If you are just starting to learn fiber photometry technology, this article is believed to be very helpful to you.

Fiber photometry is an optical method that is based on the principle of measuring the light emitted from fluorescent molecules via time-correlated single-photon counting (TCSPC)- based fiber optics. From this principle, we know that the realization of this experiment requires probe tools that can express fluorescence in the tissue, as well as a set of equipment that can transmit fluorescence and detect it.

Then we look at the fluorescence tools first. Currently the most commonly used fluorescence tools are divided into calcium indicators, neurotransmitter indicators, voltage indicators and so on. Because there are many kinds of animal models for experimental applications, and also we need to study specific brain regions or even specific kinds of cells, gene editing techniques have been widely used for probe development.

Common genetically encoded calcium indicators (GECIs) are fluorescent proteins derived from green fluorescent protein (GFP, etc.) and its variants (e.g., circularly arranged GFP, YFP, CFP, RFP, etc.) fused to calmodulin (CaM) and the M13 domain of myosin light chain kinase. When Ca²⁺ is present, it binds to CaM, leading to interaction between the M13 and CaM structural domains, triggering a structural rearrangement of cpEGFP, which enhances the green/red fluorescence signal.

The more widely used GECIs are: GCaMP, Pericams, Cameleons, TN-XXL and Twitch, among which GCaMP6, GCaMP7 are now widely used in in vivo calcium imaging studies due to its superior sensitivity. The following is a summary of the types of the common GECIs.

Figure 1

GCaMP6	GCaMP6s	High sensitivity, suitable for low frequency signals
	GCaMP6m	Moderate activity, wide range of application
	GCaMP6f	Fast dissociation, suitable for high frequency signal
jGCaMP7	jGCaMP7s	Highly sensitive and faster than GCaMP6s
	jGCaMP7f	Df/F response enhanced, suitable for single action potential or group activity experiment
	jGCaMP7b	High resting potential brightness, suitable for neuritis or nerve fibers
	jGCaMP7c	High contrast, suitable for wide range imaging
jRGECO1a	jRGECO1a	The excitation wavelength is red-shifted and can be used with GCaMP
jRCaMP1	jRCaMP1a
	jRCaMP1b
Axon-GCaMP	Axon-GCaMP	Mark the axon

The principle of neurotransmitter probes is similar to the above, in which cpEGFP is embedded in specific neurotransmitter receptors, and the binding of the receptors to neurotransmitters triggers a conformational change of the receptors into a fluorescent signal, and the real-time changes in neurotransmitter concentration can be observed with the help of imaging techniques. The neurotransmitter probes that have been developed include DA (dopamine), Ach (acetylcholine), NE (norepinephrine), 5HT (5-Hydroxytryptamine), Ado (adenosine), ATP (adenosine triphosphate), CCK (cholecystokinin), VIP (vasoactive intestinal peptide), and eCB (endocannabinoid), etc.

Because of the relatively slow signal change cycle of calcium signals as well as neurotransmitters, if we want to record the fast signal of membrane potential, we need to use relevant tools such as genetically encoded voltage indicators (GEVI). Emembrane potential changes are a direct sign of both synaptic and action potentials. In some cases, the GEVI signal is faster and more informative than measurements using GECI.

After understanding these fluorescent indicators, we need to be clear on how to express the indicators into the animal. This genetically encoded probe can be expressed in cells or mouse brain by viral injection, transfection, animal cross and other technical means. Take the brain stereotaxic injection study as an example: 1. choose the appropriate virus tool (contains the information encoded by the indicator) to inject into a specific location; 2. implant optical fiber for transmitting excitation light and collecting emission light; 3. wait for 2-3 weeks for the virus to be expressed and then test fluorescence signal.

Figure 2

Once the animals are prepared, it is now necessary to select a suitable system for collecting and analyzing the fluorescence signal. The basic components of a fiber photometry system include excitation light sources, filters, dichroic mirrors, detectors, and fiber optic accessories.

Figure 3

The GCaMP excitation wavelengths are concentrated between 450nm-500nm, while the RCaMP are concentrated between 530nm-580nm. The excitation light source needs to be selected according to the excitation wavelength range of the fluorescent indicator. Also need to choose the appropriate excitation power, for example, 20μW ~ 50μW is more appropriate for excited GCaMP, if the power is too high, it is easy to lead to signal bleaching or even quenching.
The filter needs to consider the emission wavelength range of fluorescent protein. The GCaMP emission wavelength is concentrated between 500nm-550nm, while the RCaMP is concentrated between 570nm-630nm.

Figure 4

There are many types of photodetectors, such as PMT, CCD, CMOS, photodiode, etc. It is generally believed that PMT(photomultiplier tube) is highly sensitive and suitable for detection of very weak signals, but only for single-channel experiments; CCD can be used for multi-channel recording, but the imaging speed is slow; CMOS has better cost performance and can be used for multi-channel recording, while the image processing speed is fast. Multi-channel experimental recording can use multi-branch optical fiber, through a system can record multiple brain areas or multiple animals signal changes.
Fiber optic accessories need to be selected with a black protective layer and low autofluorescence material to better avoid the interference of ambient light and autofluorescence on the signal. The choice of accessories for fiber photometry experiments can be found in the following article How to choose fiber photometry accessories for the most efficient signal transmission? – RWD Life Science (rwdstco.com)

One may wonder, if I inject two colors of fluorescent indicator in the same brain area, how does the detector distinguish between the two different signals? Don’t worry about this, the two signals can be acquired independently by controlling the excitation light and the specific opening time of the detector through software, or by choosing to add additional detectors.

As can be seen in Figure 5, the 410nm LED, 470nm LED, and 560nm LED excitation sources alternate in output at a specific frame rate (which can be set by the software). The two detectors, camera1, only synchronize the signal acquisition at the output of 410 & 470nm LEDs (corresponding to the collection of GFP signal), and camera2 only synchronize the signal acquisition at the output of 560nm LED (corresponding to the collection of RFP signal).

In RWD fiber photometry system, we used dual detector plus TDM (Time-division multiplexing) acquisition design (RWD R820 Tricolor Multichannel Fiber Photometry System – YouTube). Although more literature has confirmed that TDM has been able to avoid mutual interference of red and green fluorescence. However, the single detector uses a dual-band filter (which can be passed for both green and red), and there is a possibility that very little crosstalk can be affected. Because there is a certain possibility that the 470nm LED will excite the red fluorescent probe, thus allowing the red fluorescence to be enhanced and causing crosstalk.

So what is the role of 410nm LED? Why do I need 410 LED for simultaneous excitation when recording GCaMP signals? The fluorescence intensity of GCaMP maintains a constant state with the change of intracellular Ca²⁺ concentration when it is light excited at 405 – 420 nm wavelength. Therefore, it can be assumed to some extent that the signal change corresponding to 410 nm excitation reflects noise signals other than Ca²⁺ concentration change, such as autofluorescence, motion-induced changes, photobleaching, etc. In the data processing, the background signal can be targeted to be removed to obtain the true signal.

Figure 6

Choosing an appropriate system will help your experiment. If you want to know more about fiber photometry system, welcome to contact us!