The ability to respond adequately to injury is critical to the survival of every organism at every scale, from a single cell to multiple tissues. Wounding and its associated responses, such as phagocyte recruitment to damaged areas 1 are, therefore, significant topics in cell and tissue biology.
Wounds are sensed immediately after the tissue barrier is breached, causing a tissue gradient response involving wound contraction 2 , 3 that coordinates wound healing and subsequent regrowth 4 . Due to the mechanical nature of this contraction, tools used during experimentation must not physically impede the movement of cells near the injury site. longer-term behavior of cells near an injury site. Embedding embryos in low-melt agarose is sufficient for short-term imaging of unwounded tissue. However, the surrounding gel matrix restricts the contraction and relaxation of wounds and impedes the growth of the developing embryos over time 7 , 8 , 9 .
Microfluidics devices can immobilize zebrafish embryos at different developmental stages 10 , 11 , 12 , 13 , 14 , 15 , 16 , and some, such as the zWEDGI 7 , accommodate manual wounding. However, there are several drawbacks to available device designs. For example, many devices printed directly on plastic are incompatible with imaging on inverted microscope systems due to poor optical transparency. Furthermore, parallel channels allow multipositional imaging 10 , 11 , but the physical movement of the microscope stage introduces lag in image acquisition. Repeated iteration and optimization to solve these issues are difficult when considering the financial and technical investment required for every new design, especially for current gold-standard methods of device fabrication using polydimethylsiloxane (PDMS) soft lithography. The very first step, the creation of a master mold, often requires knowledge of 3D modeling software, access to specialized fabrication equipment, and (depending on the material used) an additional antiadhesion treatment such as silanization before the mold is ever used.
Curing the PDMS once poured takes at least one hour at high temperatures, and generally, must be done under a vacuum or with a clamp for best results, often in a clean room 7 , 17 , 18 .
As in developmental biology, these costs can rapidly prove prohibitive for experiments requiring many unique microfluidic environments across multiple models.
Of available alternative construction materials, polyethylene terephthalate (PET) is durable, nontoxic, and easy to manipulate. PET sheets are widely available plastic lamination pouches carried at most office supply stores, with pre-applied thermally-activated adhesive (generally ethylene vinyl acetate). By shaping these PET sheets using commercially available craft cutters (i.e., computercontrolled cutting plotters designed for home crafters) and adhering the layers to one another using standard thermal lamination equipment, a wide range of potential designs can be quickly generated and iterated. We have therefore adapted a previously described method of design and construction involving stacked PET 18
NOTE: Any part of this protocol may be paused at any time except for steps 1.8 and 1.9, which are sensitive to the drying time of cyanoacrylate.
1. Design the pattern for the device in the software of choice (e.g., Adobe Illustrator, Figure 1A). Depending on the age and size of the embryo, adjust the width of the channel and the size of the central imaging chamber for best fit.
1. Save each layer as an individual image file with identical dimensions. Name each file so that the thickness of the sheet to be used and the order of assembly are evident.
2. Initialize the cutting machine according to the manufacturer's instructions.
3. Upload each designed pattern to the design software for cutting.
1. In the design workspace, select the newly uploaded designs. Group patterns intended for the same thickness of PET sheet together in the same cut (Figure 2A). If necessary, readjust the size of the patterns in the design software to the correct dimensions.
2. Confirm the number and type of patterns in the cut, then select the cut material. NOTE: PET sheets may not be a material choice in the design software, including under names such as "plastic laminating pouch." Any material of similar thickness and texture (e.g., transparency sheets or non-adhesive vinyl) can be selected instead. 8. Secure the device to a 35 mm dish drilled with a ¾" hole using cyanoacrylate glue (Figure 2E,F). Adjust the shape and type of the dish according to experimental needs (e.g., any container large enough to accommodate the device-one well in a 24-well plate or a larger Petri dish). Use enough glue to seal the device fully to the dish without flooding it. NOTE: Securing the device to a holding dish is not required for device function.CAUTION: Cyanoacrylate bonds skin and eyes in seconds and irritates the respiratory system, eyes, and skin. Wear hand and eye protection and work in a well-ventilated area. 9. Waterproof the PET laminate by thoroughly coating the outer device edges with cyanoacrylate glue (Figure 2F).
Use enough glue to fully cover the outer edges. Allow the glue to cure for at least 2 h before use. NOTE: Ensure adequate room for cyanoacrylate fumes to vent. The buildup of fumes may cause cyanoacrylate to deposit on the device, obscuring optically transparent material.
1. Raise zebrafish embryos in the standard embryo-rearing medium E3 19 . If necessary, dechorionate embryos at least 1 h before imaging to allow them to flatten. This experiment used embryos at 3 dpf, but embryos can be up to 14 dpf (Figure 1E). 6. Using a hair loop, orient the embryos such that the tail of the embryo protrudes into the central imaging chamber and the yolk is securely immobilized in the wedge-shaped channel (Figure 1C). NOTE: Orienting embryos may be performed under a stereomicroscope for ease of manipulation.
7. If desired, secure the head of the embryo by adding 1.5% low-melt agarose into the holding chamber using a micropipette.
8. Move the device with embryos to the imaging system of choice to commence imaging. NOTE: Here, a laser power of 2.4 W for 30 s at 800 nm was sufficient to induce a full-thickness wound.
4. Image at room temperature using GFP (excitation 488 nm, emission 510 nm) and RFP (excitation 561 nm, emission 610 nm) channels using a frame interval of 1 min for a total of 1 h.
If necessary, release the embryos for retrieval after imaging via gentle suction near the head using a transfer pipette or swirling the dish.
1. At least 1 h before imaging, transfer the embryos to E3 containing half the standard concentration of anesthetic, in this case, 82 mg/L MS-222.
2. Select the appropriate objective for imaging (here, an inverted laser scanning confocal equipped with a 10x objective).
3. Adjust the sample focus and imaging parameters (e.g., laser power: 5, gain: 90, pinhole size: 2 AU, exposure time [scan speed: 0.5 frame/s])) for optimal signal-tonoise ratio and acquisition speed. NOTE: It is recommended that samples be prefocused and parameters for each channel on the imaging system of choice be adjusted before wounding to reduce the lag between wound formation and image acquisition. 4. Wound four Tol2(mpx:Dendra2) 23 embryos via manual transection by applying pressure with a scalpel blade across the tail fin tissue under a stereomicroscope, then return the device to the stage.
5. Capture the image on the GFP (excitation 488 nm, emission 510 nm) channel at room temperature using a 5 min frame interval for 24 h.
imaging via gentle suction near the head using a transfer pipette or swirling the dish.
For comparison of image quality produced by a laser scanning confocal with and without the aid of the RADISH, embryos expressing the intensiometric calcium biosensor GCaMP6f 22 , 24 in the outer epithelial layer were stained Embryos were wounded parallel to the sides of the central chamber using a scalpel blade (Figure 4A and Video 3) or by direct laser ablation with an IR laser (Figure 4B), then imaged through the full thickness of the tail fin fold.
To demonstrate the effectiveness of the RADISH for longerterm imaging, we investigated immune cell recruitment to the wound edge for 24 h after wounding. Four zebrafish embryos at 3 dpf expressing Tol2(mpx:Dendra2) to label neutrophils in green 23 were wounded by tail transection and imaged for 24 consecutive hours (Figure 5). Imaging artifacts typically associated with late 3 dpf to 4 dpf embryos, such as tilting and floating, were notably absent (Video 4), and tissue regrowth continued unimpeded for the experiment. Neutrophil recruitment to the wound edge continues throughout the image (Figure 5B), with most migrating cells leaving by 24 h post wounding (Figure 5C), consistent with expected inflammation resolution 25 , 26 . Copyright © 2024 JoVE Journal of Visualized Experiments jove.com September 2024 • 211 • e67313 • Page 8 of 14 Supplemental File 1: Vector and PNG files for RADISH device design as shown in Figure 1A. Please click here to download this File. Video 1: Brightfield video of manual tailfin transection using RADISH device design. Abbreviation: RADISH = Rotational Assistant for Danio Imaging of Subsequent Healing. Please click here to download this Video. Video 2: Comparison of unmounted versus mounted 3 dpf Tg(krt4:Gal4, UAS:GCaMP6f) embryos from Figure 3, acquired at 1 frame/min. Please click here to download this Video. Video 3: Calcium transients in Tg(krt4:Gal4, UAS:GCaMP6f) x (cdh1-dTomato) xt18 embryos wounded via scalpel transection or laser ablation from Figure 4, acquired at 1 frame/min. Please click here to download this Video. Video 4: Long-term imaging of leukocyte recruitment
The core premise of the PET lamination method involves cost reduction and minimization of technical barriers to entry compared to traditional means of creating microfluidic devices, such as soft lithography or PDMS molding. As such, the only critical steps in this protocol are the accurate alignment of PET layers during lamination and waterproofing of the device edges after construction. All other parts of the method may be modified according to experimental needs (e.g., if immobilization of younger or older embryos is required, the thickness of the PET layers can be adjusted to narrow or widen the channel accordingly). Designs can be created in any shape and laminated onto any base material as long as the material is heat-safe and thin enough to be sent through the laminator 18 , 27 . As PET sheets are cut using commercially available craft cutters, which read twodimensional rather than three-dimensional image files, the design and iteration of any given device require only the ability to manipulate black and white shapes in an image editing program, dramatically decreasing the difficulty. This flexibility in software choice and the commercial availability of materials used represent a dramatic increase in the accessibility of microfluidics devices in general, enabling their use in any lab or classroom for an upfront cost of less than $500 at the time of writing this paper, compared to over $2000 in starting equipment costs for PDMS mold fabrication 17 .
Furthermore, the time required to create a single useable device using this method is dramatically reduced compared to typical soft lithography. Assuming familiarity with the requisite software, one unique PET laminate microfluidic device can be conceptualized, manufactured, and immediately used in ~3 h, including the curing time of cyanoacrylate. Iteration takes even less, depending on the scale of edits needed to the original design files, and multiple variant devices can be made in parallel with minimal additional time invested and no extra equipment. Although devices created using this method are less durable than those made with PDMS for a variety of reasons (including adhesive failure and internal stresses for shapes with many layers and long aspect ratios), their inexpensive nature and scalable fabrication mean that they are far more easily replaced. As commercial craft cutters are marketed with the ability to repeat the same precise cut over and over and PET has no expiration date, the individual layers of a completed design can be pre-prepared in bulk for assembly and use at any point in the future, further reducing the overall time investment required.
However, speed and variability come at the cost of precision.
The main limitation of PET lamination compared with traditional PDMS molding is spatial resolution. Commercial craft cutters often struggle with sharp angles on shapes smaller than approximately 500 µm, and PET lamination sheets are not available at thicknesses under 33 µm (not including the thickness of the adhesive itself, which is not included in the measurement on the package), significantly limiting the scale at which this technique can be applied.
The maximum number of layers is also restricted by the size of the laminator, which may vary from manufacturer to manufacturer.
The final version of the protocol, including variations in registration and alignment technique, materials suppliers, and software, will largely depend on user preference. However, the method's accessibility and overall flexibility reduce the cost of adaptation. Individual users are
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