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Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Published: July 28, 2023 doi: 10.3791/65526
* These authors contributed equally


Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.


Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.


Calcium fluorescence imaging with genetically encoded indicators or synthetic dyes has been a powerful method of measuring neuronal activity in behaving animals, including non-human primates, rodents, birds, and insects1. The activity of hundreds of cells, up to approximately 800 µm below the brain surface, can be measured simultaneously using multi-photon imaging2,3. The activity of specific cell types can also be measured by expressing calcium indicators in genetically defined neuronal populations. Application of the imaging method for small vertebrate models opens up new possibilities in the field of neuronal computation across brain regions.

Zebrafish are a widely used model system in neuroscience research. Larval zebrafish at around 6 days post-fertilization have been used for calcium imaging due to their miniature brain and transparent body4. Juvenile zebrafish (3-4 weeks old) are also used for studying the neural mechanisms underlying sensorimotor pathways5,6. However, the maximal performance level for complex behaviors, including associative learning and social behaviors, is reached at an older age7,8. Thus, a reliable protocol is required to study multiple cognitive functions in the brains of adult zebrafish using imaging methods. While zebrafish larva and juvenile zebrafish can be embedded in agarose for in vivo imaging, adult zebrafish at 2 months or older suffer from hypoxia in such conditions and are physically too strong to be restrained by agarose. Therefore, a surgical procedure is required to stabilize the brain and enable the animal to breathe freely through the gills.

Here, we describe a head restraint protocol that involves a novel design of a single head bar. The reduced surgery time of 25 min is twice as fast as the previous method9. We also describe the design of the recording chamber (semi-hexagonal tank), the head stage and a quick-lock mechanism to combine the two parts9. Finally, the setup to present an immersive visual stimulus to study visually triggered brain activity and behaviors is also described. Overall, the procedures described here can be used to perform two-photon calcium imaging in genetically defined cell populations in a head-restrained adult zebrafish, enabling the investigation of brain activities during various behavioral paradigms.

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All animal procedures were approved and carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Academia Sinica. Details of the research tools can be found in the Table of Materials.

1. Preparation of recording chamber

  1. Prepare a semi-hexagonal tank, a base plate, and a head stage (Figure 1A; Supplementary Files 1-3). The head stage consists of two metal posts attached to a circular plate. The circular plate contains grooves that can be locked onto the base plate. After being locked together, the head stage and the base plate are laid at the bottom of the semi-hexagonal tank.
  2. Place the semi-hexagonal tank on the experimental platform of the microscope. The platform should be able to move in the X and Y directions without hitting the limit of the translation stage.
  3. Aim the 810 nm infrared (IR) light and the camera to the head stage for behavioral recording. Ensure that the field of view of the camera is large enough to fit an adult zebrafish.
    1. To prevent the two-photon laser and the visual stimulus from interfering with behavioral recording, set up an 875 nm short-pass filter and a 700 nm long-pass filter in front of the camera, respectively.
  4. To present the visual stimulus, attach back-projection films to the inner side of the three walls of the semi-hexagonal tank (Figure 1A).
  5. Aim the three projectors at the three surfaces of the tank. The three images should be aligned to form a continuous visual scene. To prevent projector lights from contaminating the calcium fluorescence signal, place a neutral density filter and a red color filter (600 nm, long-pass) in front of each projector, and place a band-pass filter (510/80 nm) in front of the photomultiplier tube (PMT).

Figure 1
Figure 1: Instruments required for head restraint surgery. (A) The quick-lock mechanism between the circular plate of the head stage and the base plate inside the semi-hexagonal tank. Computer-aided design (CAD) files of the custom-made parts can be found in Supplementary Files 1-4. (B) Ω-shaped head bar for the head restraint. (C) The three-axis micromanipulator used to position the head bar to the attachment site. Inset: orientation of the head bar in the clay. (D) Cannon to hold the fish during surgery. Inset: orientation of fish within the cannon. (E) Fish loading module and the micromanipulator used to load the fish onto the head stage. Inset: orientation of fish within the module. Please click here to view a larger version of this figure.

2. Head restraint surgery

  1. Prepare an Ω-shaped head bar (Figure 1B; Supplementary File 4) for the zebrafish head restraint. To do this, attach a piece of oil-based modeling clay to a three-axis micromanipulator. Ensure that the clay is solid enough to hold the head bar, but soft enough to detach from the head bar after head restraint.
  2. Insert an arm of the head bar into the clay and orient the head bar horizontally (Figure 1C). This will ensure that the later attachment of the head bar onto the zebrafish will be level.
    NOTE: The head bar can be made of titanium (24 mg) or stainless steel (43 mg) and can be reused for multiple experiments. The head bar material does not affect the behavior of adult zebrafish (weight ranges from 300-1,000 mg) under a head restraint.
  3. Prepare the cannon, a hollow tube for holding the fish body in place during surgery (Figure 1D).
  4. Prepare 0.03% and 0.01% tricaine methanesulfonate (TMS; see Table of Materials) in fish tank water. This will be used to anesthetize and maintain the fish in a sedated state during surgery.
  5. Prepare four tissue swabs to remove excess skin and water from the attachment sites on the skull. To prepare each swab, cut a paper towel into a 3 cm square and fold along its diagonal to produce a tube-like structure. Twist the ends of the tube into a fine point. The attachment site is very small, so a fine point allows finer control of the swab.
  6. Prepare a fish loading module for the micromanipulator (Figure 1E).
    NOTE: The module consists of two steel posts held together by a right-angle post clamp. One post attaches to the micromanipulator, while the other post holds a rotating post clamp that carries the fish. The module is used to position the fish onto the head stage with fine control. The rotating post clamp serves as a pitch adjuster to change the pitch angle of the fish.
  7. Anesthetize the fish with 0.03% TMS in fish tank water. Throughout the following steps, use a syringe to deliver 0.01% TMS in fish tank water to the mouth in short pulses every 90 s. Water flow from the perfusion should induce gill movement. This will allow the fish to survive for more than 40 min during surgery.
    NOTE: Tg[neuroD:GCaMP6f] is used due to its broad expression of the calcium indicator in the forebrain at both the larval and adult stages10.
    NOTE: Optionally, use gravity perfusion to provide constant water flow to the mouth during periods in surgery where both hands are occupied.
  8. Wrap the fish into a capsule (Figure 2A) that holds the fish inside the cannon.
    1. Wrap the anesthetized fish in a piece of moist paper tissue starting from the tip of the tail to around 1 mm caudal to the gill. Ensure that the wrapping is tight to prevent sliding of the fish out of the tissue.
    2. Wrap a soft plastic tubing with a longitudinal slit (e.g., a medium-sized heat-shrink tubing cut open) around the paper tissue to cover the fish from the end of the tail to around 2 mm caudal to the gill. The tubing ensures that the fish's body remains straight throughout the surgery.
    3. Wrap a paper towel around the tubing to a diameter that is roughly similar to the diameter of the cannon.
    4. Wrap a soft plastic tube cut from the bulb of a plastic transfer pipette around the paper towel. This ensures that the capsule can be loaded smoothly into the cannon.
    5. Load the fish into the cannon.
  9. Use a scalpel to remove the skin covering the attachment sites, two triangular areas of the skull rostrolateral to the cerebellum and above the gills (Figure 2B), then remove the skin covering the region between the attachment sites. Use tissue swabs to dry out the attachment sites and clear out any remaining skin.
    1. Use an adjustable platform to support the head during skin removal, but the platform must not contact the eyes to avoid eye injury during the operation.
      NOTE: Thorough removal of the skin at the attachment sites is crucial in reducing motion artifacts during imaging.
  10. Apply a drop of tissue glue (see Table of Materials) at the center of each attachment site using a 10 µL pipette tip.
    NOTE: Tissue glue covers the attachment sites and dries out quickly. The tissue glue provides a bonding interface between the skull and the dental cement used to glue the head bar, and prevents water from the gills from reaching the attachment site.
  11. Put the fish body in an upright position and position the head bar slightly above and caudal to the attachment sites in preparation for head bar gluing.
  12. Prepare dental cement (see Table of Materials) and use it immediately to glue the head bar to the skull (Figure 2C). The procedure is time sensitive and must be done within 45 s.
    1. Mix one small spoon of polymer with four drops of quick monomer and one drop of catalyst V. Stir the mixture evenly for 15 s.
    2. Use a micropipette and a 10 µL pipette tip to apply the mixture onto the attachment sites and the region between the sites. Avoid covering the gills and the eyes.
    3. Gently press the head bar onto the cement using the micromanipulator. Cover the head bar with the remaining cement.
    4. Wait for 12 min to allow curing of the dental cement. Avoid water contact with the cement.
  13. To improve optical access to the forebrain for calcium imaging, remove the skin above the telencephalon. Skin removal can be done in 3 min with five cuts using a scalpel (Figure 2D). Make sure that lipid droplets adhering to the surface of the skulls are removed.
  14. After curing, remove the clay from the head bar.
  15. Transfer the entire capsule from the cannon to the pitch adjuster in the fish loading module of the micromanipulator.
  16. Use the micromanipulator to position the fish. The head bar should be positioned on top of the metal posts of the head stage. Slightly increase the pitch angle of the fish so that the surface of the forebrain can be better aligned to the optical plane during two-photon imaging.
  17. Glue the head bar to the metal posts with ultraviolet (UV)-curable glue. A 15 s UV exposure is sufficient for curing.
  18. Pull out the fish from the capsule and lock the head stage onto the base plate within the semi-hexagonal tank.
  19. Immerse the animal in fish tank water. The fish should start breathing and recover from anesthesia within 1-2 min.
    NOTE: Optionally, use syringe perfusion to deliver fresh water to the mouth.

Figure 2

Figure 2: Key steps during head restraint surgery. (A) Composition of the capsule inside the cannon. (B) Attachment sites on the skull (red). Red arrows specify blood vessel sites. (C) Top: head bar attached to the fish skull. Bottom: fish loaded onto the head stage. (D) Cuts needed to remove the skin above the forebrain. Numbers denote the cutting sequence. Avoid skin removal at the marked site (arrowhead) to prevent bleeding of the animal. Please click here to view a larger version of this figure.

3. Two-photon imaging

  1. Turn on the laser and wait for 30 min for the power to stabilize. Set the wavelength to 920 nm and power to around 50 mW at the sample.
    NOTE: The laser beam controlled by the resonant scanner moves slowly at the turnaround points of the scan path. To prevent tissue damage, a Pockels cell is used to reduce laser power at the turnaround points.
  2. Place the recording chamber containing the head-restrained zebrafish on the experimental platform (Figure 3A). The platform should be able to move in the X and Y directions without hitting the limit of the translation stage.
  3. Place the objective lens as close as possible to the forebrain surface. The objective lens should be aimed at the front edge of the pupil (Figure 3B).
  4. Open the laser shutter and enable the PMT. Gradually elevate the objective lens (~1 mm) until the dorsal forebrain is revealed in the fluorescence image (Figure 3C).
  5. To increase the number of neurons recorded, use a piezo actuator to acquire images at multiple depths (six image planes, 15 µm apart). This will increase the data yield at the expense of the frame rate. Enable fast Z-axis scanning to control the piezo actuator (uniform mode, number of slices = 6, step size = 15 µm, waveform = sawtooth, flyback time = 4 ms, actuator lag = 8 ms).
  6. For behavior recording, turn on the 810 nm infrared (IR) lights on both sides of the tank. Adjust the angle to illuminate the entire body, which should be clearly visible in the camera.
  7. Turn on the projectors.
  8. Start recording data.

Figure 3
Figure 3: Setup to perform calcium imaging, behavior recording, and visual stimulus display. (A) Three projectors present a visual stimulus onto the walls of the semi-hexagonal tank. IR lights on the side are used to illuminate the body of the zebrafish. (B) Positioning of the objective lens. Left: front view. Right: side view. The distance between the 16x objective lens and the targeted brain region is around 2.5 mm. (C) Example two-photon image. Left: maximum projection of the entire dorsal forebrain in Tg[neuroD:GCaMP6f]. Right: zoomed-in image to reveal neurons across multiple brain regions. Inset: a higher magnification from a different brain region. Images are averages of 10 s of data recorded at 5Hz. Please click here to view a larger version of this figure.

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Representative Results

The protocol consists of two parts: head restraint surgery and two-photon calcium imaging of neuronal activities in the forebrain. The success of surgery is defined by the survival of the animal and the stability of the head restraint. The survival rate can be greatly improved by frequent perfusion of 0.01% TMS solution through the mouth during surgery. Fish should recover from anesthesia and breathe actively within 1-2 min after being immersed into fish tank water. Two-photon calcium imaging enables activity recording of individual neurons in the dorsal forebrain at a depth up to 200 µm from the brain surface through intact skulls (~40 µm in thickness). This imaging range covers multiple zones of the dorsal telencephalon (D), including the medial zone (Dm), the rostral part of the central zone (rDc), the caudal part of the central zone (cDc), and the lateral zone (Dl). Together they make up 30% of the telencephalon in adult zebrafish (Figure 3C). With volumetric imaging using the piezo actuator, we typically record the activity of 150 neurons in Dl or cDc, and 300 neurons in Dm and rDc in Tg[neuroD:GCaMP6f]10. Simultaneous behavioral recording is performed during brain imaging, which enables the identification of neuronal correlates of motor outputs (Figure 4).

During two-photon imaging, tail movements should not induce a visible motion artifact in the image. A small (<1 µm) and transient motion can be observed during extreme struggling. These motions are typically reversible, so imaging can be continued afterward. We also observe a slow drift (<1 µm min-1) in the lateral and axial directions9. To prevent the loss of neurons due to axial sample drift, we typically restrict our imaging session to 10 min. Photobleaching of the calcium indicator should not be observed after a 10 min imaging session under the laser power specified.

Figure 4
Figure 4: Behavior tracking and neural activity pattern in adult zebrafish. (A) An example frame of camera recording of behavior (ventral view). (B) Activity of forebrain neurons (ΔF/F, black) and tail movement intensity (blue). The intensity of tail movement was quantified by the mean of the absolute pixel-wise difference between successive video frames. Please click here to view a larger version of this figure.

Supplementary File 1: Design of the base plate. Please click here to download this File.

Supplementary File 2: Design of the circular plate. Please click here to download this File.

Supplementary File 3: Design of the semi-hexagonal tank. Please click here to download this File.

Supplementary File 4: Design of the head bar. Please click here to download this File.

Supplementary Table 1: Troubleshooting details. Please click here to download this File.

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Here, we describe a detailed protocol to restrain the head of adult zebrafish for two-photon calcium imaging. There are two critical steps to achieve a head restraint that is stable enough for laser scanning imaging. First, the head bar has to be glued to the specific attachment sites of the skulls. Other parts of the skull are often too thin to provide mechanical stability and may even be fractured during strong body movements. Second, the skin above the attachment sites has to be thoroughly removed. Residual water should also be dried thoroughly. This allows the skull to tightly bind to the tissue glue. A table that lists potential problems, their causes, and the solutions is provided (Supplementary Table 1).

Due to the curved geometry of the zebrafish forebrain, the excitation beam and resultant emission travel through different thicknesses of the brain tissue across the imaging plane. Thus, the fluorescence of neurons imaged within an optical section vary based on the neuron's distance from the brain boundary. This leads to low data yield within a recording session. The problem is particularly severe when imaging the Dl and cDc (Figure 3C). Here, we use a piezo actuator to perform recordings across multiple axial positions, which compromise the frame rate but increase the number of neurons recorded. Importantly, as neuronal activities at multiple axial positions are recorded within a single behavioral session, the data has a higher statistical power in comparison to activity data recorded from multiple behavioral sessions. Alternatively, three-photon imaging2 and adaptive optics11 can record the neuronal activities in deep tissues and relax the high curvature problem. However, the repetition rate of three-photon lasers and the heterogenous structure of skulls should be considered.

In comparison to previous methods9,12 that use multiple metal parts to stabilize the head, the current approach made a significant improvement by using a single piece of an Ω-shaped head bar. This simplified design reduces the surgery time by 50%. The protocol is also easier to learn and typically can be mastered within 2 weeks of training. Although the surgery time is largely reduced in comparison to the previous protocol9, we did not observe obvious differences in survival rate nor behavior under the head restraint. For example, the duration of time during which the animal remains active under the microscope is similar (3-8 h, depending on the animal). The amount of time required for the animal to wake up from anesthesia after the surgery is also similar (1-2 min). The transgenic line10 used in this protocol involves a single transgene that encodes the calcium indicator in the forebrain from the larval stage to adulthood, similar to the expression timeline of the triple transgenic line used in a previous study12.

Currently, the protocol has the following limitations. First, neuronal activity can be imaged through the skull at a depth up to 200 µm from the brain surface using a two-photon microscope. The image quality at various depths has been previously quantified9,12. These imaging depths allow access to the dorsal forebrain areas Dm, Dc, and Dl, but potentially exclude the ventral forebrain areas Vv, Vs, Vp, and Vd in adult zebrafish. In addition, optical access to the majority of the midbrain and the hindbrain is obstructed by the presence of the head bar and dental cement. Additional modifications to the protocol are required to perform imaging in these brain regions. Furthermore, we typically restrict an imaging session to 10 min to prevent significant sample drift. The potential cause of the drift is the weakening of the bonding between the skull and the tissue glue after vigorous tail movements. If longer recording sessions are needed, volumetric imaging with small axial steps followed by 3D image registration can be performed.

This head restraint protocol opens up several future applications. First, two-photon calcium imaging, optogenetic manipulations, electrophysiology, or even ultrasound imaging can be performed in vivo. With three-photon imaging, neuronal activity across the entire forebrain can also be recorded2. In addition, a mechanical device can be constructed to reversibly grab and release the two ends of the head bar, which enables longitudinal investigation of neural activity and circuit morphology across days. Finally, the visual display setup we described provides a 180° horizontal field of view to the animal. The setup can be used to construct an immersive virtual reality environment that interacts with head-restrained fish9. Overall, this protocol enables activity measurement of neuronal populations across the pallium of behaving adult zebrafish.

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The authors declare that they have no competing financial interests.


This work was supported by the Institute of Molecular Biology, Academia Sinica, and National Science and Technology Council, Taiwan. The Machine Shop at the Institute of Physics, Academia Sinica helped to fabricate custom-designed parts. We also want to thank P. Argast (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) for the design of the quick-lock mechanism of the head stage.


Name Company Catalog Number Comments
Acquisition card MBF Bioscience Vidrio vDAQ Microscope
Back-projection film Kimoto Diland screen - GSK present visual stimulus
Band-pass filter (510/80 nm) Chroma ET510/80m Microscope
Base plate for the semi-hexagonal tank custom made see supplemental files recording chamber
Camera filter (<875 nm) Edmund optics #86-106 Behavior recording
Camera filter (>700 nm) Edmund optics #43-949 Behavior recording
Camera lens Thorlabs MVL50M23 Behavior recording
Chameleon Vision-S Coherent Vision-S Laser
Circular plate for the head stage custom made see supplemental files recording chamber
Controller for piezo actuator Physik Instrumente  E-665. CR Microscope
Current amplifier Thorlabs TIA60 Microscope
Elitedent Q-6 Rolence Enterprise Q-6 Surgery: UV lamp
Emission Filter 510/80 nm Chroma ET510/80m Microscope
Head bar custom made see supplemental files recording chamber
Infrared light Thorlabs M810L3 Behavior recording
LED projector AAXA P2B LED Pico Projector present visual stimulus
Moist paper tissue (Kimwipe) Kimtech Science 34155 Surgery: moist paper tissue
Motorized XY sample stage Zaber X-LRM050 Microscope
Neutral Density Filters (50% Transmission) Thorlabs NE203B present visual stimulus
Ø1/2" Post Holder ThorLabs PH1.5V Surgery: hollow tube for cannon
Ø1/2" Stainless Steel Optical Post ThorLabs TR150/M Surgery: fish loading module
Objective lens 16x, 0.8NA Nikon CF175 Microscope
Oil-based modeling clay Ly Hsin Clay C4086 Surgery: head bar holder
Optical adhesive Norland Products NOA68 Surgery: UV curable glue
Photomultiplier tube Hamamatsu H11706P-40 Microscope
Piezo actuator Physik Instrumente  P-725.4CA PIFOC Microscope
Pockels Cell Conoptics M350-80-LA-BK-02 Microscope
Red Wratten filter (> 600 nm) Edmund optics #53-699 present visual stimulus
Resonant-Galvo Scan System INSS RGE-02 Microscope
Right-Angle Clamp for Ø1/2" Post ThorLabs RA90/M Surgery: fish loading module
Rotating Clamp for Ø1/2" Post ThorLabs SWC/M Surgery: fish loading module
ScanImage MBF Bioscience Basic version Microscope
Semi-hexagonal tank custom made see supplemental files recording chamber
Super-Bond C&B Kit Sun Medical Co. Super-Bond C&B Surgery: dental cement
Tricaine methanesulfonate Sigma Aldrich E10521 Surgery: anesthetic
USB Camera FLIR BFS-U3-13Y3M-C Behavior recording
Vetbond 3M 1469SB Surgery: tissue glue



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Adult Zebrafish Behaving Cognitive Functions Optical Imaging Methods Brain Activity Recording Dorsal Forebrain Head Restraint Surgery Neuron Recording Immersive Visual Environment Visually Triggered Behaviors
Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish
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Cite this Article

Bandonil, J. S., Liao, Y. H., Fathi, More

Bandonil, J. S., Liao, Y. H., Fathi, A., Huang, K. H. Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish. J. Vis. Exp. (197), e65526, doi:10.3791/65526 (2023).

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