More About Fiber Photometry: Working Principle and Process

   |  August 8, 2022

This article contains more than 3,000 words to explain the working principle, and advantages, and shows the steps of setting up the relevant experiment.

A. Fiber photometry working principle.

B. Advantages of fiber photometry.

C. How to use Fiber photometry Technology?

D. Fiber photometry system setup and record a good signal.

F. Fiber photometry data analysis.

A. Fiber photometry Working principle

The human brain has about 90 billion neurons, which are interconnected by synapses to form a complex neural network, and thus produce various complex functions. The brain can synthesize and release hundreds of neurotransmitters, and nerve signals are transmitted between neurons through the neurotransmitters released by synapses. When the nerve excitation is transmitted to the end of the synapse, it will stimulate the calcium channel on the synapse to open and promote the influx of calcium ions, and the concentration of intracellular calcium ions will increase instantly, driving the synaptic vesicles to release neurotransmitters into the synaptic space, and that will bind to the receptors on the postsynaptic membrane and transmit the neurotransmitter signals to the next neuron, thus transmitting the information step by step

Taking the detection of calcium fluorescence signal as an example, the technical principle of Fiber photometry system is to use special fluorescent indicator or fluorescence probe with the help of the strict correspondence between the change of calcium concentration and neuronal activity. The concentration of calcium in the neuron is expressed through the fluorescence intensity and captured by the fiber photometry system, so as to achieve the purpose of detecting neuronal activity.

Fiber photometry Working principle
Figure 1

In the nervous system, the intracellular calcium concentration of neurons in resting state is 50-100nM, but when neurons are excited, the intracellular calcium concentration can increase by 10-100 times, so we can label calcium ions by injecting calcium gene coding indicators (Calcium indicator, such as GCaMPs, RCaMPs, etc.).When the neural activity is enhanced, the calcium channel opens, a large amount of calcium inflows and binds with CaM, resulting in the interaction between M13 and CaM domains, leading to cpEGFP structural rearrangement, thus enhancing the green fluorescence signal.(Figure 1) Therefore, we can characterize the activity of neurons by detecting the changes of calcium signals, and then study the correlation between neuronal activity and animal behavior, and explore the regulatory mechanism behind complex behavior.

The principle of fiber photometry for the detection of neurotransmitter signals is the same as above. A new series of genetically encodable fluorescent probes, called GRAB (GPCR activation-based), has been developed. By embedding a fluorescent protein (cpEGFP) sensitive to structural changes into the neurotransmitter receptor, the research group was able to convert the chemical signal of neurotransmitter into a fluorescent signal and, in combination with existing imaging techniques, to monitor the dynamic changes of neurotransmitter concentration in real time

fiber photometry priciple
Figure 2

B. Advantages of Fiber photometry

The study of the nervous system of awake and freely moving animals is a crucial issue in contemporary neuroscience research. By detecting calcium signals associated with neuronal activity, we are able to better understand complex neural circuits. In recent years, technological developments including two-photon microscopy, single-photon microscopy, and fiber photometry have allowed us to perform real-time detection of changes in calcium signals in animals. The application of these techniques requires engineered fluorescent proteins.

Three in vivo techniques for measuring calcium
Three in vivo techniques for measuring calcium
Figure 3

In the past, most in vivo calcium imaging was done under two-photon microscopy. Two-photon microscopy is a fluorescence imaging technique in which two beams of coherent laser light are focused through the eyepiece of a microscope to a point defined by a point spread function, similar to confocal microscopy that selectively excites fluorescent-like molecules. In two-photon microscopy, the excitation light wavelength is near 700-1000 nm, close to the infrared spectrum. This is the first advantage of in vivo two-photon imaging: longer wavelengths of light scatter less in the tissue and penetrate to greater depths. The second is the good resolution that allows visualization not only of individual neurons but also of calcium activity at the ends of axons of subcellular structures.

Laser scanning techniques in two-photon microscopy are also used to detect rapid calcium dynamics in neurons during in vivo imaging, but the excitation power to distinguish signal from noise usually needs to be high enough that this causes some degree of bleaching, and photobleaching timeouts can negatively affect image quality.

Two-photon microscopy is governed by many factors in terms of imaging depth. Deeper brain regions are visualized by implanting gradient refractive index (GRIN) lenses, but due to the size of the objective, most of the cortex or other brain structures often need to be completely removed to make room for lens implantation, resulting in greater surgical damage to the animal. In most cases, it is highly advantageous to choose the surface layer of the brain as the area to be studied because of minimal damage and higher resolution.

Another disadvantage of two-photon microscopy is that the animal must be held in place by the head due to the size of the objective lens. Experimenters currently enable animals to move slightly freely by using cylindrical or spherical treadmills, or through a system of virtual reality scenarios. However, many researchers believe that head-fixed animals are under constant physical restraint and emotional stress during the experiment, and therefore cannot demonstrate that neuronal responses to the outside world are equivalent under virtual reality and free exploration. More importantly, many social behaviors, such as parent-child care, mating and fighting, cannot be studied with head-fixed experiments.

Microscope-based single-photon in vivo calcium imaging allows researchers to record calcium signals from free-ranging animals. The microscope accomplishes the observation of population neuronal activity over long periods of time by connecting to a gradient refractive index (GRIN) lens. The small size of this, device allows it to be fixed on the animal’s head without seriously impeding its movement. This tool is useful for studying development, neuroplasticity and neurodegenerative diseases.

Despite the strong utility in freely moving animals, there are still limitations to this approach. Compared to two-photon, single-photon imaging has higher background fluorescence and is more prone to light scattering. And it has limited use in behavioral experiments, such as water maze and forced swimming experiments. In addition, since the diameter of GRIN lens implant ranges from 0.5 mm to 1 mm, it will inevitably causes a certain range of tissue damage, which may affect the key circuit of the study.

The fiber photometry system is principally divided into a fluorescence excitation system and a collection system, i.e., a certain amount of calcium dye or genetically encoded calcium indicator is injected into a specific brain region of the experimental animal, and a 200-400 μm diameter fiber is implanted at the same location, which is used to transmit excitation light and collect emission light at the same time. The collected fluorescence is converted into electrical signals by the detector and then transmitted to the recording system via a data acquisition card for the purpose of real-time observation of calcium fluorescence activity in a group of nerve cells in the studied brain region. Fiber photometry uses a low-cost implanted cannula that does not need miniscope on the head. Simple animal surgery, lightweight implants, and smaller data files all contribute to long freely-behaving experiments. With high signal-to-noise ratio, and the ability to combined with a variety of behavioral paradigms.

In 2014, Professor Karl Deisseroth’s lab at Stanford University named Fiber Photometry for the first time in a published article, a method that enabled the first simultaneous tracking of animal behavior and neuronal axon terminal calcium activity. Based on the development of genetically encoded calcium indicators, experimenters can achieve cell type-specific labeling and monitor the average activity of a group of specific cell types during the completion of complex behavioral tasks in mice. The advent of this technique is a landmark for encoding behaviors such as social behavior, learned memory and fear, as well as the detection of circuit-level neural signals in pathological states.

  1. Kasey S Girven, Dennis Ryan Sparta. Probing deep brain circuitry: New advances in in vivo calcium measurement strategies[J]. ACS Chem Neurosci. 2017 Feb 15;8(2):243-251.
  2. Gunaydin LA, Grosenick L, Finkelstein JC, et al. Natural neural projection dynamics underlying social behavior[J]. Cell, 2014, 157(7): 1535-51.

C. How to use Fiber photometry Technology

Fiber photometry is performed in much the same way as optogenetics, where a fluorescent indicator (virus) is injected into a specific part of the animal, followed by the implantation of a fiber-optic cannula that transmits light. Animal surgical procedures can be referred to below.

1. Weigh each mouse prior to surgery and anesthetize the mouse. 

2. Thaw the virus on ice 
3. Use the Nanoliter micro-injector to withdraw an appropriate amount of viral vector. 
4. Shave the hair over the skull and mount the mouse on a stereotaxic apparatus.
5. Apply erythromycin ointment to maintain eye moist. 

6. Clean the skin over the skull with 3%  H202(Or iodine volts) and open the skin to expose the skull. 

7. Gently apply 3% H2O2 to the surface of the skull and remove the covering tissue. Clearly expose the bregma and lambda points.

8. Level the skull using the anterior-posterior tilt and medial-lateral tilt on the stereotaxic apparatus. 
9. Locate the appropriate location for virus injection according to a stereotaxic atlas and drill a hole (*1 mm diameter) in the skull. 

10. Remove the bone debris from the hole and puncture the dura using a syringe tip (0.4 mm) without damaging the cortex. 
11. Drill one more hole to implant a screw (or two screws) to secure the dental cement. Note: It is better to drill this hole at least 2 mm away from the hole for the optical fiber. 
12. Drive the anchoring screw into the skull using a micro-screwdriver and tweezers. Note: Avoid damage to the brain. There is no need to drive the screw deeply through the skull. 
13. Mount the virus-filled Nanoliter micro-injector to the stereotaxic arm 

14. Move the Nanoliter micro-injector to the appropriate position and lower it slowly to the target coordinates. 
15. Inject the viral vector at a rate of 30-60 nL/min to a total volume of 200 nL–500 nL 
16. After completing the injection, leave the pipette in place for 5–10 additional minutes to enable the complete diffusion of the viral solution, and then slowly withdraw it completely. 
17. Attach the stereotaxic adapter for implantable fibers to the stereotaxic arm. Insert the fiber into the stereotaxic adapter 

18. Lower the stereotaxic arm with the implantable fiber slowly to the brain region of interest. 
19. Apply a thin layer of dental cement to the skull. 
20. Apply the dental cement around the implantable optical fiber. Leave at least 4–5 mm of the ferrule of the fiber exposed (i.e., not covered by headcap cement) to ensure that the fiber can be interfaced with the patch cable 
21. Wait until the dental cement dries completely 
22. Remove the stereotaxic adapter from the implanted optical fiber by gently unscrewing it 

23. Place the mouse into a clean cage and keep it warm until it fully recovers from anesthesia. 

After two to three weeks of virus expression, the operator can connect the animal with an optical fiber to record the signal.

D. How to record a good signal?

According to the experimental procedure of fiber photometry, the following factors can be considered when the signal is not recorded well.

1. Virus.

(1) Repeated freeze-thawing will reduce virus titer and thus affect virus transfection efficiency. Therefore, in fiber photometry experiments, viruses should be taken immediately and freeze-thawing should be avoided.

2) The degree of specific binding of the virus to neurons in the target brain region will also affect the fluorescence signal. It is recommended to choose a virus tool with higher transfection efficiency.

2. Virus injection.

(1) Before virus injection, we can inject Trypan blue for preliminary experiment and improve the success rate of later experiments. Note that the difference between left and right height is less than 0.03 mm when adjusting level.

(2) Virus injection: choosing pulled glass electrodes can reduce injection damage, and for shallow brain area injection experiments, it can also reduce virus leakage. It is recommended to keep the needle for 10 min after virus injection.

(3) virus injection detection: tissue sections to see the fluorescence expression. Fluorescent cells are not visible under the microscope, perhaps because the microscope does not focus on a specific cell layer, the number of specific neurons labeled by fluorescence is too small, or the fluorescence is quenched.


If there is no obvious positive signal, the possible causes are deviations in the positioning of the brain region during surgery resulting in the wrong location of the virus injection (this is often a problem if the target region is small), the virus flowing away down the ventricle after injection, or the virus flowing out of the injection hole because the needle was not left in place after injection.

3. Implantation of fiber photometry ceramic ferrule.

(1) Virus injection and ceramic ferrule implantation can be performed in the one operation, avoiding secondary surgeries that cause damage to the model animal.

(2) Selecting the appropriate adapter for implantation procedures at different depths and multiple sites.

(3) Periodically check the fixation of the operating arm of the stereotaxic apparatus and the adapter to prevent angular deviation.

(4) The implantation position of the virus expression area and the ceramic ferrule need to be kept close to each other, and the signal strength will be significantly affected if the position exceeds 100 µm.

(Han Qin, et al. Neurophoton, 2019)
4. Consumables selection.

(1) Fiber photometry experiments are more sensitive to ambient light, it is recommended that the experiment be conducted in a relatively stable light and dim environment. Fiber photometry is more suitable to choose black ceramic ferrules with black ceramic sleeves.

(2) Larger numerical aperture supplies help fluorescence signal transmission, all the accessories supplies to maintain the same core diameter, NA value is more conducive to experimental recording.

(3) The use of high-powered lasers for optical fiber bleaching, so that the fiber and other consumables to reduce the autofluorescence, while reducing noise interference.

5. Fiber photometry system setup.

(1) Check the connection before the experiment, and use alcohol to wipe both ends of the fiber as well as the ceramic ferrules connection to avoid poor signal recording caused by dirt.

(2) The range of acquisition frame rate needs to be selected according to the fluorescent probe, when the frame rate is within the appropriate range, adjust the exposure time accordingly to help acquire a better fluorescent signal.


1. If no signal is recorded, the light source power can be increased appropriately.

2. For larger brain areas, the volume of virus injection can be increased in an appropriate amount.

3. After virus injection, you can also choose not to implant the ceramic ferrules and wait for 2-3 weeks after virus expression, and then implant the ceramic ferrules together with fiber optics into the original position. While implanting the ferrules, the calcium signal is detected with the fiber photometry system until the most obvious location of the signal is found.

E. Fiber photometry Data analysis

The original data of the fiber photometry contains information such as time axis, fluorescence values corresponding to different channels, and time stamps of event markers. Data are collected by analyzing the fluorescence change (ΔF) relative to the initial baseline fluorescence (F) and observing the signal change corresponding to the calcium transient (ΔF/F). Each peak on the ΔF /F plot represents the response of the neuron to an external light stimulus.

Given that GCaMP fluoresces only when bound to calcium ions, the two-wavelength fiber photometry system can provide critical insight into calcium dynamics by recording the calcium-independent GCaMP fluorescence.

Tri-color fiber photometry system can record red&green fluorescence signal, such as GCaMP, RCaMP, dLight, and jrGECO1a, while a unique 410nm LED is used to acquire control signals and exclude noise.

Data analysis can be done with different software, such as MATLAB, Python, or another analysis software of your choice. Here we show the basic analysis process of RWD fiber photometry software.

Take the data recorded by the RWD R820 Tricolor multichannel fiber photometry system as an example

1. Data preprocessing.

The software of R820 integrates signal acquisition and data analysis. In data analysis, the data preprocessing process includes smoothing processing, baseline correction, motion correction and other functions.

(1)Smoothing can remove too many clutter signals in the data and highlight the target peak as much as possible.

(2)Most of the baseline correction is aimed at the gradual decrease of the bleaching signal caused by the fluorescence signal recorded for a long time, or the gradual decrease of the fiber autofluorescence bleached baseline under the long-term recording. Because the data of this situation shows a downward trend as a whole, which is not conducive to the mapping and analysis of the follow-up data, so baseline correction is needed.

(3)Motion correction is used to compare the data of the 410nm channel, and the 410nm data can be used to reflect the background noise signal. Motion correction is to fit the 410nm data with the 470nm data. The real fluorescence data is obtained by removing the fluctuation of the 410nm data from the 470 data by the algorithm.

fiber photometry data analysis
Figure :Data pre-processing
2.Peak statistics

Marking the number, frequency, height and other information of peaks in a period of time is helpful for data comparison between different treatment groups, so as to get more analysis conclusions.

Before finding an effective peak, it is necessary to set the search time range, MAD (absolute median deviation) range, threshold, etc. The software will give the statistical results of the peak according to the set standard.

3. Compare

Compare fluorescence data with animal behavior data synchronously, select event markers or add event markers, and analyze and map event-related signals.

fiber photometry data analysis
Figure :Event analysis
4. Analysis

The differences of fluorescence data under different processing factors can be analyzed by comparing the data of different groups. In addition, the trajectory of animals can also be analyzed using recorded data.

fiber photometry data analysis in different groups
Figure:Analysis between different groups

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