Rapid Mounting of Zebrafish Larvae for Brain Imaging

Abstract

Brain imaging requires mounting of zebrafish larvae in a vertical position, but anesthetized or fixed larvae tend to fall on their sides without external support. Current solution is to manually hold individual larva until liquid agarose solidifies, which is time consuming, labor intensive, and unfriendly to beginners. We developed a method to form larva-shaped slots in agarose gel using a computer numerical controlled manufactured mold. Each slot nearly perfectly fits a larva in its upright position, and larvae can be easily mounted by inserting into the slots. On average, each larva can be mounted in <1 min using this method.

Introduction, Results, and Discussion

The zebrafish has become an increasingly important model organism for neuroscience research. The transparent nature of zebrafish larvae allows whole brain imaging in live or fixed intact animals.¹ Comparing between multiple brains has also been made possible: several studies have used whole-brain registration to examine functional and anatomical differences between groups of imaged brains.^2–7 Brain imaging usually requires larvae to be mounted in a vertical position in agarose, with dorsal side facing up or down depending on the purpose of the study and the microscope configuration. The brain registration methods also require mounting and imaging a large number of larvae (typically ∼20 to 30 per condition) to achieve sufficient resolution in the analysis.

However, the spindle-shaped body of zebrafish larvae makes it difficult to remain stable while sitting vertically during the mounting process. The traditional solution to this issue is by mounting just one or a few (only for highly skilled researchers) larvae at a time and manually hold each larva until the agarose gel solidifies. It is often challenging for beginners to master this technique: failure rates are usually high, with many larvae falling on their sides before agarose gel solidifies or damaged/killed during handling. Even skilled researchers can find this procedure time consuming and labor intensive. Furthermore, when imaging multiple larvae, the task of locating and orienting each larva under the microscope becomes troublesome if they are not properly aligned in the imaging chamber during the mounting process.

To solve these issues, inspired by a histology embedding technique,⁸ we developed a method for generating regularly positioned larva-shaped slots in agarose gel to facilitate rapid and convenient mounting of larvae. Each slot is generated by pouring liquid agarose around a “tooth” in the shape of a larva. The previous mold design⁸ relied on surface tension to adhere larvae to the side of each agarose slot, therefore, the size of each slot was significantly larger than a larva (width × length: 0.8 × 5.5 mm).

When trying to adopt this method for mounting, we found that the larvae frequently fell on their sides inside the agarose slots during mounting; once a larva fell on its side inside a slot, it was extremely difficult to reinstall it to a vertical position without either hurting/damaging the larva or ripping the agarose mold. Even if we were able to fix larvae in position by pouring additional agarose into the slots before they fell, due to the extra space in these slots, the larvae's head positions could easily twist or rotate before agarose solidifies.

In this study, we have improved upon the previous method by empirically adjusting the dimensions of each tooth to closely fit a 5–7 days postfertilization (dpf) larva (width × length: 0.6 × 4.5 mm) (Fig. 1A, B), enabling each slot to restrain a larva in a vertical position without squeezing it (Fig. 1F, G), thus providing superior restraining power and enhanced positional stability of larvae in all horizontal and vertical directions, preventing larvae from falling, twisting, or rotating once they are placed inside the slots.

FIG. 1.

Rapid mounting for zebrafish brain imaging using zMold. (A) The design of a “tooth” for making agarose slots. The dimensions were empirically determined to closely fit a 5–7 dpf larvae. (B) The design of the eight-teeth zMold. (C) A front view photo of the eight-teeth zMold. (D) Agarose mold made using the eight-teeth zMond in a 35 mm glass-bottomed dish. Arrows showing the indented region generated by the acrylic block and the slots generated by the teeth. (E) Image showing larvae inserted into the slots. (F) A closer look at several larvae inserted into the slots. Fixed larvae were submerged in PBS. No agarose was added to the slots. (G, H) A closer look at a slot containing a larva (G) or left empty (H). Fixed larvae were submerged in PBS. No agarose was added to the slots. The high-contrast elements and shadows in the images were due to reflection by PBS and will be eliminated after larvae are mounted by adding agarose to the slots. (I, J) Confocal imaging slices of 7 dpf triple transgenic tuba:mCar, vglut2a:GFP, and gad1b:RFP larvae, mounted using zMold (I) or using the traditional manual mounting method (J). The brain is often mounted in a tilted position using the traditional mounting method (J), which can negatively affect brain registration efforts. dpf, days postfertilization; PBS, phosphate-buffered saline. Color images are available online.

We focused on 5–7 dpf larvae because these are the developmental stages most often chosen for brain imaging studies. An arbitrary number of teeth can be placed next to each other on a base block to enable mounting of multiple larvae. A minimum distance of 1 mm is needed between each tooth for manufacturing purposes (Fig. 1B). The mold was manufactured by a computer numerical controlled (CNC) mill using acrylic as building material (Fig. 1C). We named it zMold, with z stand for zebrafish.

We tested an eight-teeth zMold that fits well on the coverslip section of a 35 mm glass-bottomed dish, a commonly used imaging chamber. By pouring 2% liquid agarose around the eight-teeth zMold and waiting for the agarose to solidify, an agarose gel containing eight slots can be generated in a matter of minutes (Fig. 1D, H). Live (anesthetized) or fixed larvae are gently pushed into each slot using a soft poking device (we use a gel loading pipette tip with part of the long tip cut off to increase its strength), with their dorsal side facing up or down depending on the user's needs (Fig. 1E–G).

After all larvae are inserted, a small amount of 1% liquid low melting point agarose was gently added to the slots to fix larvae in position. The larvae are now mounted and ready for imaging. With little practice, a first-time user can easily mount eight larvae within 5 min, averaging to <1 min per larva. Locating larvae also becomes easy during imaging, especially for microscopes equipped with a motorized stage: as each slot is perfectly aligned, after finding the first larva in the microscope's field of view, the other larvae can be located by simply moving the motorized stage 1 mm at a time in the desired direction.

We now routinely use the eight-teeth zMold to mount live and fixed larvae for brain imaging (Fig. 1I). Rotational variabilities in all axes—pitch, roll, and yaw—are consistently reduced compared with manually mounted larvae (Fig. 1J). A step file containing the eight-teeth zMold design is provided as Supplementary Data.

The zMold method is a rapid, consistent, and easy-to-learn approach for mounting zebrafish larvae for brain imaging. zMold can be built on different shapes of base block and with different number of teeth to accommodate various types of imaging chambers and applications. If needed, the tooth design can be adjusted in the future to potentially mount larvae of different developmental stages for brain imaging. In addition, although lateral mounting of larvae is considerably easier than vertical mounting since unconscious/fixed larvae naturally fall on their sides, a similar device designed specifically for lateral mounting will nevertheless be useful for improving the consistency of sample alignment, especially when a large number of larvae need to be imaged at the same time.

Authors' Contributions

Y.G. conceived the project, designed the equipment, conducted the experiments, and wrote the article. R.T.P. conceived the project, analyzed data, and revised the article.

Disclaimer

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the L.S. Skaggs Presidential Endowed Chair and by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number K99ES031050.

Supplementary Material

Supplementary Data