Fiber photometry is a relatively simple method for recording neural activity in vivo. But despite its simplicity, the best fiber photometry experiments require careful planning. There are various options for different hardware configurations, integration with behaviour systems, GEFIs, transgenic animals, and signal processing strategies. As with any methodological approach in neuroscience, a researcher’s first task is to answer a series of key questions:

1. Is fiber photometry the correct method for this scientific question? (See the first eBook in our Fiber Photometry series for an overview of this issue: An Introductory Primer on Fiber Photometry).
2. How should my photometry recording protocol be designed?
3. Which animal line and/or GEFI should I use? How should I deliver it, and to which brain regions?
4. What hardware configuration is required for this experiment?

This eBook will help you answer these questions and more by providing some in-depth tips for experimental design, validation of sensors, hardware configuration, and best practices for collecting the best possible photometry data.

Fiber photometry systems contain several key hardware components that are extremely important for researchers to have a basic understanding of. Mightex’s OASIS Photometry system is a modular, camera-based system designed to maximize user flexibility, customizability, and high-throughput experimental pipelines. Here we will review key information about each of the basic components.

Figure 4. This schematic illustrates the excitation–emission light path during a typical photometry experiment. The LED driver precisely modulates the LED light source, receiving hardware triggers and controlling trigger timing and output power. Upon activation, the LED delivers excitation light into the optical path toward the filter assembly. Within this module, dichroic and emission filters spectrally separate excitation and fluorescence signals, directing excitation wavelengths toward the sample while isolating the longer-wavelength emission containing the biological signal. The excitation light is delivered to the subject via a fiber optic patchcord connected to the photometry rig. Fluorescence emitted from the target tissue is collected through the same optical pathway, spectrally filtered to remove residual excitation light, and directed to the light detector, where the optical signal is converted into a measurable electrical output for downstream analysis.

1. Light Source

Light sources are the components that deliver excitation light into the optical path. The earliest photometry systems used lasers, which can deliver high power within a small linewidth. However, they are expensive, require enhanced safety protocols, and present a real risk of tissue heating/phototoxicity to the recording site. The OASIS Photometry system, as well as most other commercial systems, now use LEDs as the excitation light sources for recording GEFIs. LEDs deliver a broader bandwidth than lasers, but they can simply be accompanied by an emission filter to restrict the light to the intended excitation wavelength. LEDs come in many wavelengths, but photometry systems will typically use 2-3 wavelengths to account for an isosbestic channel and one or two wavelengths for recording sensor-dependent activity.

2. Light Source Driver

The light source driver provides current to drive the LED. Additionally, it triggers the LED on and off and determines how much power output is delivered. Precise control and when and how much excitation light is delivered is paramount for effective photometry experiments. Light source drivers and light sources must be compatible with respect to the maximum current output in order to sufficiently drive the LED without damaging it.

3. Dichroics and Filters

Due to the fact that multiple wavelengths of light are travelling through the optical path of the photometry rig, it is necessary to separate excitation light from emission light (the wavelength containing your signal). This is accomplished via a series of dichroic mirrors and filters. A dichroic mirror allows the passage of certain wavelengths while reflecting others. In the typical configuration for imaging a green sensor, 405 and 465 nm wavelengths are aimed at a “longpass” dichroic mirror which reflects 465 nm light and any shorter wavelengths so that these excitation wavelengths are directed into the imaging path heading towards the subject patchcord. The green emission light from the sensor, which is a longer wavelength, is able to pass through this mirror to the light detector, so that the detector sees only the emission light and none of the excitation light. However, dichroic mirrors do not reflect 100% of the light they are intended to, so an additional emission filter which only allows green light may be placed in front of the light detector to further filter the signal.

Depending on the number of wavelengths in use (including stimulation light for combined photometry + optogenetics), multiple dichroic mirrors and filters will be arranged in the light path to allow multiple excitation wavelengths to pass into the subject patchcord, with only the emission wavelengths of interest reaching the light detector.

4. Subject Patchcord

After excitation light has been generated, it must be delivered to your subject via a fiber optic connection running from the photometry rig, into the recording chamber, and into the fiber optic implant in the subject.

Fiber optic patchcords come with a variety of optical properties and various connectors. For photometry recordings, you should use a low autofluorescence fiber. Connecting the patchcord to a subject requires that one end of the patchcord has a fiber cannula with the same outside diameter as the fiber implants in your animals. This connection is secured with a metal or ceramic mating sleeve, which holds the two together via compression. The other end of the patchcord must connect either directly to the photometry system or rotary joint via an SMA connector. Patchcords with an SMA connector at both ends function to relay the optical signal to- and from the photometry system and a rotary joint. A range of core diameters is acceptable for photometry recordings (generally 200-400 µm). Larger the core diameters will offer a higher numerical aperture and more light transmission, but a larger diameter will also mean more weight and rigidness as well as more autofluorescence. The same is the case with longer versus shorter patchcords.

Subject patchcords can be single-core for imaging one site/subject, or they can be multifurcated, with multiple fiber cores to independently record multiple brain regions/animals. Multi-furcated patchcords are the means by which camera-based photometry systems are scaled to high-throughput configurations due to the ability of a single camera to detect light from multiple fiber cores. Of note, this approach means that all fiber cores receive the same pattern of LED pulses and the same level of excitation power, and LED power cannot be modulated for each animal or region. Mightex offers high-quality, multi-furcated fiber patchcords with minimal differences in light transmission across fiber cores. However, minor differences in power output tend to be inevitable, and the goal is to keep excitation power within a 5µW range.

As mentioned previously, fiber optic patchcords experience negligible power loss over the range of lengths that would be typical in a photometry recording space, so there is no downside to using long patchcords to provide as much length as is needed for your experimental setup. However, the length of the patchcord should be calibrated to allow it to lie as straight as possible, as bends in the patchcord reduce light transmission efficiency ⁹ in addition to interfering with animal behaviour.

5. Light Detector

After excitation light has been delivered to the subject and the emission light has been collected and filtered, the final hardware step is for the emission light to be collected and measured. Mightex’s OASIS Photometry system makes use of a scientific-grade CMOS camera, enabling simultaneous signal detection from multiple animals and/or brain regions without the need for multiple, expensive detectors. Our SBE-series monochromatic cameras contain a global shutter CMOS capable of hundreds of frames/second with high sensitivity.

This approach works by focusing the camera on the face of the fiber patchcord to collect fluorescence emitted from the fiber cores. In order to separate the emission responses to each excitation wavelength, light sources are cycled on and off in an interleaved fashion, such that individual frames of the recording only contain the emissions corresponding to one wavelength at a time. Because the CMOS detector contains a 2-dimensional pixel array, signals from multiple fiber cores can be recorded on one detector, enabling the use of multi-furcated fiber patchcords for high-throughput research applications at a relatively low cost.

Setting up a fiber photometry system – particularly one intended for high-throughput, simultaneous recordings – requires careful planning. Once set up, a photometry system will ideally not be disassembled or reconfigured frequently as this would require recalibration every time. This is especially the case when using multifurcated fiber bundles on a camera-based system, as disconnecting and reconnecting the fiber bundle will then require refocusing and realignment of the fiber ROIs. If your system must be portable (e.g. in the case of shared laboratory equipment/space), we recommend that you keep your system on a cart and as fully set up as possible, with multifurcated fiber branches labelled to minimize the complexity of reconnecting them to recording chambers. Your photometry system can be located on any stable surface, such as a desk, shelf, or cart, as long as you are extremely careful not to subject the fiber optic cords to physical strain such as tension or pinching. This means that you should measure your space and ensure that you have patchcords of adequate length running to your recording chambers and, if using a cart, that the cart and chambers are in a stable position so as not to tug on the patchcords or drag equipment off of surfaces.

Figure 5. This illustration demonstrates the impact of patchcord length on experimental performance and animal behavior. In the left panel, a patchcord that is too short restricts movement and places strain on the animal and patchcord. The middle panel depicts an appropriately sized patchcord, providing sufficient slack to allow naturalistic movement while minimizing tension and excess drag. In the right panel, a patchcord that is too long introduces unnecessary slack, causing the patchcord to tangle and bend at extreme angles, placing strain on both the animal and the patchcord.

Using patchcords of an appropriate length is critical both to ensure that animals can physically navigate to all areas of the behavioural apparatus and to minimize stress/bending of the patchcord. The cable should be neither excessively short (for obvious reasons) nor excessively long, because added slack will exert bending stress on the patchcord, potentially causing optical artifacts and even breaking the fiber in extreme cases. Additionally, patchcords with a standard core diameter do have a degree of bending stiffness such that a patchcord under bending stress will also exert some force on your animal, which can noticeably affect the locomotor behaviour of a 25g mouse, for example. Furthermore, an overly long patchcord may make parts of it accessible to the animal to chew on and damage. Another very important consideration is twisting of the patchcord while animals navigate the recording chamber. This is often not a significant problem in many contexts, such as shorter recordings, and can often be mitigated by using long enough patchcords to compensate. If patchcord twisting presents a problem, researchers can consider using a high-quality rotary joint, such as those offered by Mightex. Fiber optic light paths experience the most power loss at junction points, with negligible power loss over the length of a single continuous fiber, so rotary joints can in principle introduce some power loss compared with simply using a longer patchcord without a rotary joint. However, if your GEFI is reliable and you are using high-quality equipment, this should not ordinarily be a huge problem.

Pulse Patterning and Modulation

For fiber photometry to work, excitation light must be delivered in very specific temporal patterns, lest the emission signals be impossible to disentangle during analysis. The detectors used in fiber photometry systems, including Mightex’s OASIS Photometry system, are usually monochromatic in order to maximize sensitivity and dynamic range. Even if a colour camera were to be used, the activity-dependent and isosbestic channels are measured in the same range of emission wavelengths. As such, this leaves the temporal domain as the only remaining dimension along which to parse the multiple signals arising from the same fiber core. Mightex’s OASIS Photometry system uses interleaved excitation in which light sources are pulsed on and off in a staggered fashion (with the camera frame rate adjusted to match) such that each camera frame contains only a single excitation wavelength. In this case, the sample rate for each channel is a fraction of the total sample rate, which can be calculated as total frame rate multiplied by LED duty cycle.

Power Calibration

The amount of excitation power delivered by your light source must be measured and carefully calibrated. This should, at minimum, be done before every new experiment, but ideally far more often (e.g. weekly). The same power setting on an LED driver will not deliver the same intensity of excitation light across patchcords, rotary joints, and other optics, so the power must be measured at the tip of every fiber patchcord. This is not only to ensure that the intensity of excitation light is consistent between subjects, but also to continually verify that the equipment itself is intact and functioning properly. High-quality photometry equipment, when used correctly, rarely fails suddenly, but LEDs and LED drivers can be subject to performance instability or degradation. Most of all, however, the transmission efficiency of fiber optic patchcords and rotary joints can degrade substantially over time, or fail entirely, as the equipment is repeatedly exposed to the physical stresses of recording experiments. A broken fiber patchcord will often have a visible kink at the location of the break, but regardless of how visually obvious the problem is, it will manifest as a drastic drop in the baseline power detected during recording or by a light meter.

Calibrating the amount of excitation power for an experiment is a balancing act. Too little excitation power will mean that the sensor is not being sufficiently excited to reliably emit enough light to detect a signal. Too much excitation power can damage tissue, excessively bleach the sensor, and interfere with other constructs such as opsins. Moreover, higher excitation intensities will produce diminishing returns with respect to your signal:noise ratio. Determining the correct amount of excitation power is essentially a matter of finding the level of excitation power that maximizes the amplitude of sensor-dependent dynamics while minimizing photobleaching. Ali Mohebi describes an excellent method for calibrating excitation power for specific experiments ¹⁰.

The wavelength used for the isosbestic channel is usually 400-410 nm and the wavelengths for sensor-dependent activity are usually 465-470 nm (for green sensors) and/or 560 nm (for redshifted sensors). This wavelength configuration will suffice for the majority of photometry experiments. However, the landscape of GEFIs is rapidly evolving, with recent expansion into the far-red range. Mightex’s OASIS Photometry system can be customized with additional wavelengths to expand our users’ spectral palette and enable greater multiplexing. The modular nature of our hardware enables user-friendly integration of new wavelengths without the need to replace most other elements of the base system. Some additional care must be taken when combining multiple sensors, as there can be channel bleedthrough and off-target effects of excitation light. Certain fluorophores can undergo complex photoswitching in response to certain wavelengths, presenting a potential confound in experiments using multiple excitation wavelengths. For example, some redshifted GEFIs undergo an increase in baseline brightness in response to high-intensity blue light ¹¹.

Figure 6. This schematic illustrates the excitation wavelength ranges for commonly used sensors in fiber photometry: isosbestic control (400–410 nm), green fluorescent sensors (465–470 nm), red-shifted sensors (560 nm), and far-red sensors (760-765nm).

Isosbestic Channel

At this point, it is useful to provide some detail about precisely what the isosbestic channel is, in principle, and what it does in the specific case of fiber photometry. A fundamental challenge in fiber photometry is that the amount of measured fluorescence can change in response to many factors other than GEFI activity. Obtaining the activity-dependent signal from a raw photometry recording requires the removal of these activity-independent artifacts, and an isosbestic channel is the most common way of doing so.

Speaking broadly about spectroscopy, an isosbestic point is a specific wavelength (or band of wavelengths) where the absorbance of an illuminated sample exhibits no change when compared between two or more different states, such as the different phases of a chemical reaction. GCaMP will emit fluorescence to one degree or another under a range of excitation wavelengths, but when comparing between Ca²⁺-bound GCaMP and unbound GCaMP under tightly-controlled conditions (e.g. cell culture electrophysiology), there are specific wavelengths where Ca²⁺ binding leads to a change in fluorescence (i.e. around 470nm) and other wavelengths where the emitted fluorescence appears unaffected by the presence or absence of Ca²⁺ (i.e. around 405nm). Hence, the isosbestic point is a fluorescent signal that is invariant to the Ca2+ binding state of the sensor. In fiber photometry, this property allows other sources of variability in fluorescence intensity to be independently collected and later removed.

The exact isosbestic wavelength and peak excitation wavelength can vary from sensor to sensor, so the standard 400-410nm isosbestic channel used in most photometry systems may not precisely target the isosbestic point of every sensor. Although it appears to be common practice in the field to use 400-410nm isosbestic excitation across multiple sensors, some researchers may desire a more specialized selection of excitation wavelengths. The exact isosbestic points for a number of sensors have been measured empirically (see Table 1 ¹²).

Control Dyes and Sensors with No Isosbestic Point

Critically, one must verify that their GEFI of choice has an isosbestic point in the first place. There are now multiple sensors that either lack an isosbestic point, or no isosbestic point has been reported. These include some red-shifted sensors based on the fluorophore mApple ¹³ and the latest far-red dopamine sensor ¹⁴.

The typical method for empirically measuring and verifying a GEFI’s isosbestic point is to perform in vitro fluorescence imaging of GEFI-expressing cells, under a range of wavelengths, and in the presence or absence of the GEFI’s target ligand. The results of these experiments will typically be reported via a spectrum plot that looks something like (Figure 7) and shows relative fluorescence intensity or absorbance. GEFIs that have an isosbestic point will emit some fluorescence with an excitation wavelength offset from the ligand+ emission peak, and the intensity of this fluorescence will be equal with or without ligand binding.

Figure 7. Illustration of single-photon spectrum plots. A sensor is expressed in cell culture and illuminated under a range of wavelengths in the presence (solid line) or absence (dashed line) of the sensor’s ligand. In the left panel, a clear isosbestic point is visible where the sensor’s brightness is equivalent both in the presence and absence of the ligand. In the right panel, no clear isosbestic point is apparent.

The lack of an isosbestic point has major implications for how motion and photobleaching are corrected. Attempting to use a typical isosbestic wavelength (e.g. 405nm) to correct artifacts would fail to meet some of the basic assumptions of an isosbestic channel. If the GEFI emits little or no activity-independent fluorescence under 405nm illumination, then activity-independent fluctuations in GEFI brightness are unmeasurable by these means. Some signal may still be observable, but this would reflect, for example, tissue autofluorescence rather than the GEFI itself.

The current fiber photometry research literature describes at least 3 general approaches to work around this challenge. The first is to express a control dye that falls within a different spectral range from the GEFI ¹⁵ and use it similarly to an isosbestic channel. The brightness of this control dye will have no activity-dependent dynamics, but will reflect motion artifacts, pH fluctuations, and other sources of noise. A second approach, applicable in multi-colour photometry experiments, is to use the isosbestic point of one of the GEFIs to correct artifacts for the other GEFI ¹⁶. The third approach is to forego the use of a control channel entirely ¹⁴. Although this approach carries the risk of very noisy data, it can work under the right conditions, such as experiments in which an experimental manipulation causes very large changes in the signal that can overcome the noise.

Isolation of Separate Fluorescence Channels

Once the raw photometry has been collected, it must be separated into individual channels for analysis. Due to the fact that the signal is composed of emissions from a minimum of 2 excitation wavelengths through a single fiber, these signals must be separated in the temporal domain. In camera-based photometry systems such as those offered by Mightex, LEDs are pulsed on and off in an “interleaved” fashion, with one LED active at a time. The camera frame rate is also adjusted to match LED pulses so that each frame contains only one wavelength. Individual signals are generated by “deinterleaving” the raw recording, meaning that camera frames from each wavelength are separated and concatenated into separate timeseries. You can read a more detailed explanation of this on the MightexBio Blog: Fiber Photometry Data Preprocessing: Deinterleaving Signals and Fixing Dropped Frames.

Denoising and Temporal Downsampling

Raw photometry signals may be collected at sampling rates that are unnecessarily high given the underlying kinetics of a GEFI. As such, it is a common practice to temporally downsample and filter the signal of unnecessary frequencies. Photometry traces can be downsampled via decimation (keeping every e.g 10th frame and discarding the others) or binning (taking average values across blocks of multiple frames). High-frequency noise (such as electrical hum) is removed using a low-pass filter with a cutoff frequency determined relative to the speed of GEFI kinetics (e.g. 2-10 Hz for GCaMP). Multiple types of low-pass filter exist, with some being popular for use in photometry such as a Butterworth filter. Recently, researchers have begun recommending that a zero-phase filter is most advantageous due to the fact that it causes minimal distortion of the signal13. Aside from high frequency noise, other kinds of artifact arising from electrical interference can be removed using a median filter.

However you decide to filter and/or downsample your signal, there are some key points to consider. The parameters that you use to perform these procedures must account for the underlying kinetics of your GEFI of choice and the biological signal you are trying to record. Calcium dynamics are much slower than voltage dynamics, for instance, so the sampling frequency you use to record calcium will not be sufficient for voltage recordings. In any case, a useful metric here is the Nyquist theorem, which can be summarized as such: Reconstruction of a continuous signal from discontinuous samples requires that samples are collected at a minimum of twice the frequency of the signal’s fastest component. Sampling rates below this Nyquist value can lead to temporal “aliasing”. You will likely have seen examples of aliasing before, such as videos of spinning helicopter rotors where the blades appear to be immobile or move slowly or backwards. Aliasing occurs because high frequency events are only partially sampled and accumulate in the signal as low-frequency features. Although the bulk signal collected via fiber photometry is the sum of many asynchronous events across many cells, obscuring the kinetics of single cell activity, it is not inconceivable that for brief periods of reduced activity, the signal may contain only a single cell. Ideally, your denoising and temporal downsampling parameters ought to be based on the fastest components of single cell activity. It is also worth emphasizing that denoising must be done before any temporal downsampling or this can lead to aliasing.

Having followed the advice described in this eBook, you should now have much of the knowledge required to collect high-quality fiber photometry data, correctly preprocess it, and have processed traces ready for data analysis. The analysis and interpretation of fiber photometry data is a topic unto itself which will be explored in a future eBook.

If you found this eBook enlightening, we encourage you to have a look at our other eBooks, Application Notes, and to stay tuned for future releases. The Mightex team is made of experienced researchers from behavioural neuroscience and other biomedical fields. We continually produce new content to bring these fascinating topics to a wider audience and empower our customers with the knowledge to conduct the highest-quality research possible.

1. Hrvatin, S. et al. Neurons that regulate mouse torpor. Nature 583, 115–121 (2020).
2. McMahon, S. M. & Jackson, M. B. An inconvenient truth: Calcium sensors are calcium buffers. Trends Neurosci. 41, 880–884 (2018).
3. Masala, N. et al. Aberrant hippocampal Ca²⁺ micro-waves following synapsin-dependent adeno-associated viral expression of Ca²⁺ indicators. eLife (2024) doi:10.7554/elife.93804.2.
4. Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862–885 (2012).
5. Gasterstädt, I. et al. Genetically encoded calcium indicators can impair dendrite growth of cortical neurons. Front. Cell. Neurosci. 14, 570596 (2020).
6. Resendez, S. L. et al. Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nat. Protoc. 11, 566–597 (2016).
7. Yang, Y. et al. Improved calcium sensor GCaMP-X overcomes the calcium channel perturbations induced by the calmodulin in GCaMP. Nat. Commun. 9, 1504 (2018).
8. Pisanello, M. et al. The three-dimensional signal collection field for fiber photometry in brain tissue. Front. Neurosci. 13, 1–16 (2019).
9. Lustermann, B. et al. Experimental determination and ray-tracing simulation of bending losses in melt-spun polymer optical fibres. Sci. Rep. 10, 11885 (2020).
10. Mohebi, A. LED power tuning for fiber photometry – Diamonds in the rough. Diamonds in the rough https://blog.mohebial.com/led-power-tuning-for-fiber-photometry/ (2022).
11. Wu, J. et al. Improved orange and red Ca²⁺ indicators and photophysical considerations for optogenetic applications. ACS Chem. Neurosci. 4, 963–972 (2013).
12. Simpson, E. H. et al. Lights, fiber, action! A primer on in vivo fiber photometry. Neuron 112, 718–739 (2024).
13. Deng, F. et al. Improved green and red GRAB sensors for monitoring spatiotemporal serotonin release in vivo. Nat. Methods 21, 692–702 (2024).
14. Zheng, Y. et al. In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors. Science 388 (2025).
15. Meng, C. et al. Spectrally resolved fiber photometry for multi-component analysis of brain circuits. Neuron 98, 707–717 (2018).
16. Skirzewski, M. et al. Continuous cholinergic-dopaminergic updating in the nucleus accumbens underlies approaches to reward-predicting cues. Nat. Commun. 13, 7924 (2022).