Considerations:

1. If no signal is recorded, consider increasing the laser power.

2. For larger brain regions, consider increasing the amount of virus injected.

RWD Fiber Optic Cannulae with Ceramic Ferrule

Features：

• Material: Ceramic, Optical fiber

• Type: White Series/Black Series

• Applicable wavelength: 400-2200nm

• Core diameter: 100um, 200um, 300um, 400um

• Numerical aperture: 0.22, 0.39,0.50

• Outer diameter: 1.25mm (6.4mm long), 2.5mm (10.5mm long)

• Optical fiber length: 2mm-25mm (Φ1.25mm),2mm-20mm (Φ2.5mm).step

0.5mm (Other lengths can be customized)

• Low auto-fluorescence