Fiber photometry is a complex technology, involving optics, materials science, data processing and other aspects of knowledge, which easily confuses experimenters during experiment design and execution. Below are answers to some frequently asked questions about fiber photometry experiments. Hopefully, these will help your research efforts. I hope these answers can help you.

1. Whether the fiber photometry system can do multiple animal experiments?

Yes. A multi-channel fiber photometry system can record the several brain areas simultaneously. A branching optical fiber is used to record neural activities in multiple animals or multiple brain locations synchronously.

2. Do I need to wipe the fiber and cannule before the experiment?

Yes. Ceramic cannule implanted on animals’ head is easy to get dirty, and the end face of the optical fiber will also be dirty when touched by the human hand. The dirt interferes with signal transmission and experimental recording. So we recommend to wipe both end faces of the fiber and the ceramic cannule with alcohol before the experiment.

3. Do all fiber photometry experiments require virus injection?

Not necessarily. The usual procedure is to inject GCaMP virus, thereby transfecting specific cells with the virus. Animals can also be genetically modified to label specific cells with fluorescent proteins. Gcamp mice are now available as finished products.

4. What is the setting range of common parameters of optical fiber recording experiment?

For fiber end power, set it between about 20 to 60 microwatts for each channel. High-intensity illumination may lead to photobleaching. In the same experiment, the output power for both the reference light source and the excitation light source can be equivalent, as long as it does not affect data observation. The power for the reference source does not need to be set too high.

The acquisition frame rate is generally set at 40, 60, 100, up to 120 fps. Since changes in calcium signals are in an ms-level, a high acquisition frequency is unnecessary. The gain is generally set to 1. Upregulating the gain means an increase of all signal intensities, including that of noise.

5. What are the optimal frame rate and sampling rate for fiber photometry parameters?

In fiber photometry, signal acquisition frame rates are commonly described in hertz (Hz) or frames per second (FPS), indicating the times of data collection

Then, light spots will be projected to different sites on the CMOS camera target surface. By setting different ROI acquisition areas, optical signals are collected separately in each channel. Channels are independent from the interface to the light outlet. Besides, the intensity of the excitation light can be adjusted without signal interference.

7. What is the reason of using 410 nm to acquire reference excitation light?

In fiber photometry experiments, there are noises (artifacts) caused by external factors, including: autofluorescence caused by tissue damage, motion-induced changes led by animal movement, photobleaching induced by long-term imaging, and other false positive signal interference produced by non-calcium concentration changes. 410 nm excitation light will not affect the signal of fluorescent protein, but could reflect signal changes caused by the above-mentioned interference factors. Therefore, the fluorescence signal changes by 410 nm excitation light demonstrate the background noise.

8. What is the mechanism of fiber photometry system for recording neurotransmitter signals?

Most neurotransmitters have corresponding G protein-coupled receptors (GPCRs). The fluorescent proteins can combine with their receptors by genetic methods. The conformational change of a receptor after binding with its ligand (neurotransmitter) is mapped to the fluorescence signal change of the fluorescent protein. Once a specific neurotransmitter is released to activate its receptor thereby changing its conformation, corresponding conformational changes of the fluorescent protein connected to it will occur, ultimately changing its fluorescence intensity.

Thus, changes in the fluorescence intensity of these proteins can be used to mirror the dynamic fluctuations of neurotransmitter concentration.

9. What is the meaning of df/f, z-score, H eat map, and event curve mean in fiber photometry experiments?

The term df is a statistical jargon. To put it simply, df/f is the relative increase in fluorescence (relative percentage increase). Z-score, sharing the same meaning with df/f, is calculated differently. Based on the df/f data, it is denoted using the standard score formula.

A heat map is a two-dimensional matrix or table, revealing information with color changes and intuitively showing values with defined color shades. As shown in the figure below, a fluorescence heat map can clearly display the number of animals and events in different groups.

An event curve is commonly expressed by mean±sem (mean±standard error, as shown in the figure below). The darker line in the middle of the df/f curve is the mean, and the color intervals in the upper and lower regions represent bar values, which are the standard errors. Event curves are clear and intuitive, showing intergroup differences directly.

10. What are the differences between common fibers and low autospontaneous fluorescence fibers in fiber photometry system?

Common optical fibers need to be bleached with a fiber bleacher before use. It takes more than 1.5 hours for bleaching each time. After bleaching, the autofluorescence can be reduced by 50%-75%, but it will gradually recover over time. Therefore, it is necessary to repeat bleaching before the next use. Generally, this type of fiber is less costly. Low autospontaneous fluorescence fibers are made of low autofluorescence materials to ensure a low autofluorescence value, and there is no need to repeat bleaching in experiments. They are more expensive. In the case of detecting some weaker signals, low autofluorescence optical fibers are more advantageous.

11. What are the differences between a fiber photometry system and a calcium imaging miniature microscope?

Fiber photometry systems record changes of calcium or neurotransmitter signals, where results are displayed by change curves and thresholds; Miniature microscopes directly record fluorescence changes of cells as images by implanting the lens in brain area, yielding pictures or videos showing cell fluorescence signal changes.

12. What are the application scenarios of fiber photometry?

(1) Learning and memory: Alzheimer’s disease (AD), senescence, etc.;

(2) Mental disorders: depression, epilepsy, schizophrenia, etc.;

(3) Movement disorders: Parkinson’s disease (PD), Huntington’s disease, etc.;

(4) Addiction and brain reward: drug abuse, drug or alcohol addiction, sensitization, etc.;

(5) Others: pain, inflammation, waking mechanism, etc.