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 microscope objective.

Zebrafish embryos are an ideal model organism for imaging studies due to their optical transparency, genetic tractability, external fertilization and vertebrate origin. However, mounting zebrafish embryos in a specific orientation in a consistent and reproducible manner has represented a continual challenge. Mounting is especially challenging for the visualization of heart development in zebrafish embryos because cardiac morphogenesis occurs in different planes¹.

Multiple approaches have been developed to address the challenges of mounting embryos for imaging. One of the simplest methods involves mounting zebrafish embryos between two coverslips with layers of tape in between the coverslips to prevent crushing. This method allows for imaging on both inverted and upright microscopes². Zebrafish embryos can also be mounted in a highly viscous media or gel, such as methylcellulose or 0.1-0.8% low-melting-point temperature agarose, to hold embryos in specific orientations^2,3,4. These low-throughput approaches work well for many imaging projects; however, ensuring embryo integrity and reproducibly obtaining specific orientations can be troublesome. To increase imaging throughput and facilitate mounting, regularly spaced wells can be created from solidified agarose in an imaging dish^5,6,7,8. To facilitate the creation of these agarose wells, plastic molds have been created^9,10,11. While these molds can be applied to imaging along the standard dorsal-ventral-lateral axes^10,12, they are often made for specific imaging challenges, such as for use in 96-well plates¹³ or for the detection of the first heartbeat¹⁴. In specialized cases, entire imaging dishes have been created^5,15,16. For example, a microfluidic chamber (named ZEBRA) made out of PDMS facilitates both imaging and exposure of embryos to chemicals¹⁷. These molds are often made from silicone or machined from lucite^10,18,19. However, the advent of relatively inexpensive three-dimensional (3D) printers has democratized the creation of customized molds that are used to create agarose imaging wells.

3D printing, also called additive or layered manufacturing, involves producing a 3D object from a digital design by the sequential addition and fusion of individual layers^20,21. Stereolithography (SLA) utilizes ultraviolet (UV) light to cure a light-sensitive liquid resin in sequential layers²². SLA printers are cost-effective, fast, and highly accurate and can be used with a variety of resins, including those that deliver a smooth surface¹⁶. The process begins with computer-aided design (CAD) software, where digital models of the object can be designed de novo or modified from preexisting designs. The digital model is then virtually sliced into printable layers. Using these digital layers, a 3D printer constructs the object, layer by layer. Here, we present a method and framework for the custom design and creation of imaging molds using a low-cost commercially available SLA 3D-printer. Additionally, we use this framework to create molds for imaging different stages of zebrafish cardiac development on either inverted or upright microscopes and for imaging zebrafish embryos along their lateral, dorsal, or ventral axes.

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, we describe a protocol for customizing these pegs and for using this system to create agarose wells.

1. Creating a custom-designed imaging mold

1. Create a preliminary sketch of the specimen or embryo positioned in the desired orientation. Consider the desired embryo orientation and whether the microscope to be used contains upright or inverted objectives. Also consider the size of the dish, the number of embryos to be mounted, and the spacing between them.
2. Measure embryo dimensions (e.g., length, width, and height) using a microscope combined with a software tool such as FIJI²³. Use these dimensions to ensure that the embryos fit into the wells created by the pegs.
3. Identify a shape that corresponds to the embryo's geometry. Using the measurements from step 1.2 in combination with a computer-aided design (CAD) software create a peg that approximates the shape and size of the sample.
   NOTE: This shape can be created in a basic CAD software program by combining and overlapping different predesigned shapes, or the shape can be created de novo using an advanced CAD software program. Additionally, one can modify one of our predesigned pegs (see Supplemental File 1). Tapering the peg, as in Figure 1A, can help orient embryos to ensure the tissue of interest is closest to the objective.
4. Combine peg design with base design:
   1. In a CAD software program, add a custom-designed peg to one of the predesigned bases (Supplemental File 1). Then, duplicate and evenly place the pegs throughout the base for high-throughput imaging by using the tools in an advanced CAD program. Double check to ensure all pegs reach the same height away from the base.
5. Prepare the design for 3D printing:
   1. Transfer the customized mold design or one of the pre-designed molds (Supplemental File 2) to a slicer software, which converts the design into commands for the 3D printer.
   2. Within the slicer software, be sure to rotate the design so that critical components such as the pegs are positioned away from the printer stage. For the coverslip holder, which is printed on a fused deposition modeling (FDM) printer, position the print horizontally.
      NOTE: Rotation of the design will prevent warping, which can happen when delicate parts are positioned close to the printing stage.
   3. Add supports within the slicer software to ensure that delicate aspects of the mold do not break during the printing. The medium automatic support placement feature in the software along with manual curation ensures a good spread of supports without large gaps. A 100% infill strategy is used to create a solid mold.
   4. Print multiple pieces simultaneously by either using a single STL file containing all molds or arranging individual STL files together using the slicer software.
6. Printing
   NOTE: SLA printers are our preferred 3D printer type for the imaging molds due to their feasibility, precision, and their creation of smooth surfaces. An FDM printer is used to make the coverslip holder because it creates lightweight, stiffer prints than SLA printers.
   1. Start with the following print settings and optimize from there: 0.05 mm layer height; 5 bottom layer count; 2.5 s exposure time; 35 s bottom exposure time; 5 mm bottom lift and lifting distance; 60 mm/min bottom lift speed; 80 mm/min lifting speed; and 210 mm/min retract speed.
      NOTE: The print settings will likely need to be optimized by the user as they depend on resin type, room temperature, and model of printer.
7. After printing, wash the molds in water, remove the supports, and place the molds under UV light in a tissue culture hood to ensure the solidification of the resin.
   NOTE: Washing and curing the molds thoroughly is important for preventing resin from flaking off and for preventing the mold from becoming misshapen.
   Gloves and a lab coat should be worn when handling uncured resin and cleaning the molds, since uncured resin can be a skin irritant. SLA printers use UV light to cure the resin, which can be harmful to the eyes and skin. Ensure that the print chamber is securely enclosed and avoid looking directly at the UV light source.

2. A procedure for using 3D-printed molds to create agarose wells

1. Making agarose wells
   1. Make agarose wells by dissolving 2% (g/mL) agarose in E3 media (1.578 mM NaCl, 0.169 mM KCl, 0.249 mM CaCl₂*2H₂O, 0.161 mM MgSO₄*7H₂O). Place a mold in the center of a coverslip bottom dish with the arrow pointing down towards the well (see Figure 2A,B). Pipette 750 µL of 2% molten agarose/E3 along half of the imaging mold's perimeter (see Figure 2C).
      NOTE: Molten agarose should be non-viscous but cool enough to pipette without creating bubbles. We use 60 mm dishes with a 30 mm coverslip bottom well, while for molds designed for upright microscopes, a standard 60 mm dish can be used. A slightly different protocol is used for the screening mold (see below).
   2. Pipette another 750 µL of 2% molten agarose/E3 along the other half of the imaging mold's perimeter (see Figure 2D).
   3. Wait 5-7 min for the agarose to solidify, observing the transition from a clear liquid to an opaque blue gel (see Figure 2E).
   4. Add a thin layer of molten agarose around the perimeter of the solidified central agarose.
      NOTE: Although not strictly required, this step helps anchor the solidified agarose to the dish and prevent media-induced detachment.
   5. Place the outer ring (Supplemental Figure S1B) over the agarose mold assembly with the arrow pointed towards the agarose (see Figure 2F).
   6. While gently holding down on the outer ring, carefully pull straight up on the inner mold to detach it from the agarose (see Figure 2G).
      NOTE: To avoid breaking or damaging the agarose wells, do not push down too hard on the outer ring and do not twist or shift the inner mold when pulling upwards.
   7. Remove the outer ring, revealing the agarose wells in the coverslip bottom dish (Figure 2H).
   8. Inspect the agarose wells under a microscope to confirm correct sizing and production.
      NOTE: For imaging on an inverted microscope, a thin film of agarose often needs to be lifted off the bottom of the well. This can be done with forceps.
2. Modified protocol for using the screening mold
   1. Pour molten 2% agarose/E3 into the Petri dish until it is approximately halfway full.
   2. Hold the mold with the pegs facing upwards and pipet molten 2% agarose/E3 onto the pegs, until they are covered (~3 mL).
      NOTE: Ensure that bubbles are not introduced by releasing only 90% of the molten agarose that has been drawn into the pipet.
   3. Using a tilting motion, place one end and then the whole mold with pegs face down into the Petri dish containing molten agarose (Supplemental Figure S2).
   4. Allow the agarose to solidify for 5-7 min.
   5. Carefully remove the mold by using the handle to pry up one end and then the other end.

3. Mounting embryos in agarose wells with and without low-melting temperature agarose

1. Dechorionate and anesthetize embryos in a 200 μg/mL tricaine solution prior to mounting.
   NOTE: An ~400 μg/mL tricaine solution is required to stop the heart from beating.
   1. Without using low-melting-temperature agarose: Fill the imaging dish with 1x E3 and tricaine (200 μg/mL), add dechorionated anesthetized embryos to the imaging dish, use forceps to place and orient embryos in the agarose wells, and then image (see Figure 2I, J).
      NOTE: Because forceps can easily damage an embryo, an embryo poker in which 0.41 mm diameter fishing line is inserted into a capillary tube or pipette tip and secured with superglue can be used instead²⁴.
   2. Using low melting temperature agarose:
      NOTE: While the molds are designed to place embryos in specific orientations, using low melting temperature agarose to hold embryos in place can ensure that embryos do not drift during imaging or while transporting the imaging dish with the mounted embryos to the microscope. The steps below are derived from our previous protocol⁶.
      1. Create tubes of low-melting-temperature agarose by dissolving 0.1-0.8% low-melting-temperature agarose in 5 mL of E3 media and heating to 65-70 °C in a water bath. Occasionally, invert the tube until the low-melting-temperature agarose has dissolved. Aliquot the low-melting-temperature agarose solution into microfuge tubes and place them on a heat block set to 42 °C. Allow the solution to equilibrate to 42 °C for at least 1 h.
         NOTE: It is generally advisable to use a freshly made low-melting temperature agarose solution.
      2. Using a wide-bore glass Pasteur pipette, transfer a single embryo into a tube containing the low-melting temperature agarose solution. To avoid adding excess liquid to the low-melting temperature agarose solution, only transfer the embryo and liquid immediately surrounding it. Immediately transfer the embryo, which is now in low-melting agarose, to one of the agarose wells. Return the low-melting temperature agarose solution to the heat block for re-use.
      3. Using forceps or an embryo poker (see step 3.1.1), position the embryo in the well for imaging.
      4. Repeat steps 3.1.2.2-3.1.2.3 for the other wells. If the previous wells begin to dry out, use a p200 pipet to add a small amount of E3 (~50-100 μL) on top of the well.
2. Once embryos have been mounted in all wells and the low-melting temperature agarose has solidified, using a pipet slowly add the tricaine/E3 solution to the imaging dish.
3. After imaging, extract the embryos carefully from the wells using forceps and allow them to develop further to ensure imaging did not cause developmental defects.
   NOTE: The agarose wells can be removed from the imaging dish, which can be rinsed and reused. Rinsing with commercially available glass cleaner and then multiple times with ddH₂O helps to remove salt deposits on the coverslips. Although we have not experienced toxicity due to residual traces of glass cleaner, the researcher is advised to confirm these results using their own laboratory conditions.

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