A 3D-Printable device for non-invasive neuroimaging of adult zebrafish

Short Summary

This device is designed for use with an upright multiphoton microscope for neuroimaging of the adult and juvenile zebrafish. It allows for fast and reproducible mounting of fish with minimal restraint. This non-invasive approach allows for fish to be easily recovered from the imaging chamber for use in future experiments. We have demonstrated stable imaging for up to 2 hours, however Castranova et. al (DOI: 10.1242/dev.199667) have demonstrated recovery is possible from imaging sessions of 20+ hours.

This device can be 3D printed in biocompatible resin or Nylon-12 or produced in glass-filled nylon (our preference). The design features a removable restraint device which secures the fish in place via a V-shaped channel and a small well for low-melting point agarose. The use of the well confines the agarose to only the tail of the fish and allows the fish to be freed from the chamber with minimal effort. Importantly, this prevents low melting point agarose from getting near the fish’s gills. Dental wax can be affixed over top of the fish’s back for further stabilization.

The restraint device docks securely via magnets into the inner reservoir. We have designed the reservoir to include a second overflow reservoir, and two water outlet channels to prevent overflows. For unattended imaging, we recommend adding water overflow sensors. The inner reservoir also features a dock for a temperature monitor probe. The fish is intubated during the imaging period to allow for adequate oxygenation and delivery of anesthetic. To increase the ease of the intubation process, we have added a thumbscrew-adjustment on the intubation tubing support to change the position of the intubation tubing forward and backwards quickly and accurately. This allows for fine adjustments of tube length and position to accommodate different sizes of fish.

We have made three versions of the chamber available to suit multiple applications.

Standard Chamber: all imaging in Haynes et. al. 2026 was done with the standard chamber.
Multi-size Chamber: if you have a need to image large females with very prominent bellies, the multi-size chamber fits both the standard restraint device and the large size restraint device. Assembly is identical to the standard chamber, though the footprint of the restraint devices and some details of the chamber docking area are different from the standard chamber. Therefore, the restraint device from the standard chamber is not compatible and the multi-size versions of the restraint devices must be used.
Simplified Chamber: This chamber does not have an adjustable intubation tubing support, and therefore is easier and faster to assemble and only requires printing of 3 parts (the base, the clamp, and the restraint chamber). Tubing must be adjusted manually by cutting to length.