Custom 3D-Printed Molds for Zebrafish Imaging and Cardiac Development

Summary

Here we describe a method using an inexpensive stereolithographic 3D printer to create molds to facilitate the reproducible mounting of zebrafish embryos for imaging studies. Using this method, we have created molds for imaging cardiac morphogenesis in zebrafish embryos.

Abstract

Embryo mounting is one of the technical challenges researchers encounter when undertaking an imaging project. Embryos need to be oriented in a reproducible manner such that the tissue of interest is accessible to a microscope objective for the entire imaging period. To overcome this challenge, researchers can embed embryos in viscous media or create specialized dishes and casts to hold embryos in a desired orientation during imaging. Here, we describe a method for using an inexpensive stereolithographic (SLA) 3D-printer to manufacture reusable molds that create agarose wells in which embryos can be mounted for imaging. These agarose wells provide a reliable means for orienting multiple embryos for imaging. This method includes a design framework that can be easily customized for a variety of tissues, organisms, and imaging challenges. Using this method, we have created molds for imaging cardiac development in zebrafish for both upright and inverted microscopes. By utilizing materials and equipment that are accessible, this method allows researchers to easily create molds specific to their mounting needs.

Introduction

Visualizing cellular dynamics within an intact animal is essential for comprehending the pathologies of congenital diseases and the fundamental processes of development and homeostasis. This visualization encompasses imaging at multiple spatial scales to identify physiological dynamics across an entire organism as well as at the tissue, cellular and sub-cellular levels. The imaging modalities used for visualizing cellular dynamics range from immunofluorescence studies of fixed specimens to time-lapse imaging of live specimens. These imaging modalities require holding intact specimens in specific orientations, such that a region of interest is accessible to the microsc....

Protocol

The protocols, experiments, and findings described here were conducted in accordance with animal welfare regulations and guidelines, and the Institutional Animal Care and Use Committee at the University of Mississippi (protocol #24-008).

To create agarose wells of different shapes in which embryos can be oriented for imaging, we developed a design that utilizes a base attached to pegs (Figure 1A). The shape of these pegs (see arrow pointing to pegs in purple in Figure 1A) can be customized to orient embryos of different developmental stages and to focus on different tissues. Below,....

Results

The above protocol was used to design molds for imaging different stages of cardiac development in zebrafish, including cardiac fusion, heart tube formation, cardiac looping and chamber formation. Since cardiac development does not occur along the major anatomical axes of the embryo, the shape of the pegs were customized for different developmental stages in order to properly position the embryos. For example, the anterior inner placement of the zebrafish heart at 48 hours post-fertilization (hpf) relative to the spheric.......

Discussion

In this protocol, we outline a method for designing 3D-printed molds that create agarose wells which can facilitate the mounting of embryos or tissues for imaging studies. These molds can be made for imaging with either upright or inverted microscopes and the agarose wells made by using these custom molds can be used for imaging applications that range from immunofluorescence studies of fixed embryos to timelapse imaging of live embryos.

This method utilizes a foundation that contains two piec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SLA 3D Printer	Elegoo	MARS 2 PRO
Agarose	Apex Bioresearch; Genesee Scientific	20-102QD	Any brand may be substituted
Autodesk Fusion 360	Autodesk Inc.	https://www.autodesk.com/products/fusion-360/	Computer aid design (CAD) Software - more advanced
Axio-Zoom upright microscope	Zeiss	Axio Zoom.V16
Chitubox	CBD-Tech	https://www.chitubox.com/	Slicer software
Coverslip bottom dish (60 mm dish with 30 mm bottom well)	Cellvis	D60-30-1.5N
F59 Myosin heavy chain (all fast isoforms)	DHSB	F59	1:50 dilution
FDM filaments	Bambu	PLA filaments
FDM printer	Bambu	Bambu X1 Carbon FDM	0.4mm extruder nozzle
Forceps	Fisher	16-100-121	Other brands are likely to be suitable
Goat anti-mouse Alexa 488	ThermoFisher	#A28175	1:300 dilution
Imaris	Andor	10.2	Software
Inverted Confocal Microscope	Leica	Leica SP8 X
Low-melt temperature agarose	Gold Biotechnology	Catalog No: A-204-25	Other brands are likely to be suitable
Pipette pump	Bel-Art	F37898
Resin for SLA printer	Elegoo	Elegoo P05BB028	Water washable photopolymer Resin V2.0
Ricinus communis agglutinin (RCA-120)	Vector labs	#RLK-2200 (lectin kit)	1:300 dilution
TinkerCAD	Autodesk Inc.	https://www.tinkercad.com/	Computer aid design (CAD) Software - basic
Tricaine S	Western Chemical, Inc.	ANADA #200-226
Wheat germ agglutinin (WGA)	Vector labs	#RLK-2200 (lectin kit)	1:300 dilution
Wide-bore glass Pasteur pipette	Duran Wheaton Kimble (DWK)	63A53WT