Presented here is a protocol for in vivo whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy. The experimental procedure includes sample preparation, image acquisition, and visualization.
As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.
Zebrafish (Danio rerio) has been widely adopted as a model vertebrate animal in neuroscience, owing to its optical transparency at the larval stage, its fast development, its low maintenance cost, and the availability of diverse genetic tools1,2,3,4. In particular, the optical transparency of larvae combined with genetically encoded fluorescent reporters of biological events5,6,7,8,9 provides a unique opportunity to image both neuronal activity and structure at the whole-brain level10,11,12,13,14. However, even with a microscope that supports cellular resolution, the acquired images do not necessarily retain the information at the single-cell level; the optical image quality may be degraded due to the aberration introduced by the agarose gel used for sample mounting, the fish may be mounted at an angle, thus the regions of interest are not fully contained within the field of view of the microscope, and the fish may move during the recording, causing motion artifacts in the images or impeding accurate signal extraction from the images.
Thus, an effective and reproducible protocol is needed to acquire high-quality image data with minimal noise and motion. Unfortunately, publicly available protocols for imaging a whole brain of larval zebrafish in vivo15,16,17,18,19 only briefly describe the procedure, leaving substantial parts of the details, such as agarose solidification, precise mounting techniques, and sample positioning using forceps, up to each experimentalist. In addition, inconsistencies in agarose concentration and immobilization methods10,11,14,15,16,17,18,19 can lead to challenges arising from fish movement during the imaging process.
Here, a detailed protocol for whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy is provided. Figure 1 provides a graphical overview of the protocol: sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The structural and functional imaging of the larval zebrafish brain in vivo is demonstrated using a commercial confocal microscope and a custom-designed fluorescence microscope. This protocol can be tailored by practitioners for brain imaging with certain sensory stimuli or behavior contexts depending on the experimental needs and design.
The current protocol enables in vivo whole-brain imaging of larval zebrafish over an extended period (e.g., longer than 1 h) and visualizations of the acquired structural and functional imaging data.
The most critical steps are sample mounting and positioning. During sample embedding, it is crucial to prevent bubble formation and avoid dust in the agarose gel. If the gel contains air bubbles and dust, the image quality may be severely degraded. While positioning the sample using forceps, it is important to ensure that the sample is level both horizontally and vertically. Otherwise, the regions of interest may not be contained within the field of view of the microscope. In addition, this positioning should be done within a short time window before solidification of the agarose gel to avoid causing damage to its surface, as such damage compromises the image quality.
Another major challenge is to avoid motion in images that come from incomplete solidification of the agarose gel (Figure 6) and paralyzation of the zebrafish. When egg water is added to the sample before complete solidification of the agorse gel, fish move slowly both laterally and axially, manifesting as sample drift in the time-series images (Figure 6A). If the dose of paralytics is insufficient or the duration of effect ends, the sample may attempt to move, which appears as rapid twitching in the time-lapse images.
Despite the importance of the aforementioned steps for acquiring high-quality images without motion artifacts, publicly available protocols15,16,17,18,19 provide only a brief overview of the experimental procedure, lacking these details. For instance, the acquired images without immobilization protocols11 suffer from substantial motion artifacts that make the downstream image analysis challenging. By integrating essential components, such as agarose gel solidification and sample paralyzation, our protocol significantly enhances the consistency in the quality of acquired images while minimizing motion artifacts.
In summary, an optimized and reproducible experimental procedure for in vivo imaging of the larval zebrafish brain is described. The validity and reproducibility of this protocol for in vivo imaging of brain activity and structure have been confirmed in multiple settings14,18,29,30,31. The present workflow focused on the whole-brain imaging of larval zebrafish, but it can be easily applied to imaging other organs of larval zebrafish35,36.
The authors have nothing to disclose.
The zebrafish lines used for calcium imaging were provided by the Zebrafish Center for Disease Modeling (ZCDM), Korea. This research was supported by National Research Foundation of Korea (2020R1C1C1009869, NRF2021R1A4A102159411, RS-2023-00209473).
1.5 mL microcentrifuge tube | SciLab | SL.Tub3513 | To aliquot agarose gel and pancuronium bromide solution |
15 mL Falcon tubes | Falcon | 352096 | To prepare agarose gel and pancuronium bromide solution |
16× 0.8NA water dipping objective lens | Nikon | CFI75 LWD 16×W | Objective lens for whole-brain imaging |
1-phenyl 2-thiourea (PTU) | Sigma-Aldrich | P7629-10G | 200 μM of 1-phenyl 2-thiourea (PTU) |
50 mL Falcon tubes | Falcon | 352070 | To prepare egg water |
Disposable transfer pipette | SciLab | SL.Pip3032 | To transfer zebrafish larvae |
Egg water | N/A | N/A | 0.6 g sea salt in 10 L deionized water |
Forceps | Karl Hammacher GmbH | HSO 010-10 | Forceps used for sample positioning |
Low melting point agarose | Thermo Scientific | R0801 | 2% (wt/vol) agarose gel |
Napari | Napari | N/A | To visualize microscopy images in 3-D |
NIS-Elements C | Nikon | N/A | Imaging software for confocal microscope |
Pancuronium bromide | Sigma-Aldrich | P1918-10MG | 0.25 mg/mL of pancuronium bromide solution |
Petri dish, 35 mm | SPL Life Sciences | 11035 | Petri dish used for sample embedding |
Petri dish, 55 mm | SPL Life Sciences | 11050 | To prepare zebrafish larvae after screening |
Point-scanning confocal microscopy system (C2 Plus) | Nikon | N/A | Confocal microscope for whole-brain imaging |
Sea salt | Sigma-Aldrich | S9883-500G | Sea salt used for preparing egg water |